CN1285001A - Methods for simultaneous identification of novel biological targets and lead structures for drug development - Google Patents

Methods for simultaneous identification of novel biological targets and lead structures for drug development Download PDF

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CN1285001A
CN1285001A CN98813707A CN98813707A CN1285001A CN 1285001 A CN1285001 A CN 1285001A CN 98813707 A CN98813707 A CN 98813707A CN 98813707 A CN98813707 A CN 98813707A CN 1285001 A CN1285001 A CN 1285001A
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sample
probe
molecular
biological sample
library
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D·L·赫夫纳
C·M·泽普
Y·高
S·W·琼斯
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Sunovion Pharmaceuticals Inc
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Sepracor Inc
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Abstract

The combinatorial screening assays and detection methods of the present invention encompass highly diversified libraries of compounds which act as fingerprints to allow for the identification of specific molecular differences existing between biological samples. The specific molecular differences identified by the combinatorial screening assay and detection methods of the present invention are potential targets for diagnosis and development therapeutics. The methods of the invention can be used in diagnostics, drug discovery, as well as genomics and proteomics. Figure (1) which is illustrative of the methods of the invention shows the interaction between molecular probes described in Table II and a human serum sample.

Description

While identification of novel biological targets and the method that is used for the guide structure of drug development
1. invention field
The present invention relates to have simultaneously the discovery and the evaluation of the novel biological targets of critical treatment and diagnostic significance, more particularly, the present invention relates to find simultaneously and identify the novel biological targets that is used for drug development and the method for guide structure.
2. background of invention
Recently, investigators find, have unique chromosomal material circulation (Umovitz etc., Chronic Disease Syndromes Symposium, AAM, in May, 1997) in suffering from patient's blood plasma of some serious disease.Similarly, recently, different expression technology is used to proof, normal cell is compared with neoplastic cell, surpasses 500 kinds of rna transcription with visibly different horizontal expression (Zhang, L. etc., gene expression atlas in the normal and cancer cells, Science.276: 1268-72,1997).These find explanation, have a large amount of molecular differences between normal cell and abnormal cells.In fact, can suppose with having reason, exist into hundred in addition thousands of prior aries do not have detected potential source biomolecule acceptor.
The technology of using can provide valuable information for the target of treatment and diagnosis at present, and its basis is the single difference of identifying between normal and abnormal sample.But these technology can not provide the information that can identify observed difference spectrum between similar sample effectively.The collection of illustrative plates of observed difference can be for determining that more fully the special disease hypotype provides valuable information between similar sample, and they can not only help the hypotype of classifying, and can help to determine the suitable treatment regimens of special hypotype.Very clear, the discovery of novel biological targets can obtain a large amount of information, and a large amount of this targets still is not detected, and therefore is badly in need of finding their effective ways.
People early have recognized that, chemical library, no matter be artificial preparation or natural origin, available basis is screened the affinity of known organism acceptor such as protein, enzyme etc.Affine screening can be finished with following known technology, for example fluorescence polarization, scintillation proximity assay, enzyme-linked immunosorbent assay or other.Recently, it is open that a kind of porous silicon biosensor has also obtained, this method in fact can detect any can be with high-affinity and another kind of molecule bonded molecule.When biological acceptor was a kind of target that the critical treatment meaning arranged, showing had high-affinity and specific library member in diagnosis and/or drug development very high value to be arranged to this target.The development of combinatorial chemistry has increased the number of ligands that can be used for affine screening greatly.
Scintillation proximity assay (SPA), for example United States Patent (USP) 4,568, and the method for introducing in 649 is used to detect the affinity to a kind of known receptor in big chemical library.In the SPA of standard test, acceptor is with a kind of pearl mark that is loaded with scintillator, and in solution radiolabeled part screened.If the part of mark has affinity to acceptor, and combined, the scintillator on radiolabeled part of result and the bead is approaching, causes scintillator to be activated, and luminous.If the part of mark has only seldom or do not have affinity to acceptor, radio-labeled can not accumulate from radio active decay effectively near scintillator and shift energy, therefore almost detect less than luminous.The open defect of SPA is to have " noise " or background radiation activity in the system, and this is that non-specific adsorption by tagged ligand causes.For this reason, usually with bead with a kind of encapsulant such as albumin, stain remover or milk powder incubation, the sealing cause this non-specific adsorption the site.Another problem of SPA is to need to use the radioactivity material, and health has been caused threat, and this material is difficult to eliminate, and uses very expensive.
The modified form of SPA test has been used to a kind of screening procedure of state of conflict, and wherein acceptor is fixed on a kind of bead that is loaded with scintillator, places a kind of solution that contains radiolabeled this receptor substrate then.Then the part sample is joined in the mixture, can be successfully will reduce luminous quantity with the compound of substrate competition sessile receptor.At Wang, P. " target is identified, the high flux screening of pilot development and drug discovery ", Sino-American PharmaceuticalProfessionals Association (SPPA), The 5 ThRegional Symposium onDrug Discovery and Development, 1997, Kenilworth has introduced SPA and has been used for high flux screening among the NJ.
Fluorescence polarization is the another kind of frequent pilot system that a kind of specific receptor is had the compound of affinity of using, be used to identify.When fluorescence molecule is attached to an end of the oligomer that is connected with part, the rotation that has seriously limited fluorescence molecule that combines of acceptor and part.When polarized light by a kind of oligomer that contains the fluorophore mark and oligomer in conjunction with or when being adsorbed with a kind of solution of acceptor, its light that sends also is polarized.
Another kind of affine triage techniques is recently by Lin, V_Motesharei, K_Dancil, K_Sailor, M_ and Ghadiri, M. at " a kind of optical interference biosensor based on porous silicon " (A porous silicon-based optical interferometricbiosensor), Science 278, and is open among the 840-43 (1997).Briefly, this technology relates to a kind of use of thin silicon sheet, and this silicon chip is etched and produced a kind of porous surface.When rayed is radiated on the porous mass, just produced a kind of interference illustration, move during the refraction index changing of medium around its position.Use known chemical knowledge, various molecular recognition elements are adsorbed in porous surface, then with the target molecule reaction in itself and a kind of source.When the probe molecule that is fixed on porous surface combined with a kind of target molecule, the refraction index changing that is produced produced in interference illustration and moves, and this moving can be detected by a kind of electric charge link coupled instrument probe.The DNA of concentration that this transmitter can detect is very small (for example flying mole) and discern little organic molecule.The recognition component of transmitter in fact can be based on any macromolecule interaction, for example nucleic acid hybridization, enzyme substrates in conjunction with, lectin-carbohydrate interact, antibody-antigen is compound etc. in conjunction with, host-parasite.
Combinatorial chemistry can prepare the library of containing hundreds and thousands of compounds, and these compounds much may be structurally similar.Though the high flux screening program can be carried out affine screening at known target in these large-scale libraries, but people have been developed new method, these methods can provide capacity less but Matter (is seen in the library Chemical Diversity maximum, H. " the optimal variation compound of selection from structural database: the reasonable research that the two and three dimensions molecule is described ", Journal ofMedicinal Chemistry, 1997,40: 1219-1229).
Affine fingerprint detection method is used to detect small molecules difference library previously to one group of proteic binding affinity of determining.The fingerprint that screening is obtained is used to predict the affinity of single library member to other albumen or required acceptor.Compare with finger printing and with the known finger printing that can obtain, predict whether the library compound can carry out similar reaction with other compounds of desirable proteins reaction.For example, not in a big library, each part (for example to be detected itself and a kind of and special pharmaceutical active, antihistamine or anticholinergic activity) interaction of relevant known receptor, detect and just those are had to known part with the similar fingerprint of these active other compounds.(see Kauvar, L.M. etc., " by the protein-bonded part of affine fingerprint prediction energy ", Chemistry and Biology, 1995,2: 107-118; Kauvar, L.M_ " affine fingerprint ", Pharmaceutical Manufacturing International.1995,8: 25-28; Kauvar, L.M. " the figure spectrum discrimination cytotoxic chemical in the agrochemistry immunity test forward position detects ", D.Kurtz.L.Stanker and J.H.Skerritt.Editors, 1995, AOAC:Washington, D.C_305-312).
Technology of using and test at present is valuable to the known target and the acceptor of treatment and diagnosis, but they are very special in itself, thereby finite information can only be provided.For example, the emphasis of differential expression is only detecting the different levels that mRNA expresses, and it can not detect the existence of sequence difference.And this method can not provide in the relevant biosystem other to change as the information of post transcriptional modificaiton, and the latter can not cause the expression level change, and the information that can not provide adjacent cells that abnormal cells is replied in most occasions.
Therefore, the specific differences between a kind of sample of detection of biological widely of our needs and the detection method of change.A kind of like this method can also provide and help identify and be used to diagnose and the biological target of drug development and the method for guide structure.
3. invention summary
Combined sorting test of the present invention and detection method use the library of compounds of highly diverse that the mixture of complexity is explored and studied, and differentiate the particular molecule difference that exists between biological sample, and the latter can be used as the target of diagnosis or development methods of treatment.
The present invention part based on the sensitivity of applicant's design, fast, the pilot system of homogeneous, this system can use molecular probe library complicated, highly diverse that sample is estimated, explored and analyzes.Can carry out the susceptibility that high-throughout test has increased screening in a kind of mode of homogeneous.In addition, the homogeneous mode can also make interactional molecule keep their natural or activity conformation.And homogeneous pilot system of the present invention has been used strong detection system, and this detection system does not need independent step to come the detection reaction product.In a preferred embodiment, fluorescence polarization is used to the interaction between test sample and the library component.
In another embodiment, test can be carried out in a kind of mode of non-homogeneous, and one of them reactive component (for example library or sample component) releasably is connected with solid support.The reaction product that sample and library effect produce is discharged in the liquid phase, uses for example fluorescence polarization of strong detection system, and reaction product can be preferentially detected.
Test of the present invention can be used for diagnosing, drug discovery and the protein science and the genomics field of the screening of medicine and discovery, target drives, is used for identifying disease marker and medicine target.
In one embodiment, the combined sorting of hypersensitivity of the present invention test and detection method interact ligand/receptor and are used as that a kind of discovering tool comes to exist in the detection of complex biological mixture but still undetected so far acceptor.Combined sorting of the present invention test and detection method are estimated the binding interactions of the thousands of acceptor of potential in multifarious part and the biological sample, and identify that this sample uniqueness or distinctive binding interactions, the latter may be the indexs of a kind of particular pathologies (including but not limited to disease, disorder and infection).Therefore, the present invention not only identifies novel acceptor, also analyzes the specific differences that occurs between the normal and disease cell of same type and/or the tissue.Shaker test of the present invention and detection method can or focus on detecting the genotype change than existing target discover method such as genomics and provide more information with the differential expression technology that only detects DNA or RNA change; And protein science does not detect non-protein ligands, and thereby only relates to by the special sequence encoded protein.On the contrary, method of the present invention detects all types of ligand/receptor and interacts, and acceptor can be albumen, carbohydrate, nucleic acid or any molecule with shape that binding interactions can take place.
The method according to this invention, can estimate the binding affinity of the thousands of potential binding site that exists in the biological sample of diversity molecular library to complexity, and producing a binding affinity collection of illustrative plates that shows by this sample, the latter provides the fingerprint of a uniqueness for this sample.
The present invention uses multifarious library of compounds, for the biological mixture of complexity for example serum the method for fingerprint detection is provided.Provide unique fingerprint by the specificity binding interactions that produces between the molecule in library member and the biological sample for sample.This fingerprint can be identified the novel interaction that produces in a kind of particular sample; when sample related to a kind of special pathologic process, this fingerprint just can be identified the special phenotypic difference that exists between normal and the unusual ill or anosis cell of same type and/or the tissue.And, in case novel interaction is identified that the molecule that participates in it can be used as guide structure, acceptor or target, be used for diagnosis and/or drug development.
Shaker test of the present invention not only comprises identifies the new receptor of discrete, the more important thing is that also comprise the collection of illustrative plates of identifying binding interactions, the latter is the feature of an a kind of pathologic process or a class pathologic process.This binding affinity collection of illustrative plates can be more comprehensively and determines more accurately and the classification disease subtypes provides valuable information.Accurately diagnosis is not only extremely important to the detection of disease, and also extremely important to the processing and the treatment of disease, and they can both produce predictable clinical effectiveness.
Utilize principle of the present invention, can collect samples from a plurality of different individualities, and detect the interaction in itself and a kind of definite library, the data that produced can be used to produce a database that comprises all certified binding interactions.Then can with extract out and analyze from showing the relevant data of sample that a kind of individuality of studying pathologic process or disease obtains, and with database in the record that keeps compare, identify interaction or the interaction collection of illustrative plates that can predict pathologic process or morbid state.Interaction of Jian Dinging and collection of illustrative plates not only can be used for analyzing and classifying special pathologic process or morbid state like this, also can be used to organize a kind of diagnosis detecting method or develop a kind of treatment or medicine.
4. accompanying drawing summary
Fig. 1-5 is respectively the diagram of the data of embodiment 4 to 7 ineditings.
Fig. 6, probe are to the difference combination of people and bovine serum.This Figure illustrates and use the fluorescence polarization value of various probes from people and bovine serum acquisition.
The difference combination of the biological sample of listing in Fig. 7, probe 18 his-and-hers watches 7.Be described with figure from the fluorescence polarization value that each sample obtains with probe 18.
The difference combination of the biological sample of listing in Fig. 8, probe 70 his-and-hers watches 7.Be described with figure from the fluorescence polarization value that each sample obtains with probe 70.
The structure of Fig. 9, probe 147 and difference are in conjunction with collection of illustrative plates.(A) structure of probe 147 has been described.(B) this Figure illustrates the fluorescence polarization value that each sample of using probe 147 to list from table 7 obtains.
The structure of Figure 10, probe 129 and difference are in conjunction with collection of illustrative plates.(A) structure of probe 129 has been described.(B) this Figure illustrates the fluorescence polarization value that each sample of using probe 129 to list from table 7 obtains.
The structure of Figure 11, probe 135 and difference are in conjunction with collection of illustrative plates.(A) structure of probe 135 has been described.(B) this Figure illustrates the fluorescence polarization value that each sample of using probe 135 to list from table 7 obtains.
The difference combination of Figure 12, probe D6.Streptococcus aureus (MRSA), intestinal bacteria and the nonvaccinated heart and brain that use fluorescein-labeled probe D6 to measure streptococcus aureus (SA), Dimethoxyphenyl penicillin resistance leach the fluorescence polarization value of the culture sample of substratum (BHI).This Figure illustrates through each and cultivate after incubation period the fluorescence polarization value that obtains from each bacterial isolates and nonvaccinated BHI substratum.
Figure 13, serve as to classify to bacterial cultures in the basis with the fluorescence polarization value that obtains with probe D6.The result who is obtained by the test of the fluorescence polarization of 12 unknown cultures divides into groups according to their fluorescence polarization value.
Figure 14, probe are to the normally difference combination of (Lot HS300) and diabetics's serum.This Figure illustrates and use various probes from fluorescence polarization value normal and that diabetics's serum obtains.
Figure 15, probe are to the diabetes of degreasing and the difference combination of normal (Lot HS300) human serum.This Figure illustrates and use the fluorescence polarization value of various probes from the normal of degreasing and the acquisition of diabetics's serum.
5. detailed Description Of The Invention
The present invention relates to the application of the compound that can be combined with target molecule with high-affinity, this compound not only may be the guiding compound of development medicine, but also is a kind of informational molecule.
In multifarious chemical combination library, the frequency very low (may be 0.0001% or lower) that a kind of compound is combined with a kind of special target. But if a large amount of targets were arranged, this frequency could sharply increase. For example, when not being a target molecule but when 1000 target molecules may be arranged, the possibility that obtains a hit will bring up to 0.1%. Complicated biosystem contains thousands of different molecular, and majority may change in a kind of special morbid state such as prostate cancer or breast cancer in them; Therefore, it is more much higher than the possibility that obtains a single target of hit to obtain the interactional possibility of high-affinity between the information parts of the member of multifarious organic molecule combinatorial libraries and clinical sample. According to the present invention these interactions are detected and quantitatively after, just can form very complete sample collection of illustrative plates (depending on the Quantityanddiversity of system's Chinese library component of estimating).
Combined sorting test of the present invention and detection method comprise the library of compounds of highly diverse, the particular molecule difference that the latter comes the identification of organism sample room to exist as fingerprint. The particular molecule difference of identifying with combined sorting test of the present invention and detection method is the potential target of diagnosis and development therapeutic scheme. Want successfully to use combined sorting test of the present invention and detection method, need at least three parts: (1) one species diversity molecular library (probe); (2) a kind of clinical sample source (contrast and test sample); (3) a kind of sensitization test that detects library member and sample component interphase interaction.
Therefore, one aspect of the present invention is the method for analyzing a kind of pathologic process, said method comprises the acceptor that exists in the relevant biological sample of evaluation (a) pathologic process or target and (b) collection of illustrative plates of the binding interactions between part or the probe library, and wherein the collection of illustrative plates of binding interactions can provide the unique fingerprint of this pathologic process. According to the present invention, " acceptor " or " target " is that proof has binding affinity or interactional biomolecule to a kind of part or probe. The example of this acceptor or target comprise any can by differential expression or modification and can with the bioactive molecule of probe or ligand interaction, but be not limited to albumen, comprise enzyme; Lipid; Nucleic acid comprises DNA, RNA; Carbohydrate; Antigen; Antibody etc. The example of part or probe comprises natural or artificial any molecule, and it can be used for interacting with acceptor or target or in conjunction with the existence of measuring or indicating said part or probe in a kind of given biological sample.
