WO2002078634A2 - Methods for using kdop and kdop synthase inhibitors for the treatment of bacterial infection - Google Patents

Abstract

The two crystal structures of KDOPS that we present here illustrate the value of protein crystal structural analysis for characterizing the enzymatic reaction mechanism of KDOPS. Mechanistic hypotheses have underscored certain specifics of the interaction between the enzyme and the substrate, and these features in turn have provided further clarification of the details of the enzymatic reaction. The structure and function of 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDOSP) synthase, discussed herein provide a new target antibacterial drugs. KDO8P synthase catalyzes the synthesis of a unique sugar that is a required component of the outer membrane lipopolysaccharides of Gram-negative bacteria. The enzyme activity is required for the full viability of Gram-negative bacteria. Overall these disclosures provide a new method for developing new drugs, and methods of using the same to treat infections caused by bacteria that are resistant to conventional drugs.

Description

METHODS FOR USING KDOP AND KDOP SYNTHASE INHIBITORS FOR THE TREATMENT OF BACTERIAL INFECTION

FIELD OF THE INVENTION This application claims the benefit of U.S. Provisional Application No.

60/279,988, filed March 30, 2001, the contents of which are hereby incorporated by reference in their entirety into the present application. Various publications are referred to within this application. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.

The invention generally pertains to method for developing antibacterial agents and methods of making and using the same.

More particularly, the invention relates to a method of developing agents to inhibit the elaboration of 3-deoxy-D- α;z«o-2-octulosonate-8-phosphate (KDOP) in Gram- negative bacteria.

BACKGROUND OF THE INVENTION

KDOP synthase catalyzes an essential step in the biosynthesis of the lipopolysaccharide located on the outer membrane of the bacterial cell. Lipopolysaccharide biosynthesis in Gram-negative bacteria is initiated by the enzyme 3- deoxy-D- mα. ø-octulosonate- 8-phosphate (KDOP) synthase, the key enzyme in the formation of KDOP, which is an essential building block of lipopolysaccharide.

Lipopolysaccharide (LPS) is an essential component of the outer membrane of bacteria, Gram-negative bacteria in particular, and act as potent stimulators of the mammalian immune system. Importantly, in sufficient concentration in mammalian systems LPS can also lead to severe pathological disorders such as septic shock, which despite antibiotic treatment, results in the death of 25-30% of patients with septicemia. Gram-negative bacteria include among a multitude of other species and strains thereof: E. coli. 0157:H7, the culprit in illness outbreaks linked to eating undercooked hamburger; Legionella, which causes Legionnaires disease; and Vibrio, the bacterium responsible for cholera. While various classes of antibiotics have been useful in the past against Gram- negative bacteria, many species of bacteria have become resistant to traditional antibiotics such as penicillin, ampicillin, tetracycline etc. In response to this development many scientists in the field are focused on the development of endotoxin antagonists, the establisliment of structure-activity relationships and the interactions of these biomedically molecules in an effort to develop new methods of combating bacterial infections.

Lipid A is the component of LPS that is the causative agent of disorders such as septic or toxic shock and is related to other disorders such as Lyme disease. Before the discovery of the lipid A, the term "endotoxin" was used to generically describe the effect of the LPS. The lipid A from Gram(-) bacteria is heat-stable, cell-associated, pyrogenic and potentially lethal. The composition of lipid A from enteric bacteria is somewhat variable. However, it is generally recognized that such a lipid A consists of β-l,6-linked glucosamine disaccharide substituted at positions 4' and 1 by phospho-monoester groups. Fatty acids are linked to the hydroxyl and amino groups of the disaccharide to confer hydrophobicity to the lipid A. Also present in enterobacteria lipid A are amide and ester- linked D-3-hydroxy fatty acids, which consist of 14 carbons, e.g. o-hydroxy-myristic acid. The C3-OH positions of these fatty acids may be further esterified with saturated fatty acids.

Despite these general characteristics, a degree of microheterogeneity in lipid A composition occurs among diverse genera and species. Thus, Neisseria species produce 12 carbon 3-hydroxy fatty acids. Saturated fatty acid substitution varies and the C4- phosphoglucosamine disaccharide may contain a 4-amino-L-arabinose in salmonellae and Psendomonas. aeruginosa as opposed to E. coli and Shigella. A very potent and toxic lipid A is a hexacyl-l-4'-diphospholipid A. Structurally, a lipid A with one fewer or one more fatty acids will result in a biologically active, yet less toxic moiety. Removal of all fatty acids, however, deprives a particular lipid A of any biological activity. In addition, removal of either phosphate group results in significant loss of toxicity without loss of adjuvant activity. See, Zinnser, MICROBIOLOGY, (20th Ed., Appleton & Lange, Norwalk, Conn., pp. 84-86 (1992)). As discussed above, the cell-associated, heat-stable, cellular endotoxin of Gram- negative bacteria is the lipopolysaccharide (LPS). While both the O-antigen and the core regions modulate the toxic activity of the LPS, it is the lipid A region that possesses the biological activity of the endotoxin. The structure of the lipid A from enteric bacteria (e.g. E. coli) is shown in FIG.7. This structure is found in many Gram-negative bacteria, and is the minimum structure required for toxic activity. Structural variations of this molecule that lack any one of the substituent groups; e.g. lacking a phosphate or fatty acyl substituent; are less toxic or not toxic. In addition, the minimal structure for viability of the bacterium requires the addition of two KDOP residues to C-6 of the terminal glucosamine residue. In recent years, workers have discovered that endotoxin induced shock is caused by the ability of the LPS to stimulate host cells, such as macrophages, to produce excessive levels of cytokines. It is the excessive production of these cytokines, e.g. tumor necrosis factor (TNF) and interleukin I (IL-1), that results in toxic shock during bacteremia. The outside of the outer membrane of the Gram-negative bacteria contains the lipopolysaccharide "Lipid A" as a major component (often > 80%).

Lipid A has a lipid region to which is attached an inner core oligosaccharide with a relatively invariant structure. This inner core oligosaccharide elaboration begins with the synthesis of 3-deoxy-D-7«α/ o-2-octulosonate-8-phosphate (KDOP). The enzyme 3- deoxy-D-77z<_7i7._>-2-octulosonate-8-phosphate synthase or alternately KDOP synthase or KDOPS in the literature catalyzes controls the synthesis of KDOP, an unusual monosaccharide that is required for assembly of the inner core of Lipid A and that occurs naturally in very few other places in organic biochemistry. If KDOP is not present, the remainder of the extracellular oligosaccharide cannot be elaborated. Thus, bacteria that cannot make the inner core of Lipid A also cannot survive beyond one generation and thus KDOP is an essential building block required for the growth of bacteria.

Others have tried to inhibit the synthesis of either KDOPS or KDOP in the past, but the simple inhibitors all had high K_d values, approaching μM concentrations, and were thus not useful as antibacterial agents at physiologically relevant dosages.

One of the ways in which rational drug design has been added is through the efforts of crystallographers who experimentally determine the three-dimensional structure of target molecules. From this three-dimensional information workers in the field can begin to determine molecular interactions and the basis for chemical activity. SUMMARY OF THE INVENTION

Briefly stated, the current invention comprises agents capable of inhibiting the biosynthesis of lipid A through preventing the elaboration of KDOP. The invention provided herein provides a composition for antagonizing bacterial activity. This composition is comprised of a pharmaceutically acceptable carrier and an effective amount of an antagonist to the elaboration of KDOP or an inhibitor of KDOPS. In particular, the compositions provided in the current invention are effective at antagonizing Gram- negative bacteria and their elaboration of KDOP. The compositions contemplated by the current invention comprise compounds of the formula (I - IV), disclosed infra. Moreover, the current invention also provides a method for the treatment of bacterial infection comprising the administering of an effective amount of a compound capable of preventing the elaboration of the monosaccharide KDOP in bacteria.

In another embodiment, the present invention provides a method of treating bacterial infection in a subject, comprising administering to the subject an effective amount of the compound of described above.

In another embodiment, the present invention provides a method of preventing toxic shock in a subject, comprising administering to the subject an effective amount of the compound of described above.

In yet another embodiment, the present invention provides a method of treating or preventing a lipopolysaccharide mediated disorder in a subject, comprising administering to the subject a lipopolysaccharide mediated disorder inhibiting amount of the compound described above.

Thus, in particular, inhibitors of KDOPS can be used as a treatment against bacterial infection, particularly in situations in which bacteria have become resistant to available antibiotics.

A computational method for the discovery and design of therapeutic compounds is also provided. The methods used rely on an accurate representation of three-dimensional molecular spatial and improved image processing software in three- dimensional representations. The computational technique employed utilizes a software program and associated algorithms. This method is an improvement over the current methods of drug discovery which often employs a random search through a large library of synthesized chemical compounds or protein molecules for bio-activity related to a specific therapeutic use.

Other features and advantages of this invention will become apparent in the following detailed description of a preferred embodiment of this invention with reference to the accompanying drawings.

