WO2001029196A2

WO2001029196A2 - Method of selecting for an inhibitor of dtdp-6-deoxy-d-xylo-4-hexulose 3,5 epimerase (rmlc)

Info

Publication number: WO2001029196A2
Application number: PCT/GB2000/004014
Authority: WO
Inventors: James H. Naismith; Marie-France Giraud
Original assignee: The University Court Of The University Of St. Andrews
Priority date: 1999-10-19
Filing date: 2000-10-19
Publication date: 2001-04-26
Also published as: WO2001029196A3; AU7933800A; GB9924634D0

Abstract

A method of selecting for an agent which inhibits the enzyme RmlC, said method comprising the steps of providing a three-dimensional structure model of at least one active site of RmlC and using the model for designing and/or evaluating the structure of a potential inhibitory agent. The invention also relates to inhibitors of RmlC, the use of RmlC inhibitors as anti-microbial compounds and a method of selecting or identifying anti-microbial compounds.

Description

METHOD OF SELECTING FOR AN INHIBITOR OF RmlC

Field of the invention The present invention relates to a method of selecting for or identifying agents which inhibit the enzyme dTDP-6-deoxy-D-xylo-4-hexulose 3,5 epimerase (RmlC), to an inhibitor of RmlC and to its use as an anti- microbial compound. It also relates to a method of selecting or identifying anti-microbial compounds.

Background of the invention Bacterial cell-surface glycoconjugates are essential for survival of pathogenic bacteria and for interactions between bacteria and the host. Consequently, there is reason to believe that inhibitors directed against target reactions in surface-glycoconjugate assembly may provide viable alternate therapeutic approaches. However, bacterial cell surface glycoconjugate show remarkable structural diversity due to variations of the sugar components, linkages and substitutions. A successful strategy requires identification of enzymes and pathways unique to bacteria, yet present within a wide spectrum of bacterial species. One such target is the synthesis of the activated form of L-rhamnose, dTDP-L-rhamnose.

The pathway for the biosynthesis of dTDP-L-rhamnose from glucose- 1-phosphate and thymidine triphosphate requires four genes, rmlA, B, C and D. The reaction steps and the genes required for dTDP-L-rhamnose biosynthesis are set out in Fig. 1 and summarised below:

1. dTTP + D-Glc-1-P <x- dTDP-D-Glc + PP_± (glucose-1- phosphate thymidyltransferase, RmlA:EC 2.7.7.24).

2. dTDP-D-Glc → dTDP-6 -deoxy-p_-xylo-4 -hexulose (dTDP- D-glucose 4 , 6-dehydratase, RmlB : EC 4.2.1.46).

3. dTDP- 6 -deoxy-D-xylo-4 -hexulose <-> dTDP-6-deoxy-L- lyxo-4 -hexulose (dTDP-4 -dehydrorhamnose 3,5- epimerase, alternatively termed dTDP-6-deoxy-D- xylo-4 -hexulose 3,5 epimerase, RmlC:EC 5.1.3.13).

4. dTDP- 6 -deoxy-L- lyxo-4 -hexulose + NAD(P)H → dTDP-L- rhamnose + NAD(P)⁺ (dTDP-4 -dehydrorhamnose reductase, RmlD:EC 1.1.1.133).

The rhamnose biosynthesis pathway is widely distributed in gram negative and gram positive bacteria but is absent in eukaryotes and hence forms an attractive target for the development of putative therapeutic compounds. An important consideration is that there are no salvage pathways for dTDP-L-rhamnose biosynthesis and a simple route to resistance is not readily apparent. In Gram-negative bacteria, L- rhamnose is frequently found in long O-antigenic polysaccharides (O-PS) attached to lipopolysaccharides. Examples include strains of important human pathogens such as Salmonella , Shigella , Burkholderia and Yersinia , as well as plant-associated bacteria including Xanthomonas and Rhizobium . L-rhamnose is found in the capsular polysaccharides of many streptococci and in the linkage unit that joins the mycolylarabinogalactan complex to peptidoglycan in mycobacteria and in some mycobacterial glycopeptidolipids . The primary structures of many of these L-rhamnose-containing glycoconjugates have been reported (see Complex Carbohydrate Structure Database www.ccrc.uga.edu) and in some cases there is documentation of the importance of L-rhamnose and the structures containing it, in virulence and/or cell viability. However, examination of gene databases also indicates the presence of the structural genes for enzymes involved in dTDP-L-rhamnose synthesis in pathogenic bacteria where the rhamnose-containing structure is not necessarily resolved, for example in Enterococcus faecalis (Xu et al . , 1998, Infect Immun 66:4313-4323), Leptospira interrogans serovar Copenhageni ( itchison et al . , 1997, J Bacteriol 179:1262-1267). Another positive feature of the pathway is that there are four enzymes, each providing a valid therapeutic target. This broadens the range of target pathogens because some bacteria use parts of the rhamnose pathway for synthesis of other sugars. For example, the human pathogen Yersinia enterocoli tica serotype 03 utilises R lA, B, and C, in a pathway leading to the formation of 6-deoxy-L-altrose (a C-3 epimer of L-rhamnose) (Zhang et al . , 1993, Mol icrobiol 9:309-321) .

