CN1188512A - Efflux pump inhibitors - Google Patents

Abstract

Methods are provided for screening for inhibitors of microbial efflux pumps including those which export antibiotics. The screening methods are based on the increase in the intracellular concentration of a compound, such as an antibiotic, when the bacterial cells are contacted with an efflux pump inhibitor. In addition, this invention provides pharmaceutical compositions containing such efflux pump inhibitors, and methods for treating microbial infections using those compositions.

Description

Efflux pump inhibitors

Invention field

The present invention relates to distinguishing and authentication method of biocide field and potential biocide.More particularly, the present invention relates to such biocide, the adjusting of outflow pump on its mode of action and the cell and outflow pump is relevant.Background

Following background material is not considered to the prior art of unsettled claim, only is to offer help for reader's understanding.

In last half a century, microbiotic is the effective tool of treatment infectious diseases.Because the development of antibiotic therapy to the later stage 1980s, almost can be controlled bacterial infection fully in developed country.The appearance of drug-resistant bacteria especially in the later stage 1980s and the nineties early stage, is changing this situation.In main hospital and medical centre, increasing of antibiotic resistance bacterial strain is particularly general.The consequence that endurance strain increases comprises higher M ﹠ M, the longer length of patient stay and the raising of medical expense.(B.Murray.1994. " New England Journal of Medicine " (New Engl.J.Med.) 330 volume 1229-1230 pages or leaves).

The frequent use of microbiotic under hospital environment selected many antibiosis have chemical sproof bacterial population.These colonies comprise that those toxicity may be not strong, but itself have chemical sproof pathogenic bacteria to many antibiosis.These bacteriums usually infect the patient of in poor health or immunologic hypofunction.The resistance colony that occurs also comprises the pathogenic agent bacterial isolates that those have been known, and they are in the past to antibiotic sensitive.The new resistance that obtains comes from dna mutation usually, or comes from the resistance plasmid (R plasmid) that changes over to from other organism or have chemical sproof transposon.Infection with any bacterial population in the pathogenic agent of natural bacterial drug resistance or the antibiotic resistance pathogenetic bacteria is unusual difficulty with present antibiotic therapy, thereby needs to overcome the new antibiotic molecule of resistance mechanism.

Bacterium has some different mechanism to the antiviral antibiotic effect.These resistance mechanisms may be special to a certain antibiotic molecule or family, and perhaps the possibility right and wrong are special, incoherent antibiotic resistance are all had relate to.Several resistance mechanisms may be present in the single bacterial strain and these mechanism may work independently or act synergistically certain microbiotic of antagonism or one group of antibiotic effect.Concrete mechanism comprises the degraded of medicine, and enzymatically modifying causes the deactivation of medicine, and the change of drug target molecule (B.G.Spratt " science " (Science), 388 pages of 264 volumes, 1994).Yet, also exist more general mechanism of drug resistance, promptly by reducing microbiotic to intracellular transportation or improve medicine and block or reduce the approaching of microbiotic and target molecule from cell effluxing of medium outside born of the same parents.Make bacterium in the presence of one or more microbiotic, to survive thereby two kinds of mechanism all can reduce medicine in the concentration of target site, otherwise these microbiotic will suppress or kill these bacterial cells.Some bacteriums can utilize above-mentioned two kinds of mechanism and combination to have the low permeability (H.Nikaido, " science " be 264 volume 382-388 pages or leaves (Science), 1994) that active microbiotic effluxes the cell walls (comprising film) of effect.

In some cases, it is relevant with the structure of bacterium plasma membrane to come from the antibiotic resistance of low permeability.Usually, according to the structure of the plasma membrane that is wrapped in kytoplasm, bacterium can be divided into two big classes.Gram-positive (G+) bacterium has only a skim, i.e. plasma membrane.By contrast, Gram-negative (G-) bacterium has two membranes, one deck plasma membrane and one deck adventitia.The film of these bacteriums is proteinaceous lipid bilayer and can combines with other molecule.The permeability of bacterial film can influence antibiotic susceptibility/resistance, although because some antibiotic target molecules, and as penicillin-binding protein, but touch from the plasma membrane skin, but antibiotic most of target molecule all is arranged in kytoplasm or at the internal layer of plasma membrane.Thereby for the microbiotic of target molecule in plasma membrane, this microbiotic need at first pass adventitia in gram negative bacterium.If target molecule is in kytoplasm, microbiotic need pass plasma membrane in gram positive bacterium, then need pass adventitia and plasma membrane two membranes in gram negative bacterium.To all two kinds of films, microbiotic can spread through film or utilize the film movement system to pass film.

For gram negative bacterium, the fat composition of its adventitia has constituted an effective permeability barrier.The skin of this adventitia contains fat, lipopolysaccharides (LPS), and lipopolysaccharides only sees the adventitia of gram negative bacterium.The fat layer of adventitia is with quasicrystal mode height systematism and have low-down flowability.Because the low flow of adventitia fat layer, even lipophilic microbiotic can not rapid diffusion see through the fat layer.This has been demonstrate,proved by experiment shows that the hydrophobicity probe molecule seldom is assigned to the hydrophobic region of LPS, and its speed that sees through the adventitia bilayer only arrives one of percentage for seeing through 1/50th of common phospholipid bilayer (as the plasma membrane bilayer) speed.

Some microbiotic can see through film by aqueous porin passage or by special haulage system.Yet many porin channel diameters are very narrow, can not allow bigger antibiotic molecule spread effectively.In addition, many porin passages are highly hydrophilic environment, can not allow hydrophobic molecule pass through effectively.Therefore, adventitia can be used as micromolecular molecule fence, but this partial interpretation, why gram negative bacterium is more insensitive to microbiotic than gram positive bacterium usually, and why gram negative bacterium is usually to big microbiotic, as glycopeptide, have more resistance, because they can not see through adventitia.

Also provide a diffusion barrier to some microbiotic plasma membrane.Yet because the flowability of plasma membrane fat layer is greater than the flowability of gram negative bacterium adventitia, lipophilic medicine can see through the fat layer.Some other medicine, as phosphonomycin or D-seromycin, they have low-down solvability in the lipophilic environment, can utilize haulage system to pass plasma membrane, in this case, if haulage system is not synthetic, bacterium will produce resistance (Peitz etc. to medicine, 1967, " journal of biological chemistry " be 6 volumes (Biochem.J.), 2561 pages).

Number by reducing porin or the number that reduces certain porin reduce the permeability of adventitia, can reduce a certain bacterial strain to many antibiotic susceptibility, because reduced the speed that microbiotic enters cell.Yet for most antibiotics, its half balance phase is very short, if do not have other mechanism, these microbiotic can be brought into play its effect.The outflow pump is exactly an example in these other mechanism.In case enter kytoplasm or pericentral siphon, medicine will be transported back the medium of outside.This transportation mediates by the outflow pump, and they are made up of protein.Different pumps can efflux a kind of or one group of medicine specifically, can transport quinolone as the NorA system, or TetA can transport tsiklomitsin, and perhaps they can efflux many different molecules, as certain outflow pump on the Pseudomonas aeruginosa.Usually, the outflow pump contains the kytoplasm composition and molecule is transported cell needs energy.Some outflow pumps have secondary plasmalemma protein matter, and they extend pericentral siphon.Have at least some outflow pump of Pseudomonas aeruginosa to have three grades of protein that are positioned at adventitia.

The outflow pump is relevant with antibiotic resistance, because in some cases, they can remove the antibiotic molecule that major part manages to enter cell, thereby keeps antibiotic concentration in the low-down cell.Now illustrate the Pseudomonas aeruginosa mutant strain 799/61 of laboratory-induced.Can not produce any outflow pump that measures, its susceptibility to tsiklomitsin and the ring third fluorine paraxin is higher 8 to 10 times than its male parent bacterial strain Pseudomonas aeruginosa 799, and its male parent bacterial strain can synthesize the outflow pump.Equally, the null mutant mexA of the kytoplasm composition of Pseudomonas aeruginosa outflow pump, more responsive than wild-type to microbiotic.

The physiological function of outflow pump is not also understood fully.They are relevant with the tolerance of medicine, have also participated in the normal physiological activity of bacterial cell.Be encoded in outflow pump in the Pseudomonas aeruginosa mexA operon and be proved the iron ion content that is subjected in the medium and regulate and control, and regulated and control jointly with the synthetic of siderophore acceptor.Siderophore be bacterium under iron deficiency ion condition, as in the zoogenetic infection process, the necessary molecule of growing.When bacterial cell needed iron ion, they synthesized in kytoplasm and efflux.Siderophore is removed iron ion and iron ion is returned to microbe to be used for basic microbial life process in infected animals.Because in animal body, be included in the human body, do not have the free iron ion basically, the siderophore that bacterial infection produces is an important virulence factor in course of infection.

Even the organism that is coated with the tunicle with relative high-permeability usually can produce resistance by the permeability that reduces tunicle.When certain reagent mainly diffused through barrier by certain special passage, the sudden change of passage is lost may become chemical sproof actual mechanism.The 'beta '-lactam compounds imipenum of " non-classical " shows unusual anti Bacillus pyocyaneu Flugge activity, and main because this medicament can be by a special passage OprD diffusion, the physiological function of this passage is seemingly transported primary amino acid.Yet by losing the oprD passage simply, Pseudomonas aeruginosa can obtain the resistance to imipenum, at present the equal tool resistance owing to this modification of isolating most of Pseudomonas aeruginosa strain from hospital environment.In the same way, the 'beta '-lactam compounds of imitation iron chelating agent (siderophore) design sees through the known defective mutant of the special transportation of siderophore of selecting in the transportation of adventitia at them.

Put it briefly, influence the factor of the transportation (active and passive transportation) that microbiotic enters bacterial cell in the top discussion hint cell, concerning many bacterial species, be antibiotic resistance important component.General introduction

The invention provides the method for screening efflux pump inhibitors compound, these compounds can suppress the outflow pump of bacterium or other microorganism cells.The mode that these outflow pumps can energy rely on effluxes substrate molecule from kytoplasm, the substrate molecule that effluxes comprises antibacterial agent.Such efflux pump inhibitors can be used for, and for example, can allow or promote the effect of effluxing of the compound of its growth to treat infected by microbes by effect of effluxing or prevention microorganism (as the bacterium) synthetic that reduces the biocide that gives jointly.Reducing the example that this compound effluxes effect is by reducing the utilization that suppresses iron ion that effluxes of siderophore.Therefore, the present invention also provides composition that contains these efflux pump inhibitors and the method for the treatment of infected by microbes with these compositions.Thereby, the invention discloses one and be applicable to that many combating microorganisms agent have the methods of treatment of chemical sproof pathogenetic organism.Particularly suitable example is the pathogenic bacteria Pseudomonas aeruginosa in such microorganism, and it has the inherent resistance to many antiseptic-germicides commonly used.This bacterial exposure can obviously be slowed down antiseptic-germicide from effluxing of cell interior or effluxing of siderophore in certain efflux pump inhibitors.Therefore, if another kind of antiseptic-germicide and efflux pump inhibitors co-administered, this antiseptic-germicide will be accumulated to the growth of suitable concentration with the inhibition bacterial cell, otherwise owing to efflux effect, it can only keep lower intracellular concentration.According to the kind of used antiseptic-germicide, this restraining effect is attributable to bacteriostatic action or germicidal action.Although Pseudomonas aeruginosa is the example of a suitable bacterial classification, other bacterium may contain similar wide substrate pump with microbe species, and they can efflux many biocides on one's own initiative, thereby also can become suitable target.Except that above-mentioned, for some bacterial classification, efflux pump inhibitors can reduce the virulence of bacterium, for example, and by suppressing the transportation of important pathogenic factor.Be example once more with the Pseudomonas aeruginosa, the inhibition of this bacterium outflow pump has been suppressed the picked-up of iron ion, this is pathogenic to it to be very important.Related to the molecule that is called siderophore in the mechanism of bacterium iron ion transportation, it is synthesized by bacterial cell and is effluxed by the outflow pump.These siderophore molecules combine and are absorbed by bacterium with the iron ion of removing from the host tightly.In this way, obtain the required iron ion of bacterial metabolism, be maintained thereby infect.

Therefore, illustrate the effect of efflux pump inhibitors, the inhibition of Pseudomonas aeruginosa outflow pump can produce one or more following biological effects:

1. the Pseudomonas aeruginosa strain will become the antibiotic sensitive that can not be used for the pseudomonal infection treatment to those, or become more responsive to the microbiotic that can suppress the Rhodopseudomonas growth.

2. the Pseudomonas aeruginosa strain will become responsive more to the microbiotic that is used for the treatment of pseudomonal infection at present.

3. because the utilization of iron ion is hindered, the virulence of Pseudomonas aeruginosa weakens.

4. the inhibition of a pump composition may be fatal.These effects even obtain a kind of infection that can treat this bacterium.Simultaneously, as previously mentioned, similar pump also is found in other microorganism.Above some or all of these effects also can from these microorganisms, obtain, thereby they also are the suitable target compounds that detects or use efflux pump inhibitors.Therefore, term " microorganism " comprises, for example, and bacterium, fungi, yeast, and protozoon.

Therefore, first aspect the invention provides a kind of method of screening the special efflux pump inhibitors of non-tsiklomitsin.This method comprises when certain the antiseptic-germicide condition following time of bacterial growth in the inferior inhibition concentration of non-zero, determine to produce the growth of the bacterium of the special outflow pump of non-tsiklomitsin, and this antiseptic-germicide will be discharged usually by the special outflow pump of the non-tsiklomitsin in the bacterium.If be not suppressed, it is low-level that the outflow pump will make the intracellular concentration of antiseptic-germicide maintain, so antiseptic-germicide can not suppress cell.Yet, can significantly slow down or stop the compound that antiseptic-germicide effluxes if also exist, this medicament will be accumulated to a higher concentration in born of the same parents, thus antiseptic-germicide can suppress the growth of cell under higher intracellular concentration.

In certain preferred aspects, composition of outflow pump with outflow pump or have 50% amino acid sequence similarity at least by the outflow pump of Pseudomonas aeruginosa strain K385 overexpression or by the polypeptide of the part of the outflow pump of Pseudomonas aeruginosa strain PAO4098E overexpression for Pseudomonas aeruginosa mexA/mexB/oprM.Because the above-mentioned sequence similarity of this outflow pump one polypeptide fraction, this outflow pump are known as Pseudomonas aeruginosa type outflow pump.This pump will efflux one or more non-tetracycline compounds (but also can efflux tsiklomitsin), comprising, for example, the antiseptic-germicide of other type and virulence factor.In particularly preferred embodiments, the outflow pump is a Pseudomonas aeruginosa outflow pump, is naturally occurring a kind of outflow pump in Pseudomonas aeruginosa.Some this Pseudomonas aeruginosa outflow pumps are mentioned in the above.Equally in particularly preferred embodiments, bacterium is a Pseudomonas aeruginosa, for example, and bacterial strain K385 or bacterial strain PAO4098E.

Equally in an embodiment preferred, bacterium can be from other bacterial classification, as following any:

Pseudomonas aeruginosa, Pseudomonas fluorescens, pseudomonas acidovorans, Pseudomonas alcaligenes, pseudomonas putida is had a liking for maltose oligotrophy Zymomonas mobilis, the onion Burkholderia, Aeromonas hydrophila, intestinal bacteria, citrobacter freundii, Salmonella typhimurium, salmonella typhi, salmonella paratyphi, Salmonella enteritidis, shigella dysenteriae, shigella flexneri, Shigella sonnei, enterobacter cloacae, enteroaerogen, Klebsiella pneumonia, acid-producing Klebsiella bacterium, serratia marcescens, soil draw hot Frances Salmonella, morganella morganii strain, proteus mirabilis, proteus vulgaris produces the alkali Providence, providencia rettgeri, providencia stuartii, acinetobacter calcoaceticus, acinetobacter haemolyticus, yersinia entero-colitica, Yersinia pestis, artificial tuberculosis yersinia genus, middle Yersinia, Bordetella pertussis, Bordetella parapertussis, bordetella bronchiseptica, Haemophilus influenzae, Hemophilus parainfluenzae, the haemolysis hemophilic bacterium, secondary haemolysis hemophilic bacterium, Ducrey bacillus, multocida, the haemolysis pasteurella, branhamella catarrhalis, helicobacter pylori, embryo's Campylobacter, campylobacter jejuni, large intestine Campylobacter, B. burgdorferi, vibrio cholerae, Vibrio parahaemolyticus is invaded the lung legionella, Listeria monocytogenes, Diplococcus gonorrhoeae, Neisseria meningitidis, vagina Gardner Salmonella, bacteroides fragilis, bacteroides distasonis, bacterioide 3452A homology group, bacteroides vulgatus, ovum bacterioide, bacteroides thetaiotaomicron, bacteroides uniformis, bacteroides eggerthii, bacteroides splanchnicus, clostridium difficile, mycobacterium tuberculosis, mycobacterium avium, Mycobacterium intracellulare, Mycobacterium leprae, corynebacterium diphtheriae, ulcer rod bacillus, streptococcus pneumoniae, streptococcus agalactiae, streptococcus pyogenes, enterococcus faecalis, faecium, streptococcus aureus, staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcus hyicus pig subspecies, staphylococcus haemolyticus, staphylococcus hominis, Staphylococcus saccharolyticus.

Term " outflow pump " refers to a protein combination, and it can efflux substrate molecule from kytoplasm or cell pericentral siphon, and this effect of effluxing needs energy.Therefore, the outflow pump generally is positioned on the plasma membrane of cell (striding plasma membrane).Pump may stride across periplasmic space and may exist part to stride across the outflow pump of adventitia in gram negative bacterium.Specific outflow pump will comprise a polypeptide, and it has 50% amino acid sequence similarity at least with polypeptide for the part of the outflow pump of the outflow pump of the outflow pump of Pseudomonas aeruginosa mexA/mexB/oprM or Pseudomonas aeruginosa strain K385 overexpression or Pseudomonas aeruginosa strain PAO4098E overexpression.Because the outflow pump is formed the above-mentioned sequence similarity of polypeptide, such outflow pump is known as Pseudomonas aeruginosa type outflow pump.

Term " non-tsiklomitsin specificity outflow pump " refers to tsiklomitsin is not had the outflow pump of high specific (other microbiotic relatively), thereby it is not a tsiklomitsin (tsiklomitsin is a special) outflow pump.This term thereby comprise the pump (effluxing multiple compound) of extensive substrate and the compound except that tsiklomitsin is had (comprising microbiotic) pump of high specific with different structure feature.Tsiklomitsin outflow pump is relevant to the special resistance of tsiklomitsin with bacterium.(Speer etc., 1992 " clinical microbiology comment " be 5 volume 387-399 pages or leaves (Clin.Microbiol.Rev.).) as mentioned, these pumps have high specificity to tsiklomitsin, their existence makes cell have high tetracycline resistant, but they can not provide other antibiotic resistance.The gene of tsiklomitsin pump composition is found in the plasmid of Gram-negative and gram positive bacterium and can be divided into two big class: tetA (A-E) and tetK and tetL.TetA-E tetracycline resistant sex factor contains a structure gene tetA, and it is a tsiklomitsin specificity pump, and a repressor gene, tetR, and it mediates derivable tetracycline resistant.The tsiklomitsin outflow pump that belongs to this class is called tetA (A), tetA (B), and tetA (D), tetA (E) is present in enterobacteria and other gram negative bacterium.TetK is the pump relevant with the tetracycline resistant of gram positive bacterium with TetL.Its gene is regulated by the reduction of translation, with tetA class homology not.

The use of term " a kind of screening method " refers to that this method is applicable to, usually is the effect of estimating a large amount of compounds simultaneously.In the present invention, such screening is to determine that directly certain compound suppresses the ability of certain outflow pump.In this article, " screening " and detection difference to some extent, a certain method only is suitable for estimating one or more compounds in detection.

" efflux pump inhibitors " refers to disturb specifically the outflow pump to efflux its normal substrate or other compound as antibiotic compound ability.This inhibitor may have the inherent ability of antimicrobial (as antibiotic), but its main related activity is because the outflow pump suppresses active at least.Particularly interested among the present invention is that those can suppress to efflux effect or suppress the active compound of outflow pump with the extensive substrate scope that comprises antiseptic-germicide.Term " non-tsiklomitsin specificity efflux pump inhibitors " refers to suppress the efflux pump inhibitors of certain non-tsiklomitsin specificity outflow pump.Term " Pseudomonas aeruginosa type efflux pump inhibitors " refers to suppress the efflux pump inhibitors of Pseudomonas aeruginosa type outflow pump." Pseudomonas aeruginosa efflux pump inhibitors " refer to suppress to be present in Pseudomonas aeruginosa the outflow pump efflux active efflux pump inhibitors.

" potential " efflux pump inhibitors refer to by or just by the test its compound as the ability of efflux pump inhibitors, from but one " test compound " or " efflux pump inhibitors of supposition ".

Herein, about bacterium or other microbial growth, term " growth " at first refers to the breeding of microorganism, and promptly quantity increases rather than the increase of size.Therefore this term is applicable to the unicellular or cell mass of one or more types.Yet this term refers to that also the cell of growing is just carrying out its metabolic process, and is not only just to carry out taking turns breeding, as division.Certainly this term also refers to the size increases as organic multinuclear types such as some fungies, and the number of simultaneous nuclear increases.

In the context of bacterium (equally to other microorganism) cell growth, term " inhibition " refers to that the growth velocity of bacterial flora reduces.Such restraining effect can be monitored, for example, the differences in turbidity of liquid nutrient medium when existing or not existing by inhibitor, or when existing or not existing by inhibitor on the solid medium difference in size of culture plaque monitor.

About the existence of outflow pump special on the bacterium, term " excessive generation " refers to compare with this bacterial classification natural modal (being generally the mutation of non-hospital) isolate, the existence of the bacterium that specificity outflow pump number significantly increases.This term is not that the pump that only refers to outflow is formed increasing of polypeptide number, but the increasing of outflow pump number of function arranged on the phalangeal cell film.As a result, the cell of excessive generation outflow pump than this bacterium only the volume production bacterial strain of giving birth to the pump that outflows can more effectively efflux substrate molecule.