In a special embodiment, combined sorting test of the present invention and detection method can be used for detecting or analyzing a kind of pathologic process. According to this embodiment, pathologic process identified or that analyze can include but not limited to a kind of conversion phenotype, genetic defect, cancerates, cancer, tumour, genetic disorder, virus infections, bacterium infection, fungal infection or a kind of parasitic existence.
Vertebrate particularly people is very complicated on physiology. This complexity makes and diagnoses accurately very difficult and elapsed time just. For example, blood plasma contains thousands of protein, carbohydrate, lipid and nucleic acid. In these components any one or a plurality of variations or change all may have very high diagnostic significance or produce a kind of special methods for the treatment of the special disease state, but the high complexity of system has hindered the accurate determination and analysis of these changes. In one aspect, the present invention can carry out rapid analysis to very complicated biosystem, for example blood plasma or serum, tissue homogenate thing, celiolymph, urine, phlegm or other any clinical materials, include but not limited to those can with fluid form obtain or preparation material.
One embodiment of the invention are medical diagnosis. The adaptable diagnostic field of the present invention includes but not limited to detection and other diseases state and the pathologic process of the toxic side effects of the detection of early diagnosis, infection of various cancers and evaluation, treatment. For any given pathologic process, in organic complex biological system, there are a large amount of physiological changes to occur. Some change is the direct result of pathologic process, and other to be bodies reply generation to this pathologic process. Any or all these changes can be diagnosed. But unfortunately, concerning numerous diseases, diagnosable change is unknown, and before the present invention, does not also have sensitivity, method is for detection of these diagnosable changes fast. In some occasion, the diagnosis take antibody as the basis is used to detect the neoantigen relevant with special pathologic process (for example with the Cancer-Related PSA antigen of prostate), but at first identifies this antigen take antibody as basic diagnosis depends on. And antibody producing is costliness but also consuming time not only. In the present invention, the binding interactions of large, multifarious chemical library and biological sample, blood plasma (for example from the individuality with special pathologic process) compares with the sample from normal population, and difference is analyzed. This species diversity can be qualitatively or quantitative, and the result of being combined with any molecule, the example of these molecules such as protein, lipid, carbohydrate or nucleic acid, enzyme, antigen etc. The method does not have tendentiousness to the type of the molecule that it can detect. After studying or a kind of collection of illustrative plates of uniqueness can identify, this binding interactions can cause diagnosing single or a small amount of binding interactions of this pathologic process to be found. Two kinds identify the interactional method of unique combination all can be assembled into a kind of cheapness, fast, take the test of fluorescence polarization as the basis, this test can be in the clinic or other places carry out.
An application of the present invention is diagnosis and treats various types of cancers, the example of these cancers such as prostate cancer, cervical carcinoma, oophoroma. At present, the diagnosis of prostate cancer depends on take antibody and detects PSA antigen as the basis. The up-to-date evidence of other local sources shows, estimates the level of the specific cells factor and also can diagnose this disease. These two indexs may not be the earliest indexs of the prostate cancer that existed, the invention provides a kind of fast method, for the identification of other diagnosis indexs of not yet knowing. Therefore, the present invention can by with a fluorescence probe library to detect to find new diagnostic flag such as the plasma sample from the prostate cancer individuality. The discovery of using this screening test to cause then can cause the development of methods for the treatment of. Therefore, the present invention includes for the discovery of the probe of diagnostic test or kit or mark and they subsequently in the application of drug discovery.
The present invention also can be used for the detection of the diagnostic flag in the Pap smear sample. According to the present invention, the normal and unusual Pap smear sample that obtains from individuality can be screened with a label probe library, to cancerate or infectious diseases have the binding interactions of diagnostic significance can be used for take fluorescence polarization as the basis diagnosis. And, cancerate or the discovery of the relevant biomolecule that catches can be then used in the development treatment with a kind of.
The present invention can be used for being ovarian cancer screening biomarker that the latter can be used for developing a kind of diagnostic kit for ovarian cancer.Recently, ovarian cancer activation factor (OCAF) is considered to a kind of biomarker of ovarian cancer.At present, a kind of serological test at CH125 is used to detect ovarian cancer, and CH125 is a kind of biomarker of ovarian cancer.According to the present invention, fluorescence polarization test, DNA blockage test and the flicker introduced among the present invention are got close to test (SPA) and be can be used for clinical study, study the known organism mark (OCAF and CA125) of ovarian cancer and the new bio mark of evaluation ovarian cancer.These pilot systems will provide simple, quick, the responsive and accurate method of using a kind of label probe or label probe library screening clinical sample.
For example, fluorescent chemicals and the interactional fluorescence polarization value of sample (blood, serum, blood plasma or other body fluid) that obtains from the patient who suffers from ovarian cancer can with by the gynecological cancer of suffering from other kinds, non-gynecological cancer or do not have the fluorescence polarization value that the patient's of cancer clinical sample obtains and compare.Suffer from patient's the sample of ovarian cancer and other samples relatively, its polarization value changes and just shows that a kind of reagent can otherness ground combines with (a kind of) component of existing in the sample from the ovarian cancer patient.Like this, this reagent just may be a kind of useful biomarker, and ovarian cancer sample and the difference of non-ovarian cancer sample are come.Polarization value and other samples from the patient's who suffers from ovarian cancer sample do not have evident difference, just illustrate that this reagent can not distinguish ovarian cancer sample and non-ovarian cancer sample as a kind of biomarker.Be accredited as and use separately with dissimilar cancer difference bonded reagent or use, be used for detecting and diagnosing dissimilar cancers with other agent combination.And certified reagent can be used to develop the therapy of treatment cancer.
The present invention also provides the method for a kind of early stage index of identifying the diabetes outbreak.The method that is used to detect diabetes at present only develops and just useful after much infringement has taken place fully in disease.Really, diagnosis is only carried out after cardinal symptom exists.With another kind of method statement, present diagnostic method only confirms the existence of this disease.The method of introduction of the present invention can provide method as the reagent of the early stage index of diabetes for identifying.For example, the glycosylation of blood protein is a feature of diabetes, and it can produce unique binding site, and these sites can use the probe from the multifarious molecular library of a mark to detect with the method for introduction of the present invention.Those probes that are accredited as the early stage index of onset diabetes can be used for the test based on fluorescence polarization according to the present invention.This test can be in the clinic or the diagnostic test chamber as diagnostic means.
The present invention further provides evaluation and distinguish infective micro-organisms and the method for the disease that they cause.Albumen, carbohydrate and other molecules that infective micro-organisms can produce widely in its host, analyze mostly.And, responding to infection, host's organism produces unique protein such as specific antibody, specific receptors, chemokine and cytokine.All these unique molecules all are probe bonded material standed fors and thereby represent the new diagnosis target of infectious diseases.The invention provides evaluation and can distinguish the method in conjunction with material standed for of the disease that infective micro-organisms and/or they cause.That is identified can be then used in the diagnose infections disease in conjunction with material standed for, and can cause the development of pharmacological agent.Embodiment 10 has introduced the probe of finding a kind of uniqueness from a little probe library, and this probe can be distinguished the streptococcus aureus of streptococcus aureus and Dimethoxyphenyl penicillin resistance.
The present invention not only can be used for the detection of infectious bacteria, also can be used for the detection of other infectious substances such as fungi, parasite, virus and possible or even protein virus.For example, neuraminidase inhibitor is suppressed first type and Influenza B virus by development.Effective application need of these medicines has fast diagnostic kit identify the existence of influenza virus.The invention provides discovery has the tagged molecule of high-affinity to viral neuraminidase or some other virus component method.These probes can be integrated into the existence that a kind of quick diagnosis detection method detects influenza virus subsequently.
Top discussion has proved that the pilot system detection of using introduction of the present invention has and the known agent of clinical sample difference binding ability and some advantage of evaluation novel agent.Those certified reagent can use separately or with other agent combination, detect and diagnose pathologic process or disease.In case use method of the present invention to be identified, this reagent can be used as quick and cheap diagnostic means (for example, diagnostic kit) and detects and diagnose various pathologic processes and disease.
Another aspect of the present invention is to identify biological target that critical treatment and diagnostic significance are arranged and the method that develops the guide structure of medicine, comprise the probe library effect that two kinds of biological samples are identical with two kinds, wherein a kind of biological sample is contrast, detect the binding interactions between the component of every kind of probe and biological sample, and identify the binding interactions of the feature that is second kind of (non-contrast) biological sample.Part of Jian Dinging or target are a kind of biological target like this, and the probe that this biological components has an affinity is the guide structure of development medicine.
In a preferred embodiment, second kind of biological sample comprise from the individual serum of disease ill or other be the abnormal cells of same type or tissue with contrasting.Because diagnosis is usually based on a kind of detectable biological target, so this method has very high value in the diagnostic tool of the development pathologic process relevant with screened cell or tissue.And because whenever a kind of new acceptor or target are found, have the compound of affinity also to be found to this receptor, so this method is a kind of source of the guide structure of the development medicine that is used for the treatment of this pathologic process.
In another embodiment, principle of the present invention can be used for the toxicology screening of potential drug.The present invention includes the method for the toxicity characteristic of determining a kind of compound and determine that this is undertaken by the biological response of measuring toxic chemical in the method for the existence of body toxic compounds one by one.Many medicines and their meta-bolites have harmful side effect or poisonous.Press for the early stage index that to predict toxicity damage rather than only prove that infringement takes place.At present, the prediction of toxicity characteristic or side effect is very difficult in early days drug development, because it occurs in before the clinical trial.Use principle of the present invention, the existence of toxicity characteristic or lack as can predict than at present possible much morning developmental stage.Detecting the preliminary study of these early warning signals can use cell culture studies to finish as utilizing liver cell.In this method, the liver cell culture thing can be handled with known toxin, after for some time, takes out sample, detects with the diverse libraries of a tagged compound.In handling culture, occur and in contrast absent variable binding interactions may be the early stage mark of cell or tissue infringement.These probes can detect in animal model subsequently.The invention provides the method for the early stage mark of possible hurtful predictability that detection of drugs or other compounds cause like this.
According to this aspect of the invention, cultured cells or tissue are handled with known poisonous or nontoxic compound, prepare extract then from culture, and detect with a tagged compound library.This tagged compound library is detected to provide and can be reflected that cell or tissue is to poisonous or " fingerprint " that non-toxic compound is replied.Analyze any change of the finger printing relevant, and also can further classify with toxicity characteristic.Then, set up the culturing cell or the tissue of finger printing as stated above and handled, from the culture of handling, prepared extract, and detect with the tagged compound library with testing compound with unknown toxicity characteristic.Fingerprint that the cell or tissue that is produced is replied or collection of illustrative plates and the reference collection of illustrative plates that produces on this cell or tissue with known poisonous or nontoxic compound compare then, predict the essence of testing compound.
In another embodiment, principle of the present invention can be used for analyzing a kind of function of expressing protein.Genome project comprises that the Human Genome Project has produced a large amount of gene order data, and these gene orders can be cloned, and the albumen of these genes encodings can be expressed.Unfortunately, the sequence of a known gene or from this sequence express a kind of albumen can not disclose usually this proteic function, it disease effect or with this expressing protein the life cell interaction other molecules.
Recognize the shortcoming of genome in this category information is provided, several companies have accepted " protein science " method.In protein science, two dimension (2-D) polyacrylamide gel electrophoresis (PAGE) is used to according to electric charge and mass separation albumen.Then the protein graphical spectrum that is produced is compared, and attempt patient population to be classified according to the collection of illustrative plates of uniqueness.The advantage of this method is research phenotype (what is) rather than genotype (what may be).As everyone knows, the 2-D electrophoresis can not detect or solve a large amount of albumen in the unusual complex sample.Method of the present invention does not have this difficult problem, because a kind of label probe and little, medium, big target can both provide clearly resoluting signal.And the present invention is three-dimensional (3-D), and except target molecule very little (less than several kilodaltons), each binding events can both provide a resoluting signal clearly.And PAGE is designed to detect protein, and the present invention is designed to detect a kind of label probe and any greater than the binding interactions between the biomolecules of several kilodaltons.At last, carry out and analyze very consuming time, the effort and expensive of 2-D gel, and opposite, method of the present invention is very quick and cheap.Directly relatively the time, the method for the present invention that can produce prediction is better than protein science or genomics any.
The invention provides a kind of method of finding out the biological function of expressing protein.When expressing protein detects with the mark library of little organic molecule, can find that the probe of some quantity combines with these proteic binding sites.These probes itself are the potential inhibitors of these expressing proteins.These probes can join in cell culture or the other system, determine their biotic influence, and thereby can be used for determining this proteic activity.In addition, label probe can be used as a kind of artificial substrates and the test of unlabelled being at war with property of molecule finds to have the more inhibitor of high-affinity.These unlabelled molecules can detect the influence of determining these agonists or antagonist cellular function subsequently in animal model.In such a way, the technology that the function of expressing protein unknown or that do not analyze can use the present invention to introduce before a kind of is measured.
Organic genomic sequenced genes from the bacterium to people is cloned on a large scale and is expressed.The invention provides the method for illustrating these proteic functions fast and finding the clue of developing new drug thing simultaneously.In addition, the present invention includes the method for finding potential inhibitor or affinity ligands, they can be used for purifying or analyze these zymetologys or the albumen of structure function the unknown.This protein targets comprises the protein targets of identifying with genome method.
The present invention also provides such class methods, and they can be used for telling the colony of any purpose.For example this technology can be used for telling the group of carrying out clinical trial.And the present invention allows the distribution collection of illustrative plates of the early detection of pathologic process or various diseases is carried out a large amount of and cheap screening.In case the information probe is developed, the expense of screening will be very low.Screening only needs cheap fluorescence polarization instrument and probe.Probe not only prepares cheap, and single test only needs the nmole level.
A large amount of other aspects of the present invention, feature and advantage below description of Preferred Embodiments and appended claim in very obvious.But should be appreciated that the disclosure should be considered to giving an example of principle of the present invention, and should not think that the content that the present invention is introduced limits here.
5.1 the diversity ligand library and the detection thereof of probe
According to one embodiment of the invention, when biological sample such as blood, serum, body fluid such as urine, celiolymph, amniotic fluid, saliva, mucus, tissue sample, cell, virus, microorganism or organic molecule such as RNA, DNA, peptide and albumen and little organic molecule contact with one group of known agent and when producing the numerical value of one group of reflection binding interactions collection of illustrative plates, just set up " fingerprint ".According to the present invention, the diversity ligand library comprise can with acceptor or target response or bonded known agent or probe groups, it can be used to indicate acceptor or the existence of target in a kind of given biological sample.Part of the present invention or probe include but not limited to any natural or synthetic biomolecules, and can include but not limited to that nucleic acid comprises DNA or RNA, little organic molecule, peptide, glycoprotein, albumen, polysaccharide, carbohydrate or inorganic molecule.
According to the present invention, part or probe library can be used for producing difference and distinguish biological sample exactly in conjunction with collection of illustrative plates.In an embodiment preferred of the present invention, multifarious ligand library comprises known molecule, and these molecules can produce the numerical value of one group of reflection binding interactions collection of illustrative plates when contacting with a kind of biological sample.When fingerprint of the present invention is used to distinguish sample as identifying or when distinguishing a kind of special microorganism as special bacterial strain of virus, bacterium, parasite or fungi, the part diverse libraries should comprise the part or the probe of the component interaction of known energy specificity or non-specific and this microorganism.
In another embodiment of the invention, when acceptor or target when being unknown, biological sample to be measured can act in the part or the probe library of the molecule of selection at random with a kind of part that comprises, and so just can identify unique binding interactions, and the latter can identify with currently known methods subsequently.Be found the acceptor that only in testing sample, exists or in testing sample, exist and can be used as the special pathology process diagnosis relevant or the potential target of treatment with this testing sample with different concns.In addition, be found and have high-affinity and specific part that the guide structure of drug development can be provided this receptor.
Do the time spent at the part that a kind of biological sample and a stack features provide, the part that a kind of acceptor that exists in the sample is had high-affinity will combine with this molecule, form the ligand/receptor binding substances, and the latter can identify with various experimental techniques.The binding interactions that takes place in the normal or control sample can compare with the binding interactions that takes place in second sample that comprises with the abnormal cells of contrast same type or tissue, identifies two kinds of specific differences in the sample.Probe/the part that uses in the pilot system of being introduced can carry out mark, tail tag or joint, when the component in the biological sample combines with probe, just can produce a kind of detectable signal like this.Probe/part can carry out mark with marker known in the art, includes but not limited to radio isotope, fluorescence molecule, chemiluminescence compound and bioluminescent compound.
Dodge when getting close to test being used for liquid, molecule is preferably used a kind of labelled with radioisotope, includes but not limited to 32P, 35S, 125I or 131I.Radio isotope can detect with gamma counter or liquid flashing counting device.
Also available a kind of fluorescence molecule of probe/part such as fluorescein (FL), rhodamine, 4-4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid (BO or BODIPY), different sulphur flower cyanines, flower cyanines or other fluorescence dyes carry out mark.The available spectrophotofluorometer of interaction between the component of fluorescently-labeled probe/part and biological sample or preferably the fluorescence polarization by analysis of mixtures detect.
