Abstract:
The crystallized complex of NADH and Inha enzyme from Mycobacterium tuberculosis is presented. Methods of designing inhibitors to the Inha enzyme and subsequent treatment with those inhibitors of infection by M. tuberculosis are disclosed.

Description:
STATEMENT OF GOVERNMENT INTEREST 
     This invention was made with government support under NIH Grant Numbers AI33696 and AI27160. As such, the government has certian rights in the invention. 
    
    
     CROSS REFERENCE TO RELATED APPLICATION 
     This Application is a continuation of U.S. Application No. 08/307,376, filed Sep. 16, 1994, now abandoned which is a Continuation-In-Part of U.S. application Ser. No. 08/234,011 filed Apr. 28, 1994, currently pending. 
    
    
     FIELD OF THE INVENTION 
     This invention is directed to InhA enzyme crystals, to the use of said InhA enzyme crystals to determine the three dimensional structure of InhA enzyme and to the three dimensional structure of said InhA enzyme. The three dimensional structure of the InhA enzyme allows for the development of compounds which inhibit the biochemical activity of InhA enzyme in bacteria. Said compounds are developed and administered to treat bacterial infection. 
     BACKGROUND OF THE INVENTION 
     Tuberculosis remains the largest cause of death in the world from a single infectious disease and is responsible for one in four avoidable adult deaths in developing countries. Infection with drug-sensitive strains of Mycobactrium tuberculosis can be effectively cured with a combination of isoniazid, ethionamide, rifampicin and pyrazinamide. However, the emergence of multiple drug resistant strains of M. tuberculosis has resulted in fatal outbreaks in the United States. 
     Isoniazid was first reported to be active against M. tuberculosis in 1952, when it was shown to have a highly specific activity against M. tuberculosis and M. bovis, with less but considerable activity against other mycobacteria. Although isoniazid is one of the most widely used anti-tuberculosis drugs for both therapy and prophylaxis, its precise target of action on Mycobactrium tuberculosis has remained unknown. Isoniazid was first synthesized as an organic compound in 1912, but it was not until 1952 that three independent groups discovered that it had anti-tuberculosis activity. The discovery that ethionamide had anti-tuberculosis activity was predicated on the discovery that nicotinamide showed some tuberculostatic activity against M. tuberculosis. 
     Resistance to isoniazid was first reported in 1953, but in recent years has been as high as 26% in some areas of the United States. A fraction of isoniazid-resistant strains had been shown to be associated with a loss of catalase activity (see Lefford et al., Tubercle, Vol. 47, p. 109 (1966) and Stoecle et al., J. Inf. Dis., Vol. 168, p. 1063 (1993)). The catalase gene (katG) was recently cloned and deletions of this gene were shown to be correlated with isoniazid resistance in certain M. tuberculosis isolates (see Zhang et al., Nature, Vol. 358, pp. 591-593 (1992)). Furthermore, transfer of the M. tuberculosis katG gene to isoniazid-resistant M. smegmatis strains results in the acquisition of isoniazid-sensitivity, suggesting that the presence of the catalase activity results in the sensitivity of M. tuberculosis to isoniazid (see Middlebrook, Am. Rev. Tuberc., Vol. 65, pp. 765-767 and Zhang et al., Molec. Microbiol., Vol. 8, pp. 521-529 (1993)). 
     Although catalase may be important to the action of isoniazid, it does not appear to be the target of action of the drug. Isoniazid-resistance can be accounted for by the loss of catalase activity; however, only 25% of isoniazid-resistant isolates appear to be catalase-negative. Previous studies have shown that low-level isoniazid resistance correlated not with the loss of catalase activity, but rather with the co-acquisition of ethionamide resistance (see Canetti, Am. Rev. Respir. Dis., Vol. 92, p. 687 (1965); Grumbach, Rev. Tuber., Vol. 25, p. 1365 (1961); Lefford, Tubercle, Vol. 47, p. 198 (1966) and Hok, Am. Rev. Respir. Rev., Vol. 90, pp. 468-469 (1964)). 
     Drug resistance can often be mediated by the accumulation of mutations in the gene encoding the targets that result in reduced binding of drugs to their targets. For example, rifampicin resistance is often mediated by mutations in the gene encoding the β&#39; subunit of RNA polymerase. Alternatively, trimethoprim resistance can be mediated by mutations causing amplification in a target dihydrofolate reductase. 