The bacterial strain of excessive generation outflow pump thereby relative with " wild type strain ".The special outflow pump level that wild type strain produces is the common level of the natural strain isolated of this bacterial classification.But the more important thing is that the level that wild type strain produces special outflow pump is starkly lower than the level of the relevant bacterial strain of this special outflow pump of excessive generation.

Here the term of using " antiseptic-germicide " refers to the compound of the special bacteria growing inhibiting of energy.More at large, term, " biocide " refer to suppress specifically the compound of microorganism growth, thereby the explanation of this term also refers to other microorganism and biocide.Thereby this term comprises naturally occurring microbiotic and synthetic and semisynthetic compound, and these medicaments have sterilization or bacteriostatic ability.Usually, if certain antiseptic-germicide is a bacteriostatic, mean that this medicament can stop the growth of bacterial cell (but not killing bacteria) basically; If certain medicament is germ-resistant, mean that this medicament is with killing bacteria cell (can stop bacterial growth) before killing bacteria.But this term has difference clearly with the common virose compound of pair cell.Some examples of antiseptic-germicide class have quinolone (rotatory enzyme inhibitor), aminoglycoside, glycopeptide, sulfanilamide (SN), macrolide, beta-lactam and tsiklomitsin.May be antimicrobial (as antibiotic) agent when the efflux pump inhibitors among the present invention uses separately, and/or can excite the activity (improving the susceptibility of microorganism) of another kind of biocide, and/or can reduce the virulence of pathogenic agent this biocide.

A kind of " inferior inhibition concentration " of antiseptic-germicide refers to higher than zero, but is lower than the concentration of most of cell in the flora that can suppress this specific bacterial strain.(too) to other microorganism.Therefore, although a spot of high sensitive cells may be suppressed, but the growth of most cells can not be affected or only part reduction, thereby, in suitable medium, in the presence of the special antiseptic-germicide of inferior inhibition concentration, the growth of preferred special flora reduces should be no more than 50%, more preferably no more than 30%, be more preferably and be no more than 10%.But in the screening method in embodiment below 1 and 3, inferior inhibition concentration must be enough high, thereby make the inhibition of outflow pump can cause entering of corresponding growth-inhibiting and antiseptic-germicide inactivated factor.Usually, the inferior inhibition concentration of a kind of antiseptic-germicide (or biocide) is lower than minimum inhibition concentration (MIC).

No matter " comprise " back and connect what speech, " comprising " means and include but not limited to these.Therefore, the use that term " comprises " represents that the key element of listing is required or essential, but other requirement chooses wantonly, may exist, and also may not exist.No matter phrase " by ... form " what the back connects, " by ... form " mean and comprise and be limited to this.Therefore " by ... form " represent that key element is required or essential, and do not have other key element to exist." basically by ... form " refer to comprise all key elements of listing behind the phrase, but do not comprise not disturbing or improving the listed key element specific activity in this disclosure or the key element of effect of other.Therefore, phrase " basically by ... form " the listed key element of expression is required or essential, but whether other key element then influence activity of listed key element or function according to it but be optional, promptly may exist, and also may not exist.

On the other hand, the invention provides the method for a non-tsiklomitsin specificity efflux pump inhibitors of screening.In this method, the microbial culture of expressing the special outflow pump of non-tsiklomitsin is in the suitable culture medium that contains test compound and another kind of compound.This method comprises determines in the presence of test compound whether the intracellular concentration of second kind of compound (as biocide) raises.Second kind of material that compound is normally effluxed by this pump.Determining whether the concentration of second kind of compound in born of the same parents increases relates to the expression that detects the reporter gene of transcribing jointly with regulating and controlling sequence (promotor), and the expression of this regulating and controlling sequence is the intracellular concentration inductive that raise by second kind of compound.Second kind of compound in substratum with non-zero, the inferior concentration that suppresses of bacterium is existed, this can make its intracellular concentration cross low and can't induce the expression of reporter gene, is suppressed unless efflux the pump of this compound.When if certain test compound exists when not existing the expression level height of reporter gene, show that then this test compound is an efflux pump inhibitors.Second kind of compound is preferably antiseptic-germicide, but is not to be antiseptic-germicide.In certain embodiments, inserting if reporter gene is single copy karyomit(e), as inserting in the dispensable gene, is very useful.

In preferred embodiments, second kind of compound is antiseptic-germicide.In specific embodiment, what the bacterium of reorganization can be from a large amount of bacterial classifications is any, those that list for first aspect above comprising.

In some preferred embodiments, but in the induction regulating controlling sequence expression of reporter gene can induce by the raising of tsiklomitsin concentration.In those embodiments, regulating and controlling sequence may be tetA regulating and controlling sequence (as tetA (A)), and it contains the manipulation site of an energy in conjunction with TetR repressor molecule.Bacterium also can be expressed this repressor.When there be not (or tsiklomitsin concentration is very low) in tsiklomitsin, repressor will reporter gene not be expressed with handling that the site combine.Therefore, when tsiklomitsin in substratum to the bacterium of expressing the special outflow pump of non-tsiklomitsin to be inferior inhibition level when existing, horizontal dimension is held in enough low level in the born of the same parents of tsiklomitsin, reporter gene can not be expressed (or with utmost point low expression level).The active compound of pump (test compound) exists if certain can suppress to outflow, and the intracellular concentration of tsiklomitsin will raise, and TetR discharges from handling the site, and reporter gene will be expressed.Reporter gene and regulating and controlling sequence are preferred but nonessentially be inserted in the bacterial chromosome with single copy form.Usually, this gene is inserted in the dispensable gene.

In specific embodiment, the outflow pump is a Pseudomonas aeruginosa outflow pump, as MexA/mexB/oprM outflow pump or by the outflow pump of Pseudomonas aeruginosa strain K385 overexpression.The outflow pump is Pseudomonas aeruginosa type outflow pump in other specific embodiment.In specific embodiment, reporter gene a kind of enzyme of encoding, as beta-galactosidase enzymes, or other expresses the gene that easily detects product, and several such genes are known to one skilled in the art.Same in specific embodiment, used cell is a pseudomonas aeruginosa strain, but also can use other cell in other the embodiment, specifically comprises the bacterium of listing in top first aspect.

In specific embodiment, reporter gene a kind of enzyme of encoding is as beta-galactosidase enzymes; Some suitable reporter genes are known to one skilled in the art.Reporter can provide the report that a color can be surveyed in some cases, but other report also is useful.Colorimetric report i.e. also luminous reflectance of photoabsorption.Therefore, the colorimetric report may comprise the detection of the molecule (coloured molecule) with characteristic light absorption or the detection of light emission molecule (as fluorescence molecule).

The microorganism in being exposed to biocide (comprising bacterium), " intracellular concentration " of certain biocide refers to the concentration of this medicament in cell outermost tunic.Concerning the most of microbe kind, the outermost tunic is a plasma membrane, but concerning gram negative bacterium, the concentration of this medicament in periplasmic space may be its significant intracellular concentration (as to beta-lactam).Concerning gram negative bacterium, the corresponding intracellular concentration of certain antiseptic-germicide is can be near the concentration in the cell spaces of the major objective thing of this antiseptic-germicide.

Term " recombinant microorganism " refers to the operation by the people, inserted the microbial species system of certain dna structure or sequence, wherein dna structure that is inserted or sequence did not originally exist in this microorganism, perhaps were inserted into this microorganism cells or chromosomal different positions.This term does not comprise natural heredity exchange as the joint between different naturally occurring organisms.This term comprises recombinant bacteria clearly.To most of purposes, preferably inserting dna sequence dna is stable insertion, that is to say that this sequence is duplicated serially and pass to the offspring microorganism when growth.Such offspring also is " reorganization ".

Transcribe and translate the process that generates polypeptide product in " expression " phalangeal cell of term gene.In this article, this term refers to that especially expressed products has function, and says that in some sense the method that is easy to being fit to this concrete reporter detects.Therefore, concerning an enzyme reporter, product has normal enzymic activity.

Term " concentration of raising " refers to this a kind of intracellular concentration, and it shows that certain compound that is generally antiseptic-germicide is high concentration when being in a kind of second kind of compound that is generally test compound or efflux pump inhibitors and not existing.Therefore, in the description of screening method, the concentration high concentration of the intracellular concentration that a kind of antiseptic-germicide improves when not existing than test compound or known efflux pump inhibitors.The concentration of this raising may be lower than, equals or be higher than the concentration in the substratum.

Term " single copy " refers to that the nucleotide sequence of this term indication only exists with single copy form in each genome.This is different from the existence of multiple copied plasmid amplifying nucleic acid sequence clearly, can surpass one different numbers existence in individual cells in multiple copied plasmid amplifying nucleic acid sequence.

Also relevant term " karyomit(e) insertion " with the nucleotide sequence that inserts in the cell refer to that nucleotide sequence inserts and the karyomit(e) of the covalently bound cell of going into to be discussed in.This means that this sequence will duplicate jointly with the karyomit(e) other parts in cell normal replication process.Once more the explanation, this be independent of cell chromosome outside bioblast on nucleotide sequence have obviously different.

" reporter gene " is that a kind of coded product is easy to detected nucleotide sequence.The some of them reporter is an enzyme, can detect by the enzymic activity of product.A concrete example is a beta-galactosidase gene.But also have many other reporter genes to exist and known by those skilled in the art.

" regulating and controlling sequence " is can control when relevant encoding sequence expresses and with the nucleotide sequence of what horizontal expression.When relating to genetic expression, term " derivable " refers to that under certain specified conditions the level of translating of following the encoding sequence of regulating and controlling sequence improves.So under regulating and controlling sequence can be by antiseptic-germicide inductive situation, when having the antiseptic-germicide of enough high densitys, the expression level of relevant encoding sequence will improve.Such inducing action has number of mechanisms, specifically comprises derepression mechanism, and wherein in the presence of the antiseptic-germicide of enough high densitys, the repressor molecule is inactivated.

On the other hand, the invention provides a kind of method of utilizing microorganism (as bacterium) cell and can being screened efflux pump inhibitors by dyestuff, fluorophor or other compound that detects with spectrography easily (detect and maybe can survey compound) that this cell drain pump (as non-tsiklomitsin specificity outflow pump) transports.This method comprises the intracellular concentration of comparison detection compound (as dyestuff or fluorophor) when test compound exists or do not exist.If do not raise when the intracellular concentration of detection compound does not exist than test compound when test compound exists, show that this test compound is a kind of special efflux pump inhibitors.Preferred cell overexpression outflow pump.

The intracellular concentration of same preferred coloring agent or fluorescent substance can directly be determined, yet, in other embodiment, intracellular concentration is by the concentration (born of the same parents' extracellular concentration) or the indirect measurement by the concentration of dyestuff or fluorescent substance in definite extraction liquid of cell of dyestuff in definite suspension culture base or fluorescent substance.Directly determining of born of the same parents' inner dye in some cases, by the range estimation of cell (in a cell) color, can quantitatively be determined the inhibition of outflow pump.

Many different dyestuffs and fluorescent substance are known in this area.These comprise, Viola crystallina for example, Victoria Green WPB, methylene blue, benzyl purple (Benzyl Viologen), bromothymol blue, toluidine blue, methylene blue, rose-red, Alcyan indigo plant, ammoniated ruthenium oxychloride, fast green, aniline blue, dimethylbenzene green grass or young crops, tetrabromophenol sulfonphthalein, Coomassie blue, purpurum bromocresolis, tetrabromo-mcresolsulfonphthalein, trypan blue and phenol red.Suitable dyestuff or other detection compound can by judge with common method well known to those skilled in the art concrete dyestuff in whether being studied specific outflow pump or be studied in certain outflow pump transportation (effluxing) of cell make one's options.In preferred embodiments, dyestuff is Viola crystallina and Victoria Green WPB.

In yet another aspect, the invention provides a kind of screening efflux pump inhibitors method, comprise that the microorganism cells with reorganization contacts with a test compound, and determine whether the microorganism cells of reorganization grows in the presence of test compound.The microorganism of reorganization is fabricated, thereby makes that microorganism cells can be grown after the outflow pump is suppressed, and can not grow when the outflow pump is not suppressed.If the microorganism cells of reorganization is grown when test compound exists and do not grown when test compound lacks, then this test compound is an efflux pump inhibitors.This method is the positive growth screening to efflux pump inhibitors.

In concrete preferred embodiment, this method comprises that microorganism cells and a test compound, an inductor that will reorganization, the biocide of a certain concentration that is higher than the MIC of recombinant microorganism cell contact.When inducing of inactivated factor do not exist, the microorganism cells that biocide can not be recombinated effluxed or specifically inactivating, and inductor can be induced the inactivated factor of biocide.In specific embodiment, the microorganism of reorganization is a recombinant bacteria equally.Inactivated factor can be a β-Nei Xiananmei, and the antiseptic-germicide beta-lactam can be by the beta-lactam enzymatic breaking.Particularly, in one embodiment, the β-Nei Xiananmei gene can be the blaS gene, and beta-lactam is a degradable carbapenem, J.Dufresne etc. 1988, the blaS gene was identified and reported first in " biocide chemotherapy " (Antimicrob.Agents Chemother.) 32 volume 819-826 pages or leaves.And in specific embodiment, promotor is the tetA promotor, and the bacterial cell of reorganization can produce from the tetR gene the repressor of function, and is attached on the promotor.Promotor and β-Nei Xiananmei gene are preferred but nonessentially be inserted in the bacterial chromosome with single copy form.

In yet another aspect, the invention provides a kind of treatment animal microorganism and infect, as the method for infectation of bacteria, the efflux pump inhibitors by giving this class infection animal q.s is to reduce outflow pump activity, and the inhibitor here can reduce the pathogenecity of microorganism.For example, the reduction of this pathogenecity can disturb bacterium that the demand of iron ion is obtained by the transportation that suppresses siderophore.Same this pathogenecity also can be by reducing or eliminating and can cause the microbial metabolites of tissue injury influence to reduce to the host.Yet other method that reduces pathogenecity is also contained in this aspect.Animal may be, for example chicken and turkey, but be Mammals in certain preferred aspects.

Infected by microbes may be caused by bacterium in certain preferred aspects, and for example these bacteriums may be any in the bacterial species of listing in top first aspect.

In a related aspect, the invention provides a kind of method for the treatment of the animal that suffers infected by microbes with reduction outflow pump activity by the efflux pump inhibitors of giving the animal q.s.In aspect this, efflux pump inhibitors can weaken and infect relevant microorganism viability in vivo.By weakening viability in vivo, the intravital infection of the easier removing of infected animal, or microorganism even may be killed.Animal is a Mammals in specific embodiment.Equally in specific embodiment, microorganism may be any in the malignant bacteria kind, especially comprises those that list in top first aspect.

Term " viability in vivo " refers to microorganism such as bacterium survival and the energy for growth in host such as animal.Therefore, certain efflux pump inhibitors that can reduce microorganism viability in vivo can stop microbial growth, and/or kill microorganisms.Such efflux pump inhibitors thereby also be antiseptic-germicide.

In another related aspect, the present invention includes a kind of method of Mammals prophylactic treatment.In this method, to being in the efflux pump inhibitors that Mammals in infected by microbes such as the infectation of bacteria danger gives to weaken the microorganism pathogenecity.

In a related aspect, the invention provides a kind of treatment animal, specifically comprise the method for mammiferous infected by microbes, promptly with antiseptic-germicide with can improve microorganism and the efflux pump inhibitors of this antimicrobial agent sensitivity is treated suffer infected animals.In this way, can eliminate and infect relevant microorganism, use the antiseptic-germicide that is of no curative effect also can treat when perhaps being used in efflux pump inhibitors and not existing with the antiseptic-germicide of smaller dose.Therefore, this methods of treatment is particularly useful for being difficult to the independent infection that causes with the microbial strain of biocide treatment, because the dosage (may bring undesirable side reaction) that independent effect need be big, or owing to lack microbiotic with clinical efficacy.But present method also is applicable to the infected by microbes of treatment to the specific antimicrobial sensitivity, to reduce the dosage of this antiseptic-germicide.This can reduce the danger that side reaction takes place, and also can reduce the selective action to the high drug-resistance microorganism of using certain microbiotic to cause owing to continuous high dosage.Microorganism is a bacterium in specific embodiment, for example may be point out in top first aspect any.In specific embodiment, can use different antiseptic-germicides equally, these comprise quinolones, tetracyclines, glycopeptide class, aminoglycoside, beta-lactam, rifomycins, Notomycin class, Macrolide and paraxin.In specific embodiment, above microbiotic in several classes, for example a kind of below: the beta-lactam antibiotics imipenum, the part sulfomycin, inclined to one side sulfomycin, cefaclor, S 578, cefadole, rocephin, cephalo pool ketone, Kefzol, Cefixime Micronized, cefmenoxime, Cefodizime, cefonicid acid, cefoperazone, BL-S 786, cefotaxime, cefotiam, U-63196E, cefpiramide, Cefpodoxime, cefsulodin, ceftazime, cefteram, his azoles of cephalo, ceftibuten, ceftizoxime, Ro-13-9904, cefuroxime, cefuzonam, cefacetrile, Cephalexin Monohydrate Micro/Compacted, Cephaloglycin, Cephaloridine, cefoxitin, Cephapirin, Cephradine, cefmetazole, cefoxitin, cefotetan, azthreonam, carumonam, flomoxef, Shiomarin, amidine XiLin, amoxycilline Trihydrate bp, penbritin, the azlocillin, Pyocianil, benzopenicillin, Carfecillin, cloxacillin, two chlorazol XiLin, the X-1497, mezlocillin, nafcillin, Oxazacillin, penicillin G, piperacillin, the sulbenicillin, temocillin, ticarcillin, cefditoren, SC004, KY-020, Cefdinir, ceftibuten, FK-312, S-1090, CP-0467, BK-218, FK-037, DQ-2556, FK-518, cefozopran, ME1228, KP-736, CP-6232, Ro09-1 227, OPC-20000, LY206763 Macrolide azithromycin, erythromycin, erythromycin, oleandomycin phosphate, rokitamycin, Rosaramicin, Roxithromycin, troleomycin quinolones amifloxacin, cinoxacin, Ciprofloxacin, the Yi Nuosha star, fleroxacin, flumequine, Lome Norxin, nalidixic acid, norfloxicin, Ofloxacine USP 23, left-handed Norxin, quinoline acid, Pefloxacin, rosoxacin, Temafloxacin, the Tuo Shusha star, Si Basha star, clinafloxacin, PD131628, PD138312, PD140248, Q-35, AM-1155, NM394, T-3761, rufloxacin, OPC-17116, DU-6859a (identifies in Sato K. etc., " biocide chemotherapy " (Antimicrob.Agents Chemother in 1992,37 volume 1491-98 pages or leaves), DV-7751a (identifies in Tanaka M. etc., 1992 " biocide chemotherapy ", 37 volume 2212-18 pages or leaves) tetracyclines duomycin, cicloxin, doxycycline, lymecycline, methacycline, MINOCYCLINE HCL, terramycin, tsiklomitsin aminoglycoside amikacin, Arbekacin, Ambutyrosin, dibekacin, good fortune is pasted mycin, gentamicin, kantlex, meomycin, ethyl sisomicin, ribostamycin, sisomycin, spectinomycin, Streptomycin sulphate, tobramycin, clindamycin, lincomycin

In another related aspect, the present invention includes the method for Mammals prophylactic treatment.In this method, be in Mammals antiseptic-germicide and efflux pump inhibitors in infected by microbes such as the infectation of bacteria danger.

In the narration to the reaction of antiseptic-germicide, term " easily (quick) perception " refers to the susceptibility when microorganism exists antiseptic-germicide in microorganism such as bacterium.That is, improve susceptibility and mean that microorganism will be suppressed by the biocide of lower concentration in the substratum around the microorganism cells.Agent of microorganism combating microorganisms is more responsive as much as to say for this.In most of the cases the minimum inhibition concentration of this biocides (MIC) is lowered.

Here the term of using " treatment " refers to give pharmaceutical composition for preventing and/or treating purpose.Term " prophylactic treatment " refers to treatment infection as yet, but infects easily, or the patient of certain risk of infection is arranged.Term " therapeutic treatment " refers to the patient who suffers certain infection is carried out pharmacological agent.Therefore, in preferred embodiments, treatment is the synergistic agent and antibiotic (or antimicrobial) agent of uniting and give (simultaneously or successively) effective in cure amount of Mammals (for treatment or prevention purpose).

" therapeutically effective amount " or " knock-down " refers to as disclosed among the present invention, amount with efflux pump inhibitors of curative effect, or efflux pump inhibitors and biocide amount separately, curative effect is often referred to the eubolism of microorganism cells that suppresses to cause or help infected by microbes to a certain degree.Can be used for the efflux pump inhibitors of combination therapy and the dosage of biocide is effective in cure amount.Therefore, when used therapeutically effective amount refers to that those unite use, be judged as the efflux pump inhibitors of generation expectation curative effect and the amount of biocide here by clinical test results and/or animal pattern infection research.In specific embodiment, efflux pump inhibitors and biocide are by predetermined mixed, so therapeutically effective amount should be the amount of mixture.This amount and efflux pump inhibitors and antiseptic-germicide amount separately can be determined routinely by those skilled in the art, and change with used concrete efflux pump inhibitors and the different of antiseptic-germicide as the concrete bacterial strain that relates to many factors.This amount also depends on patient's height, body weight, sex, age and medical history.For prophylactic treatment, therapeutically effective amount is if there is infected by microbes to exist just with medicable amount.

Curative effect alleviates the symptom of one or more infection to a certain extent, comprises curing infecting." healing " refers to the elimination of infection symptoms, comprises and the removing of infecting other relevant remaining microorganism.But still there be (as large-scale tissue injury) in the long-term or permanent influence of some of infection after healing.

