Crystalline Structure of FABI from Burkholderia Pseudomallei

Abstract

The present invention relates to drug targets for Burkholderia pseudomallei. The invention provides a crystalline polypeptide derived from Burkholderia pseudomallei comprising the amino acid sequence set forth in SEQ ID NO: 1. Also provided are methods for co-crystallizing a binary enoyl-acyl carrier protein reductase (FabI) with a potential inhibitor of an FabI activity and for identifying an inhibitor of an activity of enoyl-acyl carrier protein reductase (FabI). A representative example of such a crystalline structure is a BpmFabI:AFN-1252 complex.

Claims

1. A crystalline structure of a binary enoyl-acyl carrier protein reductase (FabI):FabI inhibitor complex.

2. The crystalline structure of claim 1, wherein said binary complex has a space group C121 and unit cell dimensions a=134.79 , b=63.44 , c=121.84 and bond angles of ==90, =107.08.

3. The crystalline structure of claim 1, wherein said FabI is a polypeptide comprising: (a) an amino acid sequence shown in SEQ ID NO: 1; or (b) an amino acid sequence having about 95% identity with the amino acid sequence shown in SEQ ID NO: 1.

4. The crystalline structure of claim 3, wherein the polypeptide is at least 90% pure in its non-crystalline form.

5. The crystalline structure of claim 1, wherein said crystalline structure is defined by a substantial portion of atomic coordinates shown in Table 1.

6. The crystalline FabI according to claim 1, wherein the FabI inhibitor is AFN-1252.

7. The crystalline structure of claim 1, wherein said enoyl-acyl carrier protein reductase is from Burkholderia pseudomallei (BpmFabI).

8. The crystalline structure of claim 1 having a protein data base accession code 4RLH.

9. A method for co-crystallizing a binary enoyl-acyl carrier protein reductase (FabI) with a potential inhibitor of an FabI activity, comprising the step of: incubating a polypeptide having an amino acid sequence shown in SEQ ID NO: 1 with the potential FabI inhibitor to produce a binary complex with unit cell dimensions, bond angles and space group substantially identical to those of the binary crystalline complex of claim 2.

10. The method of claim 9, wherein co-crystallizing occurs in the absence of a cofactor.

11. The method of claim 10, wherein the cofactor is NADH or NADPH.

12. The method of claim 9, wherein the polypeptide has an amino acid sequence having about 95% identity with the amino acid sequence shown in SEQ ID NO: 1.

13. The FabI:FabI inhibitor binary crystalline complex produced by the method of claim 9.

14. A method for identifying an inhibitor of an activity of enoyl-acyl carrier protein reductase (FabI), comprising the steps of: generating a three-dimensional in silico model of a binary complex of FabI and a potential FabI inhibitor based at least in part on the binary complex of claim 1; and analyzing an interaction of the potential inhibitor with FabI within the complex to determine inhibitory potential.

15. The method of claim 14, comprising the steps of: inputting into the model a set of atomic structure coordinates for atoms of amino acid residues from druggable regions of FabI; inputting a set of atomic structure coordinates for the potential inhibitor; performing a fitting operation between the potential inhibitor and the druggable region of FabI; and quantifying the association between the potential inhibitor and the druggable region of FabI, thereby determining the inhibitory potential.

16. The method of claim 14, further comprising screening said potential inhibitor for inhibition of the activity of FabI.

17. The method of claim 15, wherein the atomic structures coordinates for the atoms comprising the druggable regions of FabI are shown in Table 1.

18. The method of claim 14, wherein the FabI is from Burkholderia pseudomallei (Bpm) and the identified inhibitor is a drug for a BpmFabI associated disease.

19. The method of claim 18, wherein the BpmFabI associated disease is melioidosis.

20. A crystalline structure of a binary BpmFabI:AFN-1252 complex having a protein data base accession code 4RLH.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] So that the matter in which the above-recited features, advantages and objects of the invention, as well as others that will become clear, are attained and can be understood in detail, more particular descriptions of the invention briefly summarized above may be had by reference to certain embodiments thereof that are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.

[0019] FIG. 1 is the amino acid sequence for BpmFabI (SEQ ID NO: 1).