Additional advantages of the invention will be set forth in part in the description and Figures that follow, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1A shows the superposition of the KDOPS:PEP and KDOPS:2 structures over alternate KDOPS structures (PDB code 1D9E, subunit a; and PDB code IGGO). (A) The main-chains of all four overlapped KDOPS structures are almost identical and therefore only the KDOPS :2 chain is shown in beige ribbon form for clarity. The two phosphate/sulfate ions proposed to bind to the A5P-phosphate position (at the top of the protein) and the PEP-phosphate position (in the middle of the protein) are depicted in green and brown for the 1D9E and IGGO structures respectively. Carbon backbones of the PEP and inhibitor (2) molecules are shown in yellow and cyan respectively, while oxygen, nitrogen and phosphorous atoms are shown in their default CPK colors.

Fig. IB shows a magnified view of the overlapped ligands and phosphate/sulfate ions in the active site. Colors are as described in Fig. 1 A. The phosphate/sulfate distances are 12.1 lA and 10.32A for the 1D9E (subunit a) and IGGO structures respectively, while the phosphate-phosphonate distance in the KDOPS:2 structure is 11.69A.

Fig. 2A shows an "omit map" of the refined coordinates of one of two loops in the KDOPS :PEP structure not present in the original molecular replacement search model (1D9E, subunit A). The first of two loops added in the KDOPS:PEP and KDOPS:2 structures include residues 206-217. Following positioning of the sequence in unrefined electron density maps and refinement of the structure, Fo-Fc omit maps were calculated to assure proper positioning.

Fig. 2B shows an "omit map" of the refined coordinates of one of two loops in the KDOPS:PEP structure not present in the original molecular replacement search model (1D9E, subunit A). The second of the two loops added in the KDOPS:PEP and KDOPS:2 structures include residues 245-252. Following positioning of the sequence in unrefined electron density maps and refinement of the structure, Fo-Fc omit maps were calculated to assure proper positioning.

Fig. 3 shows the positions of the active site amino acid residues which have significant conformational changes upon replacement of the substrate PEP (from the KDOPS:PEP structure -yellow) with the inhibitor 2 (from the KDOPS:2 structure - cyan). Oxygen ( red), nitrogen (blue) and phosphorous (magenta) atoms are colored to clarify the side chain positions.

Fig. 4A shows the interactions within the KDOPS active site. Interactions between

KDOPS amino acid residues and PEP in the KDOPS :PEP crystal structure. PEP carbon atoms are in yellow, all other atoms are colored according to the CPK scheme. A section of a Fo-Fc electron density omit map, contoured at 1.5σ shows unambiguous density for the PEP molecule in the configuration shown. The residues Asn62 and Arg63 which interact weakly with the PEP substrate, are depicted to show the difference between the tight contact with PEP'sf face and the weaker contact with the re face.

Fig. 4B shows the interactions within the KDOPS active site. Interactions between KDOPS residues and inhibitor 2 (cyan carbon atoms), in the KDOPS:2 structure. A section of a Fo-Fc electron density omit map, contoured at 1.0s shows unambiguous density for 2. Strong contacts between the protein and 2 are seen on both phosphate and phosphonate ends and with the carboxyl group.

Fig. 5 shows two conformers of PEP. The phosphate PO. group extends from the plane of the PEP molecule either toward the si face (A) or toward the -e face (B). One of the lone pair orbitals of the bridging (P-O-C) oxygen is oriented to either the 7-e face (A) or the si face (B) of the molecule, so that it is antiperiplanar relative to the newly generated C — C bond between C3 of PEP and Cl -aldehyde of A5P. R group represents the remaining sugar portion of A5P.

Fig. 6 shows the A5P moiety derived from the enzyme-bound inhibitor (2) structure is shown in muta-rotation between its acyclic (aldehyde) and cyclic (α- and β- furanose) forms.

Fig. 7 shows the structure of lipid A from E. coli.

Fig. 8 shows a schematic molecular model of the inner and outer membranes of E. coli K-12. Ovals and rectangles represent sugar residues, as indicated, whereas circles represent polar headgroups of various lipids.

Fig. 9 shows the structure and biosynthesis of KDOP-lipid A in E. coli. K-12. Only the KDOP and lipid A portions of LPS are required for cell growth. The red symbols indicate the relevant structural genes. A single enzyme catalyses each reaction. In almost all sequenced bacteria, the genes encoding the enzymes of lipid A biosynthesis are present in single copy. LpxA and LpxC are the most highly conserved.

Fig. 10 shows the proposed mechanism for a KD08PS- catalyzed reactions.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The following abbreviations have designated meanings in the specification:

Abbreviation Key

KDOP 3-deoxy-D-77z 7Z77ø-2-octulosonate-8-phosphate

KDOPS KDOP synthase

PEP phosphoenolpyruvate

A5P D-arabinose-5-phosphate DAHPS 3-deoxy-D-αr όz^'7zo-2-heptulosonate-7-phosphate synthase

REDOR Rotational-echo double-resonance

MurZ UDP-GlcNAc enolpyruvoyl transferase

EPSPS 5 -enolpyruvoylshikimate-3 -phosphate synthase

LPS Lipopolysaccharide PMAA Partially methylated alditol acetates

AS-PRT Anthranilate synthase-phosphoribosyl transferase complex

A Angstroms The present invention may be understood more readily by reference to the following detailed description of preferred embodiments of the invention.

Before the present products, compositions and methods are disclosed and described, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

Throughout this application, when publications are referenced, the disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

As used herein, the term "endotoxic activity" is used to describe the ability of Gram-negative bacteria to induce a variety responses in a subject or patient, such as a human or animal, where the responses can include, but are not limited to, stimulation of cytokines including, but not limited to, TNF-α, IL-1 and IL-6 as well as the stimulation of pyrogens and any other responses known in the art to be associated with endotoxic challenge or bacteremia.

A compound, molecule or composition is said to be "pharmacologically acceptable" if its administration can be tolerated by a recipient mammal. Such an agent is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in technical change in the physiology of a recipient mammal. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see REMINGTON'S PHARMACEUTICAL SCIENCES (Martin, E. W., ed., latest edition, Mack Publishing Co., Easton, Pa.). As used herein, "admix" or "admixing" refers to the contacting, optionally in liquid media, of one or more ingredients.

Mechanism of Enzyme Activity

The enzyme 3-deoxy-D-7wα7.77θ-2-octulosonate-8-phosphate (KDOP) synthase (KDOPS) catalyzes the condensation reaction between D-arabinose-5 -phosphate (A5P) and phosphoenolpyruvate (PEP) to form KDOP and inorganic phosphate (FIG. 8). This enzymatic reaction plays an essential role in the assembly process of lipopolysaccharides of most Gram-negative bacteria and is therefore an attractive target for the design of novel antibacterial drugs. Interestingly, KDOPS belongs to a family of PEP-utilizing enzymes, two of which — UDP-GlcNAc enolpyruvoyl transferase (MurZ) and 5- enolpyruvoylshikimate-3-phosphate synthase (EPSPS) — are targeted by the antibiotic fosfomycin and by the herbicide glyphosate, respectively. The last member of this family is 3-deoxy-D-α7'αέ ø-heptulosonate-7-phosphate (DAHP) synthase (DAHPS), which catalyzes a net aldol reaction similar to that of KDOPS, but between PEP and erythrose-4- phosphate (E4P) to produce DAHP. A unique mechanistic feature shared by the reactions catalyzed by these four enzymes is that while most enzymatic reactions that use PEP as a substrate proceed through cleavage of the high-energy phosphate bond (ΔGo' = -14.8 kcal/mol), the reactions of these four enzymes proceed through the unusual cleavage of the C- O bond of PEP. Additionally, because KDOP sits at the keystone position, that is at the lowest branch point of the elaborating oligosaccharide, and is required for further elaboration of the outer oligosaccharide, KDOP inhibitors can prevent the keystone branch-point of endotoxin from being assembled and (This sentence doesn't make sense) may thus prevent bacterial replication and/or the biosynthesis and harmful accumulation of endotoxin.

The last steps of E. coli lipid A biosynthesis involve the addition of laurate and myristate residues to the distal glucosamine unit (Fig. 9), generating the so-called acyloxyacyl moieties. The "late" acyltransferases require the presence of the KDOP disaccharide in their substrate. Like LpxA and LpxD, they utilize acyl-ACPs as donors . The genes encoding the lauroyl and the myristoyl transferases (htrB and msbB respectively) display some sequence similarity to each other. The msbB gene is not required for growth, but msbB mutants are greatly attenuated in their ability to activate human macrophages and to cause septic shock in animals. While the mechanisms of EPSPS and Mur Z, two enolpyruvoyl transferase enzymes, have been characterized unambigously, some mechanistic details of the reactions catalyzed by KDOPS and DAHPS are still unresolved. Early studies on KDOPS suggested that this enzyme acts upon the acyclic form of A5P and demonstrated an ordered sequence of substrate binding and product release, with PEP binding before A5P does and with P_j being released prior to KDOP. The condensation step was shown to be stereospecific, that is, with the si face of PEP attaching to the re face of the carbonyl of A5P. More recent studies using rapid-quench techniques, including the synthesis and evaluation of the first acyclic bisubstrate inhibitor (2, K_d = 0.4 μM), supported the original hypothesis of Hedstrom and Abeles, which was that the reaction pathway proceeds through an acyclic bisphosphate intermediate 1 (FIG. 9). Despite all of the above observations, none of the available evidence unambiguously identifies the true enzymatic intermediate, therefore limiting the ability of workers in the field to design inhibitors of KDOP and/or KDOPS.