Term " infected by microbes " refers to pathogenic microbes infecting host mammal.This comprises and is present in the mammalian body usually or the excess growth of the microorganism of body surface.More at large, infected by microbes may be any situation that has the subdamage host mammal of microbial population.Therefore, when excessive microbial population is present in the mammalian body or body surface, or when microorganism exist the mammiferous cell of subdamage or other to organize the time, this Mammals just " is suffering " infected by microbes.This description is particularly useful for infectation of bacteria.

Term " gives (medicine) " and " administration " refers to a kind of method that gives certain antimicrobial pharmaceutical combination preparation of Mammals, and this method is, for example local application, oral, intravenous injection, subcutaneous injection or intramuscular injection.The preferred method of administration can change with the position of the composition of the multiple factor such as medicinal compositions, potential or existing infectation of bacteria, related microorganism and the different of severity of existing infected by microbes.

Term " Mammals " is with its common biological significance.Therefore, it specifically comprises people, dog and cat, but also comprises many other species.

In yet another aspect, the present invention is a characteristic with the method that suppresses the membrane channel on the cytolemma also, and this method comprises membrane channel is contacted with the membrane channel inhibitor that inhibitor can weaken the ability that effluxes of membrane channel.In specific embodiment, a polypeptide of the outflow pump of at least one polypeptide in the membrane channel and mexA/mexB/oprM outflow pump or Pseudomonas aeruginosa strain K385 overexpression has 50% amino acid sequence similarity at least.

Here one or more types of molecules of permission that the term of using " membrane channel " refers to be positioned on the cytolemma are striden the albumen polymer that film transports.This transportation may be the passive transportation of reflection concentration gradient, or depends on the active transport of born of the same parents' self-energy supply.

" membrane channel inhibitor " is similar to efflux pump inhibitors, is a kind of compound that can slow down or stop the corresponding membrane channel of molecule utilization to carry out the cross-cell membrane transportation.

The present invention is characteristics with a kind of biocide that strengthens to the method for microbic activity also, and the non-tsiklomitsin specificity efflux pump inhibitors with outflow pump in microorganism and the cell contacts with an antiseptic-germicide in the method.Therefore, when the cell of expressing the outflow pump was handled with biocide and non-tsiklomitsin specificity efflux pump inhibitors, present method made biocide more effectively act on this cell.In specific embodiment, microorganism is a bacterium, any as in the bacterial classification of pointing out in top first aspect, antiseptic-germicide can be selected from antibiotic many structure types, for example comprises beta-lactam, glycopeptide class, aminoglycoside, quinolones, tetracyclines, rifomycins, Notomycin class, Macrolide and chloromycetin.In specific embodiment, above microbiotic in the type can be above-mentioned those.

In yet another aspect, the invention provides mammalian cell is infected the medicinal compositions of effectively treating, comprising pharmaceutical carrier and efflux pump inhibitors.It is the efflux pump inhibitors of effective antiseptic-germicide that such composition can contain when having or not other antiseptic-germicide to exist.Such composition can be used for the treatment of infection separately.In other composition, efflux pump inhibitors can improve the susceptibility of bacterium to other antiseptic-germicide, thus this composition should with such antibacterial agents.The present invention also provides the same effectively medicinal compositions of the treatment of mammalian infections, and it comprises efflux pump inhibitors and antiseptic-germicide.Same the present invention also provides the medicinal preparations that contains antiseptic-germicide, efflux pump inhibitors and carrier.

In certain preferred aspects, efflux pump inhibitors has a kind of structure shown in the following universal architecture 1-4:

R=alkyl (C wherein ₁-C ₄), fluoroalkyl (C ₁-C ₄), cross fluoroalkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), aryl (C ₆-C ₁₀), single substituted aryl (C ₆-C ₁₀) [with alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), amino, single substituted-amino be [with alkyl (C ₁-C ₄) randomly replace], two substituted-amino is [with alkyl (C ₁-C ₄) or the arbitrary combination of hydroxyl randomly replace] randomly replace], two substituted aryl (C ₆-C ₁₀) [with alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I) and amino arbitrary combination], 2-(or 3-)-thienyl, 2-(or 3-)-furyl or 2-(3-or 4-)-pyridyl.W=H, NH ₂, single ammonia that replaces is [with alkyl (C ₁-C ₄) randomly replace], two replacement ammonia are [with alkyl (C ₁-C ₄) arbitrary combination randomly replace], nitrogen heterocyclic [as the N-morpholinyl, N-piperazinyl, N-pyrrolidyl, TMSIM N imidazole base, N-pyrryl, N-pyrazolyl, N-triazolyl, or N-tetrazyl], halogen (Br, Cl, F, I), hydroxyl, alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), R ¹=(CH ₂) _nNR ^bR ^c, (CH ₂) _nNHC=(NR ^a) NR ^bR ^c, (CH ₂) _nSNHC=(NR ^a) NR ^bR ^c, (CH ₂) _nC=(NR ^a) NR ^bR ^c, (CH ₂) _nN=CNR ^bR ^c, (n=2-4); R ^a(R ^bOr R ^c)=H, alkyl (C ₁-C ₄), aryl (C ₆), substituted aryl, phenyl, substituted benzene is [with alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino randomly replaces], or R ^a+ R ^b=(CH ₂) _2-3Or-CH=CH-, X=aryl (C ₆-C ₁₀) ,-(CH ₂) _0-2Aryl (C ₆-C ₁₀), substituted aryl (C ₆-C ₁₀) [with alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino randomly replaces], replacement-(CH ₂) _0-2Aryl (C ₆-C ₁₀) [with alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino replaces on aryl], 2-(or 3-)-thienyl, 2-(or 3-)-furyl, 2-(3-or 4-)-pyridyl, benzofuryl [in any position of cumarone ring], benzothienyl [in the optional position of thionaphthene ring].Wherein exist center of asymmetry, its absolute stereo chemical property may be R or S configuration, or is racemic mixture, is included in the universal architecture description.

Meeting the chemical synthesis process that the compound of above-mentioned universal architecture can know by the technician of chemical field obtains.

" carrier " or " vehicle " is a kind ofly to be used to make the administration of compound easier, as improving the deliquescent compound or the material of above-claimed cpd.Solid carrier comprises, as starch, and lactose, Lin Suanergai, sucrose and kaolin.Liquid vehicle comprises, sterilized water for example, salt solution, damping fluid, nonionic surface active agent and edible oil such as grease, peanut oil and sesame oil.In addition, some this areas assistant agent commonly used also can be included.These and other such compound has description in the literature, as the Merck catalogue, and Merck ﹠amp; Company Rahway, NJ. the item that the mixing of heterogeneity should be noted in the medicinal compositions is existing describes, as at Gilman etc., nineteen ninety is compiled " Goodman and Gilman ' s: the pharmacy basis of treatment " (Goodman and Gilman ' s:The Pharmacological Basis of Therapeutics), the 8th edition, Pergamon Press.

In yet another aspect, the invention provides a kind of method of containing the bacterial growth of expressing non-tsiklomitsin specificity outflow pump, this method is by acting on this bacterium and non-tsiklomitsin specificity efflux pump inhibitors in the presence of the antiseptic-germicide of the concentration that is lower than bacterium MIC, and bacteria growing inhibiting.This method can be used for, and for example prevents or solve the pollution of the bacterium pair cell cultivation with outflow pump.Yet it can be used for this growth inhibiting any situation of needs.

In related aspect, the invention provides a kind of method of bacteria growing inhibiting, this method comprises the efflux pump inhibitors of expression of bacterium with a kind of component that can reduce the outflow pump contact that such inhibitor can different ways work in the regulation and control of expression.For example, it may improve the repressor branch suboutput that stops outflow pump composition to be expressed.Another kind of possible mechanism is whether inhibitor has stoped the release of repressor molecule.The example of such repressor molecule is MarR (Seoane and the Levy in the intestinal bacteria, 1994, " U.S. microbial genetics association conference summary " (Abstr.ofthe Am.Soc.for Microbiol.Gen.Meeting), Las Vegas, NV.Abstr.H-26).An example of positive regulator is that (Ahmed etc., 1994 " biology and The Chemicals " are (J.Biol.Chem.) for BmrR in the Bacillus subtilus.

In another related aspect, the invention provides a kind of method that reduces bacterial strain colony quantity, this method comprises colony contacted with the efflux pump inhibitors of a kind of composition of the outflow pump of the bacterial expression that suppresses this colony, and this outflow pump is essential to the growth of the bacterium of expressing this outflow pump.In specific embodiment, this composition is the plasma membrane composition.As mentioned above, such efflux pump inhibitors can work in many ways, includes but not limited to directly and the basal component effect, or suppresses the expression of this composition.

Term " reduces colony's quantity " and means that the bacterium of this colony will be killed.This is different from the fungistat that stops bacterial growth and breeding.According to the narration of this aspect, " basal component " of outflow pump is that bacterium is survived in vivo, i.e. the necessary composition of survival in the host.

In yet another aspect, the invention provides a kind ofly by giving the method that certain efflux pump inhibitors of animal promotes growth of animal, this efflux pump inhibitors can suppress to be present in the outflow pump that the bacterial strain in the animal is expressed, and suppresses the growth of this bacterial strain.This growth effect may be the energy expenditure that has reduced bacterium, thereby has improved the utilization of animal to food energy.Present method is applicable to, ox for example, poultry such as pig and chicken and turkey.

Show in the description of its its feature of the present invention and advantage general preferred embodiment below and the claim.The accompanying drawing summary

Fig. 1 pump of representing to outflow is divided into three major types.

Fig. 2 is the diagram of Pseudomonas aeruginosa outflow pump MexA/MexB/OprM, has shown the spatial relation of three kinds of component expections.In the figure, MexB is across plasma membrane, and MexA strides across periplasmic space, and OprM strides across adventitia.The figure on the right shows that the microbiotic that effluxes usually (as Ciprofloxacin) will be built up if test compound has suppressed to efflux effect (as by suppressing a kind of pump composition) in the bacterium kytoplasm.

Fig. 3 shown among the embodiment 1 the excessive generation transportation of the usefulness of describing Ciprofloxacin the outflow pump bacterial strain and do not produce the expected results that the bacterial strain of this outflow pump screens.The bacterial strain of excessive generation in the presence of efflux pump inhibitors, adds the Ciprofloxacin of inferior MIC concentration, with not regrowth.

Fig. 4 is the summary diagram of the report method of description among the embodiment 2.

Fig. 5 has shown the composition and the mutual relationship thereof of positive growth screening among the embodiment 3.

Fig. 6 has shown the accumulation of tsiklomitsin in born of the same parents in four kinds of pseudomonas aeruginosa strains.Supersensitivity bacterial strain 799/61 has shown the highest intracellular concentration.

Fig. 7 has shown the characteristics of four kinds of pseudomonas aeruginosa strain outflow pumps of the external evaluation that is used for L-amphetamine acyl-L-arginyl-β-naphthalene amino acid.

Fig. 8 shown L-amphetamine acyl-L-arginyl-β-naphthalene amino acid respectively with tsiklomitsin and Ciprofloxacin to each the synergistic effect in four kinds of pseudomonas aeruginosa strains identifying among Fig. 4.

Fig. 9 illustrates Pseudomonas aeruginosa strain PAO1 and after 24 hours the multiple of 13 kinds of antibiotic susceptibility is improved in the L-of different concns amphetamine acyl-L-arginyl-β-naphthalene amino acid.

Figure 10 shown Pseudomonas aeruginosa strain PAO1 and K385 when 20 μ g/ml L-amphetamine acyl-L-arginyl-β-naphthalene amino acid exists and does not exist to 13 kinds of antibiotic susceptibility (MICs).

Figure 11 has illustrated the hydrophobicity degree and the relation that is improved by the L-amphetamine acyl L-susceptibility multiple that arginyl-β-naphthalene amino acid causes of four kinds of both sexes fluoroquinolones, shows direct relation.

Figure 12 has shown that Ciprofloxacin comprises the reducing of MIC of clinical isolating and breadboard Pseudomonas aeruginosa strain to 26 strains.

Figure 13-15 has shown, the result who kills time study of 3 strain Pseudomonas aeruginosa strains.Each bacterial strain all exists or does not exist at every kind of microbiotic, has and does not have under the situation of L-amphetamine acyl-L-arginyl-β-naphthalene amino acid tsiklomitsin and Ciprofloxacin are tested.

Figure 16 has shown some bacterial classifications, and the bacterial strain of selecting from these bacterial classifications is used for the enhancement test of L-amphetamine acyl-L-arginyl-β-naphthalene amino acid to Ciprofloxacin, has pointed out part test bacterial strain that select, that demonstrate enhancement from each bacterial classification.

Figure 17 has compared L-amphetamine acyl-L-arginyl-β-naphthalene amino acid to the influence to the susceptibility of acridine orange of intestinal bacteria Acr+ and Acr-bacterial strain.

Figure 18 has shown that L-amphetamine acyl-L-arginyl-β-naphthalene amino acid is to the comparison of the influence of Ciprofloxacin susceptibility between a Pseudomonas aeruginosa strain PAO1 and a DNA gyrase mutant strain.

Figure 19 has shown three kinds of CCCP or L-amphetamine acyl-L-arginyl-β-when naphthalene amino acid exists and has produced the variation that tsiklomitsin is built up in the Pseudomonas aeruginosa strain of outflow pumps.

Figure 20 has shown the difference of the influence that CCCP and L-amphetamine acyl-L-arginyl-β-naphthalene amino acid are built up tsiklomitsin in the Pseudomonas aeruginosa strain 799/61.

Figure 21 provides a kind of structure expression of L-amphetamine acyl-L-arginyl-β-naphthalene amino acid.The physiological function of the narration outflow pump of preferred embodiment

Bacterium utilizes carrier to efflux molecule in the medium from kytoplasm outside born of the same parents.The transportation of the molecule that most of known carrier and the diffusion of film thoroughly or diffusion are very slow is relevant.The molecule that is discharged to the outer medium of born of the same parents outward comprises protein, peptide, capsular polysaccharide, oligosaccharide, siderophore and medicine.Carrier allows molecule to see through plasma membrane and see through adventitia in gram negative bacterium, and can according to its sequence homology and energy derive be divided into three major families (H.Nikaido, 1994 " science " be 264 volume 382-388 pages or leaves (Science); Dinh etc., 1994 " bacteriology magazine " are 176 volume 3825-3831 pages or leaves (J.Bacteriol.)).(see figure 1).ABC (ATP-binding cassette) family comprises medicine and enzyme carrier, and this family comprises the carrier relevant with protein excretion.For example in HlyD and the intestinal bacteria hemolysin efflux relevantly, and efflux by the AprE system at Pseudomonas aeruginosa neutral and alkali proteolytic enzyme.The multiple medicines outflow pump of finding in the mammalian cell, P-1 glycoprotein and MsrA in the streptococcus aureus belong to this class.The sub-superfamily of main promotion comprises the specificity outflow pump as Tet outflow pump, and relevant with participation medium absorbs nourishment outside born of the same parents haulage system such as Xyl system.This main promotion Zijia family is energy source with the proton motive force.The 3rd family's heavy metal tolerance/tight knot factor family comprises, the carrier relevant with the secretion of siderophore, the MexA in the Pseudomonas aeruginosa for example, the relevant carrier that effluxes with the tight knot factor in the sweet clover root nodule bacterium, with efflux relevant outflow pump with antibiotic with extensive substrate specificity, as the mex pump in the Pseudomonas aeruginosa or Acr. iron ion metabolism in the intestinal bacteria and outflow pump

Iron ion is a prevailing transition metal in the life entity, and is the basic nutrition material (Wooldridge and Williams etc., 1993 " comment of FEMS microbiology " (FEMS Microbiol.Rev) 12 volume 325-348 pages or leaves) of all pathogenic bacterias.The utilization of iron ion plays an important role in generation of infecting and development.The outer iron ion of born of the same parents combines with high-affinity iron-binding protein Transferrins,iron complexes and lactoferrin in the human body.Transferrins,iron complexes and lactoferrin help the host to resist infection by the utilization of restriction pathogenic micro-organism iron ion.Bacterium needs iron ion to survive and to breed in vivo, the different methods of obtaining iron ion thereby develop.Bacterium is synthetic and to outside media discharge iron ion is had the molecule of high-affinity, behind these molecule chelated iron ions, is transported back cell (Wooldridge and Williams, 1993) again.These molecules are commonly referred to as siderophore and are essential to the development of infectation of bacteria.Regulated by the strictness of available iron ion with the relevant system that effluxes and absorb of the synthetic of siderophore and they.Pseudomonas aeruginosa is synthesized two kinds of main siderophores, pyofluorescein and green pus bacterium chela ferritin.Two kinds of siderophores all can be removed Transferrins,iron complexes bonded iron ion (Sriyosachati and Cox,, " infecting and immunity " (Infect.﹠amp in 1986; Imm.52 volume 885-891 page or leaf; C.Wolz, 1994 " infecting and immunity " 62 volume 4021-4027 pages or leaves).Compare with green pus bacterium chela ferritin, pyofluorescein is a more effective iron ion scavenging agent and iron ion is had higher avidity that it can remove iron ion effectively from transferrin complex of protein.Pyofluorescein is a big water soluble molecules, molecular weight 1.5KD.It can not pass the fat layer diffusion of film, and need pass plasma membrane by haulage system.Although the capturing system of siderophore is existing in the literature to be described (Gensberg and Smith 1992 " general microbiology magazine " (J.Gen.Microbiol.) 138 roll up the 2381-2387 pages or leaves), its Secretory Pathway itself is just known by inference until now.The outflow pump is relevant with effluxing of siderophore, because need their bacteriums could secrete siderophore, otherwise siderophore can not be crossed adventitia and inner membrance because of its huge volume and wetting ability.In Pseudomonas aeruginosa, mexAB, the expression of oprM operon is subjected to the regulation and control of iron ion content in the substratum, and be subjected to regulation and control (Poole etc. jointly with picked-up with the generation of main siderophore pyofluorescein composition, 1993a), because the transportation of iron ion is essential (P.Sokol, 1987 " infecting and immunity " 55 volume 2021-2025 pages or leaves to the charrin's disease of success; B.Haas, 1991 " infecting and immunity " 59 volume 3997-4000 pages or leaves; Woods and Iglewski, nineteen eighty-twos " infect with immunity " 35 volume 461-464 pages or leaves), thereby so the inhibition of this pump will disturb the metabolism of bacterium iron ion to weaken its virulence.Initiatively efflux the mechanism that is used as drug tolerance

About 1980, after tetracycline resistant that S.B.Levy and colleague thereof have disclosed the escherichia coli plasmid coding was based on the outflow pump that energy relies on, the interest that resistance that the film effect of effluxing causes has begun to cause scientists was pumped or strides in the active of toxic agents.Soon the plasmid-encoded calcium tolerance that has confirmed streptococcus aureus subsequently again also is based on the mechanism of effluxing.

In of the tolerance of many organisms to many medicaments, initiatively efflux and play significant feature, this progressively gets the nod.In list at Neu three kinds clinical relevant and the permeability-related resistance mechanisms that reduce, have two kinds, possible all three kinds all mainly is owing to efflux.(i) 1987, Neal and Chater at first confirmed, streptomyces coelicolor can be protected and oneself avoid killing and wounding of methylenomycin that its produces, in the bacterial classification of the streptomyces that produces antibiotic, has identified that many microbiotic efflux gene.In them some belong to MF family, other genus ABC family.At first the (ii) a kind of novel plasmid-mediated macrolide resistance in the streptococcus aureus is thought because the permeability that reduces, be proved with initiatively efflux relevant.(iii) the modal mechanism of paraxin resistance is relevant with the enzymatic acetylize of medicine." the chemical sproof non-enzymatic mechanism of paraxin " at first also is considered to because stream effect reduction in the paraxin has been found also to cause by initiatively effluxing, because the cmLA gene among the transposon Tn 1696 shows as the carrier that initiatively outflows of MF family.And hemophilus influenzae contains a homologous chromogene, and some the non-enzymatic resistances in this (perhaps many other) bacterial classification are likely owing to initiatively efflux effect.(iv) in the staphylococcus plasmid-encoded resistance to the quaternary ammonium antiseptic-germicide be proved relevant with the effect of effluxing, i.e. the QasA-QacB carrier by the MF type and the QacC carrier of Smr type.These protein also can pump some basic dyestuffs.(v) a kind of active of fluoroquinolone norfloxicin effluxes in wild-type e. coli and finds first.Gene (norA) relevant with the similar process that initiatively effluxes is subsequently from the karyomit(e) order-checking of the resistance mutant of streptococcus aureus and be proved of coding MF family and efflux carrier.NorA can pump many kinds of fluoroquinolones.The evaluation of outflow pump

When the cell that deenergizes was built up more substrate than the cell of energizing, the outflow pump that exists in the cell can be detected.(S.B.Levy, " biocide chemotherapy " (Antimicrob.Agents Chemother.) 36 volume 695-703 pages or leaves in 1992).The outflow pump of instigating energy to rely on that deenergizes can not utilize energy.For example, this can be by making the film depolarize of having energized, eliminates proton motive force (PMF) and finish.Because many outflow pumps of having identified are driven by PMF, thereby this method is suitable.Concentration difference can directly be measured by the accumulation of material in cell, perhaps use another kind of method, the cell immigration that has accumulated certain material is not contained in the substratum of this material, energy exist or non-existent situation under, measure the speed that speed that this material loses or substratum obtain this material from cell.For the different systems that effluxes, can use different energy retarding agents, as prussiate, 2,2, 4-dinitrophenol (dnp) and carbonyl m-chloro phenylhydrazone prussiate (cccp).For some outflow pump, it effluxes activity and also can utilize and have membranin upset cystidium and study (S.B.Levy, 1992).