The also available ELISA of binding interactions between the component of probe and biological sample (enzyme linked immunosorbent assay) detects.But the probe mark or join such molecule to such as vitamin H, streptavidin or digoxin on.Probe with these molecule markers can detect with the special enzyme len antibody of this mark.In addition, probe can carry out mark with a kind of antibody, and this antibody can engage or not engage a kind of enzyme.The antibody of joining enzyme can not detect with the second antibody that has engaged a kind of enzyme.The enzyme len antibody will be in such a way and a kind of suitable substrate reactions, and it can produce a kind of detectable chemical half point that for example uses spectrophotometer, fluorophotometer or macromethod.Can be used for the enzyme of detection property traget antibody includes but not limited to malate dehydrogenase (malic acid dehydrogenase), staphylococcal nuclease, Δ 5-steroid isomerase, yeast alcohol dehydrogenase, alpha-phosphate glycerol dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase, L-Aspartase, glucose oxidase, beta-galactosidase enzymes, rnase, urase, catalase, glucose-6-phosphate dehydrogenase (G6PD), glucoamylase and acetylcholinesterase.
Method of the present invention can be used according to any technology synthetic ligand library well-known to those having ordinary skill in the art.They are preferably by using traditional liquid phase reaction or solid phase synthesis technique to prepare.Interested organic molecule can directly in solution, carry out mark as the bioactive compounds that contains first or second amino group or oh group or methylthio group or aldehyde radical or ketone group or carboxylic acid with suitable fluorescence molecule (dyestuff), prepare corresponding fluorescently-labeled part.These methods and dyestuff be at Haugland, R.P. " fluorescent probe and research compound handbook ", 6 ThIntroduce among the ED_1996.In a preferred embodiment of the invention, liquid phase is synthesized in a kind of suitable polar organic solvent or solvent mixture such as DMF, DMSO, THF and is carried out, and uses excessive slightly dyestuff to guarantee complete mark.The fluorescently-labeled probe that is produced carries out purifying with the standard technique of organic synthesis as liquid phase-liquid phase extracting, crystallization and the chromatogram (thin layer or post) of using acid or alkali.Also can use other purification process also to follow simple filtration and (see Obrecht to remove unreacted dyestuff by the introduction among the following embodiment as the liquid phase-solid phase extracting of the scavenging agent of use polymkeric substance combination, D. and Villalgordo, J.M_ " combination of the solid phase support in small molecular weight compounds library is with parallel synthetic ", Pergamon, 1998, the 3 chapters).
Figure 9881370700221
Scheme I liquid phase reaction (product scavenger resin purifying)
In one embodiment of the invention, traditional solid phase technique preparation is used in library of the present invention.Be seen in as Bodanszky, " peptide synthetic principle " (Springer-Verlag:1984); Bodanszky etc., " peptide synthetic puts into practice " are (Springer-Verlag:1984); Barany and Merrifield, " peptide: analyze, synthesize and biology ", 2 volumes, 1 chapter (Academic Press:1980); Atherton etc., Bioorg.Chem.Vol.8 (1979).This is that prior synthesizing method has several superiority because solid phase synthesis compares more.For example, can use excessive greatly reagent or initial substance to drive one reaction to finishing, and purifying can undertake by filtering simply and washing with separating, because product is attracted on the solid support.And the related locus of resin-bonded kind separates can suppress polytype intermolecular side reaction.
Introduce a kind of solid phase synthesis process that is particularly suitable for synthetic library of the present invention that is found below.This method can be formed fluorescently-labeled part diverse libraries fast and effectively, and it comprises two general step.In the first step, a kind of fluorescence dye by covalently bound to a kind of solid support.In second step, the mixture reaction of solidified dyestuff and a kind of compound or compound forms required ligand mixture, and this step can repeat repeatedly when needed.The present invention includes and use the test adhere to the library on the solid support that they are synthesized or to adhere to the library on the different solid supports.But ligand mixture preferably cuts down from upholder in the 3rd step.This available third step is included in the preferred embodiment of the synthetic method of the present invention that is shown in the scheme II:
The scheme II wherein, ﹠#60A﹠#62, ﹠#60B﹠#62, ﹠#60C﹠#62, ﹠#60D﹠#62 and ﹠#60E﹠#62 representative are suitable for the required product of general formula (b)-(g) expression or the reaction conditions that intermediate forms, frame number (i.e. []) expression available parallel and consecutive reaction, reagent and/or product.
According to the scheme II, a kind of dye molecule of general formula (a) is selected from:
X-D-Y
General formula (a) wherein D is a kind of fluorescence half point, and X and Y are the functional groups that independently is selected from halogen, alcohol radical, nitro, thiol, ether, ester, carboxylic acid, alpha-halogenated carboxylic acids derivative, amine, acid amides and protection thereof or non-protection derivative.The example of the dye molecule of general formula (a) includes but not limited to: fluorescein derivative such as dichlorotriazylaminofluorescein (DTAF), dichlorosulfofluorescein (DCSF) and nitro-fluorescein, tryptophan derivative, coumarin derivatives, naphthalene derivatives, two pyridine derivate (bpy), three pyridine derivates, Hua Jing, rhodamine and organo-metallic mixture such as Ru (bpy) 3And derivative.The selection of dye molecule depends on multiple factor, comprise such as size, solvability, susceptibility, absorption and emission wavelength to degrading under the solid state reaction condition, quantum produces and quantum produces and emission wavelength to the susceptibility of chemical environment on every side.The synthetic of most and these and other suitable combination things in these factors can be determined from document easily.Be seen in as Haugland R.P_ " fluorescent probe and research compound handbook " (6 ThEd_1996).
Equally, according to the scheme II, a kind of reaction substrate of general formula (b) is selected from
Resin-L-E
General formula (b) wherein resin is represented any solid support that is suitable for solid phase synthesis, and L is a kind of joint of supporting attached to solid phase, and E is a kind of free group that is combined on the L.Suitable solid phase support comprises such as polystyrene-divinylbenzene (PS-DVB) multipolymer and polyethylene glycerine-PEG-PS-DYB multipolymer.Have or do not have the Wang (the 4-benzyloxy benzyl alcohol of polymkeric substance combination) and the rink resin of suitable joint can be from Aldrich Chemical Co_Milwaukee, WI; Novabiochem, San Diego, CA; With Advanced Chemtech, Louisville, KY. obtains.
Joint L-E selects by following requirements, promptly it with the reaction conditions shown in combining of supporting of solid phase can be in the scheme II under cutting easily.Suitable joint is known to one skilled in the art, comprises such as halogen, thiol, alcohol, ether, ester, aldehyde, ketone, carboxylic acid, nitro, amine, acid amides, silane and their protection or unprotected derivative.This joint can use method well-known to those having ordinary skill in the art to finish with being connected of solid support.Be seen in as Bunin B.A_ " composition index ", Academic Press, 1998.
Except the criterion of introducing above, joint L-E also can select by following requirement, and promptly it will form a kind of covalent linkage with the sub-D of fluorescence half point of the dye molecule of general formula (a) under Fan Yingtiaojian ﹠#60A﹠#62, produce the curing dyestuff of a kind of general formula (c):
Resin-L-D-Y
General formula (c)
He Shidefanyingtiaojian ﹠#60A﹠#62 depends on resin, L, E and X, and it is known by those skilled in the art or can measure easily.In general, they comprise a kind of solvent that can make the resin expansion and react with X of use.Suitable solvent comprises such as dimethyl formamide (DMF), 1-Methyl-2-Pyrrolidone (NMP), tetrahydrofuran (THF) (DHF), CH 2Cl 2And composition thereof.Fan Yingtiaojian ﹠#60A﹠#62 can comprise that also for example diisopropylethylamine (DEPEA), triethylamine, Dimethylamino pyridine (DMAP) or N-methylmorphaline (NMM) come the acid that produces in the neutralization reaction to a kind of alkali.
The curing dyestuff of general formula (c) is as the basis that forms ligand library (scheme II formula of (g) expression).If but the sub-Y of reaction half point is protected, it just must go protection before other reactions.This is available, form the sub-Y ' of deprotection half point go carry out under the protection process reaction conditions that ﹠#60B﹠#62 represents in the scheme II.These conditions are difference according to the group of protection, and they are known to one skilled in the art.See Greene, T.W. and Wuts, P.G.M_ " blocking group in the organic chemistry " (2 NdEd_1991).
Then, the solidified dyestuff under Fan Yingtiaojian ﹠#60C﹠#62 with a kind of general formula E 1R 1G 1Compound reaction, produce the compound of a kind of general formula (d):
Resin-L-D-R 1-G 1
General formula (d) is E wherein 1And G 1Can be identical or different, E 1Be a kind of leavings group or blocking group, G 1Or R 1Terminal or a kind of leavings group or blocking group, R 1Representative has any chemical fragment of following feature, and it contains a protectiveness or non-protective reaction half point at least, and the latter can and/or go in suitable catalysis to make R under the protective condition 1Add on the sub-D of fluorescence half point.Suitable reaction half point includes but not limited to halogen, thiol, alcohol, ether, ester, aldehyde, ketone, carboxylic acid, nitro, amine, acid amides, silane and protectiveness thereof and non-protective derivative.He Shidefanyingtiaojian ﹠#60C﹠#62 comprises the condition that those have developed for the solid phase combinatorial chemistry.Be seen in as Brown R_ " contemporary organic synthesis ", 216 (1997); Felder, E.R. and Poppinger, D_Adv.DrugRes_30: 111 (1997); Balkenhohl, F. etc., Angew.hem.Int.Ed.Engl.35: 2288 (1996); Hermkens, P.H.H. etc., Tetrahedron 52: 4527 (1996); Hermkens, P.H.H. etc., Tetrahedron 53: 5643 (1997); Thompson, L.A. etc., Chem.Rev.96: 555 (1996); And Chem.Rev.97 (2) (1997).The example of addition reaction comprises initial amine and aldehyde addition, forms an imines, and the latter can react with multiple different half points again, comprises such as beta-lactam, tetramethyleneimine, thiozolidinones and acid amides.Acidic-group has same flexibility, and can be used to and react under the Ugi polycomponent concentrates condition such as aldehyde, amine and isonitrile, forms little acid amides or heterogeneous ring compound.
Shown in the scheme II, the curing dye molecule of general formula (c) also can with the mixture reaction of compound, every kind of these compound are all different but have general formula E 1R 1G 1Be E 1R 1G 1+ (E 1R 1G 1) '+(E 1R 1G 1) "+...+(E 1R 1G 1) i, wherein i is the number of compound in the mixture, and be one less than about 50 integer.In this case, prepare the mixture of a kind of general formula (d) compound, every kind of compound all has different R 1G 1Fragment, i.e. resin-L-D-R 1G 1+ resin-L-D-(R 1G 1) '+resin-L-D-(R 1G 1) "+...+resin-L-D-(R 1G 1) iBut the compound of general formula (c) is only preferred and general formula E 1R 1G 1The reaction of a kind of compound.
Because many pharmaceutical active compounds contain reactive half point as amine and carboxylic acid, it is considered herein that this compound is by general formula E 1R 1G 1Included, in this case, can need or not need further reaction.If but the R of the compound of general formula (d) 1Fragment is reactive half point, the addition reaction of n-1 order can be on the whole with reference to carrying out under the condition among the scheme II De ﹠#60D﹠#62 so, wherein n represents the number with the sub-D bonded of fluorescence half point half point, and be one preferably less than about 100 integer.
As mentioned above, each of these order addition reactions all can be used general formula E kR kG kSingle compound or its mixture of planting, wherein k be between 2 and n-1 between an integer, R kBe be attached to K half point on the sub-D of solidified fluorescence half point (by with k-1 half point of D bonded), E kAnd G kCan be identical or different, E kBe a leavings group or blocking group, G kBe E kTerminal or leavings group or blocking group, R kExpression has any chemical fragment of following feature, and promptly it contains one at least and can make R kAdd to reactive half point on the fixed compound.He Shidefanyingtiaojian ﹠#60C﹠#62 comprises that catalyzer, deprotection agent and other can promote R kAdd to the use of the reagent on the solidified dye composition.
Finish the solidified compound that the reaction of introducing above can form general formula (f):
Resin-L-D-R n
The mixture of the curing compound of general formula (f) or general formula (f), i.e. resin-L-D-(R 1R 2R 3R n)+resin-L-D-(R 1R 2R 3R n) '+resin-L-D-(R 1R 2R 3R n) "+... resin-L-D-(R 1R 2R 3R n) n, wherein m has maximum value I*n, and I equals to contain the E of maximum quantity compound kR kG kThe quantity of the compound in the mixture.For easy, G nFrom general formula (f), ignore (the R for example because end of part n) do not experience further addition reaction.
In the final step of scheme II, dyestuff-ligand compound cuts down from the solid phase support under Fan Yingtiaojian ﹠#60E﹠#62, produces the library of compounds of general formula (g):
P-D-R n
General formula (g) should be appreciated that wherein general formula (g) comprises by all possible compound of the generation of the reaction shown in the scheme II or the mixture of compound.Suitable cutting condition is known to one skilled in the art, and depends on the key between resin and the L.Cutting can be finished under acidity or alkaline condition, or can be by photoinduction.Multiple suitable cutting method is reported in the literature.For example, shown in the scheme III that some handle the cleavage reaction that the modified resins of general formula (f) is finished with trifluoroacetic acid (TFA) in methylene dichloride:
Scheme III wherein (j) is a kind of Wang resin derivative; (k) be a kind of Wang carbamate resins derivative; (m) be a kind of Rink resin derivative; (n) be a kind of Rink amino-acid resin derivative; (o) be a kind of trityl or chlorine triphenyl amine (being X=NH) or alcohol (being X=0) resin derivative; L 1Represent any side chain stable under the solid state reaction condition or interval.The example of suitable side chain includes but not limited to be substituted and unsubstituted alkyl, aromatic base and aralkyl.
After the cutting, preferably remove to desolvate and separate the fluorescence library.The library can be dissolved in a kind of solvent such as dimethyl sulfoxide (DMSO) (DMSO) of using in the test of the present invention of being adapted at subsequently.
The particular embodiment of the method for scheme II is shown in scheme IV-VIII.For clarity sake, these schemes do not show the reaction and the formation of mixture.But should be appreciated that each shown independent reaction all represents the possibility of massive parallel reaction.
A special embodiment of simplifying of the universal method of scheme II is shown in the scheme IV:
The scheme IV is L wherein 1Be shown in reaction conditions under can not hinder or suppress any half point of linked reaction; P represents L 1End after cutting down from solid support; R 1And R 2Can be identical or different, and can be any required half point that preferred construction and response feature are provided for the library.The example of suitable half point includes but not limited to be substituted and unsubstituted alkyl, aromatic base and aralkyl group.
According to the scheme IV, use diurethanes Wang resin or amino acid Rink resin or diamino/amino alcohol trityl/chlorine trityl resin that DTAF is cured on the solid support of the monochlorotriazylaminofluorescein resin of being given.This reaction is preferably carried out at ambient temperature.DTAF measures alkali with a kind of about 0.5 to about 3 times such as DIPEA, triethylamine, DMAP or NMM are dissolved in a kind of suitable solvent such as DMF, NMP, THF, methylene chloride or its mixture.Use excessive (between 2 to 6 times of amounts of preferably approximately), such as among DMF or the NMP, symmetric diamines replaces remaining chloro on the triazine ring, is the further synthetic new reactive group that provides.This process can use multiple different reagent to repeat repeatedly as required, then the fluorescent chemicals of generation or the mixture of compound is cut down from the reaction upholder.
Another embodiment of the universal method of scheme II is shown in the scheme V:
Figure 9881370700311
The scheme V wherein DCSF by with secondary alkylamine and preferably annular secondary amine replace a chlorine atom and be attached on the resin attached on the solid support.L like this 1A part of having formed the ring diamines.Suitable ring diamines comprises such as piperazine, homotype piperazine, 4, the two piperazines of 4 '-trimethylene and derivative and isomer.As shown in the figure, though HNR 1NH can be replaced R by any compound with suitable reactive group 1A part of also having formed the ring diamines.R 2And R 3Represent any half point that is suitable for being incorporated in the fluorescent ligand of the present invention library, and comprise side chain, be substituted and unsubstituted alkyl, aromatic base and aralkyl group etc. such as natural amino acid.
Shown in the scheme IV, by the free amine group and the amino acid whose reaction of Fmoc of (being PyBrOP/DMAP/DMF) fluorescent chemicals under the standard amide formation condition, a kind of amino acid of N-Fmoc protection is incorporated on the fluorescence resin.Remove the Fmoc group with piperidines in DMF after, new amino available the deriving such as chloride of acid, chloroform or isocyanic acid that amino acid provided produces multiple fluorescently-labeled compound.The nonrestrictive example of suitable isocyanide provides in the scheme VI:
The scheme VI is very obvious to one skilled in the art, and a large amount of to contain reactive half point (be R as other half point of amino acid, chloride of acid, chloroform and isocyanic acid 4, R 5..., R n) can be used to form and DCSF bonded part.Similarly, if the reaction conditions appropriate change, the R of scheme IV 2And R 3The chemical fragment that group connected can be made the chemical fragment of the chain extension of combination dye molecule replace by any other.
Last embodiment of the universal method of scheme II is shown in the scheme VII:
Scheme VII wherein DTAF is attached on a kind of Rink amino-acid resin, and derives with the method for introducing above subsequently.L 1, R 1And R 2Definition as above.
Except top method, the also available general solid phase synthesis technique of fluorescent mark ligand library is prepared (Obrecht, D. and Villalgordo, J.M_ " combination of the solid phase support in small molecular weight compounds library is with parallel synthetic ", Pergamon, 1998; And Bunin, B.A_ " composition index ", Academic Press, 1998).In a preferred embodiment of the invention, required compound is synthetic on solid support by the method for introducing in the document.Before cutting down from solid support, the compound on the solid support is handled the fluorescently-labeled part for preparing on the resin with suitable dyestuff.Then part is cut down from resin, obtain fluorescently-labeled part.