Crystal Structure of KDOP

One of the goals of the invention provided herein was to develop agents that would effectively inhibit the bacterial elaboration/synthesis of KDOP, thereby effectively preventing bacterial replication and accumulation of LPS, specifically the inventors sought to develop inhibitors of KDOP for use as treatment against penicillin resistant strains of bacteria. The crystal structures of 3-deoxy-D-77zα7Z77<9-2-octulosonate-8-phosphate synthase (KDOPS) from Escherichia coli complexed with the substrate phosphoenolpyruvate (PEP) and with a mechanism-based inhibitor (K_ = 0.4 μM) were determined by molecular replacement using X-ray diffraction data to 2.8 A and 2.3 A resolution, respectively. Both the KDOPS :PEP and KDOPS:inhibitor complexes crystallize in the cubic space group 123 with cell constants a = b = c =117.9 A and 117.6 A respectively and one subunit per asymmetric unit. The two structures are nearly identical, and superposition of their Cα indicates an rms difference of 0.41 A. The PEP in the KDOPS:PEP complex is anchored to the enzyme in a conformation that blocks its si face and leaves its 7"e face largely devoid of contacts. This results from KDOPS's selective choice of a PEP conformer in which the phosphate group of PEP is extended toward the si face.

Furthermore, the crystal structure reveals that the bridging (P-O-C) oxygen atom and the carboxylate group of PEP are not strongly hydrogen-bonded to the enzyme. The resulting high degree of negative charge on the carboxylate group of PEP would then suggest that the condensation step between PEP and D-arabinose-5-phosphate (A5P) should proceed in a stepwise fashion through an intermediate and transient oxo-carbenium ion at C2 of PEP. The molecular structural results are discussed in light of the chemically similar but mechanistically distinct reaction that is catalyzed by the enzyme 3-deoxy-D- αrα_>røø-2-heptulosonate-7-phosphate synthase and in light of the preferred enzyme-bound states of the substrate A5P. Recently, the first direct identification of the actual active site residues was reported. This was accomplished through the use of solid state, rotational-echo double- resonance (REDOR) NMR, in combination with mutagenesis studies. In parallel, the first X-ray crystal structure of KDOPS at 2.4 A resolution was reported. This study demonstrated that the enzyme is a homotetramer in which each monomer has the fold of a (β/α)₈barrel, similar in many respects to the previously solved X-ray structure of DAHPS. These earlier KDOPS crystals were grown in the presence of 1.4 M (NH₄)₂S0 and 0.4 M (K/ H)₃P0₄, and had two S0₄ ²7HP0 ^2" sites that were identified in the structure. These were interpreted as occupying the phosphate positions of the substrates, PEP and A5P. On the basis of this interpretation, a model structure for the active site of KDOPS was proposed in which the phosphate groups of PEP and A5P are 13.0 A apart allowing KDOP synthesis to proceed via an acyclic intermediate.

Using molecular replacement, Kretsinger and co-workers have recently solved another structure of E. coli KDOPS at 3.0 A resolution. Although in this case the crystals were grown in poly(ethylene glycol) 1500 in the presence of both substrates, PEP and A5P, neither the enzyme-bound PEP nor the bound A5P or the product KDOP could be identified. Two phosphate peaks were found in this structure as well, only 10.3 A apart, in contrast to the distance of 13.0 A found in the structure of Radaev et al.

According to the current invention we determined the structure-function relationship of KDOPS, and provide the first crystal structures of the E. coli KDOPS in binary complexes with the substrate PEP and with a mechanism-based inhibitor, at 2.8 A and 2.3 A resolution, respectively. These structures provide important information about the enzyme's active site architecture and about the interactions between the substrates and the enzyme and allow the development of improved inhibitors of KDOP elaboration for use as antibacterial agents.

Since the molecular structure of the most potent competitive inhibitor, N- phosphonomethyl-N-carboxymethyl-1-deoxy-l-aminogluco 1,6 -phosphate, known to date (K_d = 0.4 μM), combines the key features of both substrates PEP and A5P into a single molecule (FIG. 9), the structural data for the KDOPS:PEP and KDOPS:2 complexes that were obtained in the current invention are indicators of the identity of active site residues that participate in the recognition of both natural substrates by KDOPS. Analysis of these structures also provides an important basis for understanding how KDOP's three- dimensional structure dictates its catalytic mechanism.

Table 1. Diffraction data and refinement statistics.

Diffraction data KDOPS:PEP KDOPS:2

Spacegroup 123 123

Cell dimensions a= 117.9 a= 117.6

Resolution (A) 20-2.8 20-2.23

Outer shell resolution (A) 2.9 -2.8 2.3-2.23

R merge ( ^%) ^■' 6.6 9.1

Redundancy

Overall I / s 6.9 (3.2) A 17.1 (2.8) A

Completeness (%) 85.8 (85.3) A 70.6 (14.1) A

Number of residues 248 248

Number of protein atoms 2193 2193

Number of non-protein atoms

Water 15 31

Ligand 10 25

Refinement statistics

Number of reflections

Working-R set 6494 7164

Free-R set 317 360

I / s cutoff 3.0 3.0

Resolution (A) 8-2.8 8-2.3

R-factor (%) 22.8 22.5

Free R-factor (%) 28.1 25.9

Geometry

Rmsd from ideality

Bond length (A) 0.016 0.017

Bond angles (°) 2.5 2.4

Improper angles (°) 2.3 2.2 _| Dihedral angles (°) 26.1 25.7

With Figure 1 and Table 1, provided herein, it is possible to observe the overall structure of the relevant molecules. In the structures of KDOPS:PEP and KDOPS:2 complexes, the enzyme is present as a tetrameric complex, one subunit in the asymmetric unit, confirming the conclusion of Radaev et al. about the tetrameric nature of KDOPS. In contrast to previous (β/α)₈ barrel structures disclosed elsewhere (Figure 1A) the crystal forms reported herein allowed all of the amino acid residues to be visible and could be identified. Two peptide loops that were missing in the molecular replacement search model (Asp208-Gly216 and Ala247-Gly251, an active site strand) were identifiable in E_o-E_c difference maps and could be traced (Figure 2). The two structures presented in this report are almost identical with each other (rmsd values of 0.41 A or 0.45 A for α-carbons or all atoms respectively). The two structures are also very similar to the two previously solved structures; the rmsd values are 1.3 A for all comparable α-carbon atoms when our structures are aligned with the structure of Radaev et al, or with the structure of the Wagner et al. However, the current disclosure provides structures that are both more complete and contain bound substrate/inhibitor, when these regions of the protein are not included in the calculation, the α-carbon rmsd for the remaining 91% of the protein is 0.70 A.

A number of critical residues occupy significantly different positions in the structure disclosed herein, and these shifts are necessary to accommodate the presence of the substrate and inhibitor molecules in our structures (FIG. 3). In particular, a featureless strand near the active site appears to adopt a more helical conformation in our structure. Other major differences arise (FIG. 1) in surface loops, (FIG. 2) in the interdomain strand region at the C-terminus, and (FIG. 3) in the amino acid residues immediately adjacent to the unobserved, disordered segments of the previous structures.

Following coordinate refinement by simulated annealing and conjugate gradient minimization (Table 1), the interactions between the substrates and active site residues could be identified in our structure. Several residues near the A5P portion of the active site retain high temperature factors, indicating the flexibility of this region. The active site can be described as a long narrow cleft in the center of the β₈ barrel (Figure 1). The cleft is shallowest in the A5P-phosphate binding site and becomes progressively deeper towards the PΕP-phosphate binding site.

Turning to Figures 2 and 3, we note the KDOPS-PΕP interactions. The closest contacts of KDOPS with PEP appear to be salt bridges that form between the phosphate oxygens and Argl68 and His202, while Lysl38 and Glnl41 form longer range electrostatic contacts (Figure 4A). The carboxylate group of PEP lies between His202 and Asn62, and points into the cleft towards the A5P binding site. Radaev et al., proposed that three lysine residues (Lys55, Lys60 and Lysl38) might interact with the carboxylate group of PEP; however, at the present level of detail, these three residues are all too distant for the formation of hydrogen bonds to the carboxylate of PEP. The vinyl group of PEP points outwards from the active site cleft and is not within van der Waals contact distance of any nearby residue. The phosphate/sulfate ions in the Radaev et al. and Wagner et al, structures are about 3.3 A away from the PEP-phosphate and are more deeply buried in the active site cleft (Figure IB).