Another kind method, the outflow pump can be identified by the ability that they reduce the accumulation of substrate in cell.It is because they can give antimicrobial resistance that the outflow pump relevant with drug tolerance can be identified.The outflow pump relevant with multidrug-resisting has the resistance of medium level usually in the bacterium to microbiotic widely.Special resistance mechanism comprises special outflow pump, and is then much higher to antibiotic tolerance level relevant on a kind of microbiotic or the one group of structure.A large amount of incoherent microbiotic are shown low-level chemical sproof bacterial strain may express and a kind ofly initiatively efflux relevant pump with microbiotic.Whether relevant in order to identify the outflow pump with the antibiotic resistance of a given bacterial strain, should use suspection to be built up experiment by the microbiotic that the outflow pump effluxes.Should study protein graphical spectrum behind the plasmalemma protein electrophoresis to identify the appearance of new band in the proteinogram after certain proteic overexpression or the gel electrophoresis.For gram negative bacterium, also should study its outer membrane protein.Because the outflow pump is present in most of bacteriums usually, the susceptibility of microbiotic background level may be caused by naturally occurring outflow pump in the wild-type bacterium.Build up the existence of the provable outflow pump of experiment with such bacterial strain.With the separable mutant that goes out overexpression outflow pump of the substratum that contains antibiotic.These mutant have stronger resistance to microbiotic and will build up the microbiotic of lower level.They also can the overexpression plasma membrane and/or adventitia in albumen.Similarly, come out and as above study the high responsive mutant of microbiotic is also separable.These mutant will be built up microbiotic and membranin with low expression level more with higher level.With the accumulation of microbiotic in Pseudomonas aeruginosa is example, the accumulation of in four different Pseudomonas aeruginosa strains, having measured tsiklomitsin: 1) the wild type strain PAO1 of generation background level outflow pump, 2) and 3) bacterial strain PAO4098E and K385 be from PAO1, be the excessive producer of two kinds of different outflow pumps, 4) bacterial strain 799/61, but the bacterial strain of any outflow pump that does not produce detection limit and high responsive to microbiotic.The background level that tsiklomitsin is built up is relevant with the amount of the outflow pump that these bacterial strains produce.Bacterial strain 799/61 is built up more tsiklomitsin than wild-type PAO1, and the bacterial strain of two kinds of excessive generation outflow pumps is then built up the tsiklomitsin of less amount.The outer heat-extraction system of multiple medicines in the (see figure 6) bacterium

A significant development of this area is a bacterium outflow pump of having found to handle multiple medicine, and it is the vestiges of mdr system in the mammalian cell.Such as QacA, Smr, the system of QacE or MyrC can pump quaternary ammonium compound and basic dyestuff and often be called as the outer heat-extraction system of multiple medicines.But the substrate of these systems has physical similarity at least, is positively charged amphipathic molecule.On the contrary, be found in the Bmr carrier of subtilis rhodamine-6G-tolerance mutant, not only catalysed cationic type dye such as rhodamine-6G and ethidium bromide, microbiotic tetracycline (alkalescence) and T-1384 (strong basicity), efflux with the active of organic cation four benzene, also the active of catalysis paraxin (no electric charge) effluxes.Confirmed also afterwards that it pumped fluoroquinolone, the great majority in them exist with zwitterionic state under neutral pH.NorA in the streptococcus aureus is proved to be the Bmr homologue and shows as really and pumps cationic dyestuff tetracycline and paraxin, and they are all incoherent solutes of chemical structure and physical properties.

The outer heat-extraction system of another multiple medicines obtains identifying in the mutant of colibacillary anti-uncoupling agents carbonyl m-chloro phenylhydrazone prussiate (cccp).This carrier EmrB also pumps some unrelated compounds such as mercuric phenyl acetate, nalidixic acid (slightly acidic) and sulfo-cramomycin (no electric charge).

The acrA mutant of e. coli k12 is considered to produce the medicine supersensitivity owing to having improved outer membrane permeability, shows the deactivation multiple medicines and effluxes complex body AcrAE.At wild-type acrA ⁺In the bacterial strain, the accumulation of flavine steady state is very low.Because cationic dyes must concentrate,, this means that flavine must pump very actively to reply the inside negatively charged ion potential energy of striding plasma membrane in kytoplasm.In an acrA mutant, stable state is built up and have been improved 5 times at least, shows that the outer heat-extraction system of AcrAE participated in the discharge of this dyestuff.This has untied a long-standing mystery, although because carried out many researchs, do not find defective as yet at the adventitia of AcrA mutant, and has confirmed that the permeability of their adventitias is normal to a kind of probe at least.As if the substrate scope of AcrAE system is very extensive, comprises hydrophilic antibiotics such as Vulkamycin. PA-93, erythromycin (a kind of macrolide), fusidinic acid, ametycin and tsiklomitsin, and stain remover sodium lauryl sulphate (SDS).

Escherichia coli chromosome gene intrafascicular one changes marRAB, also microbiotic is widely produced tangible resistance, and these microbiotic comprise quinolones, paraxin, tsiklomitsin and beta-lactam.(at least to the resistance of fluoroquinolone and tsiklomitsin as if with efflux relevant).But MarA is the modulin of the many processes of influence, is subjected to the character and the quantity of the pump that this albumen influences not clear at present.

Recently, revealed to go out be because the low permeability of its adventitia to Pseudomonas aeruginosa to the intrinsic resistance of a large amount of biocides, also because of its outer heat-extraction system.The clinical separation strain of Pseudomonas aeruginosa, even when not containing the R plasmid shows also that the combating microorganisms agent has widely, " intrinsic " resistance of different levels.And to different medicaments such as beta-lactam, there is good dependency in paraxin between the resistance level of tsiklomitsin and fluoroquinolone.Thereby the chemical sproof different levels of inherent is considered to be caused by the corresponding difference of outer membrane permeability.Yet experimental fact has been overthrown this hypothesis.When testing the accumulation of different pharmaceutical, discovery even Pseudomonas aeruginosa wild type strain also can pump tsiklomitsin very effectively, paraxin and norfloxicin, and also this activity is relevant with this bacterial strain inherent resistance level.

The genetic identity of this outer heat-extraction system is researched and proposed by K.Poole and colleague's thereof.(Poole etc., 1993a " molecular microbiology " 10 volume 529-544 pages or leaves and Poole etc., 1993b, " bacteriology magazine " 175 volume 7363-7372 pages or leaves.) these reference do as a whole being hereby incorporated by.To Pseudomonas aeruginosa Fe ³⁺In the research of picked-up, they have cloned an operon mexA-mexB-oprM, and it is considered to work in the effluxing of siderophore pyofluorescein.(the ORFC product is accredited as OprK in the reference of Poole etc. in the above, is accredited as OprM (Poole etc., the information of not delivering) at present, and is hereby incorporated by), MexB has the type sequence of RND family carrier.After this operon was inserted into the sudden change deactivation, it is the same as with intestinal bacteria paraxin and sensitive tetracycline that this Pseudomonas aeruginosa strain almost becomes.This shows that this single outer heat-extraction system is the major cause that this strains expressed goes out general drug resistance phenomenon.

Initiatively efflux as and if also in the beta-lactam resistance of some Pseudomonas aeruginosas, work.Pseudomonas aeruginosa produces a kind of induced β-Nei Xiananmei of chromosome coding, and synergy between this enzyme and outer envelope barrier has explained that it shows as the tolerance of compound of the strong inductor of this enzyme to some.But the bacterial strain with high intrinsic resistance is the height endurability to not inducing β-Nei Xiananmei and the highly stable compound of enzymically hydrolyse being had also.And the level of the target of beta-lactam effect or β-Nei Xiananmei or character do not change in these bacterial strains.These results show that their beta-lactam resistance also causes by initiatively effluxing.But different with other hydrophobic agents, some beta-lactams can not be crossed the plasma membrane barrier, and their Substrate is in the pericentral siphon side of plasma membrane.Bringing up again Mammals mdr albumen here, to stop its substrate to stride lipid bilayer transportation significantly be useful.Give this viewpoint with strong support to the observations of the impermeable beta-lactam of bilayer.

Although the substrate specificity widely that the outer heat-extraction system of Pseudomonas aeruginosa shows is amazing a bit.But specificity is known already so widely.For example, Mammals mdr pump not only pumps basic cpd, as Zorubicin, also pumps neutral compound, as taxol and slightly acidic compound, as Plicamycin.In fact, Mammals mdr pump is suppressed by hydrophobic cynnematin, estimates that it is as its substrate analogue.Outer heat-extraction system and outer envelope barrier

Efflux carrier and be positioned at plasma membrane, therefore in gram negative bacterium, can be pumped to (see figure 2) in the pericentral siphon by imaginary medicament.If like this, the effect of effluxing just can not make these bacteriums not have to have more resistance under other factor, because the existence of outer envelope barrier makes these biocides be not easy to leave cell.A method that overcomes this barrier may be that MF and RND family in gram-negative cells many efflux carrier and some accessory proteins exist simultaneously.These albumen are considered to a kytoplasm carrier and an adventitia interchannel " bridge ", thereby medicine is can be directly outer to be discharged in the periphery medium and not to enter pericentral siphon (Fig. 2), because they are relevant with the histone in the gram negative bacterium, comprise HlyD, LktD, CyaD, AprE and CvaA, they directly efflux its substrate protein white matter to external agency as bridge and help equally.Thereby some channel proteins in accessory protein and the adventitia are possible as TolC formation mixture.Find in Pseudomonas aeruginosa that recently the mexA-mexB-oprM system has supported this model.The operon of this supposition accessory protein MexA that not only encodes, the outer membrane protein oprM that also encodes, and also this assortment of genes points out this three kinds of albumen to form mixtures, and this mixture may form a continuous passage to the outside media open (Fig. 2).In fact, OprM and outer membrane protein CyaE and PrtF show sequence homology, these outer membrane proteins may to efflux the formation of mixture relevant with three compositions, this mixture contains a cytoplasmic protein carrier, a pericentral siphon accessory protein and an outer membrane channel, they are discharged to cytolysin and proteolytic enzyme B and C in the medium outside directly respectively in bordetella pertussis and chrysanthemum Erwinia.And the deactivation of OprM is the same with the deactivation of MexA, can cause the supersensitivity to many medicaments.Now do not know also how those systems that do not contain accessory protein are discharged to medicament in the medium effectively outward.But concerning those expressed the proteic bacterium of 50kDa of such outer membrane protein such as OprM and Pseudomonas aeruginosa overexpression, adventitia provided another target compound for efflux pump inhibitors described herein.

As already mentioned, the low permeability of adventitia can not produce significant clinically resistance separately, and producing high-caliber like this resistance needs another kind of co-factor.In many systems, the factor that active efflux system is seemingly such.But this does not also mean that the adventitia barrier is inessential.Because the intracellular concentration of many medicines is interior stream and effluxes the equilibrated result, thereby in the organism as Pseudomonas aeruginosa, many probably medicaments see through the interior slowly stream effect of low permeability adventitia makes the effect of effluxing become especially effectively resistance mechanism.That is to say just have the organism such as the intestinal bacteria of high-permeability adventitia at last,, can not produce the resistance of conspicuous level, maybe can reduce the structure that it strides across the transmitance of adventitia unless medicament has big volume although have effectively outer heat-extraction system.

Aspect this, the OmpF porin that intestinal bacteria marRAB gene mutation body produces less amount is very important, they produce bigger passage between colibacillary two non-specific porins, thereby play decisive role in the osmosis of most antibiotics.Along with the reduction of interior stream effect, initiatively the effect of effluxing will produce much higher resistance.Some are plasmid-encoded in the gram negative bacterium effluxes carrier and also may utilize similar mechanism, has just reported that as far back as 1978 is suppressed an OmpF porin synthetic R plasmid.With the relevant gene of non-enzymatic paraxin resistance, efflux the homologous gene of carrier (on seeing) probably for CmlA in the hemophilus influenzae, can suppress the synthetic of the main porin of this organism.It is proteic synthetic that clone's CmlA gene can suppress the intestinal bacteria mesopore.A clinical separation strain with the chemical sproof salmonella typhi of non-enzymatic paraxin may have the transposon that contains OmpF.This ability that effluxes gene reduction outer membrane permeability by some plasmids and transposon coding is proved to be so far has only the very r factor of close limit.Combine if such activity effluxes carrier with extensive substrate, will produce great health care problem.

Usually be accompanied by the synthetic expression that effluxes carrier that suppresses of OmpF porin and improve, can under situation, take place without any hereditary change.Therefore, paraxin and tsiklomitsin improve MarA and regulate proteic transcribing, and efflux the synthetic of carrier thereby may improve one or more.Ironically, pathogenic bacteria is residing oxygen partial pressure in host tissue, and the known OmpF that can produce checks and improves resistance to some antiseptic-germicide, and this may be the result that the effect of effluxing improves.Similarly, reply microbial infection in the plant tissue and the Whitfield's ointment that produces, thereby knownly can check the synthetic of pmpF porin intestinal bacteria are temporarily improved paraxin, tsiklomitsin, the resistance of quinolones and penbritin.This medicament scope further shows a kind of intervention of and several outer heat-extraction systems.

Because the special mechanism of antibiotic resistance is considered to more important, produce more effective antibiotic effort normally the special group on antibiotic molecule modify, make them become the inert substrate of common microbiotic inactivator.The existence of more general mechanism of drug resistance forces people to reappraise this strategy.These machine-processed resistances significantly that produce clinically: what see important opportunistic pathogen Pseudomonas aeruginosa is because multiple medicines effluxes carrier and the effective coefficient result of permeability barrier to multiple antibiotic intrinsic resistance really, in showing most of The British Isles clinical separation strain that Pyocianil resistance level is improved, the raising that effluxes the carrier expression is the most probable reason of its resistance.As if concerning pharmaceutical industry, the compound that production can overcome this class mechanism will be a significant challenge, because the outer heat-extraction system of some multiple medicines can pump nearly all amphiphilic cpds.Obviously, need more information about the substrate cohesive process of these carrier.Another feasible method will be to improve the spontaneous interior stream effect of medicine, for example, thereby can be offset by interior stream effect fast by giving the enough lipotropy effects of effluxing of medicine.Really, the more lipophilic derivative of tsiklomitsin and fluoroquinolone is higher to the endurance strain activity of the gram positive bacterium that can pump these medicines.But in gram negative bacterium, more lipophilic medicine will pass through the porin passage slowlyer, thereby improve the effectiveness that lipotropy can not improve medicine.The Pseudomonas aeruginosa outflow pump that siderophore is relevant

As mentioned above, multiple medicines outflow pump is present in the Pseudomonas aeruginosa.This bacterium is a kind of important clinically pathogenic agent that many antiseptic-germicides is had intrinsic resistance.And, there is the problem that chemical sproof development brought of the medicine (as carbapenem and fluoroquinolone) of potential anti-microbial activity to occur more and more frequently to this organism usually.In addition, may be associated with the fluoroquinolone resistance to chemically incoherent antibiotic cross resistance.In vitro study with fluoroquinolone endurance strain of cross resistance shows that resistance is attributable to the reduction that medicine that the change of outer membrane permeability causes is built up.In some example, this conclusion is to produce on the evaluation of foundation of the new outer membrane protein of these mutant.

In Pseudomonas aeruginosa K385 bacterial strain, the generation of 50-KDa outer membrane protein is accompanied by the reduction to 2,2 '-two pyridines and many biocide susceptibility.(K385 bacterial strain be can at the mutant strain that contains the Pseudomonas aeruginosa siderophore defective of growing in 2,2 '-two pyridines (0.5mM), the nonferrous minimum medium) (Poole etc., 1993a).Aforesaid promise Norxin resistance Pseudomonas aeruginosa nfxB and nfxC bacterial strain, along with the generation of 54-kDa and 50-kDa outer membrane protein, also respective table reveals the susceptibility reduction to many biocides.But the reduction different with the nfxC mutant strain, that K385 and nfxB mutant strain do not show the OprD level.The Pseudomonas aeruginosa Ciprofloxacin resistance mutant that non-quinolone microbiotic is shown cross resistance also gives expression to a kind of new 54kDa outer membrane protein.At last, share in many antibiotic resistance of the Pseudomonas aeruginosa mutant strain that filters out with Meropenem or lomefloxacin (lomefloxacin) or Ofloxacine USP 23 and cefsulodin, the outer membrane protein of an about 49kDa (OprM) obtains identifying.Although above-mentioned mutant strain has some nuances on chemical sproof phenotype, the outer membrane protein of identifying in all examples may be identical.If like this, producing nfxB, nfxC, OprM and having that cause chemical sproof medicine to be built up to reduce in the mutant strain of Ciprofloxacin tolerance be not because the change of the outer membrane permeability of thinking at first probably, but efflux effect owing to antibiotic, as long as between OprM (being the ORFC product) and some bacterial outer membranes outflow chlG, have homology.Interesting is that quinolone resistance and many antibiotic resistances are also partly owing to the mechanism of effluxing in the intestinal bacteria.

Except that OprM, ORFA-ORFB-oprM (ORFC) operon also encode 40 and 108kDa, estimation be positioned two kinds of albumen on the plasma membrane.As if in view of the homology between ORFAB product and plasma membrane outflow chlG, ORFAB strides plasma membrane at antibiotic resistance and biocide and effluxes on and work.For this reason, ORFAB refers in particular to mexAB (multiple medicines efflux pump).

ORFABC (mexA-mexB-oprM) operon is regulated and control by iron ion, and OprM is derivable under certain iron ion restricted condition really.Not observing proteinic inducing action when time in the BM2 minimum medium that is grown in the iron ion shortage may be that the shortage of iron ion is not as other minimum medium in the BM2 substratum because significant iron ion pollutes in the phosphate component of BM2.This shows therefore suitable OprM (also comprise ORFABC[mexA-mexB-oprM]) expresses the stricter iron ion of needs and limits.For example, when be grown in that iron ion lacks with HEPES buffered minimum medium in the time, OprM is induced easily, this substratum is littler by the degree that iron ion pollutes.Certainly, corresponding with low iron ion content, the rate ratio of siderophore high three to four-fold in the BM2 minimum medium that iron ion lacks in this substratum.Similarly, expection can reduce the iron chelator 2,2 of available iron ion '-growing under the condition of pyridine existence to make OprM be induced.Observed OprM is subjected to Zn ²⁺The inductive phenomenon is consistent with this proteic iron regulation and control, because known Zn ²⁺Can strengthen in the Pseudomonas aeruginosa iron ion is adjusted to branch and comprises siderophore and receptor expression thereof.Zn ²⁺Siderophore is produced similar influence also be found in Pseudomonas fluorescens and Wei Nielande vinelandii.In the example of back, Zn ²⁺The output that strengthens siderophore is because Zn ²⁺Suppress the ferric iron reductase activity and caused the minimizing of ferrous ion in the kytoplasm.

ORFABC (mexA-mexB-oprM) is except being subjected to iron ion regulation and control, and this operon is also regulated and control with picked-up jointly with the generation of pyofluorescein composition, and this shows that it works in the secretion of pyofluorescein.Observed ORFABC (mexA-mexB-oprM) product is obviously consistent with homology and this conclusion of many bacterium efflux proteins.And although structurally difference is very big as the microbiotic of the substrate of the outer heat-extraction system of ORFABC (mexA-mexB-oprM), they have some common traits (aromatic ring), and great majority all have the ability that comprises iron ion in conjunction with positively charged ion.Aspect this, they are similar to the chromophoric group that contains a little tea phenol in the pyofluorescein.This shows that the drug tolerance that ORFABC (mexA-mexB-oprM) relies on is certain biocide result similar to pyofluorescein, and pyofluorescein may be the real substrate of the outer heat-extraction system of ORFABC (mexA-mexB-oprM).Obviously, ORFABC (mexA-mexB-oprM) product has substrate specificity very widely, and not only pyofluorescein, and its metabolite is the natural substrate of this outer heat-extraction system.Therefore, ORFABC (mexA-mexB-oprM) may be not only in the pyofluorescein secretion of beginning, and in the secretion of the metabolite of the pyofluorescein that utilizes again and this process thereof, also play effect.

The iron ion transportation composition superiority of substrate specificity widely also has discovery in the research of intestinal bacteria iron ion adjusting outer membrane protein Fin and Cir, it is relevant with the picked-up of having a liking for iron iron enterobactin hydrolysate that these two kinds of albumen it is reported.These albumen also can promote the microbiotic of iron content chelation group, comprise the picked-up of the beta-lactam that catechol replaces.

Noticeable high homology is the strong hint of identical function between ORFAB (mexABC) product and albumin A crA and AcrB.Although AcrA is considered to the efflux protein relevant with other biocide tolerance with acriflavine with AcrB, acriflavine can not be the normal cell substrates of these albumen.But, notice that as pyofluorescein intestinal bacteria siderophore enterobactin also is that a molecule that contains catechol is absorbing.Therefore, AcrA and AcrB may work in the secretion of enterobactin and/or its metabolite.Certified homology shows that intestinal bacteria may utilize multisystem to efflux enterobactin between ORFAB (mexAB) product and EnvCD (and AcrAB also has high homology).The Pseudomonas aeruginosa outflow pump that beta-lactam is relevant

Except mexAB-oprM pump discussed above, another Pseudomonas aeruginosa outflow pump is also relevant with the beta-lactam resistance.Because the action site of beta-lactam is positioned at the outside surface of plasma membrane, thereby beta-lactam need not enter kytoplasm and exercise its function, therefore initiatively efflux as the chemical sproof important factor of beta-lactam, and be astonishing.In addition, the existence of carboxyl means that they can not promptly spread through plasma membrane on many beta-lactams.But resistance is proved not owing to the change of the low permeability or the beta-lactam enzyme level of adventitia (Li etc., " biocide chemotherapy " (AntimicrobAgents Chemother) 38 volume 1742-1752 pages or leaves in 1994).