[0020] FIG. 2 is a generic entry detailing the tabular format of large Table 1 submitted herewith as an ASCII text file. Table 1 lists the atomic structure coordinates for the amino acid sequence (SEQ ID NO: 1) of the invention derived from x-ray diffraction from a crystal of such polypeptide. The information in this generic table corresponds to the first entry in Table 1. Record Header refers to the row type, such as ATOM. No. designates the row number. The first Atom Type column describes the atom whose coordinates are measured, with the first letter in the column identifying the atom by its elemental symbol and the subsequent letter, if present, defining the location of the atom in the amino acid residue or other molecule. Residue and residue number designates the residue of the subject polypeptide. Crystallographically, X, Y, Z define that the atomic position of the atom measured. Occ is an occupancy factor that refers to the fraction of the molecules in which each atom occupies the position specified by the coordinates. A value of 1 indicates that each atom has the same conformation, i.e., the same position, in all molecules of the crystal. B is a thermal factor that is related to the root mean square deviation in the position of the atom around the given atomic coordinate. The Chain ID column identifies each protein and ligand molecule present in the asymmetric unit using letters such as A, B, C, D and water molecules are identified by letter F. In the last Atom Type column, letter in the column identifies the atom by its elemental symbol.

[0021] FIG. 3 depicts the chemical structure of AFN-1252.

[0022] FIG. 4 is a dose-response curve for the inhibition of BpmFabI by AFN-1252.

[0023] FIG. 5A illustrates the mechanism of inhibition of BpmFabI by AFN-1252 at 300 mM crotonyl-CoA and different concentrations of NADH. The concentrations of AFN-1252 were: 0 nM (), 2.5 nM (), 5 nM (.box-tangle-solidup.), 10 nM (.Math.), 20 nM (A) and 40 nM (.diamond-solid.)

[0024] FIG. 5B illustrates the mechanism of inhibition of BpmFabI by AFN-1252 at 375 mM NADH and different concentrations of crotonyl-CoA. The concentrations of AFN-1252 were:

0 nM (), 5 nM (), 20 nM (.box-tangle-solidup.), 40 nM (.Math.) and 80 nM ()

[0025] FIG. 6 are thermal melting curves of BpmFabI alone and in presence of AFN-1252 and Triclosan

[0026] FIG. 7 is a ribbon representation of the monomer structure of BPMFabI (cyan) shown with the bound AFN-1252 (yellow) compound. The monomer composed of seven helices and seven strands is depicted in the figure. The figure was prepared using PyMol.

[0027] FIG. 8 is a ribbon diagram of BpmFabI tetrameric assembly viewed down one of 2-fold of the non crystallographic 222-fold axis. Each monomer along with the bound AFN-1252 is shown in different color.

[0028] FIG. 9 shows the active site structure of AFN-1252 bound to BpmFabI. The interacting residues seen within 3.6 from AFN-1252 are labelled. Hydrogen bonds are represented as dotted lines and water molecules as red spheres.

[0029] FIG. 10 is a cartoon diagram showing the superposed structures of BpmFabI (magenta) SA FabI (yellow) and E. coli FabI (green). The bound NAD/NADP and AFN-1252 ligands are also shown. The flexible loop (residues T194/K199) in BpmFabI was found protruding away from the active site.

DETAILED DESCRIPTION OF THE INVENTION

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs. As used in the specification and the appended claims, unless specified to the contrary, the following terms have the meaning indicated in order to facilitate the understanding of the present invention.

[0031] The singular forms a, an and the encompass plural references unless the context clearly indicates otherwise.

[0032] The term amino acid is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids. Exemplary amino acids include naturally-occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of the foregoing.

[0033] The term complex refers to a non-covalent association between at least two moieties (e.g. chemical or biochemical) that have an affinity for one another. Examples of complexes include association between antigen/antibodies, lectin/avidin, target polynucleotide/probe oligonucleotide, antibody/anti-antibody, receptor/ligand, enzyme/ligand, polypeptide/polypeptide, polypeptide/polynucleotide, polypeptide/co-factor, polypeptide/substrate, polypeptide/inhibitor, polypeptide/small molecule, and the like. Member of a complex refers to one moiety of the complex, such as an antigen or ligand. Protein complex or polypeptide complex refers to a complex comprising at least one polypeptide. The term complex is considered to be binary complex if the said non-covalent association is between two moieties.

[0034] The term druggable region, when used in reference to a polypeptide, nucleic acid, complex and the like, refers to a region of the molecule which is a target or is a likely target for binding a modulator. For a polypeptide, a druggable region generally refers to a region wherein several amino acids of a polypeptide would be capable of interacting with a modulator or other molecule. For a polypeptide or complex thereof, exemplary druggable regions including binding pockets and sites, enzymatic active sites, interfaces between domains of a polypeptide or complex, surface grooves or contours or surfaces of a polypeptide or complex which are capable of participating in interactions with another molecule. In certain instances, the interacting molecule is another polypeptide, which may be naturally-occurring. In other instances, the druggable region is on the surface of the molecule.