     Without the availability of genetic systems for the mycobacteria, the identification of the precise target of action of isoniazid and ethionamide could not be determined. Hence, it has been desirable to identify the specific point mutations that confer resistance to isoniazid and ethionamide in M. tuberculosis. The enzyme which is the target of action of isoniazid has been identified and denoted as InhA, and the gene which encodes the enzyme InhA has been denoted inhA (see Banerjee et al., Science, Vol. 263, pp. 227,230 (January 1994)). As used herein, &#34;InhA&#34; includes InhA enzyme and any mutants thereof. 
     The inhA gene shares significant homology with a gene which codes for the EnvM protein from E. coli and Salmonella typhimurium, which is known to be involved in fatty acid (lipid or mycolic acid) biosynthesis. The enzyme InhA, encoded by the inhA gene, is necessary for mycolic acid biosynthesis. 
     Mycolic acids, also referred to herein as lipids, are long chain fatty acids (60 to 80 carbons in lengths) that are major constituents of a mycobacterial cell wall. They are thought to be the chemical moieties responsible for the characteristic acid-fastness of mycobacteria and form the waxy layer of mycobacterial cells. Mycolic acids have been demonstrated to have covalent linkages to arabino-galactans and thus maintain the integrity of the mycobacterial cell wall. Inhibition in their syntheses would result in a disruption of the cell wall and the death of the mycobacteria. Since mycolic acids are unique to the mycobacteria, mycolic acid biosynthetic enzymes are excellent targets for development of drugs of use in the treatment of mycobacterial infection. However, in order to develop drugs capable of inhibiting InhA activity, it is necessary to have InhA crystals from which the three dimensional structure of InhA enzyme can be determined. 
     It is therefore an object of this invention to provide InhA enzyme crystals. 
     It is another object of this invention to provide a method of determining the three dimensional structure of InhA enzyme utilizing said crystals. 
     It is a further object of this invention to provide the three dimensional structure of InhA enzyme. 
     It is a still further object of this invention to provide a method of treating mycobacterial infection utilizing compounds which block the biochemical activity of InhA enzyme. 
     SUMMARY OF THE INVENTION 
     This invention is directed to an isolated InhA enzyme comprising a first sub-structure which is a core α/βstructure composed of six parallel β strands surrounded and interwoven by four α-helices and a second sub-structure composed of two α-helices interconnected by a loop. This invention is further directed to a method of determining the three dimensional structure of the InhA enzyme by determining the structure of InhA crystals utilizing multiple isomorphous replacement, and developing a polyalanine model of the crystals, thereby obtaining the three dimensional structure of the crystals. 
     In addition, this invention is directed to a method of treating M. tuberculosis infection comprising the determination of the three dimensional structure of InhA enzyme from M. tuberculosis, utilization of said three dimensional structure to develop a compound which binds to said enzyme and contacting said compound with said enzyme, thereby inhibiting the biochemical activity of said enzyme and treating M. tuberculosis infection. 
    
    
     BRIEF DESCRIPTION OF THE DRAWING 
     The above brief description, as well as further objects and features of the present invention, will be more fully understood by reference to the following detailed description of the presently preferred, albeit illustrative, embodiments of the present invention when taken in conjunction with the accompanying drawing wherein: 
     FIG. 1 represents a ribbon strand diagram of the three dimensional structure of InhA enzyme from M. tuberculosis. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     InhA enzyme was overexpressed in a commercially available E. coli system and purified utilizing the nucleic acid sequence of InhA. The sequence of InhA is as follows: 
     
         __________________________________________________________________________SEQ ID NO: 1__________________________________________________________________________AGCGCGACAT     ACCTGCTGCG               CAATTCGTAG                         GGCGTCAATA                                   CACCCGCAGC                                             CAGGGCCTCG                                                       60CTGCCCAGAA     AGGGATCCGT               CATGGTCGAA                         GTGTGCTGAG                                   TCACACCGAC                                             AAACGTCACG                                                       120AGCGTAACCC     CAGTGCGAAA               GTTCCCGCCG                         GAAATCGCAG                                   CCACGTTACG                                             CTCGTGGACA                                                       180TACCGATTTC     GGCCCGGCCG               CGGCGAGACG                         ATAGGTTGTC                                   GGGGTGACTG                                             CCACAGCCAC                                                       240TGAAGGGGCC     AAACCCCCAT               TCGTATCCCG                         TTCAGTCCTG                                   GTTACCGGAG                                             GAAACCGGGG                                                       300GATCGGGCTG     GCGATCGCAC               AGCGGCTGGC                         TGCCGACGGC                                   CACAAGGTGG                                             CCGTCACCCA                                                       360CCGTGGATCC     GGAGCGCCAA               AGGGGCTGTT                         