In one approach, the reactive closure of part by the solid phase support reacts in the mode of linearity synthetic with different reactive closure length by length.Final step before cutting adds dyestuff then, obtains the tagged ligand shown in the scheme VIII.In this scheme, prepared fluorescently-labeled N-hydroxyquinzolinones.Quinzolinones be modal contain one of heterocyclic biological reactivity nitrogen (see, Sinha, S. and Srivastava, M. " drug research progress ", 1994, vol.43,143-238).They show the biology and the pharmaceutical active of wide spectrum in humans and animals.They have been used as anticonvulsive agent, antibacterium and anti-diabetic reagent.Therefore fluorescently-labeled quinzolinones is very useful concerning diagnostic use and drug discovery.
The scheme VIII
In another approach, part use polycomponent concentration response such as Ugi concentrate with a kind of polymeric method preparation (Tempest, P.A. etc., Angew.Chem.Int.Ed.Engl.1996, vol.35,640-642).Make in this way, amine component is fixed on solid support such as the Rink polyimide resin, and the amino acid of aldehyde, Fmoc protection and isocyanide join in MeOH/DCM (1: 2 v/v) mixture in the expansible resin (scheme IX) in excessive mode.By being used in combination different aldehyde, acid and isocyanide, can synthesize plurality of ligand.
Figure 9881370700351
The scheme IX
5.2 biological sample
The biological mixture that the invention provides complexity maybe can carry out the method that " fingerprint " analyzed from the sample that extensive source obtains.Method of the present invention can be used to identify the special phenotypic difference that exists between the cell of normal or unusual, health or disease, non-conversion or conversion, non-infection or infection and/or tissue.Method of the present invention also can be used for identifying or distinguishing microorganism, virus, bacterium, fungi or parasitic kind.
Method of the present invention can detect all types of ligand/receptor and interact, wherein acceptor can be protein, carbohydrate, nucleic acid or any have a kind of can with the molecule of the shape of probe or ligand interaction.Be used for herein, " biological acceptor ", " acceptor ", " biological target ", " target " and " a kind of component of biological sample " are used for being included in testing sample such as disease or other abnormal cellss and/or the tissue any biomolecules of modifying such as differential expression or difference, mating surface, binding site etc.Any this acceptor all can be used as diagnosis and/or develops the target of potential methods of treatment.
Therefore, one aspect of the present invention is a kind of method of analyzing pathologic process, said method comprise identify between the acceptor that exists in the relevant biological sample of this pathologic process or target and a kind of part or the probe library the collection of illustrative plates of binding interactions, wherein this binding interactions collection of illustrative plates provides the fingerprint of a uniqueness for this pathologic process.
According to the present invention, " acceptor " or " target " is a kind of biomolecules, it is proved to be with part or probe library in a kind of testing sample binding affinity or interaction, and this binding affinity or interaction can be identical or different in testing sample and control sample.The example of this acceptor or target includes but not limited to that albumen comprises that enzyme, antigen, antibody, fat, nucleic acid comprise that DNA and RNA, carbohydrate comprise lectin, cell surface protein or acceptor or the like.
The biological sample that uses in this method can be the sample in any biomolecules source, include but not limited to biological substance for example the product of body fluid such as blood plasma, serum, urine, celiolymph, amniotic fluid, saliva, mucus, tissue sample, cell extract, in-vitro transcription and translation system (for example by King etc. at United States Patent (USP) 5, the method of submitting in 654,150 obtains) etc.And the extract that obtains from the liquid that contains bacterium, yeast, fungi, virus and protozoon etc. also can use.In these cases, a kind of albumen in the pathogenic agent or other acceptors are shown that high-affinity and specific part can disclose new target, and can be used to detect its inhibition influence this pathogenic agent.
Can obtain from extensively different sources according to the biological sample that method of the present invention is screened.For example but be not in order to limit, biological sample or mixture can obtain from patient, comprise that body fluid, serum, mucus comprise oral cavity, rectum or intestinal mucus, urine, movement etc.In addition, biological sample can comprise tissue sample, autopsy tissue, cell sample comprise medullary cell, lymphocyte, immunocyte, from the oral cavity, the mucomembranous cell that obtains of rectum or mucous membrane of small intestine etc.In another embodiment, biological sample or mixture can comprise that cell lysate or its part, carbohydrate comprise that lectin, albumen comprise that glycoprotein, cell surface receptor, peptide, nucleic acid comprise DNA or RNA etc.In another embodiment, biological sample can be or from virus, bacterium, microorganism or parasite or contain the liquid of these biological samples, for example detect the content of microorganisms in the supplied water.
Biological sample of the present invention can be available from suffering from a kind of disease, disorder or having infected the patient of the pathologic process of a kind of virus, bacterium or other microorganisms.In another embodiment, biological sample can be by being exposed to a kind of toxin with tissue, cultured cells, cell extract or pathogenic reagent prepares, perhaps by the genome of genetically engineered culturing cell, known and any given pathologic process or disorderly relevant sudden change, albumen or peptide make it to encode.
Biological substance gleanings as the clinical sample source can obtain from hospital or state-run research institution.
5.3 the interactional detection test of ligand/receptor
Consider for any pathological condition, usually can only obtain limited clinical material, the pilot system of third element of the present invention-sensitivity just must detect the binding interactions that takes place in several microlitre samples, in addition, also must detect and is lower than the affine binding interactions of the best.The non-specific binding between the acceptor that exists in part and the sample has been eliminated or greatly reduced to pilot system of the present invention.
Therefore, test of the present invention aspect provides interactional means between a kind of detection probes or part and acceptor, target or the avtive spot.In a kind of such embodiment, the test of homogeneous is used to detect acceptor or the target that exists in a kind of biological sample.This test comprises, use diverse libraries and biological sample effect in a microtitre ware or other pass equipment, and the single member in detection probes library combines with any of system components.The molecular fingerprint that collection of illustrative plates provides this sample that combines of probe library member and sample.In a preferred embodiment, the library member detects with combining with fluorescence polarization of sample component.
In another embodiment, the acceptor that exists in the detection of biological sample or the test of target comprise, probe and ligand library are connected on the structure that contains the site that can be cut by special catalyst, and the interaction that is cut between detection probes and the acceptor of this structure or support provides signal or means.More particularly, the member in part or probe library is connected on the support that has the site that can be cut by special catalyst, each side in this site all has a mark, thereby provide a kind of part/supporting structure, with part/supporting structure and a kind of biological sample effect, wherein the part of a kind of acceptor that sample is existed with affinity just combines with this receptor, and blocked the cleavage site of support.Do the time spent when part/supporting structure and special catalyst, the support that does not contain a kind of combined acceptor is cut, and complete support is just identified.
An embodiment preferred of this test further comprises, a kind of mark such as vitamin H are connected to a side identical with cleavage site on the support, and second kind of mark such as digoxin are connected to a side opposite on the support.Support is fixed on a surface, is adding catalyzer after scouring mixture with removal holder part that is cut and the part that comprises second kind of mark, complete support has just been identified in the existence of such second kind of mark.
The support that uses in the test can comprise DNA, RNA, peptide, peptide analogs, oligosaccharides or any hydrophobic or hydrophilic, synthetic or natural polymkeric substance comprises segmented copolymer.In a preferred embodiment, support is a double-stranded DNA, and works comprises article one oligonucleotide, has a complementary second oligonucleotide and a single restriction site of a coupled part.In this embodiment, special catalyst is the restriction restriction endonuclease special to this restriction site.First kind of mark can be such as vitamin H, and ligand structure can be solidificated in the surface of streptavidin or avidin bag quilt.Digoxin is a kind of preferred second mark, and anti-digoxin-peroxidase is used to indicate the existence of intact stent.
Detecting acceptor comprises in another test that a kind of biological sample exists, the member of ligand library is connected on a kind of support, handle this support with binding reagents and a kind of biological sample that a kind of support is special, and the interaction between the acceptor that exists in detector ligand and the biological sample.The support that uses in this test can comprise that DNA, RNA, peptide, peptide analogs, oligosaccharides or any hydrophobic or hydrophilic, synthetic or natural polymkeric substance comprise the obstruction multipolymer.Do not combine when in a preferred embodiment, the special binding reagents of support takes place to interact between part and acceptor with the zone of this support.The special binding reagents of support can be a kind of medicine, peptide, glycoprotein, albumen, polysaccharide, sugar or inorganic molecule.In another embodiment, support comprises a mark, and this is marked at when the special binding reagents of support combines with support and gets clogged, and entering of mark just shows not existing and the interactional existence of ligand/receptor of binding reagents that support is special like this.In a preferred embodiment, support comprises double-stranded DNA, and mark is a kind of biotinylated Nucleotide, and when receptor/ligand interacted generation, its available streptavidin or avidin detected.
Principle of the present invention in another embodiment can be used to the potential medicine is carried out the toxicology screening.At present, the drug development of the prediction of toxicity or side effect before occurring in clinical trial be unusual difficulty in early days, uses principle of the present invention, toxic existence or do not exist and can predict than present possible more Zao developmental stage.
According to this aspect of the invention, handle culturing cell or tissue, from culture, prepare extract then, detect with the tagged compound library with known toxicity or non-toxic chemical.This detection with the tagged compound library provides a kind of " fingerprint " collection of illustrative plates, and this collection of illustrative plates has reflected cell or tissue replying toxicity or non-toxic chemical.Any change of the finger printing relevant with toxicity all goes on record, and can further classify with statistical analysis or use artificial intelligence approach (neural network).Then, culturing cell or the tissue of having set up finger printing by top method handled with having unknown toxic testing compound, prepares extract from the culture of processing, and detects with the tagged compound library.Fingerprint that the cell or tissue that produces is replied or collection of illustrative plates compare with the reference collection of illustrative plates of handling this cell or tissue generation with known toxicity/non-toxic chemical then, predict the essence of testing compound.
According to the present invention, thereby chapters and sections 5.3.1 to 5.3.3 has introduced and can be used for detection probes produces binding affinity fingerprint or collection of illustrative plates to the binding affinity difference of biological sample test.
The result who obtains with the present invention can help to distinguish two kinds of sample colonies (for example normal and disease), and they can form two classifications.In first classification, one or both probes can be used to distinguish colony, and probe response can be distinguished (" Shiningpebbles ") at an easy rate between two colonies.In second classification, with two sample colony bonded minute differences can use a large amount of, distinguish with the very little probe of difference that combines of sample colony.The combination of these probes (a kind of diagnosis bunch) is for being essential for the credible difference in the unknown sample provides the statistics support.Be to produce more useful analytical system, need a large amount of probes, because all can not be subjected to having a strong impact on because of the fluctuation of the generation that suddenlys change individually in conjunction with collection of illustrative plates.In the system that only uses one or both probes, indivedual sudden changes of probe can make diagnosis more difficult.
Analyze a large amount of probes and need to use intelligentized computerized algorithm with combining of a large amount of samples.Two types analysis can be used for data.The focusing on of first kind of analysis determines which probe can be used for distinguishing the member of two kinds of sample colonies.The algorithm of this type analysis can be from the statistical analysis field (distinctive function analysis) or obtain from artificial intelligence field (neural network that supervised, feedback propagation).Operable second type analysis comprises according to detected result probe binding data classification, and whether inquiry has any similarity in based on the history (for example medical science) of the classification of corresponding probe then.Be useful on the algorithm of these type analysis in statistical analysis (main ingredient analysis) and the artificial intelligence (non-supervision, kohonen neural network).The DAP or the software package that comprise above-mentioned functions can obtain from commercial channels.For example, commercial programs SPSS (SPSS, Inc_Chicago, Illinois) and SAS (SAS Institute, Inc_Cary NC) can provide distinctive function analysis and main ingredient analysis.Several software packages such as Neuroshell 2 (Word Systems Group, Inc_Frederick, MD) or Stuttgart Neural Network Simulation (The University of Stuttgart, Stuttgart Germany) can provide feedback propagation and Kohonen neural network.
5.3.1 fluorescence polarization test
In the present invention, the fluorescence polarization test can be used to identify the binding interactions between the acceptor that exists in part and the biological sample.
Fluorescence polarization test is designed to measure combining of fluorescently-labeled compound and cold biomolecules.Fluorescence polarization test can use molecular weight to detect the interaction of itself and biomolecules up to about 10,000 fluorescently-labeled compound.The type of operable fluorescently-labeled compound includes but not limited to little organic molecule, peptide, small protein, nucleic acid, lipid and polysaccharide.Fluorescence molecule is when exciting with flat polarized light, when only they do not rotate in the time between exciting and launching, just with a fixed planar transmit light.The degree that emission light depolarizes depends on the quantity of molecule rotation, and the latter depends on bulk of molecule again.Excite and emitting fluorescence between time in, small molecules is more than macromole rotation.The top condition of this test is a tagged compound than with its bonded non-marked molecule when much smaller.Unconjugated little fluorescent mark compound rotation is rapid, and emission light depolarizes.The interaction of biomolecules and fluorescence molecule has increased effective size of fluorescent mark compound, thereby has reduced the rotation of this compound, and the latter causes launching light and keeps polarization.The polarization light intensity of emission can by insert in the detector front one movably the polarization filter measure.Intensity is in the plane surveying at 90 ° of intervals, and repeatedly specified level and vertical intensity.In some instruments, exciting filter is active and to launch filter be fixed.In other the machine, level and vertical intensity are measured simultaneously by optical fiber at some.Three company-Pan Vera, BMG Lab Technologies and LJLBiosystems sell the fluorescence polarization equipment of research grade, and Abott provides clinical labororatory's equipment.Fluorescence polarization value is determined by following equation: Fluorescence polarization value is usually divided by 1000, and represents with little polarization unit (mP).
In a kind of this test of form, a kind of fluorescence molecule such as fluorescein are connected to a length to be enough to produce on the oligomer of " promotion effect ".Then a short circuit head is connected on the fluorescence molecule, is connected on the wall of pass equipment that microtitre ware or other are applicable to fluorescence polarization with the fluorescence molecule opposing ends on the oligomer.With the part that needs screening the free end of short circuit head is modified then.Linking ligand is known in each hole.Join in each hole a kind of body source that is subjected to and incubation then.Then use polarized light fluorescence excitation molecule, measure the polarizability of polarized light-emitting.Can will reduce rotating freely of fluorescence molecule with part bonded acceptor, increase thereby can detect polarizability.On the other hand, if be not subjected to the physical efficiency binding partner, fluorescence molecule just still can rotate freely, and emission light will reduce and polarization not.For obtaining the approximation of any bind receptor to the affinity of part, from the hole, remove extract, add fresh buffer, measure the polarisation of light value of launching in each hole.Acceptor with low affinity will dissociate from acceptor, and radiative polarization will reduce.Repeat this process and just can identify ligand/receptor binding interactions with high-affinity.
5.3.2 dodging, liquid gets close to test (SPA)
Usually in the SPA test, acceptor combines with the bead that is loaded with scintillator, directly or competitively screens at the part in the solution.But also can arrange conversely, part is combined with bead, acceptor is placed solution.This change is special and combinatorial chemistry is adaptive, because in multiple synthetic schemes, the library is synthetic carries out on bead, and the needs that it can then press the SPA pilot system are saturated with scintillator.In addition, on a bead, can synthesize more than one part.In a kind of like this system, the bead that is loaded with scintillator and is coated with a kind of part immerses in a kind of liquid phase that contains the radio-labeling reagent.If the reagent of mark has affinity to a kind of part of mark, both will in conjunction with, radio-labeling reagent that is produced and the scintillator on the bead near just causing scintillator to be excited, and luminous.If the reagent of mark has only seldom or do not have affinity to a kind of part, radio-labeling will be not enough near the accumulation scintillator energy after the radio active decay be shifted.Because SPA does not need washing step, so it can detect the ligand/receptor binding interactions of low relatively affinity.
5.3.3 DNA restriction site blocking test
The basis of the principle of native system is the restriction endonuclease activity that exists or do not exist a kind of dna structure thing, and this dna structure thing is synthesized and comprises a single restriction site and a ligand-receptor system.When this works and a kind of biological mixture are done the time spent, ligand/receptor interacts and will block restriction enzyme and enter restriction site, thereby the blocking-up structure thing is hydrolyzed in this site.Can separate the works that is kept perfectly, and identify that ligand/receptor interacts.
A kind of explanation of DNA restriction site pilot system is provided here.Contain vitamin H with one at 5 ' end and hold single strand dna oligonucleotide and annealed complementary oligonucleotide that contains digoxin, make the annealed double chain oligonucleotide that is produced contain a single restriction site in the centre 3 '.The complementary oligonucleotide chain is modified contains a joint, and this joint has a terminal amino group group.The position (in this test, the position of amino group is between the 5th A of 5 ' end and the 6th T) that makes amino group not the interference-limited restriction endonuclease to the activity of this double chain oligonucleotide.The works that is produced is shown in as follows: wherein sequence GGATCC (SEQ ID--) is a restriction site.
CCTAGG
Figure 9881370700421
Amino group is modified with the part in diversity chemistry library, forms structure as follows:
Deriving of amino group can be finished when it still is connected with the CPG bead in strand oligomer synthetic process, can oligomer be discharged from bead with the alkali cutting then, then itself and complementary vitamin H-digoxin oligomer can be annealed.The another kind of method of linking ligand is that the base of can will deriving when synthesizing complementary oligonucleotide is integrated into.