In those bacteria where the contribution of the L- rhamnose-containing glycoconjugate to the biology of the cell has been investigated, the results fall into two categories. In one category the structure is required for virulence but growth of the bacterium in the laboratory is not adversely affected by its loss. After treatment with a rhamnose pathway inhibitor in vivo, the bacteria will be highly susceptible to host defences and rapidly cleared from the system. Most Gram negative examples and some of the pneumococci fall into this category. In the second category, including mycobacteria and some streptococci, the L-rhamnose- containing glycoconjugate is essential for growth under all conditions, as is detailed below.

a) L-rhamnose pathway as a target in Gram negative bacteria:

In Shigella species, L-rhamnose is found in the long- chain O-polysaccharides (O-PS) attached to the lipopolysaccharide (LPS) molecule. All O-serotypes of S . fl exneri contain L-rhamnose, as do some serotypes of S. dysenteriae (e.g. type 1) and S. boydii . It is well established that mutations resulting in loss of the 0- PS attenuate virulence in S . flexneri (Rajakumar et al . , 1994, J Bacteriol 176:2362-2373; Sandlin et al . , 1996, Mol Microbiol 22:63-73; Sandlin et al . , 1995, Infect Immun 63:229-237; Van den Bosch et al . , 1997, Mol Microbiol 23:765-775). This is evidenced by:

(i) the inability of O-PS deficient mutants to form plaques in tissue culture monolayers, due to their inability to spread to adjacent cells, following invasion; and (ii) a negative Sereny test score.

A primary effect of O-PS-deficiency is the altered localisation of IcsA, a protein that is required for intracellular movement and intercellular spread of the bacterium. Thus, while O-PS-deficient bacteria can still invade tissue-culture monolayers, they are unable to form plaques due to their inability to spread to adjacent cells (Sandlin et al . , 1996, Mol Microbiol 22:63-73; Van den Bosch et al . , 1997, Mol Microbiol 23:765-775). The requirement for O-PS in virulence of S . flexneri has been directly tested using specific mutations in the rhamnose pathway genes (Rajakumar et al., 1994, J Bacteriol 176:2362-2373; Van den Bosch et al., 1997, Mol Microbiol 23:765-775).

In Salmonella enter ica , L-rhamnose is found in a variety of O-PS from clinically relevant serotypes (e.g. serovars Typhi, Paratyphi, Enteritidis) . In E. coli , L-rhamnose is also well distributed but confined to specific O-serotypes. In both of these bacteria, the O-PS is required for resistance to complement-mediated serum killing. Extensive work with the Salmonella system indicates that the long-chain O- PS sterically hinders access of the C5b-9 complex to the outer membrane (Joiner, 1988, Ann Rev Microbiol 42:201-230). An E. coli 075 mutant with a defect in the rhamnose pathway showed >99% killing within one hour of its incubation in serum (Burns and Hull, 1998, Infect Immun 66:4244-4253). Although direct analysis of a rhamnose pathway mutant has not been done with Salmonella , mutations in other genes leading to 0-PS- deficiency result in complement sensitivity. In Burkholderia pseudomallei , mutations that eliminate the rhamnose pathway prevent formation of type II O-PS and give rise to a serum-sensitive phenotype (DeShazer et al., 1998, Mol Microbiol 30:1081-1100). It is therefore likely that many other bacteria with L- rhamnose-containing O-PS structures will also show serum resistance when the rhamnose pathway (and assembly of the protective O-PS) is inhibited.

b) L-rhamnose pathway as a target in mycobacteria:

Studies at Colorado State University (see Besra et al . , 1995, Biochemistry 34:4257-265, 6734-6743; Daffe et al., 1990, J Biol Chem 265:6734-6743; McNeil et al . , 1996, "Chemistry of the mycobacterial cell wall", pages 171-186, In (eds Rom and Garay) Tuberculosis, Little, Brown and Company, Boston, USA; McNeil et al . , 1991, J Biol Chem 266:13217-13223) revealed the presence of a specific chain of carbohydrates linking the peptidoglycan to the mycolic acids. This chain, beginning at the peptidoglycan with N-acetyl glucosamine, proceeds through L-rhamnose, D- galactofuranose, and D-arabinofuranose to the mycolic acids. From this structure came the basic hypothesis that inhibition of the insertion of any of these glycosyl residues, especially rhamnose in the present context, would inhibit growth of M. tuberculosis . Proof of this hypothesis has resulted from studies (J A Mills, M McNeil et al . , manuscript in preparation) where a temperature sensitive (TS) mutant of M. smegmatis that is complemented by the gene ( wbbL) encoding for the rhamnosyl transferase from M. tuberculosis was isolated. This observation allows for the deduction that the rhamnosyl residue must be present for the viability of M. tuberculosis . Furthermore, treatment of this TS mutant at the non- permissive temperature results in cell death. Since dTDP-rhamnose is the substrate of the rhamnosyl transferase (WbbL) it follows that its product is essential for M. tuberculosis viability. Its biosynthesis has been shown to be via RmlA-D as in other bacteria (see Ma et al . , 1997, Microbiology 143:937-945; Stern et al . , 1999, Microbiology 145:663- 671) and no other pathway than the RmlA-D for dTDP- rhamnose has ever been documented. The consensus is that accumulation of intermediates in the cell is proving lethal in these cases.

c) L-rhamnose pathway as a target in streptococci: Gene knockout experiments strongly suggest deletion of any of the rhamnose biosynthesis genes in Streptococcus Suis type II may be fatal.

In conclusion therefore the rhamnose pathway is a strong candidate for therapeutic intervention. The sugar is essential for the virulence of many gram- negative bacteria including the Shigella genus and Salmonella genus. Further it appears essential for the survival of gram positive bacteria including many Streptococci and Mycobacteria (inc M. tuberculosis) . It has been shown to be essential for survival of Pseudomonas aueriginosa .

We have determined the structure of the X-ray structure of a crystal dTDP- 6 -deoxy-D-xylo-4 -hexulose 3,5 epimerase (RmlC) . Examination and interpretation of this structure has revealed the active site pocket. It is in the active site pocket that the sugar is acted upon by the enzyme to bring about its chemical transformation. We have performed sequence alignments which show that this pocket is conserved in all RmlC sequences published or reported to date. This site is therefore highly conserved. We have also identified the nucleotide-binding site.

Summary of the invention Knowledge of these sites allows the design and optimisation of potential inhibitor molecules by rational approaches. From our studies we have identified some characteristics that an inhibitor is likely to possess. The fact that the active site pocket is very largely conserved in all known RmlC's suggests that an inhibitor will have broad specificity for other pathogenic bacteria. Thus, the term RmlC is used to encompass known RmlC together with homologues having the same activity and having similar or identical active sites.

The present invention thus provides a method of selecting for an agent which inhibit the enzyme dTDP- 6- deoxy-D-xylo-4 -hexulose 3,5 epimerase (RmlC) , said method comprising:

a) providing a three-dimensional structural model of at least one active site of RmlC; and

b) using said model for designing and/or evaluating the structure of a potential inhibitory agent.

Optionally, the structure of the agent is designed and/or evaluated with the aid of a computer.

Step b) of the method preferably includes the step of analysing the potential interaction of the agent in the active site, advantageously with the aid of a computer.

Optionally, the model may be in the form of a computer graphic file, and will usually be based upon the X-ray crystal co-ordinates of RmlC. Numerous graphic programs now exist to facilitate handling of said X-ray crystal co-ordinates and mention may be made of FRODO (Version 0), Insight and SYBYL and the like.

The structure of the agent to be tested for RmlC inhibitory activity may conveniently likewise be designed and/or evaluated in the form of X-ray crystal co-ordinates or approximations thereof.

It is further preferred that one of the active site is defined by the structure co-ordinates of RmlC amino acid residues Ser 52, Ser 54, His 63, Lys 73, Asp 84, His 120, Phe 122, Tyr 133, Ser 167 and Asp 170.

Another of the active site which can be used (the nucleotide site) is defined by the structure co- ordinates of RmlC amino acid residues Arg 24, Phe 27, Glu 29, Gin 48, Asn 50, Arg 60, Tyr 139.

According to another aspect of the invention it is provided an agent which inhibit the enzyme RmlC and which is selected according to the above method.

Advantageously the agent will comprise a negative charge and the interaction with the active site of RmlC will desirably include an association between the negative charge of the agent and at least one of the amino acid residues His 63, Lys 73 and His 120 of RmlC.