TGGCGTCGAA                                   TGTGACGTCA                                             CCGACAGCGA                                                       420CGCCGTCGAT     CGCGCCTTCA               CGGCGGTAGA                         AGAGCACCAG                                   GGTCCGGTCG                                             AGGTGCTGGT                                                       480GTCCAACGCC     GGCCTATCCG               CGGACGCATT                         CCTCATGCGG                                   ATGACCGAGG                                             AAAAGTTCGA                                                       540GAAGGTCATC     AACGCCAACC               TCACCGGGGC                         GTTCCGGGTG                                   GCTCAACGGG                                             CATCGCGCAG                                                       600CATGCAGCGC     AACAAATTCG               GTCGAATGAT                         ATTCATAGGT                                   TCGGTCTCCG                                             GCAGCTGGGG                                                       660CATCGGCAAC     CAGGCCAACT               ACGCAGCCTC                         CAAGGCCGGA                                   GTGATTGGCA                                             TGGCCCGCTC                                                       720GATCGCCCGC     GAGCTGTCGA               AGGCAAACGT                         GACCGCGAAT                                   GTGGTGGCCC                                             CGGGCTACAT                                                       780CGACACCGAT     ATGACCCGCG               CGCTGGATGA                         GCGGATTCAG                                   CAGGGGGCGC                                             TGCAATTTAT                                                       840CCCAGCGAAG     CGGGTCGGCA               CCCCCGCCGA                         GGTCGCCGGG                                   GTGGTCAGCT                                             TCCTGGCTTC                                                       900CGAGGATGCG     AGCTATATCT               CCGGTGCGGT                         CATCCCGGTC                                   GACGGCGGCA                                             TGGGTATGGG                                                       960CCACTGACAC     AACACAAGGA               CGCACATGAC                         AGGACTGCTG                                   GACGGCAAAC                                             GGATTCTGGT                                                       1020TAGCGGAATC     ATCACCGACT               CGTCGATCGC                         GTTTCACATC                                   GCACGGGTAG                                             CCCAGGAGCA                                                       1080GGGCGCCCAG     CTGGTGCTCA               CCGGGTTCGA                         CCGGCTGCGG                                   CTGATTCAGC                                             GCATCACCGA                                                       1140CCGGCTGCCG     GCAAAGGCCC               CGCTGCTCGA                         ACTCGACGTG                                   CAAAACGAGG                                             AGCACCTGGC                                                       1200CAGCTTGGCC     GGCCGGGTGA               CCGAGGCGAT                         CGGGGCGGGC                                   AACAAGCTCG                                             ACGGGGTGGT                                                       1260GCATTCGATT     GGGTTCATGC               CGCAGACCGG                         GATGGGCATC                                   AACCCGTTCT                                             TCGACGCGCC                                                       1320CTACGCGGAT     GTGTCCAAGG               GCATCCACAT                         CTCGGCGTAT                                   TCGTATGCTT                                             CGATGGCCAA                                                       1380GGCGCTGCTG     CCGATCATGA               ACCCCGGAGG                         TTCCATCGTC                                   GGCATGGACT                                             TCGACCCGAG                                                       1440CCGGGCGATG     CCGGCCTACA               ACTGGATGAC                         GGTCGCCAAG                                   AGCGCGTTGG                                             AGTCGGTCAA                                                       1500CAGGTTCGTG     GCGCGCGAGG               CCGGCAAGTA                         CGGTGTGCGT                                   TCGAATCTCG                                             TTGGCGCAGG                                                       1560CCCTATCCGG     ACGCTGGCGA               TGAGTGCGAT                         CGTCGGCGGT                                   GCGCTCGGCG                                             AAGAGGCCGG                                                       1620CGCCCAGATC     CAGCTGCTCG               AGGAGGGCTG                         GGATCAGCGC                                   GCTCCGATCG                                             GCTGGAACAT                                                       1680GAAGGATGCG     ACGCCGGTCG               CCAAGACGGT                         GTGCGCGCTG                                   CTGTCTGACT                                             GGCTGCCGGC                                                       1740GACCACGGGT     GACATCATCT               ACGCCGACGG                         CGGCGCGCAC                                   ACCCAATTGC                                             TCTAGAACGC                                                       1800ATGCAATTTG     ATGCCGTCCT               GCTGCTGTCG                         TTCGGCGGAC                                   