According to method well known in the art with to the special restriction enzyme incubation of this restriction site the time, the derived structure thing produces two parts as follows in this restriction enzyme site hydrolysis:
Figure 9881370700423
The surface reaction of hydrolysed mix and streptavidin or avidin bag quilt will cause biotin labeled part to be fixed, and the part of digoxigenin labeled is by flush away.Fixed mixture and anti-digoxin-peroxidase antibody response will produce negative findings, because the part of digoxigenin labeled is limited restriction endonuclease hydrolysis and washing subsequently and removes from mixture.
Complete part derived structure thing can be used as potential acceptor in the probe in detecting sample, and when it mixes with a kind of clinical sample, part will be captured any acceptor that following structure is had high-affinity:
Figure 9881370700431
Incubation is after for some time, and reaction mixture dilutes with a kind of suitable damping fluid, and handles with the restriction restriction endonuclease, with a kind of surperficial incubation of streptavidin bag quilt, fixes biotin molecule then.A kind of when the part on the works is had the biomolecules of high-affinity when existing, this molecule will be blocked enzyme and enter restriction site, and stop the hydrolysis of DNA support, thereby cause complete double chain oligonucleotide to be fixed.Another kind of situation is, when a kind of biomolecules can not combine with the part on the works, restriction enzyme will not be blocked, and cause DNA support hydrolysis as implied above, and the works that causes a quilt to be cut short is fixed, and discharges the part of digoxigenin labeled.A kind of standard enzyme linked immunosorbent adsorption test (ELISA) of digoxin superoxide enzyme antibody that uses can be used to detect the existence of digoxin on the streptavidin surface.The male test shows that restriction enzyme is blocked, and this shows that a molecule is attached on the joint.Like this, this method can be used to detect any part and anyly has enough affinities and size and block interaction between the acceptor that restriction enzyme enters its restriction site.
In the above in a kind of modification of the method for Jie Shaoing, one " slit " (a base deletion) is inserted in the chain of double-stranded sequence, and part is connected near this slit by as follows:
Figure 9881370700432
Handling this works with a kind of restriction endonuclease such as mung-bean nuclease or s1 nuclease will cause this works to locate hydrolysis in the slit.Hydrolysed mix and fixed avidin or streptavidin reaction are also then washed and will be removed the part that contains digoxin in the works.As mentioned above, this works will be prevented that with the acceptor that this part is had high-affinity incubation before adding nuclease this works from locating hydrolysis in the slit, and in detecting the test that digoxin exists, will obtain strong positive and reply.
In the further version of DNA restriction site test, the DNA support can be replaced by peptide, peptide analogs or any skeleton of being made up of by the polymkeric substance of the key of special enzyme or other mechanism cuttings and near the ligand/receptor interaction blocking-up that cutting can be taken place this key an energy therebetween by a kind of.For example, can use a kind of business-like poly-(glycine) or poly-(Methionin) skeleton, use peptide synthetic standard technique to insert a near chain that contains phenylalanine residue and one part therebetween.Enzyme such as Quimotrase A 4(EC3.4.21.1) can then use in the mode of the similar nuclease of introducing above.In addition, use the base of radioactivity or part in the time of can be by part relative with vitamin H on the composite structure thing and streptavidin is connected on the bead that is loaded with scintillator, this test is modified as a kind of SPA tests.
Although any test of Jie Shaoing all is suitable for using in the present invention here, this system can provide following advantage, for example, it does not need radioactivity, although radiolabeled Nucleotide can place the downstream of restriction site that this test is transformed into a kind of SPA form; This system can be by using PCR method as the detection system super sensitivity that becomes; Whole test can be carried out in the microtitre ware of streptavidin bag quilt; Only otherwise change restriction site, nucleic acid analog can be used to protect the DNA skeleton not to be subjected to the influence of the nuclease in the sample.
In the present invention, can place a pass device as 96 hole dot blotting devices the film (can be from such as PallCorporation, East Hills, NY obtains) that contains the special reaction group.Can in this facility, synthesize a library of compounds then, wherein a kind of specific functionality group that in the library is synthetic, adds and the special reaction group coupling on the film.For example, if just contain an amino group at synthetic library compound, this group will be by an amido linkage coupling.Any excessive group will seal with a kind of suitable closed reagent on the film.Then this part bonded film just can with derived in advance as with the reaction of vitamin H or radioactivity deutero-biological sample.Follow available a kind of suitable test and come detector ligand/acceptor interaction.
5.3.4 balance interference test
Though the DNA restriction site of introducing above test also can be considered to a kind of balance interference test, preferable methods is that the equilibrated between conjugated protein and its recognition sequence influences to DNA in monitoring part/interaction of molecules.For example, can use the conjugated protein UL9 of viral DNA when being positioned at the selected DNA sequence bonded to be measured compound in UL9 recognition sequence downstream in screening.The basis of this test be sequence to be measured disturb conjugated protein not with its recognition sequence bonded equilibrated ability.This test is introduced in 619 in U.S. Patent No. 5,306, and this patent is incorporated herein by reference in full at this.
According to principle of the present invention, the sequence to be measured that is positioned at UL9 recognition site downstream can be used as the skeleton of ligand library, rather than picture U.S. Patent No. 5,306,619 is equally as the protein-bonded target of DNA.A kind of supporting structure thing can be by following composition, and it contains the UL9 recognition sequence, and one of them special nucleotide base has been used such as a kind of marker such as biotin labeling, and contains a kind of part deutero-oligonucleotide, and its end is a digoxin.Combining of UL9 and recognition sequence do not disturbed in the existence of vitamin H on the known recognition sequence, and when UL9 combined with biotinylated sequence, biotin molecule was blocked and can not be fixed such as avidin or streptavidin.UL9 can and this works reaction and be incubated between UL9 and the biotinylated sequence and set up a kind of balance.A kind of biological sample can be introduced reaction mixture then, any when the part on the support is had the acceptor of affinity when existing in the sample, sequence to be measured will combine with part, disturb the balance between the conjugated protein and biotinylated sequence, thereby allow to change with the quantity of avidin or streptavidin bonded free biotin.
In a kind of version of this test method, with before compound is connected, can be with the synthetic a kind of sequence to be measured of screening sequence rather than second dna sequence dna.Any support that can allow part to connect can use.In addition, part can directly covalently boundly not need other joint or support to screening on the sequence.
Embodiment
For more fully understanding the present invention who introduces here, the following examples have been listed.To should be appreciated that these embodiment only are in order illustrating, rather than to be used for any method restriction the present invention.
It is interactional a kind of responsive method between detector ligand and the acceptor that test is got close in the sudden strain of a muscle of embodiment 1 liquid
The following examples have proved that liquid dodges the susceptibility of getting close to test (" SPA ").Present embodiment uses SPA to estimate 2, the p-aminophenyl-β-D-thiogalactoside sepharose 4B of 5-phenylbenzene oxazole (PPO)-saturated and the interaction between the beta-galactosidase enzymes.
Adding or do not adding H with the intestinal bacteria that the beta-galactosidase enzymes expression vector transforms at 37 ℃ 2 35SO 4Grow in the M9CA substratum of (0.1mCi/mL substratum).Handle the expression that culture of Escherichia coli was induced beta-galactosidase enzymes in 3 hours with 1mM IPTG.Then, the centrifugation culture of Escherichia coli is resuspended in the lysis buffer, takes turns freeze-thaw cycle by five and carries out cracking.The centrifugal cell residue of removing separates beta-galactosidase enzymes with ammonium sulfate precipitation from supernatant, then produce 80% purified product with affinity chromatography.
Estimate the beta-galactosidase enzymes and 2 of unlabelled or mark, 5-phenylbenzene oxazole (PPO)-saturated p-amino-benzene-β-D-thiogalactoside sepharose 4B (is pressed Bertoglio-Matte at United States Patent (USP) 4, the method preparation of introducing in 568,649) interaction between.P-amino-benzene-β that PPO-is saturated-D-thiogalactoside sepharose 4B is containing the PBS (8.1mmNa of 10% milk powder 2HPO 4, 1.5mM KH 2PO 4, 137mM NaCl, 2.7mM KCl, 0.5mM MgCl 2, 0.9mM CaCl 2) be incubated overnight in the solution, seal the non-special site of replying of any generation.In addition, can or even seal pearl with reagent such as albumin, stain remover from the extract of contrast solution.Then, p-amino-benzene-β-D-thiogalactoside sepharose 4B that 20 μ L PPO-are saturated and 200 μ L 35(counting that dodges in the mixture at liquid is 2.1 * 10 to the S-beta-galactosidase enzymes 5Cpm) and the unlabelled beta-galactosidase enzymes of the 1mg/mL of the PBS of 200 μ L or 200 μ L mix.In other experiments, identical cpm (6.5-7.7 * 10 5Cpm) 35The intestinal bacteria extract or the H of S mark 2 35SO 4P-amino-benzene-β-D-thiogalactoside the sepharose 4B saturated with 20 μ LPPO-mixes.Solution mixed 10 minutes, and the liquid that changes the PBS that contains 1mL 0.2% casein then over to dodges bottle.Pearl and beta-galactosidase enzymes or H 2 35SO 4Between interaction by analyzing with Beckman LS 6500 liquid flashing counting devices assessments radioactivity.
Containing 35In the sample of p-amino-benzene-β that S-beta-galactosidase enzymes and PPO-are saturated-D-thiogalactoside sepharose 4B, detected the counting (cpm of per minute 487+/-52; N=3).On the contrary, contain at the same time in the sample of the saturated p-amino-benzene-β of underlined and unlabelled beta-galactosidase enzymes and PPO--D-thiogalactoside sepharose 4B, detected 341+/-16 cpm (n=3).These results prove that unlabelled beta-galactosidase enzymes can replace the beta-galactosidase enzymes with pearl bonded, mark competitively.And these results show that SPA can be used to detect the interaction of mM.Free amino-benzene thiogalactoside is determined to have only is the Ki of 1.9 mM.In addition, this result shows, SPA can be used to detect the acceptor that has low affinity each other and the interaction between the part.In this embodiment, use PPO, pearl has reduced about 2.3 times to the affinity of beta-galactosidase enzymes.
Containing 6.5-7.7 * 10 5Cpm's 35In the sample of p-amino-benzene-β that the intestinal bacteria extract of S mark and PPO-are saturated-D-thiogalactoside sepharose 4B, about 3000 cpm have been detected.On the contrary, containing 7.7 * 10 5The H of cpm 2 35SO 4In the sample of the saturated p-amino-benzene-β-D-thiogalactoside sepharose 4B of PPO-, only detected about 670cpm.These results show by pearl and H 2 35SO 4Between the background that produces of interaction very low.Therefore, containing 35Detected signal mainly is to be produced by the interaction between the e. coli protein of pearl and mark in the sample of the intestinal bacteria extract of S mark.Pearl with 35Detected high cpm explanation does not have purification step in interacting between the intestinal bacteria extract of S mark, and the background radiation activity will be covered any specific adsorption of beta-galactosidase enzymes and SPA pearl.
Present embodiment proves that it is to detect interactional a kind of sensitization test between the part of mM level and the acceptor that test (SPA) is got close in the liquid sudden strain of a muscle.And SPA can be used for detecting the acceptor that has low affinity each other and the interaction between the part.
Embodiment 2:DNA blocking test is interactional a kind of responsive method between assessment acceptor and the part
The following examples have proved the interactional susceptibility between DNA blocking test assessment acceptor and the part.
Two kinds of oligomer that show below are by Clontech, Palo Alto, and CA is synthetic:
(SEQ.?ID_)?Ⅰ
5 ＇ BTT-TTT-TTT-TTT-TAT-ATA-GGA-TCC-TAT-ATA-TTT-TTT-TTT-TTT-TTD-3 ＇ B=vitamin H D=digoxin
(SEQ. ID_) II 5 ＇ AAA-AA-AAA-AAA-AAT-ATA-TAG-GAT-CCA-AAT-TAA-AAA-AAA-AAA-A-3 ＇ design oligomer, make it form one when annealing and are positioned at the intermediary restriction site.Use the II of 10: 1 ratios: I in annealing buffer (10mMTris-Cl[pH7.8], 0.1 M NaCl and 1.0mM EDTA) with mixture 65 ℃ of incubations 1 hour, then, make oligomer annealing 57 ℃ of incubations 3 hours, be stored in-20 ℃ then.The annealing oligomer sample of two multiple 32.8pmol is diluted in annealing buffer, with one in the repetition dilution of each gradient according to the explanation (New England BioLabs, Beverly, MA) the restriction restriction endonuclease with 40 units spends the night 37 ℃ of hydrolysis.Remaining dilution is carried out identical processing, except from the incubation mixture, omitting the restriction restriction endonuclease.Spend the night behind the incubation, sample is added in the microtitre ware of streptavidin bag quilt, (Boheringer Mannheim GmbH, Mannheim GER) analyzes according to explanation to use HRP deutero-anti digoxin antibody and ABTS substrate.Read the absorption value of sample then at the 405nm place.
The sample of handling with enzyme does not show absorption with respect to background (not having oligomer in the incubation mixture), even and the sample of not handling with enzyme also can show when being low to moderate the oligomer concentration of 3pmol and can know detected absorption.Like this, this system can detect the substrate of pmol level, and does not have background basically.
The ability of embodiment 3 restriction restriction endonuclease cutting DNA supports is subjected to the influence of the interaction of molecules of other reagent and support
Present embodiment has illustrated apart from the interaction of molecules of restriction site certain distance can influence the restriction endonuclease activity.
For the DNA oligomer being fixed on the hole wall of 96 hole titer plate, the annealed dna sample of 65.5pmol is joined in the hole of streptavidin bag quilt, wherein this annealed dna is at a terminal biotin labeling of using, at the other end digoxigenin labeled, and contain one and be positioned at the intermediary restriction site.Also set up a control wells (table 1, the 1st) that does not have DNA.Then with engage anti-digoxin-peroxidase in the damping fluid (anti--dig-POD) antibody (Boerhinger Mannheim) is handled 4 holes.Handle with the joint damping fluid that does not have antibody in three holes.Behind the incubation 1 hour, washing hole, three holes are handled 1 hour (table 1, the 3rd, 5 and 7) with the restriction restriction endonuclease of 120 units at 37 ℃.Does not handle with not containing the same solution that limits restriction endonuclease in other holes.And then washing hole, with ELISA detection complete (unhydrolysed) oligomer of standard.In the sample completely, the quantity of annealed dna represents with the Vmax of mOD/mm.
The editor as a result of this test is in following table 1.When not having DNA, the background Vmax value of reaction is 0.89.At enzyme without limits with when not having pre-treatment (table 1, band 2), completely, the Vmax value of unhydrolysed annealed dna is 30.96.On the contrary, when untreated, fixed dna used the restriction restriction endonuclease to handle, the Vmax value was 2.52 (table 1, bands 3).The reduction of Vmax shows, the hydrolysis of restriction restriction endonuclease the fixed oligomer.
Do not influence complete oligomer test (table 1, band 4,6 and 8) with anti-dig-POD pre-treatment fixed oligomer, because can obtain 34.96,45.33 and 53.71 value respectively.But, can influence restriction endonuclease activity (table 1, band 5 and 7) with anti--dig-POD pre-treatment, because before adding the restriction restriction endonuclease during pre-treatment oligomer, the hydrolysis of oligomer reduces.Subsequently with the restriction restriction endonuclease handle pretreatment sample (table 1, band 5 and 7) have 15.17 and 14.64 Vmax value respectively, and do not pass through pre-treatment but the oligomer that carries out restriction enzyme digestion has only 2.52 Vmax value.Like this, before adding the restriction restriction endonuclease, make the activity of restriction restriction endonuclease reduce by 17 times with anti--dig-POD pre-treatment oligomerization physical efficiency, this explanation is anti--activity that interaction energy influence restriction restriction endonuclease between the DNA oligomer of dig-POD and digoxigenin labeled cuts oligomer.These results prove, add the ability that can influence restriction restriction endonuclease cutting oligomer with the interactional reagent of oligomer in reaction mixture.
Table 1
Figure 9881370700491
Present embodiment proves, a kind of restriction enzyme digestion that can influence the DNA skeleton with " acceptor " of the ligand interaction that is connected on the DNA skeleton consumingly.
Embodiment 4-8 proved the susceptibility of fluorescence polarization test and biological sample to the difference of different probe in conjunction with collection of illustrative plates.The molecular probe that uses among these embodiment is listed in the table II.
The test of embodiment 4 fluorescence polarizations is an interactional responsive method between a kind of detection probes and the biological sample
The following examples prove that the component bonded ability of various probes and biological sample changes with component concentrations in this sample.And present embodiment has proved that also the difference of the component in probe and the biological sample combines.
The serial dilution thing of preparation blended human serum (Sigma Lot#116H4661) in PBS (pH 7.4), the concentration that makes the total protein in the sample is 65.6mg/mL, 13.1mg/mL, 2.6mg/mL, 0.52mg/mL and 0.1mg/mL.Adding 150 μ L PBS and 1.0 μ L then in the A1-A6 hole of 96 hole titer plate, to contain concentration be 1.0 * 10 -3The DMSO solution of the probe #5 of mg/mL (table II).Then, in A1, A2, A3, A4 and A5 hole, add the human serum solution example that 10 μ L contain 65.6mg/mL, 13.1mg/mL, 2.6mg/mL, 0.52mg/mL and 0.1mg/mL total protein respectively.Do not add human serum solution among the A6 of hole.Prepare of the repetition of B1-B6 hole as stated above as test sample in the A1-A6 hole.
Introduce similarly with top, adding 150 μ LPBS and 1.0 μ L in the C1-C6 hole, to contain concentration be 1.0 * 10 -3The DMSO solution of the probe #18 of mg/mL (table II).In C1, C2, C3, C4 and C5 hole, add the human serum solution example that 10 μ L contain 65.6mg/mL, 13.1mg/mL, 2.6mg/mL, 0.52mg/mL and 0.1mg/mL total protein then respectively.Do not add human serum solution among the C6 of hole.Prepare of the repetition of D1-D6 hole as stated above as test sample in the C1-C6 hole.