KDOPS-2 interactions

The structure of the inhibitor molecules provided herein are comprised of three unique molecular fragments: the phosphonate group, the carboxylate group, and the A5P group (Figure IB). The distance between the two phosphorous atoms of 2 in the structure of the KDOPS:2 complex is 11.8 A. This value is intermediate between those observed for the corresponding interanionic distances (phosphate/sulfate ions) in the structure of Radaev et al, and in that of Wagner et al. (Figure IB). It is expected that the observed positions of ions bound at higher ionic strength may vary slightly upon observation at reduced ionic strength and in the presence of additional constraints imposed by the remainder of the substrate. The similar general location of bound anions on the surface of the enzyme, under the variety of experimental conditions, further accentuates the positive electrostatic potential that must be present in this cavity. The phosphonate group of 2 is bound between residues Argl68, Asnl41 and His202 (Figure 4B). In the KDOPS:2 complex, residues Lysl38 and Argl68 occur in different orientations than they do in the KDOPS:PEP complex. In the present KDOPS:2 complex, N- of Argl68 lies most closely to the peripheral oxygen atom of the phosphonate group of 2, with Lysl38 directed away from this phosphonate group. In the KDOPS:PEP complex, one peripheral phosphate oxygen atom interacts with peripheral N of Argl68 and a second oxygen atom is within

3.8 A of N_ε of Lysl38 (Figure 4A, and 4B). Turning to Fig. 4A and 4B, the A5P portion of 2 is stretched along the cleft, exhibiting many van der Waals contacts with the protein. Residue Arg63 contacts this sugar portion extensively, folding over the carbon chain of the sugar and shielding sugar carbon atoms corresponding to C4 and C5 of A5P nearly completely from the solvent. Carbon atoms Cl and C2 of A5P are relatively open to the solvent on one face of 2. The phosphate group of 2, corresponding to that of A5P-phosphate, is in close contact with the guanidino group of Arg63 (Figure 4B). The conformation of this side chain differs significantly between the KDOPS:2 and KDOPS:PEP complexes (Figure 3). Residue Ser64 is about 5.0 A away from the closest phosphate oxygen of 2, and does not appear to interact with the A5P fragment of 2, in contrast to earlier indications by Radaev et al. The phosphate/sulfate ions in the Wagner et al, structure are positioned quite differently than the phosphonate and phosphate groups of the inhibitor 2. The anion in the structure of Wagner et al. that corresponds most closely to the phosphate group of A5P lies almost completely outside the cleft, nearly 6 A away from corresponding phosphate in the structure of KDOPS:2 complex (Figure IB).

Water The positions of 15 and 21 ordered, protein-bound water molecules were found in the structures of the KDOPS:PEP and KDOPS:2 complexes respectively. A single water molecule in the structure of the KDOPS:2 complex is within hydrogen-bonding distance of Asn62, which is the only protein residue that is even remotely close to the re face of the

PEP moiety of 2. The absence of a water molecule positioned more closely to C2 of PEP may indicate that this water molecule, which is mechanistically required for nucleophilic attack, may be disordered in the KDOPS :PEP structure (see the discussion section below).

The structural information achieved in this study confirms in many aspects the recently reported substrate-free structures of KDOPS. However, major differences are observed in the conformations of the active site residues responsible for the binding of both the PEP and A5P substrates (Figure 1). Visualization of the PEP and inhibitor 2 in our structures allows us to rationalize the chemically determined stereochemical course of the enzyme-catalyzed reaction. The results of such analysis lead to important implications about the mechanism of action of KDOPS.

Stereochemical Control at the PEP Binding Site Strong, well-defined electron density in the map of KDOPS :PEP complex (Figure

4 A) clearly defines the positions of the two vinylic carbon atoms and of phosphate and carboxylate groups of the enzyme-bound PEP. Inspection of the PEP binding site shows that PEP contacts the protein predominantly through its peripheral phosphate oxygen atoms, with this phosphate group anchored to the enzyme in a configuration that blocks the si face of the PEP and leaves the re face of PEP largely devoid of close contacts. By analogy with other small, highly charged substrates, this suggests that the driving force for substrate binding is largely electrostatic with substantial contributions to the free energy of interaction arising from the release of water from the solvation sphere of PEP upon going from its free to its enzyme-bound form.

Two features of PEP' s interaction with the enzyme are of importance. First, while the carboxylate oxygen atoms, the bridging oxygen atom of the phosphate, and all three of the carbon atoms are coplanar, the phosphate group extends from this plane toward the si face of the PEP molecule (Figure 4 A and Figure 5A). Such a distinct conformational selectivity of KDOPS in its recognition and binding of PEP is likely to be of some mechanistic significance. Indeed, the bridging oxygen atom appears not to be strongly hydrogen-bonded. All possible contacts of this P-O-C oxygen atom with an Oe of Asn62 at 4.3 A and toward the re face of PEP, N_η- of Argl68 (4.48 A and toward the si face), and an Na of His202 (4.36 A and toward the si face) — too far or are apparently in unfavorable orientations for hydrogen bonding (Figure 4A). This lack of direct contact to the P-O-C oxygen atom, resulting in an electron-rich bridging oxygen, may serve to favor nucleophilic attack by C3 of the vinylic double bond of PEP onto the electrophilic Cl atom of A5P (Figure 5 A). The observed conformer is also in complete accord with the observed stereochemical course of the condensation step. That is, the observed addition by the si face of PEP to the re face of the carbonyl of A5P may be rationalized by the enzyme-mediated direction of one of the lone pair sp³ orbitals of the bridging oxygen to be antiperiplanar relative to the newly generated C-C bond. Thus by stabilizing the observed conformer of bound PEP, the enzyme may activate the C3 atom of PEP to be more nucleophilic. This, indeed, can only be achieved by the conformer given in Figure 5 A, while its opposite conformer (Figure 5B) would lead to addition to the wrong (i.e. re) face of PEP.

Turning to Fig. 5, a second important feature of the interaction of PEP with KDOPS arises from how PEP's carboxylate group interacts with the protein. This carboxylate group is distant from the nearest protein residues. Atom 01 'of the carboxylate is 4.0 A away from either nitrogen atom N_ε or N₅ of His202. The 0₂ atom is positioned 3.8 A and 3.6 A away from these nitrogen atoms and lies 4.3 A away from N_ε of Gln205 (Figure 4A). Additionally, the positive charge of His202 is largely compensated by its interaction with the anionic phosphoryl group. All of these observations suggest that the carboxylate group of PEP should bear a full, albeit delocalized negative charge. Two important mechanistic advantages may be drawn from this implied ionization state of the carboxylate group of enzyme-bound PEP.

The first such advantage is that the inductive electron-donating character of the negatively charged carboxylate group is expected to increase the nucleophilicity of the double bond of PEP, an important requirement for the first event of the coupling step. Neutralization of the charge of PEP's carboxylate group (e.g. through either strong hydrogen bonding or salt bridging interactions) would have led to a strong decrease in the nucleophilicity of the double bond due to electron-withdrawing character of "neutral" carboxylic acid. The strongly reduced nucleophilicity of the olefϊn in the neutral carboxylic acid form of PEP is independently indicated (1) by the prior observation that the enolic 0-C2 bond exhibits partial double-bond character in several different small molecule crystal structures of monoionized PEP, and by crystal structure analysis of the enolase-catalyzed transformation of PEP to 2-phospho-D-glycerate (PGA). In this enolase reaction, the negatively charged carboxylate group of PEP is strongly neutralized by a catalytic Mg²⁺ ion situated at 2.4 A from both oxygen atoms of the carboxylate group. Such neutralization of the carboxylate charge leads to increased electrophilicity at C3 of PEP and thereby facilitates nucleophilic attack by water at this position.

A second advantage of maintaining a negative charge on the carboxylate group of PEP in the KDOPS reaction is that such negative charge should inductively stabilize the partial positive charge that is present on C2 of PEP in the transition state of the condensation step (FIG. 1A, FIG. 9). Similar rate enhancements due to a negatively charged α-carboxylate group are well documented in the acid-catalyzed hydrolysis of KDO-2-phosphates. Thus, although at this stage of investigation it is not clear whether the formation of new C-C and C-0 bonds during the condensation step is a synchronous or stepwise process, the current results on the position of PEP in its binding pocket would suggest that these processes should occur in a stepwise manner. The initial formation of a transient oxo-carbenium intermediate, or an early transition state having oxo-carbenium character, must be followed by the capture of water at the cationic C2 position to complete the formation of acyclic intermediate 1 (FIG. 9). The opposite sequence of steps, the initial nucleophilic attack of water at C2 of PEP and subsequent coupling of carbaniomc C3 with the carbonyl of A5P, is very unlikely because it would require a highly activated hydroxide ion and strong neutralization of PEP charges, which are not observed in KDOPS.