CGGAAGGGCC                                             CGAGCAGGTG                                                       1860CGCCCGTTCC     TGGAGAACGT               TACCCGGGGC                         CGCGGTGTGC                                   CTGCCGAACG                                             GTTGGACGCG                                                       1920GTGGCCGAGC     ACTACCTGCA               TTTCGGTGGG                         GTATCACCGA                                   TCAATGGCAT                                             TAATCGCACA                                                       1980CTGATCGCGG     AGCTGGAGGC               GCAGCAAGAA                         CTGCCGGTGT                                   ACTTCGGTAA                                             CCGCAACTGG                                                       2040GAGCCGTATG     TAGAAGATGC               CGTTACGGCC                         ATGCGCGACA                                   ACGGTGTCCG                                             GCGTGCAGCG                                                       2100GTCTTTGCGA     CATCTGCGTG               GAGCGGTTAC                         TCGAGCTGCA                                   CACAGTACGT                                             GGAGGACATC                                                       2160GCGCGGCCCC     CCGCGCGGCC               GGGCGCGACG                         CGCCTGAACT                                   GGTAAAACTG                                             CGGCCCTACT                                                       2220TCGACCATCC     GCTGTTCGTC               GAGATGTTCG                         CCGACGCCAT                                   CACCGCGGCC                                             GCCGCAACCG                                                       2280TGCGCGGTGA     TGCCCGGCTG               GTGTTCACCG                         CGCATTCGAT                                   CCCGACGGCC                                             GCCGACCGCC                                                       2340GCTGTGGCCC     CAACCTCTAC               AGCCGCCAAG                         TCGCCTACGC                                   CACAAGGCTG                                             GTCGCGGCCG                                                       2400CTGCCGGATA     CTGCGACTTT               GACCTGGCCT                         GGCAGTCGAG                                   ATCGGGCCCG                                             CCGCAGGTGC                                                       2460CCTGGCTGGA     GCCAGACGTT               ACCGACCAGC                         TCACCGGTCT                                   GGCTGGGGCC                                             GGCATCAACG                                                       2520CGGTGATCGT     GTGTCCCATT               GGATTCGTCG                         CCGACCATAT                                   CGAGGTGGTG                                             TGGGATCTCG                                                       2580ACCACGAGTT     GCGATTACAA               GCCGAGGCAG                         CGGGCATCGC                                   GTACGCCCGG                                             GCCAGCACCC                                                       2640CCAATGCCGA     CCCGCGGTTC               GCTCGACTAG                         CCAGAGGTTT                                   GATCGACGAA                                             CTCCGTTACG                                                       2700GCCGTATACC     TGCGCGGGTG               AGTGGCCCCG                         ATCCGGTGCC                                   GGGCTGTCTG                                             TCCAGCATCA                                                       2760ACGGCCAGCC     ATGCCGTCCG               CCGCACTGCG                         TGGCTAGCGT                                   CAGTCCGGCC                                             AGGCCGAGTG                                                       2820CAGGATCGCC     GTGACCGCGG               ACATCCGGGC                         CGAGCGCACC                                   ACGGCGGTCA                                             ACGGTCTCAA                                                       2880CGCATCGGTG     GCACGCTGAG               CGTCCGACAA                         CGACTGCGTT                                   CCGATCGGCA                                             ATCGACTCAG                                                       2940CCCGGCACTG     ACCGCGATGA               TCGCATCGAC                         GTGCGCGGCA                                   TTCTCGAGCA                                             CCCGCAATGC                                                       3000GCGCGATGGC     GCGTGGTCGG               GAACCCGGTG                         TTGCCGTGAC                                   GATTCGAGCA                                             ACTGCTCGAC                                                       3060GAGGCCACGG     GGCTTGGCGA               CGTCGCTAGA                         TCCCAGTCCG                                   ATGGTGCTCA                                             AGGCTTCGGC                                                       3120__________________________________________________________________________ 
    
     In order to determine the three dimensional structure of InhA enzyme, recombinant InhA from M. tuberculosis was purified. The InhA:β-Nicotinamide adenine dinucleotide, reduced and oxidized (NADH) complex was crystallized by the hanging drop vapor diffusion method, where 3 μl of protein solution (13 mg/ml InhA, in a 1:2 ratio with NADH) were mixed with 3 μl or precipitant solution (50 mM HEPES pH 7.2, 8-12% methyl pentane diol (MPD), 50 mM sodium citrate pH 6.2) on a silanized coverslip which was inverted and sealed above 700 μl of the precipitant solution. 