[0035] Druggable regions may be described and characterized in a number of ways. For example, a druggable region may be characterized by some or all of the amino acids that make up the region, or the backbone atoms thereof, or the side chain atoms thereof (optionally with or without the alpha carbon atoms). Alternatively, in certain instances, the volume of a druggable region corresponds to that of a carbon based molecule of at least about 200 amu and often up to about 800 amu. In other instances, it will be appreciated that the volume of such region may correspond to a molecule of at least about 600 amu and often up to about 1600 amu or more.

[0036] Alternatively, a druggable region may be characterized by comparison to other regions on the same or other molecules. For example, the term affinity region refers to a druggable region on a molecule (such as a polypeptide of the invention) that is present in several other molecules, in so much as the structures of the same affinity regions are the same so that they are expected to bind the same or related structural analogs. An example of an affinity region is an ATP-binding site of a protein kinase that is found in several protein kinases (whether or not of the same origin). The term selectivity region refers to a druggable region of a molecule that may not be found on other molecules, in so much as the structures of different selectivity regions are sufficiently different so that they are not expected to bind the same or related structural analogs. An exemplary selectivity region is a catalytic domain of a protein kinase that exhibits specificity for one substrate. In certain instances, a single modulator may bind to the same affinity region across a number of proteins that have a substantially similar biological function, whereas the same modulator may bind to only one selectivity region of one of those proteins.

[0037] Continuing with examples of different druggable regions, the term undesired region refers to a druggable region of a molecule that upon interacting with another molecule results in an undesirable affect. For example, a binding site that oxidizes the interacting molecule (such as P-450 activity) and thereby results in increased toxicity for the oxidized molecule may be deemed an undesired region. Other examples of potential undesired regions includes regions that upon interaction with a drug decrease the membrane permeability of the drug, increase the excretion of the drug, or increase the blood brain transport of the drug. It may be the case that, in certain circumstances, an undesired region will no longer be deemed an undesired region because the effect of the region will be favorable, e.g., a drug intended to treat a brain condition would benefit from interacting with a region that resulted in increased blood brain transport, whereas the same region could be deemed undesirable for drugs that were not intended to be delivered to the brain.

[0038] The term polypeptide, and the terms protein and peptide which are used interchangeably herein, refers to a polymer of amino acids. Exemplary polypeptides include gene products, naturally-occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants and analogs of the foregoing.

[0039] The term polypeptide of the invention refers to a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, or an equivalent or fragment thereof, e.g., a polypeptide comprising a sequence consisting of, or consisting essentially of, the amino acid sequence set forth in SEQ ID NO: 1. Polypeptides of the invention include polypeptides comprising (i) all or a portion of the amino acid sequence set forth in SEQ ID NO: 1; (ii) the amino acid sequence set forth in SEQ ID NO: 1 with 1 to about 2, 3, 5, 7, 10, 15, 20, 30, 50, 75 or more conservative amino acid substitutions; (iii) an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1; (iii) an amino acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 1. Polypeptides of the invention also include homologs, e.g., orthologs and paralogs, of SEQ ID NO: 1.

[0040] The term in silico refers to the utilization of computer modeling or computer simulation in crystalline structure analysis and drug design. Computer systems, hardware, software, algorithms, etc. on which in silico analysis is performed are well-known and standard in the art.

[0041] In one embodiment, the present invention provides a crystalline structure of a binary enoyl-acyl carrier protein reductase (FabI):FabI inhibitor complex.

[0042] In certain embodiments, the present invention provides a binary enoyl-acyl carrier protein reductase (FabI):FabI inhibitor complex that has unit cell dimensions a=134.79 , b=63.44 , c=121.84 , ==90, =107.08 with a space group C121.

[0043] In certain embodiments, the present invention provides a crystalline structure of a binary enoyl-acyl carrier protein reductase (FabI):FabI inhibitor complex where the FabI is a polypeptide comprising an amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having about 95% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0044] In certain embodiments, the present invention provides a crystalline structure of a binary enoyl-acyl carrier protein reductase (FabI):FabI inhibitor complex where the polypeptide is at least 90% pure in its non-crystalline form.

