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• W2143144919 abstract "Prevention efforts and control of tuberculosis are seriously hampered by the appearance of multidrug-resistant strains of Mycobacterium tuberculosis, dictating new approaches to the treatment of the disease. Thiolactomycin (TLM) is a unique thiolactone that has been shown to exhibit anti-mycobacterial activity by specifically inhibiting fatty acid and mycolic acid biosynthesis. In this study, we present evidence that TLM targets two β-ketoacyl-acyl-carrier protein synthases, KasA and KasB, consistent with the fact that both enzymes belong to the fatty-acid synthase type II system involved in fatty acid and mycolic acid biosynthesis. Overexpression of KasA, KasB, and KasAB in Mycobacterium bovis BCG increased in vivo and in vitroresistance against TLM. In addition, a multidrug-resistant clinical isolate was also found to be highly sensitive to TLM, indicating promise in counteracting multidrug-resistant strains of M. tuberculosis. The design and synthesis of several TLM derivatives have led to compounds more potent both in vitro against fatty acid and mycolic acid biosynthesis and in vivoagainst M. tuberculosis. Finally, a three-dimensional structural model of KasA has also been generated to improve understanding of the catalytic site of mycobacterial Kas proteins and to provide a more rational approach to the design of new drugs. Prevention efforts and control of tuberculosis are seriously hampered by the appearance of multidrug-resistant strains of Mycobacterium tuberculosis, dictating new approaches to the treatment of the disease. Thiolactomycin (TLM) is a unique thiolactone that has been shown to exhibit anti-mycobacterial activity by specifically inhibiting fatty acid and mycolic acid biosynthesis. In this study, we present evidence that TLM targets two β-ketoacyl-acyl-carrier protein synthases, KasA and KasB, consistent with the fact that both enzymes belong to the fatty-acid synthase type II system involved in fatty acid and mycolic acid biosynthesis. Overexpression of KasA, KasB, and KasAB in Mycobacterium bovis BCG increased in vivo and in vitroresistance against TLM. In addition, a multidrug-resistant clinical isolate was also found to be highly sensitive to TLM, indicating promise in counteracting multidrug-resistant strains of M. tuberculosis. The design and synthesis of several TLM derivatives have led to compounds more potent both in vitro against fatty acid and mycolic acid biosynthesis and in vivoagainst M. tuberculosis. Finally, a three-dimensional structural model of KasA has also been generated to improve understanding of the catalytic site of mycobacterial Kas proteins and to provide a more rational approach to the design of new drugs. isoniazid acyl-carrier protein cerulenin fatty-acid synthase β-ketoacyl-ACP synthase mycolate-synthesizing activity minimal inhibitory concentration thiolactomycin oleic-albumin-dextrose-catalase polymerase chain reaction Tuberculosis, in terms of infectious diseases, is the leading cause of morbidity and mortality worldwide, infecting 8 million and killing 3 million people annually (1.Snider D.E. Raviglione M. Kochi A. Bloom B.R. Tuberculosis: Pathogenesis, Protection and Control. American Society for Microbiology, Washington, D. C.1994: 3-11Google Scholar). The situation has recently been exacerbated by the human immunodeficiency virus pandemic and the increased prevalence of multidrug-resistant strains ofMycobacterium tuberculosis (2.Smith P.G. Moss A.R. Bloom B.R. Tuberculosis: Pathogenesis, Protection and Control. American Society for Microbiology, Washington, D. C.1994: 47-59Google Scholar). Vaccine prophylaxis, usingMycobacterium bovis BCG, has proven unsatisfactory in many parts of the world (3.Colditz G.A. Brewer T.F. Berkey C.S. Wilson M.E. Burdick E. Fineberg H.V. Mosteller F. J. Am. Med. Assoc. 