     Single crystals of up to 0.6 mm 3  in size were grown in this way at 19° C. within three weeks. The crystals were hexagonal in shape and were of the space group P6 2  22. The InhA crystals grown had unit cell dimensions of a=b=100.1 Å, c=140.4 Å, and α=β=90°, γ=120°. There was one monomer per asymmetric unit, and the solvent content of the crystals was approximately 60%. Two heavy atom derivatives (p-(chloromercury)- phenyl sulfonate (PCMPS), and Hg(C 2  H 3  O 2 )) were prepared and used to determine the three dimensional structure of the crystals. 
     A mercury acetate derivative of the crystals was collected after a native crystal (containing NADH) was soaked overnight in 1 mM C2H302.Hg and 10% MPD, 50 mM HEPES pH 7.2, 50 mMNa-citrate pH 6.2. The PCMPS derivative was obtained by pre-reacting the protein (13 mg/ml in 10 mM HEPES, pH 7.2, with 1:2 ratio with NADH) with 10 mM PCMPS for approximately 30 minutes at 19° C. and then crystallizing the complex under the same conditions that gave native crystals. Crystals of the InhA complex were hexagonal and isomorphous with the native form and were used in multiple isomorphous replacement (MIR) procedures to determine the three dimensional structure of the InhA enzyme. 
     Heavy derivatives (PCMPS, mercury acetate, and lead acetate) of the P6 2  22 crystals of InhA were used to determine the three dimensional structure of InhA. The lead derivative was collected after a native crystal, originally produced in the presence of a 2:1 NADH:protein ratio, was soaked overnight in 1 mM C 4  H 6  O 4  Pb in 0.1M Na acetate, 0.1M Na HEPES, 10% MPD (methyl pentane diol), pH 6.5. The mercury derivative was collected after a native crystal grown in the same fashion was soaked overnight in 1 mM C 4  H 6  O 4  Hg in 0.1 Na citrate, 0.1M Na HEPES, 10% MPD, pH 7.2. The PCMPS derivative was obtained by mixing the protein (10 mg/mL in 10 mM HEPES, pH 7.2) with a 6-fold molar excess of PCMPS overnight at 19° C. and then crystallizing the complex under the same conditions that gave native crystals. A heavy atom derivative of InhA with PCMPS can also be obtained by utilizing the same procedure as in the lead acetate experiment, but with lower metal occupancy. Crystals of the InhA-PCMPS were hexagonal with the native form and were used in the MIR procedures. 
     Heavy atom binding positions were found using Patterson maps. The heavy atom binding positions (as calculated from difference Patterson maps) were refined by an iterative series of phase refinement, using the package PHASES (W. Furey, VA Medical School and University of Pittsburgh, Pa.), and XtalView (see McRee et al. (1993)), running on a Silicon Graphics. Iris computer. Solvent flattening (Wang, 1985) procedures, as implemented in PHASES, were used to further improve the MIR phases. From the resulting electron density map (up t 2.8 Å), a partial model of InhA was built. 
     All data sets were collected on a Siemens multiwire area detector, using a Rigaku RU-200 rotating anode X-ray source operating at 55 kV and 85 mA. Data were reduced using the Siemens package XENGEN (Siemens Analytica X-ray Instruments, Inc., Madison, Wis.) on a Silicon Graphics Iris computer. For the native data set, the R-merge on intensities was 9.6% to 2.2 Åfor 23880 reflections (81% complete). The PCMPS derivative had an R-merge on intensities of 13.9% for 26375 reflections to 2.5 Åresolution. The HG(C 2  H 3  O 2 ) derivative had an R-merge on intensities of 14.3% for 26261 reflections at 2.5 Åresolution. 
     The three dimensional structure of InhA was determined using multiple isomorphous replacement data collected from the derivatives. Table 1, below, summarizes the statistics for phase determination. 