By top introduction probe #20-24 (table II) is similarly handled.In Fluorolite FPM-2 fluorescence polarization microtitre system 96 hole titer plate are carried out reading then, the fluorescence polarization that is obtained (mP) data repeat averaged with experiment, and total protein is mapped.The result as shown in Figure 1, it the proof different probes have nothing in common with each other with combining of human serum component.And the component concentrations that exists in the human serum can influence mP value (measurement of probe bonded).
Embodiment 5 probe #25 are to the blended human serum with to the dose response of streptavidin-HRP joiner
Present embodiment proves that probe can be used to distinguish biological sample.And present embodiment proves that also probe is complied with the different and different of this concentration of component with the component bonded ability of biological sample.Can for determining to observe the strong binding interactions with a kind of probe when having serum protein, we have measured probe #25 (table II) to the PHS with to the dose response of streptavidin-HRP joiner.The serial dilution thing of preparation blended human serum (Sigma Lot#116H4661) in PBS (pH=7.4), the concentration that makes the total protein in the sample is 65.6mg/mL, 13.1mg/mL, 2.6mg/mL, 0.52mg/mL and 0.1mg/mL.Adding 150 μ LPBS and 1.0 μ L then in the A1-A6 hole of 96 hole titer plate, to contain concentration be 1.0 * 10 -3The DMSO solution of the probe #25 of mg/mL.Then, in A1, A2, A3, A4 and A5 hole, add the human serum solution example that 10 μ L contain 65.6mg/mL, 13.1mg/mL, 2.6mg/mL, 0.52mg/mL and 0.1mg/mL total protein respectively.Do not add human serum solution in the A6 hole.Prepare the repetition of B1-B6 and C1-C6 hole as stated above as test sample in the A1-A6 hole.
The serial dilution thing of preparation streptavidin-HRP joiner (Sigma 59420) in PBS (pH=7.4), the concentration that makes the total protein in the sample is 1.0mg/mL, 0.1mg/mL, 0.01mg/mL, 0.001mg/mL, 0.0001mg/mL and 0.00001mg/mL.Adding 150 μ L PBS and 1.0 μ L then in the A1-A7 hole of another piece 96 hole titer plate, to contain concentration be 1.0 * 10 -3The DMSO solution of the probe #25 of mg/mL.Then, in the A1-A6 hole, add the human serum solution example that 10 μ L contain 1.0mg/mL, 0.1mg/mL, 0.01mg/mL, 0.001mg/mL, 0.0001mg/mL and 0.00001mg/mL total protein.Do not add human serum solution among the A7 of hole.。Prepare of the repetition of B1-B7 hole as stated above as test sample in the A1-A7 hole.With Fluorolite FPM-2 fluorescence polarization microtitre systems measurement or measure the fluorescence polarization of sample in two 96 hole titer plate, the fluorescence polarization that is obtained (mP) data repeat averaged with experiment then, and to the log mapping of total protein.
Data as shown in Figure 2, these data declarations, a little less than the combination very of probe #25 to serum protein, under this experimental conditions, observe probe in conjunction with the serum protein that needs about 100 μ g.On the contrary, probe #25 and streptavidin-HRP joiner be combined in the streptavidin that there is 1.0 μ g-HRP joiner the time just can finish.Therefore, probe #25 has higher affinity to streptavidin-HRP comparison human serum.
Embodiment 6 uses probe #25 to detect streptavidin-HRP joiner in blended people and bovine serum
The following examples prove, in the blended biological sample, the component that probe is had high-affinity can be detected when the lower concentration.
For determine that can a spot of conjugated protein by force (streptavidin-HRP joiner) be detected when having serum protein, in the blended human serum, add streptavidin-HRP joiner, and detect replying probe #25 (table II).At first, adding 150 μ LPBS and 10 μ L in the A1-A3 hole of one 96 hole titer plate, to contain concentration be 1.0 * 10 -3The DMSO solution of the probe #25 of mg/mL.Then, adding 10 μ L concentration in all three holes is the human serum solution example (by obtaining by 1: 5 dilute Si gma pooled serum Lot#116H4661 in PBS) of 13.1mg/mL total protein.Like this, the total amount of human serum is 131 μ g in each hole.131 μ g serum proteins and three repetitions of probe #25 bonded are measured in these three hole representatives.
Then, adding 150 μ L PBS and 1.0 μ g in the B1-B3 hole of 96 hole titer plate, to contain concentration be 1.0 * 10 -3The DMSO solution of the probe #25 of mg/mL.Adding 10 μ L concentration in the B1-B3 of hole is the human serum solution example (by obtaining by 1: 5 dilute Si gma pooled serum Lot#116H4661 in PBS) of 13.1mg/mL total protein and streptavidin-HRP joiner solution example that 10 μ L contain the 0.1mg/mL total protein.Human serum protein's total amount is 131 μ g in each hole, and the total amount of streptavidin-HRP joiner is 1.0 μ g.Probe #25 and protein bound three repetitions of 1.0 μ g streptavidin-HRP joiners are measured in B1-B3 representative in hole when having 131 μ g serum proteins.
The fluorescence polarization of sample reads in Fluorolie FPM-2 fluorescence polarization microtitre system in the titer plate.The polarization that is obtained (mP) data are averaged with three mensuration, design a kind of figure and are presented at when having streptavidin-HRP joiner and serum protein the change that polarization (mP) takes place when having only serum protein.The result as shown in Figure 3, it shows that proteic being combined in of probe #25 and 1.0 μ g streptavidin-HRP joiners is easy to when having 131 μ g serum proteins detect.These results show, the fluorescence polarization test is the bonded sensitivity method between detection probes and the sample composition that exists with μ g/mL concentration.
It is the serum structure " fingerprint " that has or do not have streptavidin-HRP joiner that embodiment 7 uses probe #1-25
The following examples prove, a small amount of fingerprint that can change biological sample by force in conjunction with the existence of component.
At first, add the DMSO solution of the probe #1-12 of 150 μ LPBS and 1.0 μ L in the A1-A12 hole of 96 hole titer plate, the concentration of every kind of probe is 1.0 * 10 -3Mg/mL.This makes hole A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11 and A12 contain 1.0 * 10 of probe #1, #2, #3, #4, #5, #6, #7, #8, #9, #10, #11 and the #12 of 1.0 μ L (table II) respectively -3Mg/mL solution and 150 μ L PBS.B1-B12 handles with same way as in the hole, as the revision test of probe 1-12.
Secondly, add the DMSO solution of the probe #13-24 of 150 μ LPBS and 1.0 μ L in the C1-C12 hole of same 96 hole titer plate, the concentration of every kind of probe is 1.0 * 10 -3Mg/mL.This makes hole C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11 and C12 contain 1.0 * 10 of probe #13, #14, #15, #16, #17, #18, #19, #20, #21, #22, #23 and the #24 of 1.0 μ L (table II) respectively -3Mg/mL solution and 150 μ L PBS.D1-D12 handles with same way as in the hole, as the revision test of probe 13-24.Then, in hole E1 and E2, add 1.0 * 10 of 150 μ LPBS and 1.0 μ L probe #25 -3Mg/mL DMSO solution.Then, add the PHS (Sigma Lot#116H4661) of 10 μ L in the A1-A12 on 96 orifice plates, B1-B12, C1-C12, D1-D12 and the E1-E2 hole by dilution in 1: 10.Total protein content in the 10 μ L serum is 65.6 μ g.
Then, read the fluorescence polarization in each hole on 96 orifice plates in Fluorolite FPM-2 fluorescence polarization microtitre system, the polarization that is obtained (mP) value is mapped on bar figure, is used for showing " fingerprint " of this serum sample.The results are shown in Fig. 4.
Then, in each hole, add streptavidin-HRP joiner solution that 10 μ L contain the 0.1mg/mL total protein.Total streptavidin-HRP joiner the albumen that is added in each hole is 1.0 μ g.Then, plate is reading in Fluorolite FPM-2 fluorescence polarization microtitre system, and the polarization that is obtained (mP) value is mapped being shown on the bar figure of Fig. 5.This Figure illustrates " fingerprint " of the serum sample that is added with streptavidin-HRP joiner.This result shows, can detect a small amount of streptavidin-proteic existence of HRP joiner in serum sample, and this albumen can change observed serum fingerprint.These presentation of results can use the fluorescence polarization experiment sieving can distinguish the probe of normal and unusual component in the biological sample.
Embodiment 8-9 proves that the fluorescence polarization test can be used to screen the probe library that complex mixture can be separated from each other.
Embodiment 8 can with the evaluation of people and bovine serum difference bonded probe
Use fluorescence polarization determination table II listed 4,4-two fluoro-5,7-dimethyl-4-boron-3a, the compound 1-19 of 4a-diaza-s-indacene-3-propionic acid (BO) mark (molecular probe, Eugene, OR) and the interaction between foetal calf serum or the human serum.Human serum (Sigma Lot#116H4661) contains the 65.6mg/mL total protein, and foetal calf serum (Sigma Lot#96H4615) contains the 47.3mg/mL total protein.For reducing the influence of total protein concentration, the foetal calf serum of 10 μ L and the human serum of 47.3/65.6 * 10 μ L=7.2 μ L are used in this experiment.
The PBS that in A1-A10, B1-B10, C1-C9, D1-D9, E1-E10, F1-F10, G1-G9 and the H1-H9 hole of 96 hole titer plate, adds 150 μ L.Add dimethyl sulfoxide (DMSO) (DMSO) solution of 1.0 μ L compound 1-10 in hole A1-A10, B1-B10, C1-C9, D1-D9, E1-E10, F1-F10, every kind of compound concentrations is 1.0 * 10 -3Mg/mL.In this mode, each contains PBS and 1.0 μ L1.0 * 10 of 150 μ L hole A1, B1, E1 and F1 -3The DMSO solution of the compound #1 of mg/mL.Similarly, hole A2, B2, E2 and F2 contain PBS and 1.0 μ L1.0 * 10 of 150 μ L -3The DMSO solution of the compound #2 of mg/mL, hole 3-10 also are like this.In hole C1-C9, D1-D9, G1-G9 and H1-H9, add dimethyl sulfoxide (DMSO) (DMSO) solution of 1.0 μ L compound 11-19, every kind of compound concentrations is 1.0 * 10 -3Mg/mL.In this mode, each contains PBS and 1.0 μ L1.0 * 10 of 15 μ L hole C1, D1, G1 and H1 -3The PBS of mg/mL and 1.0 μ L1.0 * 10 -3The DMSO solution of the compound #12 of mg/mL.Hole 13-19 also is like this.Then this plate emission filter that excites filter and 530nm with 485nm in Fluorolite FPM-2 fluorescence polarization microtitre system is carried out reading.Polarization that is obtained (mP) and intensity data are represented blank reading.
Then, in containing hole A1-A10, the B1-B10 of PBS and aforesaid probe, C1-C9, D1-D9, add 7.2 μ L human serums (Sigma Lot#116H4661).Like this, A1-A10 and B1-B10 series are the revision test of compound 1-10 in human serum, and serial C1-C9 and D1-D9 are the revision test of compound 11-19 in human serum.Similarly, the foetal calf serum (Sigma Lot#96H4615) that in containing hole E1-E10, F1-F10, G1-G9 and the H1-H9 of PBS and aforesaid probe, adds 10 μ L.Like this, like this, E1-E10 and F1-F10 series are the revision test of compound 1-10 in foetal calf serum, and serial G1-G9 and H1-H9 are the revision test of compound 11-19 in foetal calf serum.Then by the top reading of data in the Fluorolite FPM-2 fluorescence polarization microtitre system that is presented in.The polarization (mP) and the intensity data that produce are shown in Fig. 6.MP value to every kind of probe is made bar figure.The value of preceding two bars every kind of probe of representative revision test in human serum, the value of latter two every kind of probe of representative revision test in foetal calf serum.This mP is data represented from mark emission polarising degree.Higher reading shows that tagged compound combines with a macromole, and lower readings signify tagged compound bonded degree is lower.We as can be seen, compound #2 and #18 in human serum with a kind of macromole bonded degree than high in foetal calf serum.
Embodiment 9 can with the evaluation from the biological sample difference bonded probe of different mammal species
The following examples have proved that biological sample can be distinguished from each other, and comprise distinguishing kind and kind, disease and non-disease, and the basis of this differentiation is that the difference that produces during with the probe library screening when them is in conjunction with collection of illustrative plates.
In 96 hole titer plate,, and use Fluorolite FPM-2 fluorescence polarization microtitre systematic survey fluorescence polarization with every kind of sample and the fluorescently-labeled molecular combinations listed in the 1 μ L table 3.Fluorescence labeling probe below using in the fluorescence polarization test: myo-inositol-1-phosphoric acid (probe 18, Molecular Probes), final concentration that final concentration is about the BODIPY mark of 0.18nM are the fluorescein-labeled phenytoin (probe 70 of 10.5nM; Sigma), the final concentration fluorescein-labeled probe 129 (Sepracor), the final concentration that the are about 0.096 μ g/mL fluorescein-labeled probe 135 (Sepracor), the final concentration that are about 0.078 μ g/mL is about the fluorescein-labeled probe 147 (Sepracor) of 0.09 μ g/mL.Measure interactional fluorescence polarization value between every kind of probe and blood plasma or the cell extract.
The biological sample list that uses in table 3 test
Figure 9881370700551
Based on fluorescence polarization value, the myo-inositol of BODIPY mark-1-phosphoric acid (probe 18) has proved with difference from different types of sample and has combined (Fig. 7).For example, use probe 18, the fluorescence polarization value of blended human plasma is higher 3 times than the fluorescence polarization value of blended bovine serum or blended foetal calf serum approximately.But, in fluorescence polarization value, almost do not observe difference from the sample of identical type.For example, use probe 18, in blended WKY rat plasma and blended SHR rat plasma, do not detect the difference of tangible fluorescence polarization value.These results show that probe such as myo-inositol-1-phosphoric acid can be used for obtaining relevant to different types of information.
The fluorescence polarization value of the phenytoin of fluoresceinization (probe 70) shows that it also is a kind of probe (Fig. 8) that can be used for distinguishing from different types of blood plasma.For example, the fluorescence polarization value of the pooled human plasma of usefulness probe 70 acquisitions is more much higher than other samples.In addition, between blended bovine serum and blended foetal calf serum, also detect the notable difference of fluorescence polarization value.These presentation of results, the phenytoin of probe such as fluoresceinization not only can be used for distinguishing from different types of sample, also can be used to distinguish from same kind but the sample of different development stage.
Probe with similar structures also can be used for distinguishing from different types of sample.Probe 147,129 structurally similar with 135 (Fig. 9 A, 10A and 11A) all uses solid state chemistry to give up and make up on a fluorescein skeleton, and they can provide enough information for the classification from the blood samples of 6 kinds of primates of listing in table 3.Probe 147 and 129 proofs, with fluorescence polarization value behind primate (mankind and non-human) the blood plasma incubation than with non-human primate animal plasma (Fig. 9 B and 10B) incubation after high.But, probe 129 with from the blood plasma incubation of monkey after than with blood plasma incubation from the people after the polarization signal height (Figure 10 B) that shows.Behind probe 135 and the cercopithecus aethiops plasma sample incubation observed fluorescence polarization value than with from high (Figure 11 B) behind other monkeys or people's the blood plasma incubation.Therefore, can to probe 129,135 and 147 and the difference of primate plasma sample be incorporated into edlin, thereby can use these information to come exactly sample each other to be distinguished.
Embodiment 10 can distinguish the evaluation of probe of the streptococcus aureus of streptococcus aureus and Dimethoxyphenyl penicillin resistance
Below test be used for identifying the probe of streptococcus aureus (MRSA) and the intestinal bacteria (E.coli) can be used to distinguish streptococcus aureus (S.aureus), Dimethoxyphenyl penicillin resistance.
The heart and brain leach liquor substratum that the streptococcus aureus (MRS) and the intestinal bacteria (E.coli) of streptococcus aureus (SA), Dimethoxyphenyl penicillin resistance are inoculated in 25ml or 35ml volume (BHI, Difco) in.Culture is incubated to the OD of intestinal bacteria, SA and MRSA on 37 ℃ of shaking tables 660nmValue reaches 0.344,0.411 and 0.367 respectively.Above-mentioned culture OD 660nmAny difference of value illustrates in table.In this experiment, in 96 hole fluorescence polarization titer plate of black, the 5ul culture sample is diluted among 100ul phosphate-buffered salt (PBS)+0.1% N gamma Globulin BGG.Then, in cell suspension, add the 5ul dilution, final concentration be about 10nm, from the fluorescence labeling probe in library, with the incubation time of plate in 37 ℃ of incubations 30 minutes or indication.After the incubation, use the fluorescence polarization quantity in each sample of Fluorolite FPM-2 fluorescence polarization microtitre systems measurement.Every kind of probe is carried out the multiple fluorescence polarization determination.
The fluorescence labeling probe that uses in the fluorescence polarization experiment is bought from the outside or is synthetic at Sepracor.Table 4 has been listed fluorescent mark, probe source and the probe number of probe title, use.