The suggested stepwise mechanism for KDOPS is further indicated by results obtained through mechanistic experiments with 2, with analogues of PEP, and with intramolecular models of the KDOPS-catalyzed reaction. Furthermore, the same oxo- carbenium ion transition-state of PEP has been suggested earlier to account for the enzymatic reactions of MurZ and EPSPS. These enzymes catalyze the enol ether transfer from PEP to their respective co-substrate alcohols, and represent a different and distinct class of enzymatic reaction involving the same C-0 bond cleavage of PEP and the same stereospecific 2-si face addition of an electrophile at C3 of PEP, that is observed for KDOPS.

This commonality in stereochemical course of the reaction further strengthens the mechanistic links between these two distinct classes of enzymes and may also indicate a structural and evolutionary relationship between these two classes. The current disclosure therefore provides that although PEP-bound structures are not yet available for EPSPS and MurZ, the recognition and binding of PEP by these enzymes should resemble that by KDOPS. Moreover, although the C-0 bond cleavage of PEP represents an unusual class of PEP reaction, the stereospecific 2-si face addition at C3 of PEP is consistent for nearly all PEP-utilizing enzymes. Surprisingly, in contrast to the similarities observed within the other class of PEP- utilizing enzymes, comparison of the KDOPS-bound PEP to the PEP molecule bound by the structurally and mechanistically most closely related enzyme, DAHPS, reveals that DAHPS binds to the opposite conformation of PEP (Figure 5B). Although the latter structure was determined for DAHPS:PEP:Pb²⁺ complex, in which the conformation of PEP may be dramatically perturbed by the unique chemical properties of Pb²⁺, it is clear that resolution of this apparent discrepancy between the binding of PEP by KDOPS and by DAHPS awaits further experiments.

Catalytic Strategies of KDOPS and DAHPS

The recent insightful analysis of the structural and evolutionary relationship between KDOPS and DAHPS led Radaev et al., to conclude that these proteins evolved from a common ancestor and that they follow identical catalytic strategies. In contrast, based on the different structural motifs for the recognition and binding of PEP by these two enzymes, we suggest that the initial elementary steps of their catalytic mechanisms might be also very different. Indeed, earlier studies by DeLeo et al., and recently by Wagner et al., have suggested that C2 of PEP serves as an electrophilic center in the DAHPS-catalyzed reaction. Regionally specific attack by water at this center was proposed to be the initial catalytic step, followed by addition by C3 of PEP to Cl of E4P. This mechanism differs from that in the KDOPS reaction, in which C3 of PEP instead appears to function as a nucleophile in the first step. Thus, the functioning of PEP either as a C2-electrophile (in DAHPS) or as a C3 -nucleophile (in KDOPS), must be exquisitely controlled by the interaction of PEP with these enzymes. This postulated mechanistic difference between these two enzymes — despite the apparent chemical similarity in the catalyzed reactions — in fact can rationalize the unique requirement for metal ions by DAHPS, but not by KDOPS.

The A5P Binding Site

KDOPS acts upon the acyclic (aldehyde) form of A5P, and that initial binding of either PEP or A5P leads to kinetically competent catalysis. In addition, the recent solid- state REDOR NMR data on the binary KDOPS :A5P complex strongly suggested that both the α- and β-furanose forms of A5P, which constitute more than 97% of its anomers in solution, are the primary forms which bind to KDOPS. The rationalization for these accumulated observations was that bound cyclic A5P should be able to open up to its acyclic form before the chemistry of the C-C coupling step occurs. The structural data that we present here for the A5P region of the KDOPS :2 complex strongly supports this assumption.

First, while the two hydroxyl groups corresponding to C2-OH and C3-OH of A5P portion of the inhibitor are close to or within hydrogen-bonding range of a few possible hydrogen bond donors and acceptors on the enzyme, two other hydroxyl groups corresponding to Cl-OH and C4-OH have no neighboring hydrogen bonding atoms from the protein closer than 4 A away (Figure 4B). The structure implies that, aside from the ionic interactions with the C5-phosphate group, the C2-OH and C3-OH may participate in recognition of A5P through possible hydrogen-bonding interactions. The C4-OH participates through a van der Waals contact with C« of Asn62, and the involvement of the

Cl-OH appears to be less direct. In general, much, though not all, of the A5P portion of 2 fits rather snugly into the active site pocket, indicating a large degree of van der Waals contact. This suggests that much of the free energy of interaction of A5P with KDOPS may be entropic in origin, arising from the release of water from the hydration zone of free A5P, again a common situation for small hydrophilic ligands. Thus the orientation of A5P appears to be driven by electrostatic and complementary van der Waals interactions with the protein.

The results of some earlier experimental observations of the interactions of deoxy analogues of A5P with KDOPS can be rationalized with regard to the geometry of this interaction surface. The analogue 4-deoxy A5P serves as a substrate with a /_caι value that is similar to that of A5P, while 2-deoxy A5P and 3-deoxy A5P do not function either as substrates or inhibitors of the enzyme. The hydroxyl groups corresponding to C2-OH and C3-OH of A5P, both interact predominantly with the one peptide turn (residues Glyl51- Prol 52) that becomes ordered in the structures of both the PEP and 2 complexes. However, those corresponding to Cl-OH and C4-OH of A5P interact mainly with residues from the loop including Arg63 that folds over the top of 2 in our structure. The results with the deoxy analogues therefore suggest that interaction with the initially disordered 147-152 turn is crucial for the proper recognition of A5P or its analogues by KDOPS and that some of the favorable binding energy may arise from interaction with, concomitant ordering of, and increased intramolecular hydrogen bonding within the strand that ends at this turn.

Turning to Fig. 6, while the openness of the region surrounding the Cl hydroxyl group predisposes KDOPS to be less sensitive to variation at this position in the substrate, this openness also provides sufficient free space to accommodate binding by the acyclic form of A5P, or by either of A5P's cyclic furanose forms (Figure 6). Indeed, the Cl, which represents the anomeric carbon of A5P, is in only 3.6 A away from the hydroxyl oxygen at C4, and is thus sufficiently close to rationalize KDOPS 's recognition of the ring opened form or of either furanose form. In addition, since the values of the rate constants for the ring opening of free A5P in solution are one order of magnitude greater than the k_ca\ for KDOPS, the enzyme need not activate bound anomeric forms of A5P for ring opening. Thus, if we assume that the position of Cl hydroxyl of A5P moiety in KDOPS:2 closely mimics the position of A5P carbonyl in its bound acyclic form (KDOPS :A5P), then KDOP's active site should allow the ring closure and the ring opening of A5P to proceed spontaneously, with little conformational mobility at the active site. A definitive confirmation of the A5P's binding features awaits the determination of the X-ray structure of a KDOPS :A5P complex; however, the present structural data in conjunction with our veiy recent observations by solid-state REDOR NMR suggests that A5P's dynamic mutarotation (Figure 6), an interconversion between cyclic and acyclic forms, should be possible at the enzyme active site.

Chemical Compounds of Interest

In a further aspect, there is provided a method for the treatment of bacterial infection in a human or non-human mammal, which method comprises the administration of an effective, non-toxic and pharmaceutically effective amount of an antibacterial inhibitor or KDOP, or a pharmaceutically acceptable derivative thereof. Through the methods provided herein the following molecules have been determined to desirable characteristics as KDOP inhibitors, therefore having antibacterial activity. These molecules can be of the formula (I):

or a pharmaceutically acceptable salt thereof, and/or a pharmaceutically acceptable solvate thereof, wherein X_! represents O, NH, CH₂, CHF, CF₂, N-Aryl, N-Glycosyl, N- (CH₂CH₂0)_n - alkyl, N-(CH₂CH₂0)_n - aryl, N-(CH₂CH₂0)_n - glycosyl, and N-alkyl; X₂ represents O, NH, CH₂, CF₂, N-Alkyl, N-Aryl, N-Glycosyl, N-(CH₂CH₂0)_n - alkyl, N- (CH₂CH₂0)_n - aryl, and N-(CH₂CH₂0)_n - glycosyl; X₃ represents N, CH, N-(CH₂CH₂0)_n - alkyl, N-(CH₂CH₂0)_n - aryl, and N-(CH₂CH₂0)_n - glycosyl; X₄ represents O, NH, CH₂, CHF, CF₂, N- Alkyl, N-Aryl, and N-Glycosyl; Ri represents 2-glycosyl, 1 -glycosyl, aryl, alkyl, 0(CH₂CH₂0)_n-(2-glycosyl), and (CH₂CH₂0)-(1 -glycosyl); and n independently is an integer from 1 to 6 inclusive.

The compounds of formula (I), and the pharmaceutically acceptable salts thereof, may exist in one of several tautomeric forms, all of which are encompassed by the present invention as individual tautomeric forms or as mixtures thereof.