     
                                           TABLE 1__________________________________________________________________________HEAVY ATOM DERIVATIVES OF INHAFROM MYCOBACTERIUM TUBERCULOSISHEAVY ATOM  CONCENT              R.sub.sym                 R.sub.merge                     EXT. Diffr. (Å)                             N.sup.o SITES                                   PHASING POWER__________________________________________________________________________Hg(C.sub.2 H.sub.3 O.sub.2).sub.2       1 mM   0.143                 0.106                     2.5     1     1.55PCMPS CO-CRYSTAL       2 mM   0.139                 0.107                     2.5     4     1.60__________________________________________________________________________ 
    
     Data produced a mean figure of merit of 0.499 for 11061 phased reflections with F &gt;1δ. Solvent flattening (Wang, 1985) procedures, as implemented in PHASES, were used to further improve the MIR phases. From the resulting electron density map, a partial polyalanine model was built using the program TOM, a derivative of FRODO (Jones, 1985), displayed on an Iris Graphics workstation. 
     The polyalanine model was refined using molecular dynamics and energy minimization (see Brunger et al. (1987)). In the first step, the simulated annealing procedure &#34;slow cool&#34; (see Brunger (1992)) was used. Electron density maps (both 2|F o  -F c  | and |F o  -F c  |) were using the atomic coordinates of the polyalanine model. Subsequently, the use of a combination of the MIR map and combined maps (maps obtained combining model-based and MIR phases) allowed for the tracing of the complete model and the incorporation of the complete amino acid sequence, as well as the bound NADH moiety. 
     It was determined by the inventors that recombinant InhA from M. tuberculosis is a single-domain enzyme, shown as a ribbon strands diagram in FIG. 1. Two substructures can be identified in the protein. The first substructure is a core α/β structure composed of six parallel β strands surrounded and interwoven by four α-helices, harboring the N-terminal section of the macromolecule. The second substructure is a C-terminal region, composed mainly of two α-helices interconnected by a short loop. The topology of substructure 1 emulates that of the dinucleotide binding fold of many dehydrogenases in that it contains a twisted β-sheet in the middle, surrounded by α-helices. 
     This substructure can be divided into two sections. The first section consists of two β strands (B-1 and B-2) and two short α-helices (A-1 and A-2). This section is connected to the second section of the fold by a third β strand (B-3), which crosses over to the other side of the structure. The second part of the fold consists of an α-helix (A-3), connected by a long loop to the 4th 14-residue β strand (B-4). A fourth α-helix (A-4) connects into a fifth β strand (B-5), which is followed by a 25-residue α-helix (A-5). This structure then connects into a sixth β strand (B-6), which is the last secondary structural motif in the nucleotide binding fold. The second part of the nucleotide-binding fold is unusual in that the helices are of very long nature. The longest α-helix, A-5, may be interacting with the carboxyl terminal helices. 
     A short loop connects the nucleotide binding fold to the carboxyl terminal domain, which consists of a short β strand (B-7) followed by two helices (A-6 and A-7) interconnected by a 5-residue loop. The C-terminal portion of the molecule consists of two other α-helical structures. 
     The active site of InhA lies on a cavity on the surface of the molecule, formed by the carboxyl termini of the β sheets which participate in the α/β core and two α-helices, A-5 and A-6. NADH lies in an extended conformation along the top of the carboxyl termini of the core sheet, in a binding manner which is commonly observed in dinucleotide binding enzymes. The substrate binding site is in the hydrophobic cavity composed of helices 4, 5 and 6, which are highly rich in hydrophobic residues. The hydrophobic nature of this cavity likely renders it optimal for the accommodation of the lipid substrate in close proximity to the nicotinamide moiety of NADH. It is also likely that the three aforementioned helices form a core which acts as a flexible diaphragm which expands upon substrate binding. 
     The three dimensional structure of InhA enzyme can be utilized to develop compounds which bind to InhA enzyme thereby inhibiting the biochemical activity of InhA enzyme, such as mycolic acid biosynthesis. Specifically, compounds can be designed which bind to the active site and/or the NADH region on the InhA enzyme to inhibit the biochemical activity of the InhA enzyme. Hence, the compounds which are developed utilizing the three dimensional structure of InhA enzyme can be administered to treat M. tuberculosis infection. 
     Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of various aspects of the invention. Thus, it is to be understood that numerous modifications may be made in the illustrative embodiments and other arrangements may be devised without departing from the spirit and scope of the invention. 