Table 4 fluorescence labeling probe Fang Ku
Figure 9881370700581
According to fluorescence polarization value, have four probes (11A, 10B, 6D and 10E) show with aureus strains combine contrast and BHI substratum (inoculating) or intestinal bacteria sample be combined with difference (table 5).Use probe 10B, 10E and 6D to repeat the fluorescence polarization test and can be used for distinguishing SA and MRSA to determine them.Probe 10B and 10E can not show with the difference of the streptococcus aureus (MRS) of streptococcus aureus (SA) and Dimethoxyphenyl penicillin resistance and combine.Use probe 10B or 10E, in SA, MRSA, intestinal bacteria and nonvaccinated BHI media samples, do not obtain tangible fluorescence polarization value difference (table 6).Therefore, probe 10B and 10E can not be used for distinguishing SA and MRSA, intestinal bacteria and nonvaccinated BHI substratum.
Table 5: the fluorescence polarization value of the microbial culture matter sample that detects with fluorescent tag molecule
Figure 9881370700611
Table 6 probe 10B and 10E can not be used to distinguish bacterial isolates
Figure 9881370700612
The result who is obtained by the fluorescence polarization test of using probe 6D shows that this probe can be used to distinguish SA and MRSA, intestinal bacteria and nonvaccinated BHI substratum (table 7; Fig. 2).Compare with the streptococcus aureus (MRS) of p-methoxy-phenyl penicillin resistance, nonvaccinated BHI substratum and intestinal bacteria (E.coli), (SA) can obtain higher fluorescence polarization value from streptococcus aureus.The difference of this fluorescence polarization value have only when bacteria samples from incubation just can detect (table 7 during the culture more than 5 hours; Figure 12).Only when the culture incubation time was 7.5 hours or 24 hours, the SA sample just showed the fluorescence polarization value height than MRSA sample.The fluorescence polarization value that obtains from the BHI substratum is only when the culture incubation time is 7.5 hours or 24 hours, just than low from the fluorescence polarization value of SA and MRSA acquisition.On the contrary, intestinal bacteria show minimum fluorescence polarization value always.Therefore, the binding ability of probe 6D and bacteria samples component changes with the increase of bacterial cultures incubation time.
The fluorescence polarization level that is obtained by probe 6D increases in the SA sample in time, but reduces in time in other samples.For example, colibacillary fluorescence polarization value from the 211.8+ of the sample that obtains by 1 hour culture of incubation/-8.8mP reduce to the 173.4+ of the sample that obtains by 24 hours culture of incubation/-3.1mP (table 7).On the contrary, the fluorescence polarization value of SA from the 233.3+ of the sample that obtains by 1 hour culture of incubation/-5.1mP be increased to the 279.0+ of the sample that obtains by 24 hours culture of incubation/-3.3mP (table 7).These presentation of results, probe 6D preferentially combines with some unknown component of SA, and along with the increase of incubation time, the expression of these components also increases.And these results show that also probe 6D can be used for distinguishing SA and MRSA.
Present embodiment proves that this pilot system can be used for screening the probe that can distinguish bacterial isolates.In the present embodiment, from a little library that contains 80 kinds of fluorescence labeling probes, found the probe (6D) of the employed three kinds of bacterial isolateses of introducing above being used for distinguishing of experiment.
Table 7: probe 6D can be used to distinguish bacterial isolates
Figure 9881370700631
Embodiment 11 probe 6D distinguish the ability of streptococcus aureus from other bacterial isolateses
Following experiment is used for determining whether probe 6D can be used for distinguishing streptococcus aureus (SA) from other bacteriums.
With 12 cultures of 5 kinds of different bacterium bacterial strains be inoculated in the 35ml volume heart and brain leach liquor substratum (BHI, Difco) in.Though the kind of every kind of culture is unknown to the experimenter, but known 4 kinds of cultures contain streptococcus aureus (SA), 2 kinds of cultures contain the streptococcus aureus (MRSA) of p-methoxy-phenyl penicillin resistance, 2 kinds of streptococcus aureuses (QRSA) that contain the quinoline resistance, 2 kinds of faeciums (VREF) that contain the vancomycin resistance.Culture is incubated overnight on 37 ℃ of shaking tables.The 2nd day, each took out the sample of 7 5ul from 12 cultures, and in 96 hole fluorescence polarization titer plate of black it is diluted among 100ul phosphate-buffered salt (PBS)+0.1% N gamma Globulin BGG.Then, the fluorescein-labeled Sep0119288 of the about 10nM probe of 5ul 6D is joined in the cell suspension, and with the incubation time of plate in 37 ℃ of incubations 30 minutes or indication.Behind the incubation, use the fluorescence polarization quantity in each sample of FluoroliteFPM-2 fluorescence polarization microtitre systems measurement.12 kinds of the highest and minimum polarization readings of not identifying 7 samples of every kind of culture are removed, 5 of being left are worth averaging and standard deviation.
Fluorescence polarization value to the bacterial cultures sample determination that contains bacterial cell is shown in table 8.According to fluorescence polarization value the fluorescence polarization test-results of 12 kinds of cultures is divided into groups.These groupings are shown in Figure 13.Every kind of grouping all is equivalent to a bacterial isolates (table 9).The SA culture has the highest fluorescence polarization value, and intestinal bacteria have minimum fluorescence polarization value (Figure 13).Therefore, use this simple fluorescent probe, the culture of 12 kinds of the unknowns can be divided into exactly 5 bacterial strains, and SA can be distinguished from other organisms that this research is used.
Table 8: 12 kinds of fluorescence polarization values of not determining culture that use probe 6D to detect
Figure 9881370700651
Table 9. is divided into 5 groups corresponding to bacterial isolates according to fluorescence polarization value with 12 kinds of unidentified bacterial culturess
Unknown Organism
1，9 QRSA
2，4，6，10 SA
3，7 MRSA
5，12 E.coli
8，11 VREF
Present embodiment proves, the invention provides a kind of method of distinguishing bacterial isolates.And present embodiment shows that technology of the present invention can be used for detecting and the diagnose infections microorganism.
Embodiment 12: ligand library synthetic
Method 1: the liquid phase of fluorescently-labeled amine is synthetic
Primary amine and secondary amine use FITC (1.2 times) at room temperature using FITC (1.2 times) to handle (if use amine salt, the triethylamine that adds equivalent dissociates amine) under the situation that has lutidine (DMAP) in DMF or DMSO respectively respectively.After about 24 hours, the amine of 2 times of amounts is removed resin, and ((tris-(2-aminoethyl)-amine polystryrene HL resin) joins in the reaction mixture.After 24-48 hour, filtering mixt is removed resin, and the filtrate that further will contain the fluorescent mark part of purifying with DMSO is diluted to the required concentration of screening.Table 10 has been listed according to the scheme I and has been used liquid phase synthetic fluorescent mark amine.
Table 10
The list of table 10 fluorescent mark amine
Figure 9881370700681
Figure 9881370700701
Method 2: amine carboxylamine Wang resin synthetic
With Wang resin (25g, 20mmol, 0.8mmol/g carrying capacity) and carbonyldiimidazole (CDI) (20g, 120mmol) in exsiccant THF (180ml) room temperature vibration 2 days.Mixture is filtered, and with DMF, THF and CH 2Cl 2Thorough washing resin, dry resin, thus obtain imidazole carbonyl Wang resin (27g).
In independent reaction tubes, use piperazine (1.7g then, 20mmol), homotype piperazine (2.0g, 20mmol) or 4,4 '-trimethylenedipiperidine (4.2g, 20mmol) THF/DMF (1: 1, at room temperature handled imidazole carbonyl Wang resin (5g, 4mmol, 0.8mmol/g carrying capacity) 40ml) 20 minutes.Use DMF, THF and CH then 2Cl 2Thorough washing resin, vacuum-drying obtains corresponding amine carboxylamine Wang resin respectively.
Method 3: by the DTAF on the synthetic Wang resin of amine carboxylamine joint
Amine carboxylamine Wang resin (1.0 times of amounts) and DTAF hydrochloric acid (2.5 times of amounts) were vibrated 12-17 hour in room temperature under the situation that has diisopropylethylamine (DIPEA) (4.0 times of amounts) in DMF.Filter resin, and with DMF, THF and CH 2Cl 2Thorough washing resin, vacuum-drying obtains the monochlorotriazylfluorescein on the Wang resin.
Method 4: by the DTAF on synthetic trityl of amine joint or the chlorine trityl resin
With trityl amine or 2-chlorine trityl polyimide resin (Novabiochem) (0.12mmol, 1.0 doubly measure) and DTAF (0.3mmol, 2.5 doubly measure) in DMF (ml), under the situation that has DIPEA (0.48mmol, 4.0 times of amounts), vibrated 24-40 hour in room temperature.Resin DMF, THF and CH 2Cl 2Thorough washing resin, vacuum-drying produces the monochlorotriazylfluorescein on the trityl resin.
Method 5: the DTAF on the synthetic RINK resin
Handled Rink amide resins (1g, 0.8mmol, 0.8mmol/g carrying capacity) 2 hours with 30% piperidine solution among the DMF (5ml), the Rink amide resins that is produced washs with DMF, and is then with the DCM washing, dry then.Then with the DTAF among Rink amide resins (0.8mmol) and the 5ml DMF (2.5 times of amounts, 2mmol) and DIPEA (4.0 times of amounts 3.2mmol) were vibrated 24 hours.Use the DMF/THF washing resin, with the DCM washing, vacuum-drying obtains the monochlorotriazylfluorescein on the Rink resin then.
Similarly, the Fmoc-amino on the Rink resin (under the peptide synthesis condition of standard as DIC/DMAP/DCM by the preparation of Rink polyimide resin and Fmoc-amino acid) is handled with piperidines in DMF and is removed the Fmoc group.In DMF, at room temperature handled amino acid Rink resin 24 hours with the DTAF of 2.5 times of amounts and the DIPEA of 4.0 times of amounts then.After washing and the drying, just obtain the monochlorotriazylfluorescein on the Rink resin.
Method 6: the reaction of fluorescence joint and a kind of diamines
Monochlorotriazylfluorescein on a kind of solid phase support such as Wang resin or the trityl resin at room temperature handled in DMF 24-40 hour with a kind of symmetrical diamines of 4.0 times of amounts, use the DMF/MeOH washing resin then, then wash with DCM, vacuum-drying obtains the amino fluorescein that a kind of solid phase is supported.
Method 7: prepare a kind of fluorescein-labeled carbamide compound library
Three seed amino acid Rink resins (are the L in the scheme I 1Be CH 3, isobutyl-, benzyl) (each 500mg, 0.4mmol, 0.8mmol/g carrying capacity) use DTAF (2.5 times of amounts) to handle respectively according to the introduction in the top method 5 in independent test tube, obtains three kinds of different monochloro fluorescein derivatives on the resin.Use piperazine (each 4.0 times of amount) and 4 then respectively, 4 '-trimethylenedipiperidine (4.0 times of amounts) handles these resins according to method 6, and (each 250mg 0.2mmol), obtains 6 kinds of fluorescein-labeled resins of amino.(each 25mg 0.02mmol) reacts with parallel mode in THF with 10 kinds of isocyanides (each 3.0eq) respectively, obtains 60 kinds of fluorescent mark carbamide compounds on the resin with these 6 kinds amino fluorescein resins then.Use 30% TFA/DCM cutting resin then, follow vacuum-drying, obtain the fluorescence carbamide compound (analyzing confirmation) of 60 kinds of separate marking, they are dissolved in the DMSO of 1ml, be used for biological screening with HPLC/MS.
Method 8: DCSF is connected on WANG resin or the trityl resin by the amine joint
(4mmol, 0.8mmol/g) (2.0 times of amounts, 8mmol) (2.0 times of amounts were vibrated 2-3 hour in room temperature under situation 8mmol) there being DIPEA in 50ml exsiccant DMF with DCSF with amine carboxylamine Wang resin.Use DMF, MeOH then, then use the DCM washing resin, drying obtains the monochlorosulfofluorescein on the Wang resin.
In a similar manner, in DMF, handle a kind of piperazine trityl resin existing under the situation of DIPEA, obtain the monochlorosulfofluorescein on the trityl resin with DCSF.
Method 9:monochlorosulfofluorescein and the reaction that encircles diamines
In the DMF of 10ml, handled 24 hours with a kind of ring diamines such as piperazine (4.0mmol, 3.0 times of amounts) from the fluorescein on the Wang resin of method 8 (1.33mmol, 1.0 times of amounts).Use the DMF/MeOH washing resin then, then with the DCM washing, vacuum-drying obtains the amino sulfofluorescein on the Wang resin.
Method 10: the library of preparation sulfofluorescein mark
The amino sulfofluorescein Wang resin that contains different diamines joint (for example piperazine, homotype piperazine and 4, the composition of 4 '-trimethylenedipiperidine) exists a kind of coupling reagent such as diisopropylcarbodiimide (DIC) (10 times of amounts) and DMAP (5.0 times of amounts) at room temperature to handle in DMF/DCM in a kind of parallel mode 24-48 hour with different organic acid (10 times of amounts).After amine is by complete acidylate (then downcut low amounts of resin and carry out HPLC), use the DMF/MeOH washing resin, then with DCM washing, vacuum-drying.Use 30% TFA/DCM cutting resin then, then vacuum-drying obtains required fluorescein-labeled compound.
Method 11: fluorescein-labeled QUINZALINONE's is synthetic
The 1st step, O-azanol (the reference: Floyd of Wang resin-bonded, C.D. etc., Tetrahedron Lett.1996, vol.37,8045) (2.0g, 1.6mmol, 0.8mmol/g carrying capacity) with isatoic acid anhydride (6.4mmol, 4.0 times of amounts) in the DMF of 25ml under the situation that has DMAP (0.8mmol, 0.5 times of amount) 65-70 ℃ of oscillation treatment 26 hours.Filter the resin compound that is produced then, and thoroughly wash with DMF, MeOH and DCM, drying obtains the N-azanol on the resin.
The 2nd step; N-azanol resin (0.04mmol; 50mg; 0.8mmol/g the carrying capacity) a-amino acid (0.2mmol of the first group of Fmoc protection that obtains with the commercial channel; 5.0 doubly the amount), coupling reagent PyBrOP (Bromo-tris-pyrrolidino-phosphoniumhexafluorophosphate; 0.2mmol, 5.0 times of amounts) and DMAP (0.2mmol, 5.0 times of amounts) in dimethyl amine (DMAC) solvent (0.6ml) at 60-65 ℃ of oscillation treatment 20-24 hour.With resin filter, and thoroughly wash with DMF, MeOH and DCM, drying obtains the quinazolinone on the resin.Then resin is suspended from 30% piperidines of (0.6ml) among the DMF,, after filtering and washing, obtains the amino quinazolinone resin of free, be used for the connection of next reactive module to remove the Fmoc blocking group from amino group.
The 3rd step; amino quinazolinone (50mg on the resin; 0.04mmol) with the a-amino acid (0.2mmol of second group of Fmoc protection; 5.0 doubly the amount), coupling reagent DIG (diisopropylcarbodiimide; 0.2mmol; 5.0 doubly the amount) and DMAP (0.2mmol, 5.0 times of amounts) in DMF (0.6ml), at room temperature handled 20-24 hour.Thoroughly wash drying then with resin filter, and with DMF, MeOH and DCM.Then resin is suspended from 30% piperidines of (0.6ml) among the DMF,, after filtering and washing, obtains the free amine group resin, be used for the connection of dyestuff to remove the Fmoc blocking group from amino group.
The 4th step, aminoresin (0.04mmol) DTAF-HCl dyestuff (Dichlorotriazylamino fluorescein monohydropylethylamine, 0.08mmol, 2.0 doubly measure) and DIPEA (diisopropylethylamine, 0.16mmol, 4.0 times of amounts) and in DMF (0.6ml) at room temperature oscillation treatment 20-24 hour.Thoroughly wash then with resin filter, and with DMF, MeOH and DCM, drying obtains the quinazolinone on the fluorescently-labeled resin.
In the 5th step, fluorescently-labeled resin (0.04mmol) was with 30% trifluoroacetic acid (TFA) of (1.0ml) among the DCM at room temperature oscillation treatment 1-2 hour.This mixture is filtered, and with the DCM washing resin of 0.5ml.Collect the filtrate that merges, drying is carried out in vacuum-evaporation, obtains required fluorescently-labeled quinazolinone part, and the latter dissolves in DMSO and is used for screening.
Use the amino acid of different isatoic acid anhydrides and Fmoc-protection to carry out above-mentioned reaction in a parallel manner, just can prepare above-mentioned fluorescently-labeled quinazolinone ligand library.
Method 12: use the UGI coupling synthetic ligands on the solid phase
Rink polyimide resin (1.0 times of amounts) (handling business-like Rink polyimide resin preparation) with 25% piperidines among the DMF MeOH/DCM (1: 2, expand in v/v).The amino acid (10 times of amounts) that adds acid anhydride (10 times of amounts) and Fmoc-protection.Mixture at room temperature vibrated 1-2 hour.Add isocyanide (10 times of amounts) then.Mixture at room temperature vibrated 20-24 hour.Then with resin filter, and with DMF, MeOH and DCM thorough washing, and dry.Resin is then handled with 25% piperidines among the DMF, obtains containing the resin of a free amine group group.The DIPEA that doubly measures of DTAF that this resin is doubly measured with the 2.0-3.0 among the DMF and 4-6 handles then, after the filtration and washing of routine, obtains the fluorescently-labeled part on the resin.Also dry with TFA/DCM then from the resin cutting, obtain required fluorescently-labeled part.
Embodiment 13: use fluorescence polarization to distinguish and identifier's serum sample
The following examples explanation uses the present invention to distinguish and/or identify two or more human serum samples.For example, when sample screened with a probe library, according to the difference in conjunction with collection of illustrative plates that is produced, normal and unusual serum can be distinguished mutually.In addition, use suitable probe solution, the present invention can be used to diabetes serum and normal or other non-diabetic serum differences.Following discussion has illustrated the general method that can be used for distinguishing the human serum sample.