[0045] In certain embodiments, the present invention provides a crystalline structure of a binary enoyl-acyl carrier protein reductase (FabI):FabI inhibitor complex where the crystalline structure is defined by a substantial portion of atomic coordinates shown in Table 1.

[0046] In certain embodiments, the present invention provides a crystalline structure of a binary enoyl-acyl carrier protein reductase (FabI):FabI inhibitor complex where the FabI inhibitor is AFN-1252.

[0047] In certain embodiments, the present invention provides a crystalline structure of a binary enoyl-acyl carrier protein reductase (FabI):FabI inhibitor complex where the enoyl-acyl carrier protein reductase is from Burkholderia pseudomallei (BpmFabI).

[0048] In certain embodiments, the present invention provides a crystalline structure of a binary enoyl-acyl carrier protein reductase (FabI):FabI inhibitor complex that has a protein data base accession code 4RLH.

[0049] In an aspect of these embodiments, the present invention provides a crystalline structure of a binary enoyl-acyl carrier protein reductase (FabI):FabI inhibitor complex comprising the amino acid sequence set forth in SEQ ID NO: 1; where the crystalline structure comprises unit cell dimensions a=134.79 , b=63.44 , c=121.84 and bond angles of ==90, =107.08 with a space group C121.

[0050] In another aspect of these embodiments, the present invention provides a crystalline structure of a binary enoyl-acyl carrier protein reductase (FabI):FabI inhibitor complex comprising the amino acid sequence having at least about 95% identity with the amino acid sequence set forth in SEQ ID NO: 1; where the crystal comprises unit cell dimensions a=134.79 , b=63.44 , c=121.84 and bond angles of ==90, =107.08 with a space group C121.

[0051] In another aspect of these embodiments, the present invention provides a crystalline structure of a binary enoyl-acyl carrier protein reductase (FabI):FabI inhibitor complex where the polypeptide is at least 90% pure in its non-crystalline form; where the crystal comprises unit cell dimensions a=134.79 , b=63.44 , c=121.84 and bond angles of ==90, =107.08 with a space group C121.

[0052] In certain preferred embodiments, the present invention provides a crystalline FabI from organism Burkholderia pseudomallei wherein the crystal comprises unit cell dimensions a=134.79 , b=63.44 , c=121.84 , ==90, =107.08 with a space group C121.

[0053] In certain preferred embodiments, the present invention provides a crystalline FabI from organism Burkholderia pseudomallei wherein the crystal is in binary complex with a FabI inhibitor, preferably AFN-1252.

[0054] In another embodiment, the present invention provides a crystalline structure of a binary BpmFabI: AFN-1252 complex having a protein data base accession code 4RLH.

[0055] In yet another embodiment, the present invention provides a method for co-crystallizing a binary enoyl-acyl carrier protein reductase (FabI) with a potential inhibitor of an FabI activity, comprising the step of incubating a polypeptide having an amino acid sequence shown in SEQ ID NO: 1 with the potential FabI inhibitor to produce a binary complex with unit cell dimensions, bond angles and space group substantially identical to those of the binary crystalline complex as described supra.

[0056] In certain embodiments, the present invention provides a method for co-crystallizing a binary enoyl-acyl carrier protein reductase (FabI) with a potential inhibitor of an FabI activity where co-crystallizing occurs in the absence of a cofactor.

[0057] In certain embodiments, the present invention provides a method for co-crystallizing a binary enoyl-acyl carrier protein reductase (FabI) with a potential inhibitor of an FabI activity where co-crystallizing occurs in the absence of the cofactor NADH or NADPH.

[0058] In certain embodiments, the present invention provides a method for co-crystallizing a binary enoyl-acyl carrier protein reductase (FabI) with a potential inhibitor of an FabI activity where the polypeptide has an amino acid sequence having about 95% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0059] In aspects of these embodiments, the present invention provides a method for co-crystallizing a binary enoyl-acyl carrier protein reductase (FabI) with a potential inhibitor of an FabI activity where the crystalline FabI has unit cell dimensions a=134.79 , b=63.44 , c=121.84 , ==90, =107.08 with a space group C121 in the absence of cofactors.

[0060] In aspects of these embodiments, the present invention provides a method for co-crystallizing a binary enoyl-acyl carrier protein reductase (FabI) with a potential inhibitor of an FabI activity where the crystalline FabI has unit cell dimensions a=134.79 , b=63.44 , c=121.84 , ==90, =107.08 with a space group C121 in the absence of NADH.