Experiment shows in the Pseudomonas aeruginosa of high drug-resistance two kinds of plasmalemma proteins and a kind of outer membrane protein high expression level.These proteic expression levels are higher than the relevant bacterial strain that does not have high drug-resistance significantly.As if in addition, supersensitivity mutant strain K799/61 lacks active efflux system, because it can not pump tsiklomitsin and paraxin basically, the albumen of these hint three kinds of excessive generations in the high drug-resistance bacterial strain is compositions of Pseudomonas aeruginosa outflow pump system.This work materials and methods commonly used is seen 1994 " biocide chemotherapy " 38 volume 1742-1752 pages or leaves such as " biocide chemotherapy " (Antimicrob.Agents Chemother) 38 volume 1732-1741 pages or leaves in 1994 such as Li and Li.All these documents are hereby incorporated by.

These proteinic aminoacid sequences are easy to determine with method well-known to those having ordinary skill in the art.For example, every kind of albumen all can produce separation and purification the bacterial strain of evaluation of this outflow pump from high level.The aminoacid sequence of the every kind of protein purification amino acid sequencing method of available standards is subsequently determined.Any in the same available many different methods of the every kind of proteic nucleotide sequence of encoding determined easily.A kind of method is that the aminoacid sequence of a protein part is convertible into the oligonucleotide probe (the preferred sequence of using low degeneracy of the code) of a cover degeneracy, about eight to 20 Nucleotide of each in them.The probe of such cover degeneracy is used to survey the encoding sequence of this polypeptide total length thereupon.In case detect, this encoding sequence can be the gene of given polypeptide to confirm it with common recombinant technology operation and order-checking.Another kind method is, in the outflow pump defective bacterial strain corresponding to losing or the encoding sequence of the outflow composition of defective can be identified and separate and check order.Although for example understand these methods, other method also can be used effectively, and may be preferred under specific situation.Outflow pump indispensable gene

The outflow pump is important to the Pseudomonas aeruginosa cell.Existing good circumstantial evidence shows that the inhibition pair cell of outflow pump kytoplasm composition is lethal.The unique method that produces invalid mexB mutant strain be utilize pyofluorescein produce defective bacterial strain or can not be in the substratum that iron ion lacks the bacterial strain of growth and iron ion metabolism damage.(Poole etc., 1993 " molecular microbiology " are 10 volume 529-544 pages or leaves (Mol.Microbiol.)) if the mexB mutant strain in fact is lethal, the inhibitor of the pump kytoplasm composition that outflows so may be germ-resistant.The inhibition of outflow pump

The present invention shows the outflow pump in the pathogenic bacterium, and the existence of especially many substrate outflow pumps can be used on SCREENED COMPOUND with in the method for finding efflux pump inhibitors.Shown in this term, also described in general introduction, such inhibitor can reduce the outflow pump effluxes antiseptic-germicide from kytoplasm ability.The present invention shows that such screening method can be the bacterium design with multiple medicines outflow pump, and Pseudomonas aeruginosa outflow pump especially is as the outflow pump of overexpression among mexA/mexB/oprM and bacterial strain K385 and the PAO4098E.

A kind of method is can slow down antiseptic-germicide effluxing from bacterial cell based on efflux pump inhibitors.Significantly during cell growth inhibiting, the outflow pump makes this medicament keep low-down concentration in cell but this antiseptic-germicide concentration is very low in growth under the bacterium that contains the pump that outflows can outflowed the situation that antiseptic-germicide that pump effluxes exists by this.But when certain efflux pump inhibitors also exists and the enough high energy of its concentration when suppressing this outflow pump active significantly, it can improve the intracellular concentration of antiseptic-germicide.The raising of antiseptic-germicide concentration can cell growth inhibiting.The cytostatic ability of antiseptic-germicide can confirm that wherein a kind of high level produces the outflow pump with two relevant bacterial strains, and another kind does not produce.Therefore, the concentration of used antiseptic-germicide must be enough high to suppress high-levelly to produce the growth of the bacterial strain of outflow pump.But also must be enough low so that the growth of the bacterial strain of high-level generation outflow pump can not obviously be suppressed.Thereby the compound that suppresses high level outflow pump strain growth is accredited as the efflux pump inhibitors of supposition.The growth-inhibiting that suppresses because of the outflow pump can be by more same bacterial strain in the efflux pump inhibitors existence of supposition but there is not the growing state under the condition of antiseptic-germicide to further specify.If the growing state when the inhibitor of supposition and the growing state of antiseptic-germicide in the presence of simultaneously obviously exist not as the inhibitor that has only supposition, growth-inhibiting is likely because the outflow pump suppresses so, accounts for major part at least.In some embodiment of this screening method, it is very useful using the bacterial strain of excessive generation outflow pump.The inferior inhibition concentration of antiseptic-germicide should be enough high to suppress the growth of this bacterium wild type strain so.

Another screens the method for the inhibitor of extensive substrate outflow pump such as Pseudomonas aeruginosa outflow pump, be based on the use of reporter gene, the expression of reporter gene is subjected to the control of a regulating and controlling sequence, and this regulating and controlling sequence can be induced by sufficiently high certain compound of concentration such as antiseptic-germicide (or analogue of a non-activity).The reporter gene that has derivable regulating and controlling sequence in this method inserts in the dispensable gene of bacterial chromosome with single copy form.Therefore, when having bacterial growth that the reporter gene structure inserts in the following time of condition of efflux pump inhibitors compound and certain antiseptic-germicide (its concentration is not enough to obvious bacteria growing inhibiting when the other factor lacks) existence, the intracellular concentration of this antiseptic-germicide will improve.The rising of this antiseptic-germicide intracellular concentration is because efflux the reduction of efficient, as described in top method.But before antiseptic-germicide reaches growth inhibiting intracellular concentration, the concentration of rising will be induced the expression of reporter gene.The discovery of expressing shows that the function of the pump that outflows is suppressed, and promptly just screened compound is an efflux pump inhibitors.

Well-known regulatory mechanism has been used in a design of this method, and wherein the expression of reporter gene is subjected to inducing of tsiklomitsin.In this mechanism, tsiklomitsin combines with tetR repressor molecule tightly, has stoped tetR to handle combining of site with tetA.Do not have tetR to handle combining of site with tetA, detectable product will be transcribed and be translated subsequently to the reporter gene encoding sequence in this operator gene downstream.(Kirsch etc., 1991 " microbiotic magazine " are 44 volume 210-217 pages or leaves (J.Antibiotics)).As mentioned above, one contains the tetR gene, and the structure of tetA regulating and controlling sequence and reporter gene encoding sequence is inserted in the dispensable gene of Pseudomonas aeruginosa.The cell of these reorganization can be used for screening subsequently as mentioned above and have the active compound of efflux pump inhibitors.

Aforesaid method and Rothstein etc. " antiseptic-germicide chemotherapy " (Antimicrob.AgentsChemother.) the described work of 37 volume 1624-1629 pages or leaves differ widely.This report has been described a kind of with the method for the tetR gene on the plasmid together with the technology screening intestinal bacteria tsiklomitsin specificity efflux pump inhibitors of identifying new tsiklomitsin.

The third screening method report that is suppressed as the outflow pump by the spectral detection of the compound (detectable compound) of outflow pump transportation in the cell.In this method, the cell with this outflow pump contacts with detectable compound.When efflux pump inhibitors does not exist, but the intracellular concentration of detection compound intracellular concentration of this compound when suppressing compound that the outflow pump effluxes effect and exist compare, will maintain lower concentration.Therefore, if but when test compound exists, detect the rising of detection compound intracellular concentration.This shows that this test compound has suppressed the activity that effluxes of pump, thereby may be efflux pump inhibitors.But perhaps can determine born of the same parents' extracellular concentration (concentration in the suspension medium) of detection compound.Thereby can calculate intracellular concentration or directly utilize born of the same parents' extracellular concentration.Do not compare when not existing with efflux pump inhibitors, the existence of inhibitor makes still less dyestuff or fluorescence mass-energy build up in cell, thereby born of the same parents' extracellular concentration of dyestuff or fluorescent substance will be lower.

The 4th kind of report that screening method suppresses as the outflow pump with positive growth.Inducing of the microbiotic deactivation thing that this screening method stimulates based on the outflow pump.Therefore microorganism can grow in the presence of the microbiotic of inhibition concentration.The outflowed intracellular concentration of the compound that pump effluxes of this antibiotic deactivation thing raises and induces.Be suppressed (as being suppressed by the test compound) if efflux the outflow pump of this compound, the deactivation thing will be expressed (or activation), produce the antibiotic intracellular concentration that suppresses before reducing, and this will cause detectable growth.

An embodiment of this screening method is used the tetA promotor with sensitive tetracycline the same in top second screening method.But, this promotor connecting one have only when the tetA promotor by the gene that can grow after inducing.An example of this gene is the blaS gene, and its coding is had a liking for the L-1 β-Nei Xiananmei of maltose oligotrophy Zymomonas mobilis (xanthomonas maltophilia).The product of this gene can provide the required positive of this screening method to select.This fusion gene will place karyomit(e) to obtain genetic stability.The L-1 β-Nei Xiananmei is had whole functions after inducing in Pseudomonas aeruginosa, and carbapenem then can not be secreted by the outflow pump of Pseudomonas aeruginosa.β-Nei Xiananmei on the pseudomonas karyomit(e) can not hydrolysis carbapenem (J.Trias, " biocide chemotherapy " (Antimicrob.Agents Chemother.) 33 volume 1201-1206 pages or leaves in 1989).

The screening strain will be at the tsiklomitsin of lower concentration, and there are growth down in test compound and carbapenem.If the outflow pump is suppressed, the intracellular concentration of tsiklomitsin will raise, thereby induce the tetA promotor, and the expression, L-1 β-Nei Xiananmei that are blaS immediately are with the hydrolysis carbapenem, thereby cell can be grown.If pump is not suppressed, the intracellular concentration of tsiklomitsin will can not raise, and cell also just can not synthesize the L-1 β-Nei Xiananmei, just can not grow in the presence of carbapenem yet.

This class screening method also can be simply provide the function of titration necessary amounts by the concentration that changes carbapenem in the substratum, and the system of the high flexible of the compound that a kind of searching has different inhibition abilities is provided.Retardance outflow pump can not change the tolerance of imipenum, because this substrate can not be by the identification of outflow pump, if pump is suppressed, it can not change to some extent yet.Effect-the regulatory mechanism of pump

The normal function that efflux pump inhibitors can be by suppressing pump or the normal expression of pump or both are suppressed and suppress the pump that outflows.In order to study the regulation and control of pump, the existence of pump is with monitored on the film.For example the electrophoretic technique of available standards is monitored it, and the Western that utilizes protein staining technology (Coomassie blue or silver dye) commonly used or utilize the anti-antibody that outflows the pump composition to carry out standard analyzes, and can be observed the band of pump composition correspondence.The mechanism of inhibitor effect will further be studied at transcriptional level, for example analyze, or the control by reporter gene being in outflow pump startup down and the activity of detection reporter gene encoded protein matter by utilizing the probe that from the gene order of coding outflow pump composition, obtains to carry out Northern with the known array in the gene.The explanation of screening source compound and substructure research method

Method of the present invention is applicable to that screening multiplely may have the active source compound of efflux pump inhibitors.Compound with different sink carries out preliminary screening, but present method also is applicable to other compound library.Such storehouse can be the natural product storehouse, combinatorial libraries, or other small molecules storehouse.In addition, also can be tested from the compound in commercialization source.The commercially available analogue of the efflux pump inhibitors that this test is particularly useful for having identified.

Treat that by at first limiting SCREENED COMPOUND is those compounds with preferred structure feature, the compound with known structure of the commercial source pump screening active ingredients that can be used to effectively outflow.For example, at first attempt setting up a Ya Ku who is used to screen, it will increase can suppress the outflow pump structure.The strategy of this trial comprises the commercially available compound that obtains to contain common constructional feature in the Mammals efflux pump inhibitors.In order to quicken this trial, (MDLInfbrmation Systems Inc.) is used to commercially available chemical reagent catalog data base (MDLInformation Systems, two-dimentional substructure research Inc.) to the ISIS computer program.This database contains the structure and the purchasing order information of nearly 175,000 kinds of commercially available compounds.Other is open, commercially available chemical database also can use.The general introduction of success compound

The instantiation compound that is accredited as efflux pump inhibitors with the method among the present invention is L-phenylalanyl-L-arginyl-β-naphthalene amino acid.Describe among the external feature of some of this compound embodiment 7 below.

This compound can be considered to have the universal architecture of following structure 1-4 representative:

R=alkyl (C wherein ₁-C ₄), fluoroalkyl (C ₁-C ₄), cross fluoroalkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), aryl (C ₆-C ₁₀), single substituted aryl (C ₆-C ₁₀), [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), amino, single substituted-amino [is randomly used alkyl (C ₁-C ₄) replace], two substituted-aminos [are randomly used alkyl (C ₁-C ₄) combination replace], or hydroxyl replaces], two substituted aryl (C ₆-C ₁₀), [alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I) and amino arbitrary combination], 2-(or 3-)-thienyl, 2-(or 3-)-furyl, or 2-(3-or 4-)-pyridyl.W=H, NH ₂, single substituted-amino [is randomly used alkyl (C ₁-C ₄) replace], two substituted-aminos [are randomly used alkyl (C ₁-C ₄) arbitrary combination replace], nitrogen heterocyclic [as the N-morpholinyl, N-piperazinyl, N-pyrrolidyl, TMSIM N imidazole base, N-pyrryl, N-pyrazolyl, N-triazolyl or N-tetrazyl], halogen (Br, Cl, F, I), hydroxyl, alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄).R ¹=(CH ₂) _nNR ^bR ^c, (CH ₂) _nNHC=(NR ^a) NR ^bR ^c, (CH ₂) _nSNHC=(NR ^a) NR ^bR ^c, (CH ₂) _nC=(NR ^a) NR ^bR ^c, (CH ₂) _nN=CNR ^bR ^c, (n=2-4), R ^a(R ^bOr R ^c)=H, alkyl (C ₁-C ₄), aryl (C ₆), substituted aryl, phenyl, substituted-phenyl [is randomly used alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino the replacement], perhaps R ^a+ R ^b=(CH ₂) _2-3Or-CH=CH-, X=aryl (C ₆-C ₁₀) ,-(CH ₂) _0-2Aryl (C ₆-C ₁₀), substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino the replacement], replacement-(CH ₂) _0-2Aryl (C ₆-C ₁₀) [with alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F, I) or amino replace at the aryl position], 2-(or 3-)-thienyl, 2-(or 3-)-furyl, 2-(3-or 4-)-pyridyl, benzofuryl (in any position of cumarone ring), benzothienyl (in any position of thionaphthene ring).Wherein have asymmetric center, its absolute stereo chemical property may be R or S configuration, may be dl-mixture maybe, is included in the general structure.

Although L-phenylalanyl-L-arginyl-β-naphthalene amino acid is an efflux pump inhibitors, suppress active related compound but still need find to have enhanced physiology profile and efflux.Usually, the general method of identifying the related compound with better medicinal properties is an analogue of preparing or obtain many initial compounds.Exist trickle textural difference between these compounds.These analogues are tested subsequently to determine whether they have kept activity.Identify following factor, as tolerance to degraded, serum combination, total toxicity and solvability.According to the result of these analyses, can determine a preliminary structure-activity relationship (SAR), reflected the relation between special constructional feature or substituting group and activity level and some other factors relevant with using intention (for example as the treatment compound).This information can be used for instructing directly further preparation and test of analogue subsequently, to find the compound of the comprehensive characteristics with improvement.For L-phenylalanyl-L-arginyl-β-naphthalene amino acid, with reference to top general formula, a reasonably first one step process of preparation analogue is that the different analogue of a series of W of preparation is to find out compound more stable in serum.Therefore, as implied above, such cover analogue will carry out the analysis of the medicinal properties of screening active ingredients and improvement.Pharmaceutical composition and administering mode

For the specific compound of efflux pump inhibitors can pass through itself, or by with antibacterial agents, or become pharmaceutical compositions to give the patient with suitable carriers or mixed with excipients.Efflux pump inhibitors has two kinds of different modes at least with antibacterial agents.A kind of is that a certain amount of efflux pump inhibitors and a certain amount of antiseptic-germicide are mixed into mixture, as solution or pulverulent mixture.In such mixture, the quantitative proportion of inhibitor and antiseptic-germicide can change to be fit to the concrete treatment of share and expecting.Share in the mode at second kind, inhibitor and antiseptic-germicide covalent attachment, and this bonded molecule can be broken apart in cell.But term " share " possibility that also can refer to other, comprises the administration in turn of inhibitor and another kind of antiseptic-germicide.In addition, the form administration that efflux pump inhibitors and/or another kind of antiseptic-germicide can the medicine precursors, promptly this compound gives the patient with the form that could form functional form after modified in cell.When the disorderly patient of treatment interest (interest), can give such medicament with therapeutically effective amount, dosage refers to that the compound of this consumption will cause the improvement of symptom or the prolongation of patient lifetime, and can comprise the elimination of infected by microbes.

The toxicity of such compound and curative effect can the pharmacology method with standard be determined in cell cultures or laboratory animal, for example, determine LD ₅₀(50% colony's lethal dose) and ED ₅₀(50% colony's dosage).Dosage ratio between toxicity and curative effect is therapeutic index, and can be expressed as LD ₅₀/ ED ₅₀The ratio form.Compound with big therapeutic index is preferred.The data that obtain from these cell culture experiments and zooscopy can be used for preparing the dosage range that is used for the people.The dosage of preferred this compounds is in ED ₅₀In the very little or avirulent circulation composition scope.According to used medicine type and used administering mode, dosage can change in this scope.The serum-concentration of preferred efflux pump inhibitors treatment should be in 0.1-100 μ g/ml scope.

For any compound that is used for method of the present invention, its dosage can be from cell culture test according to a preliminary estimate.For example, can determine that in animal model certain dosage is to obtain round-robin plasma concentration scope, the IC in this scope ₅₀Determine by cell culture test.Such information can be used for further accurately determining the effective dose in the people.Levels of drugs in the blood plasma can be measured, and for example measures with the HPLC method.

In a more preferred embodiment, the efflux pump inhibitors in the pharmaceutical composition has the structure shown in the aforesaid general structure.

Appropriate preparation, the situation that administering mode and dosage will be looked the patient by concrete doctor makes one's options.(seeing " the pharmacy bases of treatment " such as Fingl (THE PHARMACOLOGICAL BASISOF THERAPEUTICS) 1975, the chapter 1 page 1).It should be noted that the attending doctor should know because how toxicity or tissue injury and this reach and when stop, interrupt or adjust administration.On the contrary, the attending doctor also should know when clinical response is not enough and therapeutic dose should be mentioned higher level (preventing toxicity).For example the severity of condition can partly be evaluated by the prognostic evaluation method of standard.And dosage and possible administration frequency will be with patient's age, body weight with to the different of the reaction of medicine and change.Also can be used for veterinary science with top similar program.

According to the special infection of being treated, can prepare such medicament and systematically or administration partly.Make up a prescription and medication is found in " Remington's Pharmaceutical Science " (Remington ' sPharmaceutical Sciences), the 18th edition, Mack Publishing Co.Easton.PA (nineteen ninety).Suitable administering mode comprises the oral cavity, rectum, and through skin, vagina, through mucous membrane, or enterally administering; Parenterai administration comprises muscle, and is subcutaneous, intramedullary injection, and in the sheath, in the direct ventricle, intravenously, intraperitoneal, in the nose, or intraocular injection, only exemplify a part.

In order to inject, medicament of the present invention should be mixed with the aqueous solution, the damping fluid such as Hank ' the s solution of preferred physical compatibility, Ringer ' s solution, or physiology salt buffer.For administration, in preparation, used the permeate agent that is suitable for barrier to be infiltrated through mucous membrane.Such permeate agent is known in the art.

Be used to realize that with disclosed among the present invention the compound of inventing is mixed with the medicament that is fit to the whole body administration with medicinal carrier, also within the scope of the invention.Select correct carrier and suitable manufacturing process, composition of the present invention especially is mixed with the composition of solution, can be through parenterai administration, and as by intravenous injection.These compounds can be mixed with easily with pharmaceutical carrier well known in the art and be suitable for oral medicament.Such carrier can make compound of the present invention be mixed with can be by the tablet of the patient's who is treated oral absorption, pill, capsule, liquid, glue, syrup, slurry, suspension etc.

The pharmaceutical composition that is suitable among the present invention comprises that those contain the composition that effective amount of actives can reach required purpose.Significant quantity determine what those skilled in the art accomplished easily, especially referring to after the detailed disclosure that provides here.Except activeconstituents, these pharmaceutical compositions can contain suitable pharmaceutical carrier, comprise vehicle and auxiliary, and they can help active compound is processed as medicinal preparations.The preparation of preparing for oral administration can be tablet, drageeing, the form of capsule or solution.Pharmaceutical composition among the present invention can be made in a known manner, for example utilizes traditional mixing, dissolving, and granulation, sugar-coatization suspends (levitating), emulsification, the capsule parcel wraps methods such as (entrapping) or freeze-drying.

The pharmaceutical preparation that is used for parenterai administration comprises the aqueous solution of active compounds in water-soluble form.In addition, the suspension of active compound also can be made into suitable oily injectable suspensions.Suitable lipophilic solvent or solvent comprise fatty oil such as sesame oil, or synthetic fatty acid ester such as ethyl oleate or triglyceride, or liposome.Water miscible injectable suspensions can contain the material that improves suspension viscosity, Xylo-Mucine for example, sorbyl alcohol, or dextran.Also randomly containing suitable stabilizers and solubility promoter in the suspension makes compound can form highly spissated preparation.

Being used for oral medicinal preparations can randomly smash mixture to pieces by active compound is combined with solid excipient, the processing granular mixture, and obtain after adding suitable auxiliary, as required, can be made into tablet or sugar-coat nuclear.Suitable vehicle is weighting agent particularly, as sugar, comprises lactose, sucrose, N.F,USP MANNITOL, or sorbyl alcohol; Cellulosics such as W-Gum, wheat starch, rice fecula, horse official seal sweet potato starch, gelatin, tragakanta, methylcellulose gum, hydroxyprolyl-methylcellulose gum, Xylo-Mucine, and/or polyvinylpyrrolidone (PVP).As required, can add decomposition agent, as cross-linked polyvinylpyrrolidone, agar or algin or its salt are as algin sodium.