Will be from the fluorescence labeling probe of three different libraries plates (XN1192, XN1043-58, GY1175-96 and plate 1) and available from Western States Plasma Company (Fallbrook, serum sample mixing CA).Serum sample comprises from the blended diabetes serum of three diabetics (proving diabetes by reference laboratory) with from pooled serum (the Lot HS300 of normal individual; SeraCare, Oceanside, California).
At first, every kind of fluorescence labeling probe of 2.5ul (1-10nM) is mixed in 96 orifice plates with 95ul damping fluid (PBS+O.03% lithium dodecyl sulfate) and 5ul serum.Then with mixture 37 ℃ of incubations 30 minutes.20, centrifugal 3.5 hours of 000xg, and get 5ul lower floor, flaxen serum layer carries out the probe binding analysis with the serum sample of indication.Use the fluorescence polarization of FluoroliteFPM-2 fluorescence polarization every kind of probe of microtitre systems measurement and every kind of sample.Every kind of probe is to three repetitions of every kind of sample detection, and asks for average fluorescence polarization value (mP).
In the preliminary screening of using 80 kinds of fluorescein-labeled probes to carry out, probe shows evident difference in the fluorescence polarization value that obtains from two kinds of serum samples.List in the table 11 with table 12 in the probe described from the fluorescence polarization value of normal pooled serum (Lot HS300) and diabetes serum acquisition, showing evident difference (Figure 14).There are 10 kinds of fluorescence polarization values that in diabetes serum, obtain higher in 12 kinds of probes listing in the table 11 than what in normal serum (LotHS300), obtain.These results show that probe 58A2,58A3,58A6,58B6,58B7,58B11,58H8,96G3,54G3 and P184 some composition preferential and in the diabetes serum combines.
Figure 9881370700751
From the discrepant possible reason of fluorescence polarization value of normal (Lot HS300) and the acquisition of diabetes serum sample is that the lipid content in the diabetes serum is than high in the normal serum.For determining whether the lipid content in the diabetes serum influences the fluorescence polarization value that is obtained, by centrifugal serum sample is sloughed lipid, and the same probe of listing in the use table 11 is measured fluorescence polarization value. list in table 12 with the fluorescence polarization value that these probes obtain, and the mode with figure is described in Figure 15. use probe 58A2,58A3,58A6,58B6,58B7,58B11,58H8,96G3, as if the difference degree between the fluorescence polarization value that 54G3 and P1B4 obtain in diabetes serum and normal serum (Lot HS300) bigger when serum is sloughed lipid.The fluorescence polarization value of degreasing diabetes serum is than unskimmed diabetes serum height.These presentation of results, the lipid content in the diabetes serum can not be interpreted as any diabetes serum than normal serum (Lot HS300) more preferably bonding probes 58A2,58A3,58A6,58B6,58B7,58B11,58H8,96G3,54G3 and P184.And these results show that the lipid that exists in the diabetes serum can reduce the binding affinity of the another kind of component that exists in probe 58A2,58A3,58A6,58B6,58B7,58B1l, 58H8,96G3,54G3 and P1B4 and the diabetes serum.
For probe 56C6 and 54E8, observed difference is not compared and is changed in the fluorescence polarization value difference between degreasing diabetes serum and the degreasing normal serum (LotHS300) and the serum that contains lipid at two kinds.But removing the lipid that contains in the serum deprivation has influenced the fluorescence polarization value difference that use probe 54G3 and P1B4 obtain greatly from normal (Lot HS300) and diabetes serum.When having lipid, use fluorescence polarization value that these two kinds of probes obtain in diabetes serum apparently higher than the value that obtains from normal serum (Lot HS300).But in degreasing serum, the fluorescence polarization value that use probe 54G3 and P1B4 obtain from normal (Lot HS300) and diabetes serum much at one.These presentation of results probe 54G3 and P1B4 combine with a kind of lipid soluble ligand in the diabetes serum.Degreasing is to the influence explanation of probe bonded, probe at least with the combining of three kinds or more target.
Generally speaking, the result who introduces above proves that the test based on fluorescence polarization that the human serum sample can use the present invention to introduce is distinguished from each other.And the method that present embodiment uses can be used for identifying the probe that can distinguish normal and diabetes serum.
The table II
The compound of BO-among the embodiment 4-8 and FL-mark
Table II (continuing)
The compound of BO-among the embodiment 4-8 and FL-mark
Figure 9881370700791
Table II (continuing)
The compound of BO-among the embodiment 4-8 and FL-mark
Table 12
The structure of the probe that uses among the present invention
In the scope of the particular embodiment that the present invention is not limited to here introduce, these embodiments just are used for illustrating all respects of the present invention.In fact, except the content that shows here and introduce, according to the introduction and the accompanying drawing of front, various modifications of the present invention are very obvious to one skilled in the art.These are revised also in the scope of the claim of appendix.
Here all reference of quoting all here are incorporated herein by reference when being used for all purposes in full.

Claims (57)

1. analyze the method for the sample of forming by the mixture of molecular components, comprising:
(a) this sample and a kind of molecular probe library are acted under the permission molecular probe binding partner bonded condition special with it; And
(b) use a kind of single member of pilot system detection molecules probe library of homogeneous to combine with any of molecular components in the sample, its Chinese library member provides a kind of molecular fingerprint with the collection of illustrative plates that combines of sample for this sample.
2. the process of claim 1 wherein that the member in molecular probe library is labeled.
3. the method for claim 2, wherein mark is a fluorescence.
4. the method for claim 3, its Chinese library member detects with combining with fluorescence polarization of sample component.
5. the method for claim 2, wherein molecular probe and a kind of photoactivation functional group are crosslinked.
6. the method for claim 5, wherein functional group is activated, and forms a kind of chemical bond between a kind of component of probe and sample.
7. the method for claim 2, its middle probe is with a kind of scintillator mark, the component radio-labeling of sample.
8. the method for claim 7, the interaction between the component of its middle probe and biological sample causes light emission.
9. claim 1 or 4 method, the member of its Chinese library is a biomolecules.
10. the method for claim 9, the biomolecules in its Chinese library is glycoprotein, lipoprotein, albumen, polypeptide, peptide, peptide analogs, amino acid, polysaccharide, oligosaccharides, sugar, polynucleotide, oligonucleotide, Nucleotide, nucleosides, DNA or a kind of derivative, RNA or a kind of derivative, fat, glycolipid, lipopolysaccharides, organic molecule or inorganic molecule.
11. the method for claim 10, the biomolecules in its Chinese library are acceptor, part, antibody, antigen, epi-position, somatomedin, cytokine or chemokine.
12. the method for claim 1 or 4, wherein sample is patient samples or clinical sample.
13. the method for claim 1 or 4, wherein sample is the extract of product, virus, microorganism or the pathogenetic organism of body fluid, urine, lymph, whole blood, serum, blood plasma, red corpuscle, white corpuscle, tissue, cell, cell extract, in-vitro transcription or translation.
14. the method for claim 13 wherein will use molecular probe library molecular fingerprint that produces in sample and the molecular fingerprint that uses the molecular probe library to produce in the negative control sample to compare.
15. the method for claim 14, wherein the negative control sample is from a normal donor.
16. the method for claim 13 wherein will use molecular probe library molecular fingerprint that produces in sample and the molecular fingerprint that uses the molecular probe library to produce in positive control sample to compare.
17. the method for claim 16, wherein positive control sample is from a sick or affected donor.
18. the method for claim 1 or 4, wherein molecular components is glycoprotein, lipoprotein, albumen, polypeptide, peptide, peptide analogs or amino acid in the sample.
19. the method for claim 1 or 4, wherein the molecular components in the sample is polynucleotide, oligonucleotide, Nucleotide, nucleosides, DNA or derivative or RNA or a derivative.
20. the method for claim 1 or 4, wherein the molecular components in the sample is polysaccharide, oligosaccharides or sugar.
21. the method for claim 1 or 4, wherein the molecular components in the sample is lipid, glycolipid, lipopolysaccharides or lipoprotein.
22. the method for claim 1 or 4, wherein the molecular components in the sample is organic molecule or inorganic molecule.
23. the method for claim 1 or 4, wherein the molecular components in the sample is acceptor, part, antibody, antigen, epi-position, somatomedin, cytokine or chemokine.
24. the test of an analysis of biological samples comprises:
(a) with biological sample and the effect of fluorescently-labeled probe library; And
(b) binding interactions of every kind of fluorescence labeling probe of detection and biological sample.
25. a method of distinguishing biological sample comprises:
(a) with biological sample and identical fluorescence labeling probe library effect;
(b) binding interactions of every kind of probe of detection and every kind of biological sample, and
(c) identify a kind of binding interactions collection of illustrative plates that biological sample can be distinguished from each other.
26. an evaluation has the biological target and the method that is used for the guide structure of drug development of critical treatment and diagnostic significance, comprising:
(a) with two kinds of identical probe libraries and two kinds of biological sample effects, wherein first kind of biological sample is contrast;
(b) binding interactions between the component of every kind of probe of pilot system detection of use homogeneous and biological sample; And
(c) identify the second kind of distinctive probe of biological sample/biological components binding interactions, wherein the component of identifying like this is a kind of biological target, and is a kind of guide structure of development medicine to the probe that this biological components has an affinity.
27. the test of the sample that an analysis is made up of the molecular components mixture, said test comprises:
(a) member with the molecular probe library is connected on such support, and this support has the site that an energy is cut by a kind of special catalyzer, and each side in the site respectively has a mark, thereby molecular probe/supporting structure thing is provided;
(b) with molecular probe/supporting structure thing and sample combination, wherein the molecular probe that a kind of molecular components that exists in the sample is had an affinity combines with this sample component, forms a kind of mixture that can block the cleavage site of support;
(c) in mixture, add special catalyst, the support that does not comprise a bonded sample component is cut; And
(d) detect release or mixture the reservation on support of unconjugated molecular probe from the support;
A kind of molecular fingerprint that combines the collection of illustrative plates sampling of its Chinese library member and sample.
28. the test of claim 21, its medium-height trestle comprise DNA, peptide, peptide analogs or other polymkeric substance.
29. the test of claim 21 wherein combines with this receptor the part that a kind of acceptor that exists in the biological sample has an affinity, and the special reagent of blocking-up support is near support.
30. the test of claim 21, the special binding reagents of its medium-height trestle are a kind of medicine, peptide, oligopeptides, polypeptide, albumen, glycoprotein, lipoprotein, sugar, oligosaccharides, polysaccharide, organic or inorganic molecule.
31. interactional stent area with between part and the acceptor does not take place and combines in the test of claim 21, the special binding reagents of its medium-height trestle.
32. the test of claim 21, its medium-height trestle comprises a kind of mark, and this is marked at and conjugated proteinly is closed when combining with the screening sequence, just shows near this mark that the special binding reagents of support does not exist and ligand/receptor interacts like this and exists.
33. test according to claim 21, wherein part and first mark are connected the same side of support, and test further is included in first mark support is fixed, add the catalyzer after scouring, remove the loose part of the support that is cut, this part that is cut comprises second mark, and wherein complete support is identified by the existence of second mark.
34. the test of claim 27, its medium-height trestle is made up of double-stranded DNA, and mark is a kind of biotinylated Nucleotide.
35. a method of distinguishing the biological sample of same type comprises:
(a) member in identical probe library is connected to contains a cleavage site and on all markd support in these both sides, site;
(b) with biological sample and identical probe library and identical specificity catalyst action;
(c) binding interactions of every kind of probe of detection and every kind of biological sample; And
(d) identify the collection of illustrative plates to make the binding interactions that biological sample is distinguished from each other.
36. the test of claim 30, it further comprises uses a kind of contrast biological sample and identifies the distinctive probe of non-contrast biological sample/biological sample component binding interactions, wherein the component of identifying like this is a biological target, and the probe that this biological components is had affinity is the guide structure of development medicine.
37. the method for complex sign ligand library comprises:
(a) a kind of fluorescence dye is fixed on a kind of solid support by a kind of mode of connection of cutting, forms first kind of product mixtures;
(b) mixture with said first kind of product mixtures and one or more compounds or compound reacts time enough at a certain temperature, forms second kind of product mixtures; And
(c) reaction mixture is cut down from solid support, form the tagged ligand library; Wherein fluorescence dye comprises two reactive half point, and they are independently selected from halogen, sulfenyl, alcohol, ether, ester, aldehyde, ketone, carboxylic acid, nitro, amine, acid amides, silane and their protection or unprotected derivative.
38. the method for claim 37, wherein fluorescence dye is a kind of rhodamine derivative, flower cyanines derivative, fluorescein derivative, tryptophan derivative, coumarin derivatives, naphthalene derivatives, two pyridine derivate, three pyridine derivates, organo-metallic mixture.
39. the method for claim 37, wherein solid support contains a joint, and joint is a kind of halogen, sulfenyl, alcohol, ether, ester, aldehyde, ketone, carboxylic acid, nitro, amine, acid amides, silane and their protection or unprotected derivative.
40. the method for the ligand library of complex sign comprises:
(a) by a kind of joint that cuts compound is fixed on the solid support;
(b) a kind of fluorescence dye is connected on the fixed compound; And
(c) downcut molecule-reacted fluorogenic dye product from the solid phase support, produce fluorescently-labeled ligand library.
41. a method of analyzing the toxic characteristic of test agent comprises:
(a) with two kinds of identical probe libraries and two kinds of biological sample effects, wherein first kind of biological sample is contrast, and the test agent effect of second kind of biological sample;
(b) binding interactions between every kind of probe of detection and the biological sample; And
(c) identify the second kind of distinctive probe of biological sample/sample binding interactions.
42. in biological sample, measure the method that toxic agent exists, comprising:
(a) with two kinds of identical probe libraries and two kinds of biological sample effects, wherein first kind of biological sample is the positive control of handling with toxic agent;
(b) detect binding interactions between every kind of probe and the every kind of biological sample; And
(c) in second kind of biological sample, identify probe/toxic agent binding interactions as the feature of positive control sample.
43. the method for claim 41 or 42, the binding interactions between its middle probe and the biological sample use a kind of pilot system of homogeneous to detect.
44. the method for claim 43, its middle probe library is by fluorescence or chemiluminescent labeling.
45. the method that the albumen of analytic sample is formed comprises and uses the method for claim 1 or 26 to prepare glycoprotein in the sample, lipoprotein, protein, polypeptide, peptide, peptide analogs or amino acid whose molecular fingerprint.
46. in patient, diagnose the illness or the method for illness, comprising:
(a) use the molecular fingerprint of the method preparation of claim 45 from the biological sample of patient's acquisition; And
(b) molecular fingerprint of patient samples and the molecular fingerprint of positive or negative control sample are compared.
47. the method for prediction medicine or result of treatment in patient comprises:
(a) use the molecular fingerprint of the method preparation of claim 45 from the biological sample of patient's acquisition; And
(b) with the molecular fingerprint of patient samples with (ⅰ) to medicine or treatment is replied and to medicine or treatment do not reply, (ⅱ) compare the molecular fingerprint that medicine or treatment produce the patient of adverse reaction.
48. the method for monitoring therapeuticing effect in patient comprises:
(a) before treatment and after for some time, use the molecular fingerprint of the method preparation of claim 45 from the biological sample of patient's acquisition; And
(b) molecular fingerprint that patient samples is produced and the molecular fingerprint of positive or negative control sample compare.
49. the method that the Nucleotide of analytic sample is formed comprises that the method for using claim 1 or 26 prepares the molecular fingerprint of polynucleotide in the sample, oligonucleotide, Nucleotide, nucleosides, DNA or a kind of derivative, RNA or a kind of derivative.
50. in patient, diagnose the illness or the method for illness, comprising:
(a) use the molecular fingerprint of the method preparation of claim 49 from the biological sample of patient's acquisition; And
(b) molecular fingerprint of patient samples and the molecular fingerprint of positive or negative control sample are compared.
51. the method for diagnostic medicine or result of treatment in patient comprises:
(a) use the molecular fingerprint of the method preparation of claim 49 from the biological sample of patient's acquisition; And
(b) with the molecular fingerprint of patient samples with (ⅰ) to medicine or treatment is replied and to medicine or treatment do not reply, (ⅱ) compare the molecular fingerprint that medicine or treatment produce the patient of adverse reaction.
52. the method for monitoring therapeuticing effect in patient comprises:
(a) before treatment and after for some time, use the molecular fingerprint of the method preparation of claim 49 from the biological sample of patient's acquisition; And
(b) molecular fingerprint that patient samples is produced and the molecular fingerprint of positive or negative control sample compare.
53. the test kit of the molecular components of an analytic sample comprises:
(a) a kind of molecular probe library; With
(b) a kind of molecular components bonded means homogeneous or liquid phase that detect in library member and the sample.
54. according to the test kit of claim 53, its a kind of fluorescent mark in middle probe library, detection means is a kind of fluorescence polarization instrument.
55. the test kit of claim 54, each member that it further is included as the library provides a kind of reaction vessel instrument, makes it to be suitable for carrying out the test of homogeneous.
56. the test kit of claim 54, it further comprises a kind of support, and the member in library is connected with this support releasedly.
57. the test kit of claim 54, wherein the molecular probe library is selected to the molecular components combination relevant with diabetes, infectious diseases, inflammatory disease, heart trouble, neoplastic disease, autoimmune disease and central nervous system disease.
CN98813707A 1997-12-18 1998-12-18 Methods for simultaneous identification of novel biological targets and lead structures for drug development Pending CN1285001A (en)

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