In a preferred embodiment, the agent will include an apolar region located within the apolar cavity of the active site formed between amino acid residues nos . 131, 122 and 75.

In a yet further embodiment, the agent will include a region able to intercalate between the apolar amino acid residues nos Phe 27 and Tyr 139.

According to a further aspect of the invention it is provided a method of selecting an anti-microbial (anti- bacterial or anti-fungal) compound, said method comprising following the steps outlined above, and including the step of selecting an agent that binds to the active site of RmlC sufficiently tightly to impede the biosynthesis of rhamnose and thus growth of the micro-organism.

Optionally, the agent selected may be added to the enzyme RmlC and the enzymic activity (and hence the degree of inhibition by the agent) determined.

A further aspect of the invention relates to the use of the RmlC inhibitor defined above as anti-microbial agent, and more particularly as an agent effective against gram-negative bacteria, mycobacteria and streptococci.

According to a further aspect of the present invention it is provided an anti-microbial compound having the characteristics of the agent mentioned above. Preferably, such compound has the following characteristics: a) a negative charge which on binding of the agent to RmlC is positioned to interact with His 63, Lys 73 and His 120 of RmlC;

b) an apolar region, which on binding of the agent to RmlC is positioned in the locality of amino acid nos 131, 122 and 75; and

c) an intercalating region, which on binding of the agent to RmlC is positioned between amino acid nos Phe 27 and Tyr 139.

The active site of RmlC and interactions with its natural substrate are further illustrated by the Figures.

Brief description of drawings

Figure 1 : is a schematic representation of the rhamnose pathway showing the transformation carried out by each enzyme RmlA, RmlB, RmlC and RmlD during the conversion of glucose 1-phosphate.

Figure 2 : shows a proposed mechanism for the epimerisation of the dTDP-6-deoxy-D-xylo-4-hexulose . The order of epimerisation at the C3 and C5 position is not known. The key residues are identified.

Figure 3 : shows the amino acids alignment of RmlC enzymes from other organisms. The absolute conservation of the active site residues should be noted. The last sequence is a monoepimerase (only epimerises the 3 position) . Neither Tyr 133 nor Asn 50 are found in this enzyme, both these residues are essential for epimerisation at the 5 position.

Figure 4a : shows the amino acid residues which form the main active site, a sulphate ion found in the crystal is shown.

Figure 4b : Shows the predicted orientation of the substrate molecule in the main active site. This prediction is based upon the structure of an enzyme substrate mimic complex and sequence alignments.

Figure 5 : is a cross eye stereo view of the main active site of RmlC.

As shown in Figures 4 to 5 , the main active site of RmlC is formed from residues Ser 52, Ser 54, His 63, Lys 73, Asp 84, His 120, Phe 122, Tyr 133, Ser 167 and Asp 170 which line the main active site pocket. The stereo-view of Figure 5 shows that these residues are clustered inside the two β- sheets of RmlC monomers. The residues His 63, Lys 73 and His 120 form a positively charged hole. This hole stabilises the keto function of the sugar. An inhibitor will require a partial or full negative charge to bind strongly to these residues. The Aspartic acid residues are bound to the Histidine residues, making the Histidine residues more basic. The Histidine residues act as the bases to extract the protons from the sugar ring at positions C3 and C5 (see Figures 4 to 5) .

Residues Phe 131, Phe 122 and Val 75 form a significant apolar cavity and bind the C5 substituent methyl group of the sugar ring. (These residues are not absolutely conserved in all enzymes but replacement residues are always hydrophobic . ) This cavity can be exploited for inhibitor design, significant gain in inhibitor potency would be obtained by designing an inhibitor with an apolar group to interact with this cavity. There is another large cavity at the 04 (of the sugar) binding site. This cavity is large and has a completely conserved serine (Ser 167) residue in it. The residues surrounding the approach to the catalytic His residues (63 and 12) bond donors and acceptors. A high degree of inhibitor specificity will come from designing an inhibitor to interact with these residues.

The nucleotide site is formed by Arg 24, Phe 27, Glu 29, Gin 48, Asn 50, Arg 60, Tyr 139. The nucleotide specificity is governed by residues which interact with the ring of the nucleotide. Not all RmlC enzymes use thymine as the nucleotide. Effective inhibitors designed against this site will intercalate between the aromatic residues Phe 27 and Tyr 139 (both absolutely conserved) .

Two approaches have been developed to determine the activities of RmlC and D. One involves assay of RmlC and D in combination and the other uses a combined assay for RmlB, C and D.

a) RmlC and RmlD :

The specific assay for determination of RmlC typically contains 45 mM potassium phosphate buffer, pH 7.0 , 9 mM MgCl₂, 0.18 mM dTDP- 6 -deoxy-D-xylo-4 -hexulose , 0.072 mM NAD(P)H, a 20-fold molar excess of RmlD for determination of RmlC, and an appropriate volume of RmlC in a total volume of 0.5 ml .