[0061] In aspects of these embodiments, the present invention provides a method for co-crystallizing a binary enoyl-acyl carrier protein reductase (FabI) with a potential inhibitor of an FabI activity where the crystalline FabI has unit cell dimensions a=134.79 , b=63.44 , c=121.84 , ==90, =107.08 with a space group C121 in the absence of NADPH.

[0062] In aspects of these embodiments, the present invention provides a method for co-crystallizing a binary enoyl-acyl carrier protein reductase (FabI) with AFN-1252 where the crystalline FabI having unit cell dimensions a=134.79 , b=63.44 , c=121.84 and bond angles of ==90, =107.08 with a space group C121 comprising placing the crystalline FabI in a solution comprising the AFN-1252.

[0063] In yet another embodiment, the present invention provides a FabI:FabI inhibitor binary crystalline complex produced by the method described supra.

[0064] In yet another embodiment, the present invention provides a method for identifying an inhibitor of an activity of enoyl-acyl carrier protein reductase (FabI), comprising the steps of generating a three-dimensional in silico model of a binary complex of FabI and a potential FabI inhibitor based at least in part on the binary complex as described supra; and analyzing an interaction of the potential inhibitor with FabI within the complex to determine inhibitory potential.

[0065] In certain embodiments, the present invention provides a method for identifying an inhibitor of an activity of enoyl-acyl carrier protein reductase (FabI), comprising the steps of inputting into the model a set of atomic structure coordinates for atoms of amino acid residues from druggable regions of FabI; inputting a set of atomic structure coordinates for the potential inhibitor; performing a fitting operation between the potential inhibitor and the druggable region of FabI; and quantifying the association between the potential inhibitor and the druggable region of FabI, thereby determining the inhibitory potential.

[0066] In certain embodiments, the present invention provides a method for identifying an inhibitor of an activity of enoyl-acyl carrier protein reductase (FabI) as described supra further comprising screening the potential inhibitor for inhibition of the activity of FabI.

[0067] In certain embodiments, the present invention provides a method for identifying an inhibitor of an activity of enoyl-acyl carrier protein reductase (FabI) where the atomic structures coordinates for the atoms comprising the druggable regions of FabI are shown in Table 1.

[0068] In certain embodiments, the present invention provides a method for identifying an inhibitor of an activity of enoyl-acyl carrier protein reductase (FabI) where the FabI is from Burkholderia pseudomallei (Bpm) and the identified inhibitor is a drug for a BpmFabI associated disease.

[0069] In certain embodiments, the present invention provides a method for identifying an inhibitor of an activity of enoyl-acyl carrier protein reductase (FabI) where the FabI is from Burkholderia pseudomallei (Bpm) and the identified inhibitor is a drug for melioidosis.

[0070] In an aspect of these embodiments, the present invention provides a method for identifying an inhibitor of an activity of enoyl-acyl carrier protein reductase (FabI) where the FabI crystal comprises unit cell dimensions a=134.79 , b=63.44 , c=121.84 , ==90, =107.08 with a space group C121 to treat a Burkholderia associated disease.

[0071] In an aspect of these embodiments, the present invention provides a method for identifying an inhibitor of an activity of enoyl-acyl carrier protein reductase (FabI) where the FabI crystal comprises unit cell dimensions a=134.79 , b=63.44 , c=121.84 , ==90, =107.08 with a space group C121 to treat melioidosis.

[0072] In certain embodiments, the present invention provides a method for identifying or designing an inhibitor of the activity of BpmFabI for the treatment of BpmFabI associated diseases, particularly melioidosis, including: a) supplying a computer modeling application with a set of structure coordinates of a complex, including at least a portion of a druggable region from a BpmFabI; b) supplying the computer modeling application with a set of structure coordinates of a chemical entity; and c) determining whether the chemical entity is expected to bind to the complex, wherein binding to the complex is indicative of potential modulation of the activity of a BpmFabI.

[0073] In certain embodiments, the present invention provides a method for identifying or designing an inhibitor of the activity of BpmFabI, supplying a computer modeling application with a set of structure coordinates of a complex wherein the complex comprises at least a portion of a druggable region of a BpmFabI; supplying the computer modeling application with a set of structure coordinates for a chemical entity; evaluating the potential binding interactions between the chemical entity and active site of the molecular complex; structurally modifying the chemical entity to yield a set of structure coordinates for a modified chemical entity, and determining whether the modified chemical entity is expected to bind to the complex, wherein binding to the complex is indicative of potential modulation of the BpmFabI.