Should provide suitable bag quilt on the sugar-coat nuclear, for this purpose, available spissated sugar soln wherein optionally comprises gum arabic, talcum powder, polyvinylpyrrolidone, carbopol gel, polyoxyethylene glycol, and/or titanium dioxide, lacquer solution and appropriate organic solvent or solvent mixture.Dyestuff or pigment can be used in tablet or the coated tablet bag quilt to differentiate or to describe the feature of the various combination of active compound medicament.

Be used for oral medicinal preparations and comprise the capsule of the sucking fit of making and the softness made from gelatin and softening agent such as glycerine and sorbyl alcohol, airtight capsule with gelatin.The capsule of sucking fit can contain and weighting agent such as lactose, and tackiness agent such as starch, and/or lubricant such as talcum powder or Magnesium Stearate blended activeconstituents also optionally contain stablizer.In soft capsule, active compound dissolves in or is suspended in the suitable liquid, as fatty oil, and whiteruss, or liquid macrogol.Also can add stablizer in addition.EXAMPLE Example 1: efflux pump inhibitors screening-growth-inhibiting screening

This test is the screening based on the molecule that can strengthen the antiseptic-germicide effect.Two pseudomonas aeruginosa strains in this screening method, have been used.Bacterial strain K385 is by the isolating a kind of mutant strain with multidrug resistance of K.Poole, its energy overexpression outflow pump, bacterial strain K613, a kind of oprM ∷ Hg mutant strain (Poole etc., 1993a of susceptibility; Poole etc., 1993b).Underproof unknown molecular is added in the substratum, and substratum is inoculated with bacterial strain K385.If the test molecules in inhibiting outflow pump, thereby it will strengthen the effect of antiseptic-germicide and suppress grow.When not existing, also tests by antiseptic-germicide these compounds to detect their inherent bacteriostatic activities.Screening method

The fresh inoculation liquid of bacterial strain K385 is diluted to 1/50 with same substratum subsequently 35 ℃ of overnight incubation in the Mueller-Hinton broth culture.Cultivate that culture reaches 0.2-0.3 in the OD at 600nm place value after about 60 minutes.Mueller-Hinton broth culture with new preparation is diluted to 1/50 then.Every hole contains the culture inoculation of the microtiter plate of 50 μ l Mueller-Hinton substratum with 50 μ l dilution, contains the test compound of 20 μ g/ml and the Ciprofloxacin of 0.5 μ g/ml in 50 μ l substratum.Contain the Mueller-Hinton broth culture of same volume and test compound but do not have the second cover microtiter plate of Ciprofloxacin also to inoculate with the culture of 50 μ l dilution.Initial growth conditions is to contain the test compound of 10 μ g/ml and the Ciprofloxacin of 0.25 μ g/ml in the 100 μ l Mueller-Hinton broth cultures.Titer plate places 35 ℃ of incubators to hatch 20 hours.Growing state is measured under 600nm with micro plate readout instrument (Thermomax micro plate readout instrument, Molecular Devices).Used following contrast: nonvaccinated Mueller-Hinton meat soup, be grown in the K385 bacterial strain in the Mueller-Hinton broth culture that contains 0.25 μ g/ml Ciprofloxacin and do not contain Ciprofloxacin, be grown in the K613 bacterial strain in the Mueller-Hinton broth culture that contains 0.25 μ g/ml Ciprofloxacin and do not contain Ciprofloxacin.Nonvaccinated Mueller-Hinton meat soup is used for establishing the growth baseline.Test compound was tested with double when Ciprofloxacin existed.Compare the growing state of bacterial strain K385 in the presence of 0.25 μ g/ml Ciprofloxacin, the 10 μ g/ml L-phenylalanyl-addings of L-arginyl-β-naphthalene amino acid have strengthened the effect of Ciprofloxacin.In the OD600 that is surveyed, growth has been suppressed 99%.When Ciprofloxacin did not exist, 10 μ g/ml L-phenylalanyl-L-arginyl-β-naphthalene amino acids were 1% to the inhibition of growth.

Bacterial strain PAO4098E with the excessive generation another kind outflow pump different with the K385 bacterial strain can carry out similar screening.The concentration of testing the used Ciprofloxacin of toughener in this screening method is 0.2 μ g/ml rather than 0.25 μ g/ml.

If test compound suppresses the outflow pump, the intracellular concentration of Ciprofloxacin will raise, and the cell growth will be suppressed.The compound that strengthens the Ciprofloxacin effect will be identified and further be estimated.

As discussed above, in preferred embodiments, the microbiotic that utilizes other bacterial classification of expressing the outflow pump and effluxed by these outflow pumps also can carry out similar screening.The intracellular concentration of 2: the second kinds of screening methods of embodiment-measurement tsiklomitsin

A kind of more special method is with the basis that is expressed as with tetR regulatory domain control reporter gene.TetR is a known regulator control system, and to tsiklomitsin concentration sensitivity, it can induce synthesizing of TetA when tsiklomitsin concentration in the cell raises.If the outflow pump is blocked, the intracellular concentration of tsiklomitsin will raise, and will be induced by the protein of TetR control.

TetR and control region thereof are cloned, and a tetA-lacZ fusion gene also is under the control of TetR.This construction will be inserted in the nonessential chromogene of Pseudomonas aeruginosa, being utilized as the basis and will setting up a kind of screening method with this bacterial strain.It is different that this construction and Rothstem etc. report, i) fusion gene makes up at the different positions of tetA, ii) do not design for seeking the special efflux pump inhibitors of tsiklomitsin, and be to seek many substrates efflux pump inhibitors to design, iii) this construction will insert in karyomit(e) rather than the plasmid.

Bacterial strain PAO1 will be used for this screening method.It will contain above-mentioned construction in a dispensable gene.PAO1 will be grown in the substratum of the tsiklomitsin that contains inferior inhibition concentration and beta-galactosidase enzymes substrate.Before inoculation or culture begun the growth after, underproof compound will add in the substratum.If test compound has suppressed the outflow pump, the intracellular concentration of tsiklomitsin will raise, and LacZ will be synthesized.The available beta-galactosidase enzymes substrate that exists of LacZ detects.Embodiment 3: the accumulation of the third screening method-reagent that adds lustre to

A kind of special screening method is to add lustre in the Pseudomonas aeruginosa of non-growth or other bacterium or the absorption of fluorescent reagent (or other detectable compound).(present embodiment after this will only refer to dyestuff.) inhibition of outflow pump of one or more types increasing of will causing that dyestuff builds up.Therefore, if the outflow pump is blocked, the available metric measurement thereby the intracellular concentration of dyestuff will raise, perhaps in some cases, available eye estimate.

Pseudomonas aeruginosa strain PAM1001 will be used to the screening method of present embodiment, but the Pseudomonas aeruginosa strain identical or other outflow pump of other overexpression or other bacterial classification with interested outflow pump (as other non-tsiklomitsin specificity outflow pump) also can be used for present method equally.PAM1001 is grown in the standard medium, and cell is by centrifugal results.Cell transfer is to the Eppendorf centrifuge tube and with the optical density(OD) of suitable damping fluid resuspension to the best.Before cultivating for some time at ambient temperature, dyestuff and compound to be tested all add in the substratum.The centrifugal back of suspension cell precipitation is used the dyestuff in spectrophotometric analysis (program A) or the extracting sedimentation cell and is measured the amount of dyestuff with spectrophotometry or spectrophotofluorimetry (program C).As mentioned above, also can measure dye strength (program B) in the suspension substratum.In this case, if cell is built up the more dyestuff of volume, the amount of dyestuff and photoabsorption thereof will descend in the supernatant, and the method case description of these three kinds of programs is as follows:

Cell preparation: with frozen PAM1001 streak inoculation in the Mueller-Hinton agar plate and in 35 ℃ of overnight incubation.Gather in the crops 2-4 clone, be used for being seeded to the pipe that contains 2mlMueller-Hinton meat soup, in rotary shaker with 200-250rpm, 35 ℃ of overnight incubation.In a 250ml erlenmeyer flask, overnight culture is diluted to 40ml at 1: 100, and under above-mentioned isometric growth condition, grows to middle logarithmic phase (equaling 1) with the Perkin ElmerLamda Bio spectrophotometric instrumentation 600nm OD of place value with identical substratum.Room temperature low speed (4000g20 minute) centrifugal collecting cell and 0.05M potassium phosphate buffer (pH6.9) washing that contains 0.2% glucose with 40ml once, with 1: 10 just volume be resuspended in the identical damping fluid.Zhi Bei cell is used among three kinds of programs in this way.

The change of program A-cell color: cell is diluted to 1OD under 600 nm in containing the Eppendorf pipe (1.5ml size) of above-mentioned damping fluid.It is 1ml that the test compound of Viola crystallina (final concentration 1 μ g/ml) and suitable concn adds the Guan Zhongzhi final volume.Cell suspension in room temperature incubation 5 minutes and in the Eppendorf whizzer with 14, centrifugal 2 minutes of 000rpm.If the outflow pump is suppressed, cell precipitation will become red-purple.When efflux pump inhibitors L-phenylalanyl-L-arginyl-β-naphthalene amino acid adds to 40 μ g/ml.The precipitation that is exposed to the cell suspending liquid in Viola crystallina and L-phenylalanyl-L-arginyl-β-naphthalene amino acid becomes purple, and is not exposed to the precipitation and the nondiscoloration of the cell suspending liquid in Viola crystallina and the L-phenylalanyl-L-arginyl-β-naphthalene amino acid.The change of program B-supernatant photoabsorption: in containing the Eppendorf pipe (1.5ml size) of above-mentioned damping fluid with cell dilution to 600nm 0.5OD.Viola crystallina (final concentration 8 μ g/ml) and L-phenylalanyl-L-arginyl-β-naphthalene amino acid adds in the pipe, final volume 1ml.With cell suspension incubation 5 minutes, with 14, centrifugal 2 minutes of 000rpm collected the photoabsorption that supernatant is surveyed the 590nm place in the Eppendorf whizzer in room temperature.The sample result of this program is as follows:

Be exposed to the photoabsorption (photoabsorption of 100%=Viola crystallina in not celliferous damping fluid) of the cell conditioned medium liquid of Viola crystallina (8 μ g/ml) and efflux pump inhibitors L-phenylalanyl-L-arginyl-β-naphthalene amino acid
				Bacterial strain	Efflux pump inhibitors μ g/ml	% photoabsorption (n=3)	????SD
?PAM1001	????40	????30	????12.09
				?PAM1001	????0	????91	????351

The extracting of dyestuff in the program C-cell precipitation: cell dilution is to 600nm 0.5OD in containing the Eppendorf pipe (1.5ml size) of above-mentioned damping fluid.Viola crystallina (16 μ g/ml final concentration) and 100 μ g/ml L-phenylalanyl-L-arginyl-β-naphthalene amino acid adds Guan Zhongzhi final volume 0.5ml.Cell suspension is in room temperature incubation 5 minutes, in the Eppendorf whizzer with 14, centrifugal 2 minutes of 000rpm.Washed cell once and with it is resuspended in the 20 μ l damping fluids; Add 400 μ l1-propyl alcohol, incubation 10 minutes is also centrifugal.Collect supernatant and survey the Viola crystallina level in 590nm with spectrophotometry.Sample result is as follows:

The per-cent (100%=is acellular) of Viola crystallina accumulation amount when efflux pump inhibitors L-phenylalanyl-L-arginyl-β-naphthalene amino acid exists and do not exist
			Bacterial strain	Inhibitor exists	Inhibitor does not exist
????PAM1001	????60.16	????10.63

The known efflux pump inhibitors of above-mentioned usefulness proves that as the result of the example program of the screening method of test compound dyestuff accumulation sieve method can be used for identifying other efflux pump inhibitors.Embodiment four kinds of screening methods-positive growth screening in 4: the

This test is a positive growth screening: promptly show the existence of efflux pump inhibitors by the growth of bacterial cell.This test has utilized the β-Nei Xiananmei gene that is subjected to derivable control region control.For this screening method, one contains the tetR gene, and the construction of tetA promotor and blaS gene is inserted in the dispensable gene in the Pseudomonas aeruginosa strain PAO1 karyomit(e), and the blaS genes encoding is from the β-Nei Xiananmei of xanthomonas maltophilia; Carbapenem is effective substrate of this β-Nei Xiananmei.It can be the β-Nei Xiananmei of effective substrate with carbapenem that bacterial strain PAO1 can not produce, and in addition, carbapenem can not be effluxed by the outflow pump of Pseudomonas aeruginosa.When the β-Nei Xiananmei that can effectively utilize carbapenem does not exist, thereby the carbapenem of high density will overcome the growth that the low permeability of Pseudomonas aeruginosa adventitia suppresses bacterium.Therefore, if the blaS expression of gene do not induced since carbapenem exist the Pseudomonas aeruginosa cell with death.Yet, if tsiklomitsin exists with low-down level in substratum, and efflux pump inhibitors exists with the concentration of the outflow pump that can effectively suppress to efflux tsiklomitsin, and the intracellular concentration of tsiklomitsin will raise, thereby induce the blaS expression of gene, make bacterium to grow the hydrolysis carbapenem.

The activity of the also titratable weak efflux pump inhibitors of this test.Utilize the carbapenem of different concns in the substratum can finish this titration.Insufficient inhibitor can not be induced the blaS expression of gene fully, thereby can not cause the positive cell growth under the high carbapenem concentration, because a small amount of derivative β-Nei Xiananmei can not the hydrolysis q.s carbapenem, but under low carbapenem concentration, can induce enough β-Nei Xiananmeis, the carbapenem that hydrolysis is enough is grown cell.Therefore, this test provides the method that detects weak efflux pump inhibitors and strong efflux pump inhibitors.Embodiment 5: the evaluation of target compound

In order to assess the effect of outflow pump in pathogenesis, we have obtained the mexA and the oprK of coding outflow pump composition among the PAO1.Group displacement method structure with standard obtains this bacterial strain.The null mutation bacterial strain that can not obtain mexB may be because this sudden change be lethal.MexA and the strain of oprK null mutation are more responsive to microbiotic, and will be used for animal model to test the virulence of these mutant strains the mouse of neutrophilic leukocyte minimizing.Embodiment 6: the evaluation of successful compound

The compound that proves efflux pump inhibitors by one or more above-mentioned screening methods (growth-inhibiting, reporter is induced, or positive growth screening) screening will detect the ability of its enhancing Pseudomonas aeruginosa to irrelevant microbiotic promise Norxin and sensitive tetracycline.Subsequently, with the microbiotic of certain limit, as tsiklomitsin, fluoroquinolone, beta-lactam and aminoglycoside are determined the enhancement of these molecules, also will detect the anti-microbial activity of efflux pump inhibitors compound.

And, also will detect these compounds to other pump (as the MexC-MexD-OprL system, NorA, TetA, restraining effect MDR), compounds identified also will detect the ability that it suppresses the outflow pump at molecular level.Embodiment 7: a kind of successful compound-L-phenylalanyl

The evaluation of-L-arginyl-β-naphthalene amino acid

With the screening of the growth-inhibiting described in the embodiment 1, compound L-phenylalanyl-L-arginyl-β-naphthalene amino acid is accredited as efflux pump inhibitors.Select one group of compound earlier according to the structure of compound and the therepic use suitability of expection, in these compounds, screen.The structure of this compound as shown in figure 21.After this evaluation, this compound is subjected to the 7 described external evaluations as embodiment.Embodiment 8: successful compound-L-phenylalanyl-L

The character A.L-phenylalanyl-L-arginyl-β-steaming acid amides of-arginyl-β-naphthalene amino acid is used to identify the existence of outflow pump.

L-phenylalanyl-L-arginyl-β-naphthalene amino acid can be used for identifying the existence of the outflow pump that is subjected to its inhibition.This antibiotic enhancement that effluxes based on the pump that suppressed by L-phenylalanyl-L-arginyl-β-naphthalene amino acid of identifying, or identify by the substrate such as the proteinic secretion of monitoring pump.B. external brief introduction (profile)

L-phenylalanyl-L-arginyl-β-naphthalene amino acid has improved the bacterial strain K385 of Pseudomonas aeruginosa wild-type PAO1 bacterial strain and the excessive generation of two kinds of outflow pumps and the PAO4098E susceptibility (Fig. 7 and 8) to tsiklomitsin and Ciprofloxacin.Utilize checker test, also proved the synergy that these three bacterial strains share L-phenylalanyl-L-arginyl-β-naphthalene amino acid and tsiklomitsin or Ciprofloxacin clearly.L-phenylalanyl-L-arginyl-β-naphthalene amino acid is to the mutant strain K799/61 of the outflow pump defective not influence (Fig. 8) of susceptibility to tsiklomitsin and Ciprofloxacin.L-phenylalanyl-L-arginyl-β-naphthalene amino acid also demonstrates the inherent anti-microbial activity under high density (32 μ g/ml), the MIC of four bacterial strains of Pseudomonas aeruginosa is as shown in table 2.It should be noted that two kinds of excessive producers of outflow pump are to L-phenylalanyl-L

The susceptibility of arginyl-β-naphthalene amino acid is starkly lower than the mutant strain (Fig. 8) of wild-type or outflow pump defective.

Utilize Pseudomonas aeruginosa PAO1 and K385, we have shown that L-phenylalanyl-L-arginyl-β-naphthalene amino acid also has many microbiotic (tsiklomitsins that pumped specifically by Pseudomonas aeruginosa of enhancing, paraxin, piperacillin, ceftazime and comprise Ciprofloxacin and seven kinds of fluoroquinolones of promise Norxin) active function, but, non-Pseudomonas aeruginosa MDR/ pyofluorescein outflow pump (Li etc., 1994a; Li etc., the 1994b) MIC of the medicine of substrate such as imipenum and gentamicin is at L-phenylalanyl-L-arginyl-β-still remain unchanged when naphthalene amino acid exists.

Fig. 9 illustrates the multiple raising of Pseudomonas aeruginosa strain PAO1 to every kind of tested antibiotic susceptibility, and Figure 10 has listed their MIC.The proportional relation of reduction degree (Figure 11) that the relative hydrophobicity of four kinds of both sexes fluoroquinolones and L-phenylalanyl-L-arginyl-β-naphthalene amino acid cause to the MIC of Pseudomonas aeruginosa strain PAO1 and K385.

L-phenylalanyl-L-arginyl-β-naphthalene amino acid has reduced Ciprofloxacin to all laboratory strains of survey Pseudomonas aeruginosa and the MIC of clinical separation strain (n=26).18 clinical separation strains and 8 laboratory strains are arranged in this strains.Use the broth culture micro-dilution method, studied this strains Ciprofloxacin and Ciprofloxacin and susceptibility that the L-phenylalanyl-L-arginyl-β-naphthalene amino acid share.Ciprofloxacin is 2 μ g/ml to the MIC90 of this strains.And add after final concentration is L-phenylalanyl-L-arginyl-β-naphthalene amino acid of 20 μ g/ml, then reduce to 0.125 μ g/ml (Figure 12).

With three bacterial strains of Pseudomonas aeruginosa, PAO1, K385 and PAO4098E have carried out the tsiklomitsin of L-phenylalanyl-L-arginyl-β-naphthalene amino acid and inferior inhibition concentration or the time kill studies that Ciprofloxacin share.Studies show that final concentration is the adding of L-phenylalanyl-L-arginyl-β-naphthalene amino acid of 20 μ g/ml, has recovered the bacteriostatic activity of tsiklomitsin to these three kinds of bacterial strains fully.Equally, when having 20 μ g/ml L-phenylalanyl-L-arginyl-β-naphthalene amino acid, the Ciprofloxacin that is low to moderate 0.2 μ g/ml all has fungicidal activity (Figure 13-15) to the bacterial strain of all tests.

We confirm that also the kind of most of enterobacteriaceaes and Rhodopseudomonas can be by L-phenylalanyl-L-arginyl-β-naphthalene amino acid improve to the susceptibility of Ciprofloxacin.L-phenylalanyl-L-arginyl-β-naphthalene amino acid has strengthened the effect of Ciprofloxacin to the clinical separation strain of streptococcus aureus.Its MIC reduces to 4 μ g/ml (Figure 16) from 16 μ g/ml.

We have also studied the influence of L-phenylalanyl-L-arginyl-β-naphthalene amino acid to intestinal bacteria AcrAB pump defective mutant, and this bacterial strain obtains by transposon mutagenesis in the laboratory of H.Nikaido.We find that L-phenylalanyl-L-arginyl-β-naphthalene amino acid has improved the susceptibility of the e. coli k12 of parent strain NH817-positive phenotype of a kind of Acr to acridine orange.The bacterial strain NH818 of AcrAB pump defective type is more responsive to acridine orange than parent strain, and its sensitivity levels is not subjected to L-phenylalanyl-L-arginyl-β-naphthalene amino acid to influence (Figure 17).

L-phenylalanyl-L-arginyl-β-naphthalene amino acid shows and can strengthen the anti-effect that has the Pseudomonas aeruginosa strain (PAO236 NalA) of known DNA gyrase sudden change of Ciprofloxacin.The overall sensitivity of gram negative bacterium combating microorganisms agent is determined the Relative Contribution of the avidity of target compound by outer membrane permeability (comprising the effect of effluxing) and medicine.If the effect of effluxing of quinolone is affected in the NalA bacterial strain, cause that the necessary apparent quinolone concentration of growth-inhibiting will reduce, cause the susceptibility of quinolone is strengthened (Figure 18).