     
         __________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 1(2) INFORMATION FOR SEQ ID NO: 1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 3120(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: both(ii) MOLECULE TYPE: DNA(A) DESCRIPTION:(iii) HYPOTHETICAL: No(iv) ANTI-SENSE:(v) FRAGMENT TYPE:(vi) ORIGINAL SOURCE: inhA operon(A) ORGANISM: M tuberculosis(B) STRAIN:(C) INDIVIDUAL ISOLATE:(D) DEVELOPMENTAL STAGE:(E) HAPLOTYPE:(F) TISSUE TYPE:(G) CELL TYPE:(H) CELL LINE:(I) ORGANELLE:(vii) IMMEDIATE SOURCE: M tuberculosis(viii) POSITION IN GENOME:(A) CHROMOSOME/SEGMENT:(B) MAP POSITION:(C) UNITS:(ix) FEATURE:(A) NAME/KEY:(B) LOCATION:(C) IDENTIFICATION METHOD:(D) OTHER INFORMATION:(x) PUBLICATION INFORMATION: None(A) AUTHORS:(B) TITLE:(C) JOURNAL:(D) VOLUME:(F) PAGES:(G) DATE:(H) DOCUMENT NUMBER:(I) FILING DATE:(J) PUBLICATION DATE:(K) RELEVANT RESIDUES IN SEQ ID NO:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:AGCGCGACATACCTGCTGCGCAATTCGTAGGGCGTCAATACACCCGCAGCCAGGGCCTCG60CTGCCCAGAAAGGGATCCGTCATGGTCGAAGTGTGCTGAGTCACACCGACAAACGTCACG120AGCGTAACCCCAGTGCGAAAGTTCCCGCCGGAAATCGCAGCCACGTTACGCTCGTGGACA180TACCGATTTCGGCCCGGCCGCGGCGAGACGATAGGTTGTCGGGGTGACTGCCACAGCCAC240TGAAGGGGCCAAACCCCCATTCGTATCCCGTTCAGTCCTGGTTACCGGAGGAAACCGGGG300GATCGGGCTGGCGATCGCACAGCGGCTGGCTGCCGACGGCCACAAGGTGGCCGTCACCCA360CCGTGGATCCGGAGCGCCAAAGGGGCTGTTTGGCGTCGAATGTGACGTCACCGACAGCGA420CGCCGTCGATCGCGCCTTCACGGCGGTAGAAGAGCACCAGGGTCCGGTCGAGGTGCTGGT480GTCCAACGCCGGCCTATCCGCGGACGCATTCCTCATGCGGATGACCGAGGAAAAGTTCGA540GAAGGTCATCAACGCCAACCTCACCGGGGCGTTCCGGGTGGCTCAACGGGCATCGCGCAG600CATGCAGCGCAACAAATTCGGTCGAATGATATTCATAGGTTCGGTCTCCGGCAGCTGGGG660CATCGGCAACCAGGCCAACTACGCAGCCTCCAAGGCCGGAGTGATTGGCATGGCCCGCTC720GATCGCCCGCGAGCTGTCGAAGGCAAACGTGACCGCGAATGTGGTGGCCCCGGGCTACAT780CGACACCGATATGACCCGCGCGCTGGATGAGCGGATTCAGCAGGGGGCGCTGCAATTTAT840CCCAGCGAAGCGGGTCGGCACCCCCGCCGAGGTCGCCGGGGTGGTCAGCTTCCTGGCTTC900CGAGGATGCGAGCTATATCTCCGGTGCGGTCATCCCGGTCGACGGCGGCATGGGTATGGG960CCACTGACACAACACAAGGACGCACATGACAGGACTGCTGGACGGCAAACGGATTCTGGT1020TAGCGGAATCATCACCGACTCGTCGATCGCGTTTCACATCGCACGGGTAGCCCAGGAGCA1080GGGCGCCCAGCTGGTGCTCACCGGGTTCGACCGGCTGCGGCTGATTCAGCGCATCACCGA1140CCGGCTGCCGGCAAAGGCCCCGCTGCTCGAACTCGACGTGCAAAACGAGGAGCACCTGGC1200CAGCTTGGCCGGCCGGGTGACCGAGGCGATCGGGGCGGGCAACAAGCTCGACGGGGTGGT1260GCATTCGATTGGGTTCATGCCGCAGACCGGGATGGGCATCAACCCGTTCTTCGACGCGCC1320CTACGCGGATGTGTCCAAGGGCATCCACATCTCGGCGTATTCGTATGCTTCGATGGCCAA1380GGCGCTGCTGCCGATCATGAACCCCGGAGGTTCCATCGTCGGCATGGACTTCGACCCGAG1440CCGGGCGATGCCGGCCTACAACTGGATGACGGTCGCCAAGAGCGCGTTGGAGTCGGTCAA1500CAGGTTCGTGGCGCGCGAGGCCGGCAAGTACGGTGTGCGTTCGAATCTCGTTGGCGCAGG1560CCCTATCCGGACGCTGGCGATGAGTGCGATCGTCGGCGGTGCGCTCGGCGAAGAGGCCGG1620CGCCCAGATCCAGCTGCTCGAGGAGGGCTGGGATCAGCGCGCTCCGATCGGCTGGAACAT1680GAAGGATGCGACGCCGGTCGCCAAGACGGTGTGCGCGCTGCTGTCTGACTGGCTGCCGGC1740GACCACGGGTGACATCATCTACGCCGACGGCGGCGCGCACACCCAATTGCTCTAGAACGC1800ATGCAATTTGATGCCGTCCTGCTGCTGTCGTTCGGCGGACCGGAAGGGCCCGAGCAGGTG1860CGCCCGTTCCTGGAGAACGTTACCCGGGGCCGCGGTGTGCCTGCCGAACGGTTGGACGCG1920GTGGCCGAGCACTACCTGCATTTCGGTGGGGTATCACCGATCAATGGCATTAATCGCACA1980CTGATCGCGGAGCTGGAGGCGCAGCAAGAACTGCCGGTGTACTTCGGTAACCGCAACTGG2040GAGCCGTATGTAGAAGATGCCGTTACGGCCATGCGCGACAACGGTGTCCGGCGTGCAGCG2100GTCTTTGCGACATCTGCGTGGAGCGGTTACTCGAGCTGCACACAGTACGTGGAGGACATC2160GCGCGGCCCCCCGCGCGGCCGGGCGCGACGCGCCTGAACTGGTAAAACTGCGGCCCTACT2220TCGACCATCCGCTGTTCGTCGAGATGTTCGCCGACGCCATCACCGCGGCCGCCGCAACCG2280TGCGCGGTGATGCCCGGCTGGTGTTCACCGCGCATTCGATCCCGACGGCCGCCGACCGCC2340GCTGTGGCCCCAACCTCTACAGCCGCCAAGTCGCCTACGCCACAAGGCTGGTCGCGGCCG2400CTGCCGGATACTGCGACTTTGACCTGGCCTGGCAGTCGAGATCGGGCCCGCCGCAGGTGC2460CCTGGCTGGAGCCAGACGTTACCGACCAGCTCACCGGTCTGGCTGGGGCCGGCATCAACG2520CGGTGATCGTGTGTCCCATTGGATTCGTCGCCGACCATATCGAGGTGGTGTGGGATCTCG2580ACCACGAGTTGCGATTACAAGCCGAGGCAGCGGGCATCGCGTACGCCCGGGCCAGCACCC2640CCAATGCCGACCCGCGGTTCGCTCGACTAGCCAGAGGTTTGATCGACGAACTCCGTTACG2700GCCGTATACCTGCGCGGGTGAGTGGCCCCGATCCGGTGCCGGGCTGTCTGTCCAGCATCA2760ACGGCCAGCCATGCCGTCCGCCGCACTGCGTGGCTAGCGTCAGTCCGGCCAGGCCGAGTG2820CAGGATCGCCGTGACCGCGGACATCCGGGCCGAGCGCACCACGGCGGTCAACGGTCTCAA2880CGCATCGGTGGCACGCTGAGCGTCCGACAACGACTGCGTTCCGATCGGCAATCGACTCAG2940CCCGGCACTGACCGCGATGATCGCATCGACGTGCGCGGCATTCTCGAGCACCCGCAATGC3000GCGCGATGGCGCGTGGTCGGGAACCCGGTGTTGCCGTGACGATTCGAGCAACTGCTCGAC3060GAGGCCACGGGGCTTGGCGACGTCGCTAGATCCCAGTCCGATGGTGCTCAAGGCTTCGGC3120__________________________________________________________________________