These molecules can be of the formula (II):

or a pharmaceutically acceptable salt thereof, and/or a pharmaceutically acceptable solvate thereof, wherein Xi represents O, NH, CH₂, CHF, CF₂, N-Aryl, N-Glycosyl, N- (CH₂CH₂0)_n - alkyl, N-(CH₂CH₂0)_n - aryl, N-(CH₂CH₂0)_n - glycosyl, and N- alkyl; X₂ represents O, NH, CH₂, CF₂, N-Alkyl, N-Aryl, N-Glycosyl, N-(CH₂CH₂0)_n - alkyl, N- (CH₂CH₂0)_n - aryl, and N-(CH₂CH₂0)_n - glycosyl; X₃ represents N, CH, N-(CH₂CH₂0)_n - alkyl, N-(CH₂CH₂0)_n - aryl, and N-(CH₂CH₂0)_n - glycosyl; X₄ represents O, NH, CH₂, CHF, CF₂, N- Alkyl, N-Aryl, N-Glycosyl; Ri represents 2-glycosyl, 1 -glycosyl, aryl, alkyl, 0(CH₂CH₂0)_n-(2-glycosyl), and (CH₂CH₂0)-(l-glycosyl); and n independently is an integer from 1 to 6.

The compounds of formula (II), and the pharmaceutically acceptable salts thereof, may exist in one of several tautomeric forms, all of which are encompassed by the present invention as individual tautomeric forms or as mixtures thereof.

These molecules can be of the formula (III):

or a pharmaceutically acceptable salt thereof, and/or a pharmaceutically acceptable solvate thereof, wherein Xi represents O, NH, CH₂, CHF, CF₂, N-Aryl, N-Glycosyl, N- (CH₂CH₂0)_n - alkyl, N-(CH₂CH₂0)_n - aryl, N-(CH₂CH₂0)_n - glycosyl, andN- alkyl; X₂ represents O, NH, CH₂, CF₂, N-Alkyl, N-Aryl, N-Glycosyl, and N-(CH₂CH₂0)_n - alkyl, N-(CH₂CH₂0)_n - aryl, and N-(CH₂CH₂0)_n - glycosyl; X₃ represents N, CH, N- (CH₂CH₂0)_n - alkyl; N-(CH₂CH₂0)_n - aryl, and N-(CH₂CH₂0)_n - glycosyl; represents O, NH, CH₂, CHF, CF₂, N-Alkyl, N-Aryl, N-Glycosyl; Rj represents 2-glycosyl, 1- glycosyl, aryl, alkyl, 0(CH CH₂0)_n-(2-glycosyl), and (CH₂CH₂0)-(1 -glycosyl); and independently is an integer from 1 to 6 inclusive.

The compounds of formula (III), and the pharmaceutically acceptable salts thereof, may exist in one of several tautomeric forms, all of which are encompassed by the present invention as individual tautomeric forms or as mixtures thereof.

These molecules can be of the formula (IV):

or a pharmaceutically acceptable salt thereof, and/or a pharmaceutically acceptable solvate thereof, wherein Xi represents O, NH, CH₂, CHF, CF₂, N-Aryl, N-Glycosyl, N- (CH₂CH₂0)_n - alkyl, N-(CH₂CH₂0)_n - aryl, N-(CH₂CH₂0)_n - glycosyl, and N- alkyl; X₂ represents O, NH, CH₂, CF₂, N-Alkyl, N-Aryl, N-Glycosyl, N-(CH₂CH₂0)_n - alkyl, N- (CH₂CH₂0)_n - aryl, and N-(CH₂CH₂0)_n - glycosyl; X₃ represents N, CH, N-(CH₂CH₂0)_n - alkyl, N-(CH₂CH₂0)_n - aryl, and N-(CH₂CH₂0)_n - glycosyl; X4 represents O, NH, CH₂, CHF, CF₂, N-Alkyl, N-Aryl, N-Glycosyl; R₁ represents 2-glycosyl, 1 -glycosyl, aryl, alkyl, 0(CH₂CH₂0)_n-(2-glycosyl), and (CH₂CH₂0)-(1 -glycosyl); and n independently is an integer from 1 to 6 inclusive.

The compounds of formula (IV), and the pharmaceutically acceptable salts thereof, may exist in one of several tautomeric forms, all of which are encompassed by the present invention as individual tautomeric forms or as mixtures thereof.

For the above Formula's the 2-glycosyl substituent of Ri can consist of the following derivatives (a-c):

(a)

or a pharmaceutically acceptable salt thereof, and/or a pharmaceutically acceptable solvate thereof, wherein X₅ represents O, NH, CH₂, CHF, CF₂, N-Aryl, N-Glycosyl, N- (CH₂CH₂0)_n - alkyl; N-(CH₂CH₂0)_n - aryl, N-(CH₂CH₂0)„ - glycosyl, and N- alkyl.

For the chemical compounds of formula's I-IV, an alkyl group refers to a straight chained or branched group having 2 to 10 carbon atoms. Optionally, the alkyl group can be substituted with one to three heteroatoms such as nitrogen, oxygen and sulfur. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracyl and derivatives thereof. Acceptable derivatives of the above compounds, as provided by the the current invention, would include all tautomeric forms, ionization states thereof, zwitterions, and counterions thereof.

Treatment Methods

Methods of treating septic shock or bacterial infection in a subject using the compositions of the present invention are provided. In particular, the composition methods provided herein may aid in the development of antibacterial agents such as those disclosed herein above. The agents disclosed herein function in a way that antagonizes the elaboration of KDOP. It is contemplated that the present invention is directed to human and animal subjects or patients. The compositions and products of the present invention may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, topically, transdermally, or the like, although parenteral intravenous administration is typically preferred, especially in acute cases of endotoxicosis. The exact amount of such compositions and products required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the disease that is being treated, the particular compound used, its mode of administration, and the like. Thus, it is not possible to specify an exact amount. However, an appropriate amount may be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein and optimization procedures known in the art. Depending on the intended mode of administration, the products and compositions of the present invention can be formulated into pharmaceutical compositions in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, lotions, creams, gels, or the like, preferably in unit dosage form suitable for single administration of a precise dosage. The compositions will include, as noted above, an effective amount of the selected compound in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc.

For solid compositions, conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc. an active compound as described herein and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see REMINGTON'S PHARMACEUTICAL SCIENCES (latest edition). For oral administration, fine powders or granules may contain diluting, dispersing, and/or surface active agents, and may be presented in water or in a syrup, in capsules or sachets in the dry state, or in a non-aqueous solution or suspension wherein suspending agents may be included, in tablets wherein binders and lubricants may be included, or in a suspension in water or a syrup. Where desirable or necessary, flavoring, preserving, suspending, thickening, or emulsifying agents may be included. Tablets and granules are preferred oral administration forms, and these may be coated.

Parenteral administration, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system, such that a constant level of dosage is maintained. See, e.g., U.S. Pat. No. 3,710,795, which is incorporated by reference herein.

Formulation

The dosage administered will be a therapeutically effective amount of active ingredient and will, of course, vary depending upon known factors such as the pharmacodynamic characteristics of the particular active ingredient and its mode and route of administration; age, sex, health and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the effect desired. Usually a daily dosage (therapeutic effective amount) of active ingredient can be about 5 to 400 milligrams per kilogram of body weight. Ordinarily, 10 to 200, and preferably 10 to 50, milligram per kilogram per day given in divided doses 2 to 4 times a day or in sustained release form is effective to obtain desired results.

Dosage forms (compositions) suitable for internal administration contain from about 1.0 to about 500 milligrams of active ingredient per unit. In these pharmaceutical compositions, the active ingredient will ordinarily be present in an amount of about 0.05- 95% by weight based on the total weight of the composition.

The active ingredient can be administered orally in sold dosage forms such as capsules, tablets and powders, or in liquid dosage forms such as elixirs, syrups, emulsions and suspensions. The active ingredient can also be formulated for administration parenterally by injection, rapid infusion, nasopharyngeal absorption or dermoabsorption. The agent may be administered intramuscularly, intravenously, or as a suppository. Additionally, gene therapy modes of introduction can be used to target the introduction of the compound. The skilled artisan readily recognizes that the dosage for this method must be adjusted depending on the efficiency of delivery. Gelatin capsules contain the active ingredient and powdered carriers such as lactose, sucrose, mannitol starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.

Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration contain preferably a water soluble salt of the active ingredient, suitable stabilizing agents and, if necessary, buffer substances. Antioxidizing agents such as sodium bisulfate, sodium sulfite or ascorbic acid either alone or combined are suitable stabilizing agents. Also used are citric acid and its salts and sodium EDTA. In addition, parenteral solutions can contain preservatives such as benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Suitable pharmaceutical carriers are described in Remington 's Pharmaceutical Sciences, a standard reference text in this field.

Additionally, standard pharmaceutical methods can be employed to control the duration of action. These are well known in the art and include control release preparations and can include appropriate macromolecules, for example, polymers, polyesters, polyaminoacids, polyvinyl, pyrolidone, ethylenevinylacetate, methyl cellulose, carboxymethyl cellulose or protamtine sulfate. The concentration of macrocolecules as well as the methods of incorporation can be adjusted in order to control release. Additionally, the agent can be incorporated into particles of polymeric materials such as polyesters, polyaminoacids, hydrogels, poly (lactic acid) or ethylenevinylacetate copolymers. In addition to being incorporated, these agents can also be used to trap the compound in microcapsules.