Tsiklomitsin is built up the activity that experiment is used for measuring Pseudomonas aeruginosa outflow pump.This measuring [ ³H]-accumulation of tsiklomitsin in bacterium, and make its optimization to measure the accumulation of tsiklomitsin in Pseudomonas aeruginosa.The accumulation of in four kinds of different pseudomonas aeruginosa strains, having measured tsiklomitsin: 1) the wild type strain PAO1 of generation basal level outflow pump, 2) from the bacterial strain PAO4098E and the K385 and 3 of two kinds of different outflow pumps of PAO1 and excessive generation) bacterial strain 799/61, but a kind ofly can not produce the outflow pump of detection limit and the bacterial strain responsive excessively to microbiotic.

Bacterium at synthetic outflow pump, PAO1 in the cell suspension of PAO4098E or K385, adds the normal function that L-phenylalanyl-L-arginyl-β-naphthalene amino acid suppresses the outflow pump, in that 1mM or 0.1mM L-phenylalanyl-L-arginyl-β-when naphthalene amino acid existed, tsiklomitsin was built up and is significantly improved.Similar suppressor mode (Figure 19) also can occur after CCCP adds in the cell suspension, CCCP is a proton conductor, and it can destroy by the proton gradient of outflow pump as the source energy.

When measuring the tsiklomitsin accumulation in the bacterial strain 799/61 that can not produce the outflow pump, the adding of L-phenylalanyl-L-arginyl-β-naphthalene amino acid does not change the steady-state level that tsiklomitsin is built up.The tsiklomitsin that the adding of proton conductor CCCP has reduced in the bacterial strain 799/61 is built up.When CCCP adds in the cell suspension and pH reaches balance on the film both sides, will reach a new tsiklomitsin homeostasis.When CCCP exists, add L-phenylalanyl-L-arginyl-β-naphthalene amino acid in the cell suspension and can not change the steady-state level that tsiklomitsin is built up, positive according to expectation such it be the specificity efflux pump inhibitors (Figure 20) that does not influence proton gradient.

This result clearly illustrates that L-phenylalanyl-L-arginyl-β-naphthalene amino acid has suppressed the function of outflow pump in the Pseudomonas aeruginosa.L-phenylalanyl-L-arginyl-β-naphthalene amino acid can not influence proton gradient, can not be as the proton uncoupling agents.Embodiment 9: the interior evaluating of successful compound

The bacterium efflux pump inhibitors is at first in external evaluation.Those demonstrate can effectively suppress to outflow pump and those and demonstrate with microbiotic that synergistic compound is selected to come out to carry out to assess in the body.To carry out efficacy test with the method for standard.Can carry out preliminary efficacy assessment (M.G.Bergeron.1978, Scand.J.Infect.Dis. supplementary issue 14 volume 189-206 pages or leaves with mouse septicemia model; S.D.Davis, 1975, " biocide chemotherapy " be 8 volume 50-53 pages or leaves (Antimicrob.AgentsChemother.)).Hyperlethal bacterium is used to attack these rodents in this model.Changing in treatment time and the microbiotic dosage one or binomial treats.In these experiments, the dosage of microbiotic and efflux pump inhibitors is all different.Synergistic agent (efflux pump inhibitors) share with microbiotic and uses microbiotic to compare obvious raising of the deadly provide protection of infecting separately and is indicated as positive findings.

Second kind of used efficacy models is mouse soft tissue infection model (Vogelman etc., 1988, " infectious diseases magazine " be 157 volume 287-298 pages or leaves (J.Infect.Dis.)).In this model, with the back leg muscle of the infectation of bacteria anesthetized mice of suitable titre.Mouse is (-4 ,-2 and 0 days with the treatment of 125mg/kg endoxan) or the immunocompetent mouse that neutrophil reduces.Infective dose is generally every animal 10 ⁵-10 ⁶Clonogenic unit.Efflux pump inhibitors and/or antibiotic share the treatment carried out can be after infection, also can be before infection.The propagation of bacterium (or dead) in the monitoring leg muscle in the full time scope.Effectively share than independent use microbiotic and demonstrate higher activity.Activity is defined in the reduction of tested rate of bacterial growth in the rat tissue.

Another model that is used to estimate the efflux pump inhibitors effect be the diffuser casing model (Malouin etc., nineteen nineties " infecting and immunity " (Infect Immun) 58 volume 1247-1253 pages or leaves; Day etc., " infection magazine " are 2 volume 39-51 pages or leaves (J.Infect); Kelly etc., 1989 " infecting and immunity " be 57 volume 344-350 pages or leaves (Infect.Immun)).In this model, in rodentine peritoneal cavity, insert a diffuser casing with surgical operation.This chamber can be made up of the polypropylene cylinder, and the two ends of cylinder cover with semi-permeable membranes.The peritoneal fluid that diffuses into or go out cell provides nutrition for microorganism.Compare when the propagation of bacterium is with use microbiotic separately when microbiotic/efflux pump inhibitors exists and do not exist.Share and use separately antibiotic distribution of dose to be used to assess the effect of biocide/share.

The 3rd model that is used for the severe test that efflux pump inhibitors/microbiotic share is endocarditis model (J.Santoro and M.E.levinson, 1978 " infect and immunity) " (InfectImmun.) 19 volume 915-918 pages or leaves).Rat and rabbit all can be effectively used to this model.The effect that efflux pump inhibitors and microbiotic share compares with using antibiotic effect separately.Terminal point is generally the survivaling cell that remains in the heart vegetation when treatment finishes.

The infection model example that is provided and nonrestrictive, other model is suitable to be used for specific microbial infection.Especially, in some cases the cell infection model can be used to substitute animal model.

Embodiment described herein and not meaning that limits the invention.It will be appreciated by those skilled in the art that the present invention can be with many bacterial strains or kind, or other cell type is implemented.

Other embodiment is included in the following claim.

Claims (79)

1. the method for the non-tsiklomitsin specificity efflux pump inhibitors of screening when the antiseptic-germicide that is included in inferior inhibition concentration exists, determines whether the non-tsiklomitsin specificity of potential efflux pump inhibitors suppresses the growth of bacterium

Wherein said bacterium produces a non-tsiklomitsin specificity outflow pump.

2. the process of claim 1 wherein the non-tsiklomitsin specificity of the excessive generation of said bacterium outflow pump.

3. the method for claim 2, the growing state of the bacterium that further comprises more excessive generation outflow pump and another kind of bacterium that can not excessive generation outflow pump,

If the growth of the said second kind of bacterium of growth fraction of the bacterium of wherein excessive generation outflow pump is subjected to suppressing greatly, this test compound is a non-tsiklomitsin specificity efflux pump inhibitors so.

4. the method for the non-tsiklomitsin specificity efflux pump inhibitors of screening comprises

When test compound existed and do not exist, the bacterial cell that will have non-tsiklomitsin specificity outflow pump contacted with the dyestuff or the fluorescent substance that are transported by this outflow pump; With

The intracellular concentration of dyestuff or fluorescent substance when relatively this test compound exists and do not exist;

Compare when not existing when wherein this test compound exists, the intracellular concentration of dyestuff or fluorescent substance raises and shows that this test compound is a non-tsiklomitsin specificity efflux pump inhibitors.

5. the method for claim 4, wherein said dyestuff is selected from Viola crystallina and Victoria Green WPB.

6. the method for claim 4, determine the change in concentration of dyestuff in the suspension culture base wherein said relatively comprising.

One kind the screening non-tsiklomitsin specificity efflux pump inhibitors method,

Comprise and determining when second kind of compound of test compound and inferior inhibition concentration exists express in the recombinant bacteria of non-tsiklomitsin specificity outflow pump one, whether the intracellular concentration of second kind of compound raises,

Wherein said determine to comprise the expression that detects the reporter gene that is subjected to second kind of compound concentration rising inductive regulating and controlling sequence control and

If the expression level of reporter gene is not higher when not existing than it when wherein this test compound exists, this test compound is a non-tsiklomitsin specificity efflux pump inhibitors so.

8. the method for claim 7, wherein said second kind of compound is antiseptic-germicide.

9. the concentration that the method for claim 8, wherein said regulating and controlling sequence are raise by tsiklomitsin is induced.

10. the method for claim 9, wherein regulating and controlling sequence is the tetA regulating and controlling sequence, it contains a manipulation site in conjunction with TetR.

11. the method for claim 7, wherein said reporter gene is expressed a kind of enzyme.

12. the method for claim 11, wherein said reporter gene is a beta-galactosidase gene.

13. the method for claim 7, wherein said reporter gene provide the colorimetric report.

14. a method of testing efflux pump inhibitors comprises the steps:

A. will express the outflow pump the recombinant microorganism cell contact with a test compound and

B. determine when this test compound existed, whether the microorganism cells of reorganization grew,

Wherein be suppressed the back when not being suppressed when the outflow pump, said microorganism cells will be grown better,

When wherein if this test compound exists when not existing said recombinant microorganism cell grow better, this test compound is an efflux pump inhibitors so.

15. the method for claim 14 comprises this a recombinant microorganism cell and a test compound, an inductor contacts with a biocide that is higher than the concentration of the non-MIC of inducing of this recombinant microorganism cell,

Comprise further whether the intracellular concentration of determining inductor when test compound exists raises,

Wherein the intracellular concentration of inductor rising is induced the inactivated factor that produces said biocide.

16. the method for claim 15, wherein said recombinant microorganism cell is a recombinant bacterial cell, and said biocide is an antiseptic-germicide.

17. the method for claim 16, wherein recombinant bacterial cell contains the dna structure of artificial insertion, and this structure contains an intracellular concentration inductive promotor that raise by this inductor, transcribe with the sequence of a coding antiseptic-germicide inactivated factor,

Wherein the intracellular concentration of inductor rising induces the sequence of coding antiseptic-germicide inactivated factor to express.

18. the method for claim 17, wherein inductor is induced the β-Nei Xiananmei expression of gene, and said antiseptic-germicide is a beta-lactam.

19. the method for claim 18, wherein inductor is a tsiklomitsin, and the β-Nei Xiananmei gene is the blaS gene, and beta-lactam is a carbapenem.

20. the method for claim 17 or 19, wherein promotor is the tetA promotor, and recombinant bacterial cell is expressed the tetR gene.

21. the method for claim 18 or 19, wherein β-Nei acyl enzyme gene is that karyomit(e) inserts.

22. any one method among the claim 1-21, the pump that wherein outflows is Pseudomonas aeruginosa type outflow pump.

23. the method for claim 22, the pump that wherein outflows are Pseudomonas aeruginosa outflow pump.

24. the method for claim 23, wherein bacterium is a Pseudomonas aeruginosa.

25. the method for claim 24, wherein Pseudomonas aeruginosa is bacterial strain K385 or PAO4098E.

26. any one method among the claim 1-21, below wherein at least a bacterium is selected from:

Pseudomonas aeruginosa, Pseudomonas fluorescens, pseudomonas acidovorans, Pseudomonas alcaligenes, pseudomonas putida, have a liking for maltose oligotrophy Zymomonas mobilis, onion Burkholderia, Aeromonas hydrophila, intestinal bacteria, citrobacter freundii, Salmonella typhimurium, salmonella typhi, salmonella paratyphi, Salmonella enteritidis, shigella dysenteriae, shigella flexneri, Shigella sonnei, enterobacter cloacae, enteroaerogen, Klebsiella pneumonia, acid-producing Klebsiella bacterium, serratia marcescens, soil draws hot Frances Salmonella, morganella morganii strain, proteus mirabilis, proteus vulgaris, produce the alkali Providence, providencia rettgeri, providencia stuartii, acinetobacter calcoaceticus, acinetobacter haemolyticus, yersinia entero-colitica, Yersinia pestis, artificial tuberculosis yersinia genus, middle Yersinia, Bordetella pertussis, Bordetella parapertussis, bordetella bronchiseptica, Haemophilus influenzae, Hemophilus parainfluenzae, haemolysis hemophilic bacterium, secondary haemolysis hemophilic bacterium, Ducrey bacillus, multocida, haemolysis pasteurella, branhamella catarrhalis, helicobacter pylori, embryo's Campylobacter, campylobacter jejuni, the large intestine Campylobacter, B. burgdorferi, vibrio cholerae, Vibrio parahaemolyticus, invade the lung legionella, Listeria monocytogenes, Diplococcus gonorrhoeae, Neisseria meningitidis, vagina Gardner Salmonella, bacteroides fragilis, bacteroides distasonis, bacterioide 3452A homology group, bacteroides vulgatus, the ovum bacterioide, bacteroides thetaiotaomicron, bacteroides uniformis, bacteroides eggerthii, bacteroides splanchnicus, clostridium difficile, mycobacterium tuberculosis, mycobacterium avium, Mycobacterium intracellulare, Mycobacterium leprae, corynebacterium diphtheriae, ulcer rod bacillus, streptococcus pneumoniae, streptococcus agalactiae, streptococcus pyogenes, enterococcus faecalis, faecium, streptococcus aureus, staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcus hyicus pig subspecies, staphylococcus haemolyticus, staphylococcus hominis, Staphylococcus saccharolyticus.

27. any one method among the claim 1-21, the pump that wherein outflows are selected from the outflow pump of mexA/mexB/oprM and Pseudomonas aeruginosa strain K385 overexpression.

28. any one method among the claim 14-21, wherein recombinant bacteria is from the Pseudomonas aeruginosa strain, bacterial strain K385, or bacterial strain PAO4098E.

29. a method for the treatment of infected by microbes in the animal comprises the efflux pump inhibitors that suffers infected animals to be enough to reduce the active amount of outflow pump,

Wherein efflux pump inhibitors reduces the interior viability of pathogenic or body of microorganism in this animal.

30. a method for the treatment of infected by microbes in the animal comprises the efflux pump inhibitors that suffers certain biocide of infected animals and be enough to reduce the active amount of outflow pump,

Wherein efflux pump inhibitors improves the susceptibility of microorganism to this biocide.

31. the prophylactic treatment method of an animal comprises that the animal of infected by microbes danger efflux pump inhibitors is arranged, wherein this efflux pump inhibitors reduces the pathogenic of microorganism in animal.

32. the prophylactic treatment method of an animal comprises certain biocide of animal and efflux pump inhibitors that infected by microbes danger is arranged, wherein efflux pump inhibitors improves the susceptibility of microorganism to this biocide.

33. any one method among the claim 29-32, wherein said animal is a Mammals.

34. any one method among the claim 29-32, wherein said infected by microbes is relevant with bacterium.

35. a method that strengthens the biocide antimicrobial acivity comprises microorganism is contacted with the non-tsiklomitsin specificity efflux pump inhibitors that can effectively suppress the amount of outflow pump in this microorganism with this biocide.

36. the method for claim 35, wherein said microorganism is a bacterium.

37. the method for claim 34 or claim 36, wherein said infectation of bacteria is with to be selected from following a kind of bacterium relevant:

Pseudomonas aeruginosa, Pseudomonas fluorescens, pseudomonas acidovorans, Pseudomonas alcaligenes, pseudomonas putida is had a liking for maltose oligotrophy Zymomonas mobilis, the onion Burkholderia, Aeromonas hydrophila, intestinal bacteria, citrobacter freundii, Salmonella typhimurium, salmonella typhi, salmonella paratyphi, Salmonella enteritidis, shigella dysenteriae, shigella flexneri, Shigella sonnei, enterobacter cloacae, enteroaerogen, Klebsiella pneumonia, acid-producing Klebsiella bacterium, serratia marcescens, soil draw hot Frances Salmonella, morganella morganii strain, proteus mirabilis, proteus vulgaris produces the alkali Providence, providencia rettgeri, providencia stuartii, acinetobacter calcoaceticus, acinetobacter haemolyticus, yersinia entero-colitica, Yersinia pestis, artificial tuberculosis yersinia genus, middle Yersinia, Bordetella pertussis, Bordetella parapertussis, bordetella bronchiseptica, Haemophilus influenzae, Hemophilus parainfluenzae, the haemolysis hemophilic bacterium, secondary haemolysis hemophilic bacterium, Ducrey bacillus, multocida, the haemolysis pasteurella, branhamella catarrhalis, helicobacter pylori, embryo's Campylobacter, campylobacter jejuni, large intestine Campylobacter, B. burgdorferi, vibrio cholerae, Vibrio parahaemolyticus is invaded the lung legionella, Listeria monocytogenes, Diplococcus gonorrhoeae, Neisseria meningitidis, vagina Gardner Salmonella, bacteroides fragilis, bacteroides distasonis, bacterioide 3452A homology group, bacteroides vulgatus, ovum bacterioide, bacteroides thetaiotaomicron, bacteroides uniformis, bacteroides eggerthii, bacteroides splanchnicus, clostridium difficile, mycobacterium tuberculosis, mycobacterium avium, Mycobacterium intracellulare, Mycobacterium leprae, corynebacterium diphtheriae, ulcer rod bacillus, streptococcus pneumoniae, streptococcus agalactiae, streptococcus pyogenes, enterococcus faecalis, faecium, streptococcus aureus, staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcus hyicus pig subspecies, staphylococcus haemolyticus, staphylococcus hominis, Staphylococcus saccharolyticus.

38. the method for claim 34 or 36, wherein efflux pump inhibitors is non-tsiklomitsin specificity efflux pump inhibitors.

39. the method for claim 34 or 36, wherein efflux pump inhibitors is a Pseudomonas aeruginosa type efflux pump inhibitors.

40. any one method among claim 30 and the 32-39, wherein said infected by microbes is an infectation of bacteria, and said biocide is an antiseptic-germicide.

41. the method for claim 40, wherein said antiseptic-germicide is a quinolone.

42. the method for claim 40, wherein said antiseptic-germicide is a tsiklomitsin.

43. the method for claim 40, wherein said antiseptic-germicide is a beta-lactam.

44. the method for claim 40, wherein said antiseptic-germicide is a Notomycin.

45. the method for claim 40, wherein said antiseptic-germicide is a paraxin.

46. the method for claim 40, wherein said antiseptic-germicide is a glycopeptide.

47. the method for claim 40, wherein said antiseptic-germicide is an aminoglycoside.

48. the method for claim 40, wherein said antiseptic-germicide is a macrolide.

49. the method for claim 40, wherein said antiseptic-germicide is a rifomycin.

50. a method that suppresses membrane channel function on the microorganism cells film comprises membrane channel is contacted with the membrane channel inhibitor, wherein inhibitor reduces the ability that effluxes of membrane channel.

51. the method for claim 50, wherein a polypeptide of the outflow pump of membrane channel polypeptide and mexA/mexB/oprM outflow pump or Pseudomonas aeruginosa K385 overexpression has 50% amino acid sequence similarity at least.

52. the pharmaceutical composition that can effectively treat the infected by microbes of animal contains efflux pump inhibitors.

53. the pharmaceutical composition of claim 52, the infectation of bacteria that can effectively treat animal.

54. the pharmaceutical composition of claim 52, the infected by microbes that can effectively treat animal.

55. one kind when the pharmaceutical composition that gives can effectively treat when animal is enough to reduce the active amount of outflow pump the infected by microbes of animal, comprises efflux pump inhibitors and biocide.

56. the pharmaceutical composition of claim 55, wherein said composition can effectively be treated mammiferous infected by microbes, and wherein said microorganism is a bacterium.

57. the pharmaceutical composition of claim 56, it can treat the infectation of bacteria of animal effectively, and wherein biocide is an antiseptic-germicide.

58. one kind to be enough to reduce the pharmaceutical composition that can effectively treat the infected by microbes of animal when the active amount of outflow pump gives animal with biocide, comprises a kind of pharmaceutical carrier and a kind of efflux pump inhibitors.

59. the pharmaceutical composition of claim 58, the infectation of bacteria that can effectively treat animal, wherein biocide is an antiseptic-germicide.

60. the pharmaceutical composition of claim 59 can effectively be treated mammiferous infected by microbes, wherein said microorganism is a bacterium.

61. the pharmaceutical composition of claim 52,55 or 58, wherein said efflux pump inhibitors has any structure among the structure 1-4:

R=alkyl (C wherein ₁-C ₄), fluoroalkyl (C ₁-C ₄), cross fluoroalkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F, or I), aryl (C ₆-C ₁₀), single substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F, I), and amino, single substituted-amino [is randomly used alkyl (C ₁-C ₄) replace], two substituted-aminos [are randomly used alkyl (C ₁-C ₄) replacement of arbitrary combination], or hydroxyl replaces], two substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F, I) and amino arbitrary combination replace], 2-(or 3-)-thienyl, 2-(or 3-)-furyl, or 2-(3-or 4-)-pyridyl.W=H, NH ₂, single substituted-amino [is randomly used alkyl (C ₁-C ₄) replace], two substituted-aminos [are randomly used alkyl (C ₁-C ₄) arbitrary combination replace], nitrogen heterocyclic [as the N-morpholinyl, N-piperazinyl, N-pyrrolidyl, TMSIM N imidazole base, N-pyrryl, N-pyrazolyl, N-triazolyl, or N-tetrazyl], halogen (Br, Cl, F, I), hydroxyl, alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), R ¹=(CH ₂) _nNR ^aR _b, (CH ₂) _nNR ^bR ^c, (CH ₂) _nNHC=(NR ^a) NR ^bR ^c, (CH ₂) _nSNHC=(NR ^a) NR ^bR ^c, (CH ₂) ⁿC=(NR ^a) NR ^bR ^c, (CH ₂) _nN=CNR ^bR ^c, (n=2-4); R ^a(R ^bOr R ^c)=H, alkoxyl group (C ₁-C ₄), aryl (C ₆), substituted aryl, phenyl, substituted-phenyl [is randomly used alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino the replacement], perhaps R ^a+ R ^b=(CH ₂) _2-3Or-CH=CH-, X=aryl (C ₆-C ₁₀) ,-(CH ₂) _0-2Aryl (C ₆-C ₁₀), substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino the replacement], replacement-(CH ₂) _0-2Aryl (C ₆-C ₁₀) [with alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino in the replacement of aryl position], 2-(or 3-)-thienyl, 2-(or 3-)-furyl, 2-(3-or 4-)-pyridyl, benzofuryl [in the optional position of cumarone ring], benzothienyl [in the optional position of thionaphthene ring].Wherein have center of asymmetry, its absolute stereo chemical property may be R or S-configuration, or is racemic mixture.