[0074] In certain embodiments, the present invention provides a method for obtaining structural information about a molecule or a molecular complex of unknown structure comprising: (a) crystallizing the molecule or molecular complex; (b) generating an x-ray diffraction pattern from the crystallized molecule or molecular complex; (c) applying at least a portion of the structure coordinates set forth in Table 1 to the x-ray diffraction pattern to generate a three-dimensional electron density map of at least a portion of the molecule or molecular complex whose structure is unknown.

[0075] Provided herein are crystalline structures comprising an enzyme enoyl-acyl carrier protein reductase (FabI) from, for example, Burkholderia pseudomallei (BpmFabI) as solved and described herein. The BpmFabI crystal provides information about the structure of the polypeptide comprising the FabI, such as shown in SEQ ID NO: 1, and druggable regions or domains and the like contained therein, all of which could help in the rational design and development of new FabI inhibitors to treat diseases associated with B. pseudomallei. Also provided are methods utilizing the crystalline structures to design modulators of one or more of their biological activities. In particular, the present invention provides a use of crystalline structure of BpmFabI to design therapeutic and diagnostic molecules. Moreover, modulators, inhibitors, agonists or antagonists against the polypeptides comprising the crystalline structure, or biological complexes containing them, or orthologous thereto, may be used to treat any disease or other treatable condition of a patient, including humans and animals, and particularly to treat a disease caused by or associated with Burkholderia pseudomallei.

[0076] The present invention demonstrates that AFN-1252 is a potent inhibitor of BpmFabI. Co-crystal structure and thermofluor data demonstrated that AFN-1252 forms a stable binary complex with BpmFabI. Kinetic studies show that AFN-1252 can compete with NADH, but the binding is uncompetitive with crotonyl-CoA. The results of the binding studies and identification of key interactions of AFN-1252 with BpmFabI are useful in designing and developing more potent BpmFabI inhibitors to treat, for example, but not limited to, melioidosis.

[0077] The following examples are used to illustrate the certain aspects and embodiments of the present invention in more details, and are not intended to limit the invention in any way.

Example 1

Inhibitory Potential of AFN-1252 on BpmFabI

a) Expression and Purification of BpmFabI Isoform 1

[0078] Burkholderia pseudomallei FabI (Isoform1) gene corresponding to amino acids 1 to 263 was synthesized and subcloned into pET21a vector. Transformants of E. coli BL21DE3 star strain containing pET21a-BpmFabI (isoform 1) was induced with 0.5 mM IPTG for 16 h at 18 C. Cell pellet was washed with lysis buffer (20 mM Tris pH 8.0, 500 mM NaCl, 1 mM PMSF and 5 mM Imidazole) and treated with 50 g/ml lysozyme for 30 min. Cells were lysed by sonication and the cell debris clarified by centrifugation at 12000 rpm for 40 min at 4 C. The supernatant was bound to Ni-NTA beads pre-equilibrated with 20 mM Tris pH 8.0, 500 mM NaCl and 5 mM imidazole buffer, and eluted with 500 mM imidazole. The fractions containing BpmFabI passed through Superdex-75 column in 20 mM Tris pH 8.0, 500 mM NaCl, 5 mM Imidazole.

b) BpmFabI Enzyme Inhibition Assay for AFN-1252

[0079] The potency of AFN-1252 to inhibit BpmFabI was evaluated in a spectrophotometric assay by monitoring the oxidation of the cofactor NADH. Buffer used for the assay was 30 mM PIPES, pH 6.8, containing 150 mM NaCl and 1 mM EDTA. The Michaelis Menton constant (Km) of crotonyl-CoA was determined from the enzyme activity at increasing concentrations of this cofactor (data not shown). AFN-1252 was pre-incubated with BpmFabI for 30 minutes and the reaction was started by adding substrate mix containing crotonyl-CoA (300 M) and NADH (375 M). The oxidation of NADH was monitored by following the decrease of absorbance at 340 nm. IC.sub.50 value was determined by fitting the dose-response data to sigmoidal dose response (variable slope) curve using Graphpad Prism software V4. To determine the mechanism of binding, kinetic studies were carried out at different concentrations of inhibitor and varying the concentration of NADH at a fixed concentration of crotonoyl CoA (300 M) and also by varying the concentrations of crotonoyl CoA keeping NADH concentration fixed at 375 M. Lineweaver-Burk plots were subsequently generated to determine the mechanism of binding of AFN-1252 to BpmFabI. Since AFN-1252 is reported to be a specific inhibitor of Gram-positive S. aureus, it was surprising to see that it is active against Gram-negative B. pseudomallei bacterium.