Useful pharmaceutical dosage forms for administration of the compounds of this invention can be illustrated as follows. Capsules: Capsules are prepared by filling standard two-piece hard gelatin capsulates each with 100 milligrams of powdered active ingredient, 175 milligrams of lactose, 24 milligrams of talc and 6 milligrams magnesium stearate. Soft Gelatin Capsules: A mixture of active ingredient in soybean oil is prepared and injected by means of a positive displacement pump into gelatin to form soft gelatin capsules containing 100 milligrams of the active ingredient. The capsules are then washed and dried. Tablets: Tablets are prepared by conventional procedures so that the dosage unit is 100 milligrams of active ingredient. 0.2 milligrams of colloidal silicon dioxide, 5 milligrams of magnesium stearate, 275 milligrams of microcrystalline cellulose,l 1 milligrams of cornstarch and 98.8 milligrams of lactose. Appropriate coatings may be applied to increase palatability or to delay absorption. Injectable: A parenteral composition suitable for administration by injection is prepared by stirring 1.5% by weight of active ingredients in 10% by volume propylene glycol and water. The solution is made isotonic with sodium chloride and sterilized. Materials and Methods

Protein Purification and Crystallization The wild type KDOPS (specific catalytic activity 9 units/mg) was isolated from the overproducing E. coli DH5α (pJUl) strain, as previously described. For the production of selenomethionine-labeled KDOPS ([Se-Met]KDOPS), competent E. coli methionine auxotroph cells [strain B834(λDΕ3) (Novagen)] were transformed with plasmid pJUl containing the kdsA gene. Using the protocol of optimization of growth conditions developed in this laboratory, we observed that over 100 mg of homogeneous [Se-

MefjKDOPS could be obtained per liter of culture. The molecular mass difference between [Se-Met]KDOPS (31 311.46 ± 8.66) and the native KDOPS (30 842.38 ± 4.97) was determined to be 470.8 mass units (VG BioQ electrospray spectrometer), indicating 100% substitution of Met with Se-Met residues. The /_caι value for [Se-Met]KDOPS (5.3 s^" ') was about 29% lower than that of the native enzyme (7.1 s^"1). Both the native and the [Se-Met]KDOPS exhibited similar K_m values for PEP binding (7.1 and 7.9 μM, respectively). These kinetic data clearly demonstrate that the incorporation of Se-Met into KDOPS does not significantly alter either KDOPS 'catalytic activity or its substrate specificity.

Purified [Se-Met]KDOPS was dialyzed against 0.1 M Tris-HCl buffer (pH 7.2) containing 0.02% β-mercaptoethanol and 0.02% sodium azide, for three changes over 12 h, and was then concentrated to about 45 mg/mL with an Amicon Centricon-10 microconcentrator. All crystals were grown at 23° C by vapor diffusion in hanging drops. Drops were prepared by mixing 2 μL of the above concentrated enzyme solution with 2 μL of the reservoir solution. The reservoir solution contained either inhibitor 2 (0.053 mM) or the substrate PEP (10 mM) in sodium 2-(N-morpholino)ethanesulfonate (MOPS) buffer (61 mM, pH 6.1) with glycerol (25% v/v) as cryosolvent and PEG 400 (10%, v/v). Cubic crystals of the [Se-Met]KDOPS:PEP complex in the space group 123, with cell constants α = _> = c = 117.9 A, α = β = γ = 90° appeared within several days. Crystals were flash frozen in a stream of N₂, X-ray data extending to 2.8 A were collected at -180°C using the inverse-beam method on an R-axis II imaging plate system. A complete set of data was collected from a single crystal.

The crystals of [Se-Met]KDOPS-2 complex also belong to the space group 723 with cell constants a = b = c = 117.6 A, α=β=v=90. X-ray data extending to 2.3 A were collected at -180°C at an energy of 12 660eV on Beamline X4A at the Brookhaven National Synchrotron Line Source. This Se-Met MAD (multiple wavelength anomalous diffraction) data set could not be used to determine MAD phases because of missing data at other wavelengths. X-ray data sets were processed using the programs DENZO and SCALEPACK. The structure was solved by molecular replacement using AMORE (21) with the ligand-free structure of KDOP synthase (subunit A) as the search model. The crystallographic model was refined using X-PLOR.

A computational method for the discovery and design of therapeutic compounds is provided. The methods used rely on an accurate representation of three-dimensional molecular spatial information and improved image processing software. The computational technique employed utilizes a software program and associated algorithms. This method is an improvement over the current methods of drug discovery which often employs a random search through a large library of synthesized chemical compounds or protein molecules for bio-activity related to a specific therapeutic use.

Thus, it is to be understood that the embodiments of the invention herein described are merely illustrative of the application of the principles of the invention. Reference herein to details of the illustrated embodiments are not intended to limit the scope of the claims, which themselves recite those features regarded as essential to the invention. It will be evident from the foregoing description that changes in the form, methods of use, and applications of the elements of the disclosed ligands or methods of determining their chemical structure or modification thereof may be resorted to without departing from the spirit of the invention, or the scope of the appended claims.

Thus, it can be appreciated that a computational method and an apparatus therefore have been presented which will facilitate the discovery of novel bio-active and/or therapeutic molecules, these methods rely on the use of a computational methods employing a general recursive method for determining the macromolecular crystallographic phases of molecules so as to recognize and predict ligand binding affinity.

Accordingly, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

Citations Incorporated Herein by Reference

1. Ray, P. H. (1980) J. Bacteriol. 141, 635-644.

2. Unger, F. M. (1981) Adv. Carbohydr. Chem. Biochem. 38, 323-388. 3. Haslam, E. (1993) in "Shikimic Acid. Metabolism and Metabolites," Wiley, New York.

4. Walsh, C. T., Benson, T. E., Kim, D. H., and Lees, W. J. (1996) Chem. Biol. 3, 83- 91.

5. Anderson, K. S., and Johnson, K. A. (1990) Chem. Rev. 90, 1131-1149. 6. Brown, E., Marquardt, J., Lee, J., Walsh, C. T., and Anderson, K. S. (1994)

Biochemistry 33, 10638-10645.

7. Kohen, A., Jakob, A. and Baasov, T. (1992) Eur. J. Biochem. 208, 443-449.

8. Kohen, A., Berkovich, R., Belakhov, V. and Baasov, T. (1993) Bioorg. Med. Chem. Lett. 3, 1577-1582. 9. Dotson, G. D., Nanjappan, P., Reily, M. D., and Woodard, R. W. (1993) Biochemistry 32, 12392-12397.

10. Liang, P., Lewis, J., Anderson, K. S., Kohen, A., D'Souza, , W. F., Benenson, Y., and Baasov, T. (1998) Biochemistry 37, 16390-16399.

11. Du, S., Faiger, H., Belakhov, V., and Baasov, T. (1999) Bioorg. Med. Chem. 7, 2671-2682.

12. Hedstrom, L. and Abeles, R. (1988) Biochem. Biophysics. Res. Commun. 157, 816- 820.

13. Kaustov, L., Kababya, S., Du, S., Baasov, T., Gropper, S., Shoham, Y., and Schmidt, A. (2000) J. Am. Chem. Soc. 122, 2649-2650. 14. Kaustov, L., Kababya, S., Du, S., Baasov, T., Gropper, S., Shoham, Y., and Schmidt, A. (2000) Biochemistry 39, 14865-14876.

15. Radaev, S., Dastidar, P., Patel, M., Woodard, R. W., and Gatti, D. L. ( 2000) J. Biol. Chem. 275, 9476-9484.

16. Shumilin, I. A., Kretsinger, R. H., and Bauerie, R. H. (1999) Structure 7, 865-875. 17. Wagner, T., Kretsinger, R. H., Bauerie, R. H., and Tolbert, W. D. (2000) J. Mol.

Biol. 301, 233-238. 18. Mechaly A., Teplinsky, A., Belakhov, V., Baasov, T., Shoham, G., and Shoham, Y. (2000) J. Biotech. 78, 83-86.

19. Otwinowski, Z. (1993) DENZO: An Oscillation Data Processing Program for Macromolecular Crystallography. New Haven, CT, Yale Univ. Press. 20. Otwinowski, Z. (1993) SCALEPACK: Software for the Scaling Together of

Integrated Intensities Measured on a Number of Separate Diffraction Images. New Haven, CT, Yale Univ. Press.

21. Navaza, j. (1994) Acta Cryst. A50, 157- 163.

22. Brunger, A. T. (1992) X-PLOR: A System for X-ray Crystallography and NMR. New Haven CT, Yale Univ. Press.

23. Burgi, H. B., Dunitz, J. D, Lehn, J. M., and Wipff, G. (1974) Tetrahedron 30, 1563-1572.

24. Baasov, T., and Kohen, A. (1995) J. Am. Chem. Soc. 117, 6165-6174.

25. Souhassou, M., Schaber, P. M., and Blessing, R. H. (1996) Acta Cryst. B52, 865- 875.

26. Zhang, E., Brewer, j. M., Minor, W., Carreira, L. A., and Lebioda, L. (1997) Biochemistry 36, 12526-12534.

27. Benenson, Y., Belakhov, V. and Baasov, T. (1996) Bioorg. Med. Chem. Lett. 6, 2901-2906. 28. Du, S., Plat, D., Belakhov, V., and Baasov, T. (1997) J. Org. Chem. 62, 794-804.