62. the pharmaceutical composition of claim 61, wherein said efflux pump inhibitors has structure 1,

R=alkyl (C wherein ₁-C ₄), fluoroalkyl (C ₁-C ₄), cross fluoroalkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F, or I), aryl (C ₆-C ₁₀), single substituted aryl (C ₆-C ₁₀) [with alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), amino, single substituted-amino [is randomly used alkyl (C ₁-C ₄) replace], two substituted-aminos [are randomly used alkyl (C ₁-C ₄) arbitrary combination replace], or hydroxyl replaces arbitrarily], two substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I) and amino arbitrary combination replace], 2-(or 3-)-thienyl, 2-(or 3-)-furyl, or 2-(3-or 4-)-pyridyl.W=H, NH ₂, single substituted-amino [is randomly used alkyl (C ₁-C ₄) replace], two substituted-aminos [are randomly used alkyl (C ₁-C ₄) arbitrary combination replace], nitrogen heterocyclic [as the N-morpholinyl, N-piperazinyl, N-pyrrolidyl, TMSIM N imidazole base, N-pyrryl, N-pyrazolyl, N-triazolyl, or N-tetrazyl], halogen (Br, Cl, F, I), hydroxyl, alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), R ¹=(CH ₂) _nNR ^aR _b, (CH ₂) _nNR ^bR ^c, (CH ₂) _nNHC=(NR ^a) NR ^bR ^c, (CH ₂) _nSNHC=(NR ^a) NR ^bR ^c, (CH ₂) _nC=(NR ^a) NR ^bR ^c, (CH ₂) _nN=CNR ^bR ^c, (n=2-4), R ^a(R ^bOr R ^c)=H, alkyl (C ₁-C ₄), aryl (C ₆), substituted aryl, phenyl, substituted-phenyl [is randomly used alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F, or I), or amino the replacement], perhaps R ^a+ R ^b=(CH ₂) _2-3Or-CH=CH-, X=aryl (C ₆-C ₁₀) ,-(CH ₂) _0-2Aryl (C ₆-C ₁₀), substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino the replacement], replacement-(CH ₂) _0-2Aryl (C ₆-C ₁₀) [with alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, f or I), or amino in the replacement of aryl position], 2-(or 3-)-thienyl, 2-(or 3-)-furyl, 2-(3-or 4-)-pyridyl, benzofuryl [in any position of cumarone ring], benzothienyl [in any position of thionaphthene ring].Wherein have center of asymmetry, its absolute stereo chemical property may be R or S-configuration, or is racemic mixture.

63. the pharmaceutical composition of claim 62, wherein R=alkyl (C ₁-C ₄), fluoroalkyl (C ₁-C ₄), cross fluoroalkyl (C ₁-C ₄), aryl (C ₆-C ₁₀), single substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), amino, single substituted-amino [is randomly used alkyl (C ₁-C ₄) replace], two substituted-aminos [are randomly used alkyl (C ₁-C ₄) arbitrary combination replace], or hydroxyl replaces], two substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I) and amino arbitrary combination replace], 2-(or 3-)-thienyl, 2-(or 3-)-furyl, or 2-(3-or 4-)-pyridyl.W=H, NH ₂, single substituted-amino [is randomly used alkyl (C ₁-C ₄) replace], two substituted-aminos [are randomly used alkyl (C ₁-C ₄) arbitrary combination replace], nitrogen heterocyclic [as the N-morpholinyl, N-piperazinyl, N-pyrrolidyl, TMSIM N imidazole base, N-pyrryl, N-pyrazolyl, N-triazolyl, or N-tetrazyl], halogen (Br, Cl, F, I), hydroxyl, alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), R ¹=(CH ₂) _nNR ^bR ^c, (CH ₂) _nNHC=(NR ^a) NR ^bR ^c, (CH ₂) _nSNHC=(NR ^a) NR ^bR ^c, (CH ₂) _nC=(NR ^a) NR ^bR ^c, (CH ₂) _nN=CNR ^bR ^c, (n=2 ,-4), R ^a(R ^bOr R ^c)=H, alkyl (C ₁-C ₄), aryl (C ₆), substituted aryl, phenyl, substituted-phenyl [is randomly used alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino the replacement] or R ^a+ R ^b=(CH ₂) _2-3Or-CH=CH-, X=aryl (C ₆-C ₁₀) ,-(CH ₂) _0-2Aryl (C ₆-C ₁₀), substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino the replacement], replacement-(CH ₂) _0-2Aryl (C ₆-C ₁₀) [with alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino in the replacement of aryl position], 2-(or 3-)-thienyl, 2-(or 3-)-furyl, 2-(3-or 4-)-pyridyl, benzofuryl [in any position of cumarone ring], benzothienyl [in any position of thionaphthene ring].Wherein have center of asymmetry, its absolute stereo chemical property may be R or S configuration, or is racemic mixture.

64. the pharmaceutical composition of claim 63, wherein R=alkyl (C ₁-C ₄), fluoroalkyl (C ₁-C ₄), aryl (C ₆-C ₁₀), single substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), amino, single substituted-amino [is randomly used alkyl (C ₁-C ₄) replace], two substituted-aminos [are randomly used alkyl (C ₁-C ₄) arbitrary combination replace] replace], 2-(or 3-)-thienyl, 2-(or 3-)-furyl, or 2-(3-or 4-)-pyridyl.W=H, NH ₂, single substituted-amino [is randomly used alkyl (C ₁-C ₄) replace], two substituted-aminos [are randomly used alkyl (C ₁-C ₄) arbitrary combination replace], nitrogen heterocyclic [as the N-morpholinyl, N-piperazinyl, N-pyrrolidyl, TMSIM N imidazole base, N-pyrryl, N-pyrazolyl, N-triazolyl, or N-tetrazyl], R ¹=(CH ₂) _nNR ^bR ^c, (CH ₂) _nNHC=(NR ^a) NR ^bR ^c, (CH ₂) _nC=(NR ^a) NR ^bR ^c, (CH ₂) _nN=CNR ^bR ^c, (n=2-4); R ^a(R ^bOr R ^c)=H, alkyl (C ₁-C ₄), aryl (C ₆), perhaps R ^a+ R ^b=(CH ₂) _2-3Or-CH=CH-, X=aryl (C ₆-C ₁₀) ,-(CH ₂) _0-2Aryl (C ₆-C ₁₀), substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, f or I), or amino the replacement], replacement-(CH ₂) _0-2Aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino the replacement], 2-(or 3-)-thienyl, wherein there are center of asymmetry in 2-(or 3-)-furyl, or 2-(3-or 4-)-pyridyl, and its absolute stereo chemical property may be R or S configuration, or is racemic mixture.

65. the pharmaceutical composition of claim 61, wherein said efflux pump inhibitors has structure 2, wherein R=aryl (C ₆-C ₁₀), single substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), amino, single substituted-amino [is randomly used alkyl (C ₁-C ₄) replace], two substituted-aminos [are randomly used alkyl (C ₁-C ₄) arbitrary combination replace], or hydroxyl replaces], 2-(or 3-)-thienyl, 2-(or 3-)-furyl, or 2-(3-or 4-)-pyridyl, R ¹=(CH ₂) _nNR ^bR ^c, (CH ₂) _nNHC=(NR ^a) NR ^bR ^c, (CH ₂) _nSNHC=(NR ^a) NR ^bR ^c, (CH ₂) _nC=(NR ^a) NR ^bR ^c, (CH ₂) _nN=CNR ^bR ^c, (n=2-4), R ^a(R ^bOr R ^c)=H, alkyl (C ₁-C ₄), aryl (C ₆), substituted aryl, phenyl, substituted-phenyl [is randomly used alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino the replacement] or R ^a+ R ^b=(CH ₂) _2-3Or-CH=CH-, X=aryl (C ₆-C ₁₀) ,-(CH ₂) _0-2Aryl (C ₆-C ₁₀), substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino the replacement], replacement-(CH ₂) _0-2Aryl (C ₆-C ₁₀) [with alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino at the aryl substitution in ring], 2-(or 3-)-thienyl, 2-(or 3-)-furyl, 2-(3-or 4-)-pyridyl, benzofuryl [on any position of cumarone ring], benzothienyl [on any position of thionaphthene ring].Wherein have center of asymmetry, its absolute stereo chemical property may be R or S-configuration, or is racemic mixture.

66. the pharmaceutical composition of claim 65, wherein R=aryl (C ₆-C ₁₀), single substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), amino, single substituted-amino [is randomly used alkyl (C ₁-C ₄), replace], two substituted-aminos [are randomly used alkyl (C ₁-C ₄) arbitrary combination replace], or hydroxyl replaces], 2-(or 3-)-thienyl, 2-(or 3-)-furyl, or 2-(3-or 4-)-pyridyl.R ¹=(CH ₂) _nNR ^bR ^c, (CH ₂) _nNHC=(NR ^a) NR ^bR ^c, (CH ₂) _nSNHC=(NR ^a) NR ^bR ^c, (CH ₂) _nC=(NR ^a) NR ^bR ^c, (CH ₂) _nN=CNR ^bR ^c, (n=2-4), R ^a(R ^bOr R ^c)=H, alkyl (C ₁-C ₄), aryl (C ₆), substituted aryl, phenyl, substituted-phenyl [is randomly used alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino the replacement] or R ^a+ R ^b=(CH ₂) _2-3Or-CH=CH-, X=aryl (C ₆-C ₁₀) ,-(CH ₂) _0-2Aryl (C ₆-C ₁₀), substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino the replacement], replacement-(CH ₂) _0-2Aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino the replacement], 2-(or 3-)-thienyl, 2-(or 3-)-furyl, 2-(3-or 4-)-pyridyl, cumarone [on any position of cumarone ring], thionaphthene [on any position of thionaphthene ring].Wherein have center of asymmetry, its absolute stereo chemical property may be R or S-configuration, or is racemic mixture.

67. the pharmaceutical composition of claim 66, wherein R=aryl (C ₆-C ₁₀), single substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), amino, single substituted-amino [optional alkyl (C ₁-C ₄) replacement], two substituted-aminos [are randomly used alkyl (C ₁-C ₄) arbitrary combination replace], or hydroxyl replaces], 2-(or 3-)-thienyl, 2-(or 3-)-furyl, or 2-(3-or 4-)-pyridyl.R ¹=(CH ₂) _nNR ^bR ^c, (CH ₂) _nNHC=(NR ^a) NR ^bR ^c, (CH ₂) _nC=(NR ^a) NR ^bR ^c, (CH ₂) _nN=CNR ^bR ^c, (n=2-4), R ^a(R ^bOr R ^c)=H, alkyl (C ₁-C ₄), aryl (C ₆), perhaps R ^a+ R ^b=(CH ₂) _2-3Or-CH=CH-, X=aryl (C ₆-C ₁₀) ,-(CH ₂) _0-2Aryl (C ₆-C ₁₀), substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino the replacement], replacement-(CH ₂) _0-2Aryl (C ₆-C ₁₀) [with alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino in the replacement of aryl position], 2-(or 3-)-thienyl, 2-(or 3-)-furyl, 2-(3-or 4-)-pyridyl, wherein have center of asymmetry, its absolute stereo chemical property may be R or S-configuration, or is racemic mixture.

68. the pharmaceutical composition of claim 61, wherein said efflux pump inhibitors has structure 3

R=alkyl (C wherein ₁-C ₄), aryl (C ₆-C ₁₀), single substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), amino, single substituted-amino [is randomly used alkyl (C ₁-C ₄) replace], two substituted-aminos [randomly the arbitrary combination with alkyl replaces] replace], two substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I) and amino arbitrary combination replace], 2-(or 3-)-thienyl, 2-(or 3-)-furyl, or 2-(3-or 4-)-pyridyl.W=H, NH ₂, single substituted-amino [is randomly used alkyl (C ₁-C ₄) replace], two substituted-aminos [are randomly used alkyl (C ₁-C ₄) arbitrary combination replace], nitrogen heterocyclic [as the N-morpholinyl, N-piperazinyl, N-pyrrolidyl, TMSIM N imidazole base, N-pyrryl, N-pyrazolyl, N-triazolyl, or N-tetrazyl], R ¹=(CH ₂) _nNR ^bR ^c, (CH ₂) _nNHC=(NR ^a) NR ^bR ^c, (CH ₂) _nC=(NR ^a) NR ^bR ^c, (CH ₂) _nN=CNR ^bR ^c, (n=2-4), R ^a(R ^bOr R ^c)=H, alkyl (C ₁-C ₄), aryl (C ₆), substituted aryl, phenyl, substituted-phenyl [is randomly used alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I) replaces] or R ^a+ R ^b=(CH ₂) _2-3Or-CH=CH-, X=aryl (C ₆-C ₁₀) ,-(CH ₂) _0-2Aryl (C ₆-C ₁₀), substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino the replacement], replacement-(CH ₂) _0-2Aryl (C ₆-C ₁₀) [with alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino in the replacement of aryl position], 2-(or 3-)-thienyl, 2-(or 3-)-furyl, 2-(3-or 4-)-pyridyl, wherein have center of asymmetry, its absolute stereo chemical property may be R or S-configuration, or is racemic mixture.

69. the pharmaceutical composition of claim 68, wherein R=alkyl (C ₁-C ₄), aryl (C ₆-C ₁₀), single substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), the amino replacement], two substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), amino arbitrary combination replaces], 2-(or 3-)-thienyl, 2-(or 3-)-furyl, or 2-(3-or 4-)-pyridyl.W=H, NH ₂, single substituted-amino [is randomly used alkyl (C ₁-C ₄) replace], two substituted-aminos [are randomly used alkyl (C ₁-C ₄) arbitrary combination replace], nitrogen heterocyclic [as the N-morpholinyl, N-piperazinyl, N-pyrrolidyl, TMSIM N imidazole base, N-pyrryl, N-pyrazolyl, N-triazolyl, or N-tetrazyl], R ¹=(CH ₂) _nNR ^bR ^c, (CH ₂) _nNHC=(NR ^a) NR ^bR ^c, (CH ₂) _nC=(NR ^a) NR ^bR ^c, (CH ₂) _nN=CNR ^bR ^c, (n=2-4), R ^a(R ^bOr R ^c)=H, alkyl (C ₁-C ₄), aryl (C ₆), substituted aryl, phenyl, substituted-phenyl [is randomly used alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄) replace] or R ^a+ R ^b=(CH ₂) _2-3Or-CH=CH-, X=aryl (C ₆-C ₁₀) ,-(CH ₂) _0-2Aryl (C ₆-C ₁₀), substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino the replacement], replacement-(CH ₂) _0-2Aryl (C ₆-C ₁₀) [with alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄) replace at the aryl position], 2-(or 3-)-thienyl, 2-(or 3-)-furyl, wherein there is center of asymmetry in 2-(3-or 4-)-pyridyl, and its absolute stereo chemical property may be R or S-configuration, or is racemic mixture.

70. the pharmaceutical composition of claim 69, wherein R=alkyl (C ₁-C ₄), aryl (C ₆-C ₁₀), 2-(or 3-)-thienyl, 2-(or 3-)-furyl, or 2-(3-or 4-)-pyridyl, W=H, NH ₂, single substituted-amino [is randomly used alkyl (C ₁-C ₄) replace], two substituted-aminos [are randomly used alkyl (C ₁-C ₄) arbitrary combination replace], nitrogen heterocyclic [as the N-morpholinyl, N-piperazinyl, N-pyrrolidyl, TMSIM N imidazole base, N-pyrryl, N-pyrazolyl, N-triazolyl], R ¹=(CH ₂) _nNR ^bR ^c, (CH ₂) _nNHC=(NR ^a) NR ^bR ^c, (CH ₂) _nC=(NR ^a) NR ^bR ^c, (CH ₂) _nN=CNR ^bR ^c, (n=2-4), R ^a(R ^bOr R ^c)=H, alkyl (C ₁-C ₄), aryl (C ₆), substituted aryl, phenyl, perhaps R ^a+ R ^b=(CH ₂) _2-3Or-CH=CH-, X=aryl (C ₆-C ₁₀) ,-(CH ₂) _0-2Aryl (C ₆-C ₁₀), substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄) replace], 2-(or 3-)-thienyl, 2-(or 3-)-furyl, wherein there is center of asymmetry in 2-(3-or 4-)-pyridyl, and its absolute stereo chemical property may be R or S-configuration, or is racemic mixture.

71. the pharmaceutical composition of claim 61, wherein said efflux pump inhibitors has structure 4

R=alkyl (C wherein ₁-C ₄), aryl (C ₆-C ₁₀), single substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), amino, single substituted-amino [is randomly used alkyl (C ₁-C ₄) replace], two substituted-aminos [are randomly used alkyl (C ₁-C ₄) arbitrary combination replace] replace], two substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I) and amino arbitrary combination replace], 2-(or 3-)-thienyl, 2-(or 3-)-furyl, or 2-(3-or 4-)-pyridyl.R ¹=(CH ₂) _nNR ^bR ^c, (CH ₂) _nNHC=(NR ^a) NR ^bR ^c, (CH ₂) _nC=(NR ^a) NR ^bR ^c, (CH ₂) _nN=CNR ^bR ^c, (n=2-4), R ^a(R ^bOr R ^c)=H, alkyl (C ₁-C ₄), aryl (C ₆), substituted aryl, phenyl, substituted-phenyl [is randomly used alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I) replaces], perhaps R ^a+ R ^b=(CH ₂) _2-3Or-CH=CH-, X=aryl (C ₆-C ₁₀) ,-(CH ₂) _0-2Aryl (C ₆-C ₁₀), substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino the replacement], replace (CH ₂) _0-2-aryl (C ₆-C ₁₀) [with alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino in the replacement of aryl position], 2-(or 3-)-thienyl, 2-(or 3-)-furyl, 2-(3-or 4-)-pyridyl, wherein have center of asymmetry, its absolute stereo chemical property may be R or S-configuration, or is racemic mixture.

72. the pharmaceutical composition of claim 71, wherein R=alkyl (C ₁-C ₄), aryl (C ₆-C ₁₀), single substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), the amino replacement], two substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), amino arbitrary combination replaces], 2-(or 3-)-thienyl, 2-(or 3-)-furyl, or 2-(3-or 4-)-pyridyl, R ¹=(CH ₂) _nNR ^bR ^c, (CH ₂) _nNHC=(NR ^a) NR ^bR ^c, (CH ₂) _nC=(NR ^a) NR ^bR ^c, (CH ₂) _nN=CNR ^bR ^c, (n=2-4), R ^a(R ^bOr R ^c)=H, alkyl (C ₁-C ₄), aryl (C ₆), substituted aryl, phenyl, substituted-phenyl [is randomly used alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄) replace], perhaps R ^a+ R ^b=(CH ₂) _2-3Or-CH=CH-, X=aryl (C ₆-C ₁₀) ,-(CH ₂) _0-2Aryl (C ₆-C ₁₀), substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄), halogen (Br, Cl, F or I), or amino the replacement], replacement-(CH ₂) _0-2Aryl (C ₆-C ₁₀) [with alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄) replace at the aryl position], 2-(or 3-)-thienyl, 2-(or 3-)-furyl, wherein there is center of asymmetry in 2-(3-or 4-)-pyridyl, and its absolute stereo chemical property may be R or S-configuration, or is racemic mixture.

73. the pharmaceutical composition of claim 72, wherein R=alkyl (C ₁-C ₄), aryl (C ₆-C ₁₀), 2-(or 3-)-thienyl, 2-(or 3-)-furyl, or 2-(3-or 4-)-pyridyl.R ¹=(CH ₂) _nNR ^bR ^c, (CH ₂) _nNHC=(NR ^a) NR ^bR ^c, (CH ₂) _nC=(NR ^a) NR ^bR ^c, (CH ₂) _nN=CNR ^bR ^c, (n=2-4), R ^a(R ^bOr R ^c)=H, alkyl (C ₁-C ₄), aryl (C ₆), substituted aryl, phenyl, perhaps R ^a+ R ^b=(CH ₂) _2-3Or-CH=CH-, X=aryl (C ₆-C ₁₀) ,-(CH ₂) _0-2Aryl (C ₆-C ₁₀), substituted aryl (C ₆-C ₁₀) [randomly use alkyl (C ₁-C ₄), alkoxyl group (C ₁-C ₄), alkylthio (C ₁-C ₄) replace], 2-(or-3-)-thienyl, 2-(or-3-)-and furyl, 2-(3-or 4-)-pyridyl.Wherein have center of asymmetry, its absolute stereo chemical property may be R or S-configuration, or is racemic mixture.

74. antibiotic preparation that contains antiseptic-germicide, efflux pump inhibitors and pharmaceutical carrier.

75. a method that suppresses to express the bacterial growth of non-tsiklomitsin specificity outflow pump, the antiseptic-germicide that is included in the concentration that is lower than bacterium MIC exists down, this bacterium is contacted with non-tsiklomitsin specificity efflux pump inhibitors,

76. the method for a bacteria growing inhibiting comprises bacterium is contacted with efflux pump inhibitors, wherein this inhibitor is reduced in the expression of a certain outflow pump component of normal expression in this bacterium.

77. a method that reduces by a bacterial strain colony comprises this colony is contacted with efflux pump inhibitors that wherein this inhibitor is suppressed at a certain component of the outflow pump of expressing in this bacterial population, this component is essential to the bacterial growth of expressing this outflow pump.

78. the method for claim 77 should component be the plasma membrane composition of outflow pump wherein.

79. a method that promotes growth of animal comprises giving this animal efflux pump inhibitors,

Wherein this efflux pump inhibitors suppress outflow pump that bacterial strain in this animal expresses and

Wherein this efflux pump inhibitors reduces the growth of bacterial strain in this animal.

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