[0080] As shown in FIG. 4, AFN-1252 inhibited BpmFabI with an IC.sub.50 of 9.6 nM. Lineweaver-Burk plots were generated at different concentrations of AFN-1252 to understand the mechanism of inhibition of AFN-1252 in the presence of NADH and crotonyl-CoA. The concentration of either crotonyl-CoA or NADH was varied, keeping the other constant. The Lineweaver-Burk plot for AFN-1252 binding to BpmFabI at 300 M crotonyl-CoA and varying concentrations of NADH is shown in FIG. 5A. Increase in Km (seen as decrease in 1/[NADH]) with increasing concentrations of AFN-1252, and a corresponding decrease in Vmax (seen as increase in 1/Abs value) suggested mixed inhibition of BpmFabI in which AFN-1252 could bind to enzyme directly, and also to BpmFabI-NADH complex. The Lineweaver-Burk plot for AFN-1252 binding to BpmFabI at 375 uM NADH and varying concentrations of crotonyl-CoA is shown in FIG. 5B. Decrease in Km with corresponding decrease in Vmax suggested that AFN-1252 was uncompetitive with crotonyl-CoA for BpmFabI binding and the inhibitor was bound to BpmFabI-crotonyl-CoA complex.

c) Thermofluor Assay

[0081] Binding of AFN-1252 to BpmFabI was also monitored by thermofluor assay. AFN-1252 stabilized the enzyme as determined by the increase in melting temperature of 12 C. (FIG. 6 & Table 2). NADH did not have any effect on the Tm of BpmFabI or BpmFabI:AFN-1252 complex. For comparison, a similar experiment was carried out with Triclosan. Interestingly, Triclosan stabilized BpmFabI only in the presence of NADH (Tm of 9 C.); neither NADH nor Triclosan alone stabilized the protein. This data suggests the formation of a ternary complex of BpmFabI-Triclosan-NADH, indicating that AFN-1252 binding mechanism is different as compared to that of Triclosan. The results are given in Table 2.

TABLE-US-00001 TABLE 2 Stabilization effect of Triclosan/AFN-1252 on BpmFabI Ligand Tm ( C.) NADH 0 AFN-1252 12.76 1.26 AFN-1252 + NADH 11.61 0.10 Triclosan 0 Triclosan + NADH 8.49 0.32

Example 2

X-Ray Crystallography

a) Crystallization of BpmFabI Isoform 1

[0082] Co-crystals of BpmFabI with AFN-1252 were obtained using hanging drop vapor diffusion technique. Concentrated protein (15 mg/ml) was incubated overnight with 0.5 mM AFN-1252 and 1 mM NADH in a reservoir buffer containing 0.1 M MES pH 5.0, 0.1 M NaCl, 10% PEG 3350.

b) X-Ray Data Collection and Structure Determination

[0083] Co-crystals of BpmFabI with AFN-1252 were flash frozen at 100K using 20% glycerol as cryo-protectant. The diffraction data was collected using in-house Rigaku RU300 X-ray generator with R-AXIS IV++detector to a maximum resolution of 2.3 . Data indexing, integration and scaling were performed using DENZO and SCALEPACK. The structure was solved by molecular replacement (MR) method using the search model with PDB Code 3EK2. Alternate cycles of restrained refinement and manual rebuilding were performed with the programs REFMAC 5.2.0001 and Coot respectively. Five percent of the reflections were randomly excluded from the refinement to monitor the free residual-factor (R.sub.free). A summary of the data reduction and structure refinement statistics is provided in Table 3.

TABLE-US-00002 TABLE 3 Diffraction data and structure refinement statistics of BpmFabI: AFN-1252 Complex Space Group C121 Cell Parameters () a = 134.79, b = 63.44, c = 121.84, = = 90, = 107.08 Resolution range () 40-2.26 Total no. reflections 114,107 Unique reflections 42,792 Completeness (%) 93.11 (89.9)a* Rsym 0.062 (0.157) I/I 11.8 (2.6) Multiplicity 2.7 (2.7) Refinement Resolution () 40-2.26 No. of reflections 40,619 Completeness (%) 92.48 (79.28) Rwork/Rfree 0.161/0.250 r.m.s. deviations Bond lengths () 0.015 Bond angles () 1.855 Ramachandran plot Residues in the most favored region 96.5 (%) Residues in the allowed region (%) 3.5 a*Values corresponding to the outermost shell are given within parentheses.