29. Kim, D. H., Lees, W. J., Haley, T. M., and Walsh, C. T. (1995) J. Am. Chem. Soc. 177, 1494-1502.

30. Albeg, D. G., Lauhon, C. T., Nyfeler, R., Faasler, A., and Bartlett, P. A. (1992) J. Am. Chem. Soc. 114, 3535-3546. 31. Rose, L A. (1972) CRC Crit. Rev. Biochem. 7, 33-57.

32. Matsumura, H., Tereda, M., Shirakata, S., Inoue, T., Yoshinaga, T., Izui, K., and Kai, Y. (1999) FEBSLett. 458, 93-96.

33. Larsen, T. M., Laughlin, L. T., Holden, H. M., and Rayment, I. (1994) Biochemistry 33, 6301-6309. 34. Herzberg, O., Chen, C. C. H., Kapadia, G., Mcguire, M., Carroll, L. G., Noh, S. J., and Dunaway-Mariano, D. (1996) Proc. Nat. Acad. Set USA 93, 2652-2657. 35. DeLeo, A. G., Dayan, J., and Sprinson, D. B. (1973) Biochem. Biophys. Res. Commun. 26, 187-192.

36. Wagner, T., Shumilin, I. A., Bauerie, R., and Kretsinger, R. H. (2000) J. Mol. Biol. 301, 389-399. 37. Ray, P. H., Kelsey, J. E., Bigham, E. C, Benedict, C. D., and Miller, T. A. (1983) ACS Symp. Ser. 237, 141-170.

Claims

CLAIMSWhat is claimed is:

1. A composition comprising a structurally modified molecule wherein said molecule prevents the biosynthesis of Lipid A by preventing the elaboration of KDOP in the outer membrane of GramNegative Bacteria.

2. A compound having the following formula:

wherein X_λ is O; NH; CH₂; CHF; CF₂; N-Alkyl; N-Aryl; N-Glycosyl; N-(CH₂CH₂0)_π - alkyl; N-(CH₂CH₂0)_n - aryl; or N-(CH₂CH₂0)_n - glycosyl; wherein X₂ is O; NH; CH₂; CF₂; N-Alkyl; N-Aryl; N-Glycosyl; N-(CH₂CH₂0)_n - alkyl; N- (CH₂CH₂0)_n - aryl; or N-(CH₂CH₂0)_n - glycosyl; wherein X₄ is O; NH; CH₂; CHF; CF₂; N-Alkyl; N-Aryl; or N-Glycosyl; wherein R] is 2-glycosyl; 1 -glycosyl; aryl; alkyl; 0(CH₂CH₂0)_n-(2-glycosyl); or (CH₂CH₂0)-(l-glycosyl); wherein each n independently is an integer from 1 to 6 inclusive; or a pharmaceutically acceptable salt thereof;

3. A compound of claim 2 wherein R₁ is 2-glycosyl or 0(CH₂CH₂0)_n-(2- glycosyl).

4. A compound of claim 3 wherein the 2-glycosyl substituent of Ri is selected from the group consisting of:

37

SSL-DOCSl 1203383 v₂ (a)

( )

(c)

wherein X₅ is O; NH; CH₂; CHF; CF₂; N-Alkyl; N-Aryl; N-Glycosyl; N-(CH₂CH₂0)_n - alkyl; N-(CH₂CH₂0)_n - aryl; or N-(CH₂CH₂0)_n - glycosyl.

5. A composition comprising the compound of claim 2 and a pharmaceutically acceptable carrier.

6. A method of preventing KDOP elaboration in GramNegative Bacteria in a subject that comprises administering a therapeutically effective amount of the compound of claim 2 to the subject.

7. A method of treating a bacterial infection in a subject that comprises administering a therapeutically effective amount of the compound of claim 2.

8. A method of treating a subject suffering from septic shock that comprises administering to the subject a therapeutically effective amount of the compound of claim 2.

9. A compound having the following formula:

wherein X₂ is O; NH; CH₂; CF₂; N-Alkyl; N-Aryl; N-Glycosyl; N-(CH₂CH₂0)_n - alkyl; N- (CH₂CH₂0)_n - aryl; or N-(CH₂CH₂0)_n - glycosyl; wherein X₃ is N; CH; N-(CH₂CH₂0)_n - alkyl; N-(CH₂CH₂0)_n - aryl; or N-(CH₂CH₂0)_n - glycosyl; wherein X₄ is O; NH; CH₂; CHF; CF₂; N-Alkyl; N-Aryl; or N-Glycosyl; wherein Ri is 2-glycosyl; 1 -glycosyl; aryl; alkyl; 0(CH₂CH₂0)_n-(2-glycosyl); or (CH₂CH₂0)-(l-glycosyl); wherein each n independently is an integer from 1 to 6 inclusive; or a pharmaceutically acceptable salt thereof.

10. A compound of claim 9 wherein Ri is 2-glycosyl or 0(CH₂CH₂0)_n-(2- glycosyl).

11. A compound of claim 10 wherein the 2-glycosyl substituent of Ri is selected from the group consisting of:

(a)

(b)

(c)

wherein X₅ is O; NH; CH₂; CHF; CF₂; N-Alkyl; N-Aryl; N-Glycosyl; N-(CH₂CH₂0)_n alkyl; N-(CH₂CH₂0)_n - aryl; or N-(CH₂CH₂0)_n - glycosyl.

12. A composition comprising the compound of claim 9 and a pharmaceutically acceptable carrier.

13. A method of preventing KDOP elaboration in GramNegative Bacteria in a subject that comprises administering a therapeutically effective amount of the compound of claim 9 to the subject.

14. A method of treating a bacterial infection in a subject that comprises administering a therapeutically effective amount of the compound of claim 9.

15. A method of treating a subject suffering from septic shock that comprises administering to the subject a therapeutically effective amount of the compound of claim 9.

16. A compound having the following formula:

wherein X₂ is O; NH; CH₂; CF₂; N-Alkyl; N-Aryl; N-Glycosyl; N-(CH₂CH₂0)_π - alkyl; N- (CH₂CH₂0)„ - aryl; or N-(CH₂CH₂0)_n - glycosyl; wherein X4 is O; NH; CH₂; CHF; CF₂; N- Alkyl; N-Aryl; or N-Glycosyl; wherein each n independently is an integer from 1 to 6 inclusive; or a pharmaceutically acceptable salt thereof.

17. A composition comprising the compound of claim 16 and a pharmaceutically acceptable carrier.

18. A method of preventing KDOP elaboration in GramNegative Bacteria in a subject that comprises administering a therapeutically effective amount of the compound of claim 16 to the subject.

19. A method of treating a bacterial infection in a subject that comprises administering a therapeutically effective amount of the compound of claim 16.

20. A method of treating a subject suffering from septic shock that comprises administering to the subject a therapeutically effective amount of the compound of claim 16.

21. A compound having the following formula:

wherein X₂ is O; NH; CH₂; CF₂; N-Alkyl; N-Aryl; N-Glycosyl; N-(CH₂CH₂0)_n - alkyl; N- (CH₂CH₂0)_n - aryl; or N-(CH₂CH₂0)_n - glycosyl; wherein X₃ is N; CH; N-(CH₂CH₂0)_n - alkyl; N-(CH₂CH₂0)_n - aryl; or N-(CH₂CH₂0)_n - glycosyl; wherein ₄ is O; NH; CH₂; CHF; CF₂; N-Alkyl; N-Aryl; or N-Glycosyl; wherein Ri is 2-glycosyl; 1 -glycosyl; aryl; alkyl; 0(CH₂CH₂0)_n-(2-glycosyl); or (CH₂CH₂0)-(l-glycosyl); wherein each n independently is an integer from 1 to 6 inclusive; or a pharmaceutically acceptable salt thereof.

22. A compound of claim 21 wherein Ri is 2-glycosyl or 0(CH₂CH₂0)_n-(2- glycosyl).

23. A compound of claim 22 wherein the 2-glycosyl substituent of Ri selected from the group consisting of:

(a)

(b)

(c)

wherein X₅ is O; NH; CH₂; CHF; CF₂; N-Alkyl; N-Aryl; N-Glycosyl; N- (CH₂CH₂0)_n - alkyl; N-(CH₂CH₂0)_n - aryl; or N-(CH₂CH₂0)_n - glycosyl.

24. A composition comprising the compound of claim 21 and a pharmaceutically acceptable carrier.

25. A method of preventing KDOP elaboration in GramNegative Bacteria in a subject that comprises administering a therapeutically effective amount of the compound of claim 21 to the subject.

26. A method of treating a bacterial infection in a subject that comprises administering a therapeutically effective amount of the compound of claim 21.

27. A method of treating a subject suffering from septic shock that comprises administering to the subject a therapeutically effective amount of the compound of claim 21.