Assays for RmlD were done using a similar reaction mixture but incorporating a 100-mold molar excess of RmlC.

Time absorption plots were recorded at 25°C in a Beckman DU65 spectrophotometer or a Hitachi U-2010 spectrophotometer. Enzyme activities were calculated from the linear decrease of the absorption at 340 nm. Assays with low amounts of NAP(P)H were analysed in a Hitachi F-2000 spectrofluorometer with an excitation at 340 nm and recording emission at 460 nm.

b) RmlB, C and D:

The reaction mixture consists of 20 nmol of TDP-Glc (0.002 ml of 10 mM TDP-Glc), 10 nmol NADP (0.01 ml of 1 mM NADPH) , 0.006 ml RmlB (0.5 mg/ml), 0.0044 ml RmlC (0.3 mg/ml), 0.0045 ml RmlD (0.22 mg/ml), 0.001 ml of 0.1 M MgCl₂, and 50 mM HEPES buffer pH 7.6 to total volume of 0.05 ml. RmlB, RmlC, RmlD and NADPH are pre- incubated at room temperature for 30 min, and then the MgCl₂, and the buffer (HEPES) is added. (Incubation of RmlB with NAD overnight is important prior to assay.) Finally, the TDP-Glc is added the last to start the reaction. The reaction is incubated at room temperature. The light absorption was measured at 340 nm every 20 min.

Inhibitors will be tested against either of the two assay conditions described. Compounds which inhibit the enzyme will be co-crystallised with RmlC and the X- ray structure determined. The compound will then be redesigned according to the criteria outlined earlier to more optimise its interaction with the protein. Thus a cycle of design, test, co-crystallisation, redesign will follow.

Claims

Claims :

1. A method of selecting for an agent which inhibits the enzyme RmlC, said method comprising the steps of :

b) using said model for designing and/or evaluating the structure of a potential inhibitory agent .

2. The method of Claim 1, wherein the structure of said agent is designed and/or evaluated with the aid of a computer.

3. The method of Claim 1 or 2 , wherein step b) comprises the step of analysing the potential interaction of said agent in said active site.

4. The method of any one of Claims 1 to 3 , wherein said model is in the form of a computer graphic file, based upon the X-ray crystal co-ordinates of RmlC.

5. The method of any one of Claims 1 to 4 , wherein the structure of said agent is designed and/or evaluated in the form of X-ray crystal co-ordinates or approximations thereof.

6. The method of any one of Claims 1 to 5, wherein said active site is defined by the structure co- ordinates of RmlC amino acid residues Ser 52, Ser 54, His 63, Lys 73, Asp 84, His 120, Phe 122, Tyr 133, Ser 167 and Asp 170.

7. The method of any one of Claims 1 to 6 , wherein said active site is defined by the structure co- ordinate of RmlC amino acid residues Arg 24, Phe 27, Glu 29, Gin 48, Asn 50, Arg 60 and Tyr 139.

8. A method of selecting an anti-microbial compound, said method comprising selecting for agents which inhibit the enzyme RmlC according to the method described in any one of Claims 1 to 7, said method comprising the step of:

i) selecting an agent that binds to the active site of RmlC sufficiently tightly to impede the biosynthesis of rhamnose and thus growth of the microbe.

9. An inhibitor of RmlC, said inhibitor being selected according to the method of any one of Claims 1 to 8.

10. An inhibitor of RmlC, said inhibitor comprising a negative charge which interacts with at least one of the amino acid residues His 63, Lys 73 and His 120 of RmlC.

11. An inhibitor of RmlC, said inhibitor comprising an apolar region able to be located within the apolar cavity of the active site formed between amino acid residues nos. 131, 122 and 75.

12. An inhibitor of RmlC, said inhibitor comprising a portion able to intercalate between the apolar amino acid residues Phe 27 and Tyr 139.

13. An inhibitor of RmlC having the following characteristics:

a) a negative charge which on binding of the agent to RmlC is positioned to interact with the amino acid residues His 63, Lys 73 and His 120 of RmlC;

b) an apolar region, which on binding of the agent to RmlC is positioned in the locality of amino acid residues nos 131, 122 and 75;

c) an intercalating region, which on binding of the agent to RmlC is positioned between amino acid residues nos Phe 27 and Tyr 139.

15. The use of a RmlC inhibitor of Claims 9 to 14 as an anti-microbial agent.