Example 3

Analysis of X-Ray Structure of BpmFabI

a) Monomer and Quaternary Structures of the BpmFabI

[0084] The BpmFabI monomer contains a single domain composed of a seven-stranded parallel -sheet (1, 2, 3, 4, 5 6, 7) sandwiched by three -helices from the top (1, 2, 3) and three from the bottom (4, 5, 6). Another helix, 7 is located at the C terminal tail of the protein (FIG. 7). The overall fold is common to dinucleotide-binding FabI enzymes and resembles the typical Rossmann fold. The structural superposition showed an r.m.s. deviation of 1.047 , 0.87 and 1.07 respectively for Bpm (256 Ca atoms; PDB; 3EK2), Ec (251 Ca atoms; PDB; 4JQC) and ScFabI (242 Ca atoms; PDB; 4FS3) crystal structures.

[0085] The crystallographic asymmetric unit consists of four protomers arranged as a tetramer with an approximate 222 symmetry (FIG. 8). The elements involved in the tetramer formation include the 7 strand, helices 4 and 5, and the C-terminal tail which cross over to the neighboring monomer. The helices 4 and 5 from the two monomers interact with each other to create a strong dimer. The 7 strand from the seven-stranded n-sheet of each dimer associates to form an extended 14 stranded -sheet, which facilitate the formation of a stable tetrameric assembly. This larger assembly may represent the biologically active form of BpmFabI. This is further supported by analytical gel filtration experiments which indicated a tetrameric protein in solution.

b) Binding Site Structure of AFN-1252 in BpmFabI

[0086] The electron density for AFN-1252 was clearly visible in the active site cavity in all four subunits. The flexible loop containing protein residues 194 to 204 accommodates the AFN-1252 molecule in the binding pocket. AFN-1252 binding to BpmFabI is mediated through multiple chemical interactions (FIG. 9). The tetrahydronaphthyridone moiety of the molecule makes two hydrogen bonds to the main chain atoms of Ala95. A water mediated interaction is observed between the main chain NH atom of Arg97 and the CO group of napthyridinone ring. The amide CO group of the inhibitor also forms hydrogen bonds with the side chain of Tyr156 and a water mediated interaction with Lys163 side chain. The 3-methylbenzofuran moiety is located in a hydrophobic pocket created by the side chains of Ile100, Tyr146, Tyr156, Pro191, Phe203 and Ile206.

[0087] Comparison of crystal structures of AFN-1252:BpmFabI complex with the apo-BpmFabI (PDB-3EK2) revealed no major conformational changes except in the active site region. Near the active site, the loop containing residues Thr194-Lys199 adopts ordered conformation as a result of AFN-1252 binding, whereas the same loop is highly disordered in the apo structure. Thus the change in loop conformation appears to have been induced by the AFN-1252 binding. Further, involvement of this flexible loop in the formation of binary/ternary complexes across FabIs is well established.

[0088] From this analysis, it was found that AFN-1252 formed binary complex with BpmFabI which was compared with reported ternary complexes of AFN-1252 and co-factor bound to Ec (PDB-4JQC) and SaFabI (PDB-4FS3) wherein strong hydrogen bonds and - contacts were observed between the co-factor and AFN-1252. Comparison of AFN-1252 bound binary and ternary complex structures revealed no major conformational changes due to the presence or absence of the co-factor in the active site (FIG. 10). Thus the BpmFabI binary complex structure establishes that an inhibitor binding is feasible in the absence of the cofactor. This finding suggests that inhibitors for BpmFabI can be designed by considering interactions solely with protein residues. The binary BpmFabI:AFN-1252 comples was deposited and assigned protein data base accession code 4RLH.

[0089] The co-crystal structure of AFN-1252: BpmFabI complex shows that the inhibitor binds to the enzyme as a binary complex. This binding mode is different from the reported ternary complex of EcFabI/SaFabI with Triclosan and NADH. Binding of AFN-1252 to FabI is mediated by hydrogen bonding interactions and further stabilized by - interactions. AFN-1252 stabilized the enzyme in the absence of NADH which was in contrast with Triclosan wherein stabilization effect on BpmFabI was observed only when both NADH and Triclosan were present. Mixed mode of inhibition observed in kinetic studies also shows that AFN-1252 can compete with NADH for BpmFabI binding site.

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[0130] All publications and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.