1994; 271: 698-702Crossref PubMed Scopus (1695) Google Scholar). Recent research has focused on understanding the molecular basis of drug resistance in M. tuberculosis, and a great deal of progress has been made in this regard in relation to several major anti-tuberculous drugs, including rifampicin (4.Telenti A. Imboden P. Marchesi F. Lowrie D. Cole S.T. Colston M.J. Matter L. Schopfer K. Bodmer T. Lancet. 1993; 341: 647-650Abstract PubMed Scopus (1085) Google Scholar), streptomycin (5.Finken M. Kirschner P. Meier A. Wrede A. Bottger E.C. Mol. Microbiol. 1993; 9: 1239-1246Crossref PubMed Scopus (301) Google Scholar), pyrazinamide (6.Scorpio A. Zhang Y. Nat. Med. 1996; 2: 662-667Crossref PubMed Scopus (599) Google Scholar), ethambutol (7.Belanger A.E. Besra G.S. Ford M.E. Mikusova K. Belisle J.T. Brennan P. Inamine J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11919-11924Crossref PubMed Scopus (390) Google Scholar), and isoniazid (INH)1 (8.Zhang Y. Heym B. Allen B. Young D. Cole S. Nature. 1992; 358: 591-593Crossref PubMed Scopus (1086) Google Scholar, 9.Banerjee A. Dubnau E. Quémard A. Balasubramanian V. Ulm K.S. Wilson T. Collins D. De Lisle G. Jacobs Jr., W.R. Science. 1994; 263: 227-230Crossref PubMed Scopus (1207) Google Scholar, 10.Mdluli K. Slayden R.A. Zhu Y. Ramaswamy S. Pan X. Mead D. Crane D.D. Musser J.M. Barry III, C.E. Science. 1998; 280: 1607-1610Crossref PubMed Scopus (373) Google Scholar).Mycolic acids are high molecular weight α-alkyl, β-hydroxy fatty acids with the general structure R-CH(OH)-CH(R′)-COOH, where R is a “meromycolate” chain consisting of 50–56 carbons and R′ is a shorter aliphatic branch possessing 22–26 carbons (11.Brennan P.J. Nikaido H. Annu. Rev. Biochem. 1995; 64: 29-63Crossref PubMed Scopus (1545) Google Scholar). Mycolic acids are key components of the mycobacterial cell wall and may play a role as an effective lipophilic barrier to the penetration of some antibiotics (11.Brennan P.J. Nikaido H. Annu. Rev. Biochem. 1995; 64: 29-63Crossref PubMed Scopus (1545) Google Scholar). Considering the importance of mycolic acids in bacterial survival, enzymes involved in the metabolism of these specific molecules represent attractive targets for the design of new anti-mycobacterial agents. Although there has been controversy about the mechanism of action of INH, it is clear that disruption of mycolic acid biosynthesis is one of its earliest detectable effects. This is apparently achieved through inhibition of inhA, an enoyl-acyl carrier protein (ACP) reductase, a key enzyme involved in the biosynthesis of fatty acids and mycolic acids (9.Banerjee A. Dubnau E. Quémard A. Balasubramanian V. Ulm K.S. Wilson T. Collins D. De Lisle G. Jacobs Jr., W.R. Science. 1994; 263: 227-230Crossref PubMed Scopus (1207) Google Scholar). However, mutations within katG, which encodes a catalase-peroxidase enzyme, lead to the majority of INH-resistant isolates (8.Zhang Y. Heym B. Allen B. Young D. Cole S. Nature. 1992; 358: 591-593Crossref PubMed Scopus (1086) Google Scholar), demonstrating that INH is a prodrug and that an activated metabolite is responsible for its mode of action (12.Blanchard J.S. Annu. Rev. Biochem. 1996; 65: 215-239Crossref PubMed Scopus (227) Google Scholar). Presumably, inhA is the primary target for the activated form of INH, and indeed, mutations in the inhA gene account for some cases of INH resistance inM. tuberculosis. More recently, mutations have also been observed within clinical M. tuberculosis isolates resistant to INH, traceable to kasA, which encodes a β-ketoacyl-ACP synthase, another key enzyme involved in mycolic acid biosynthesis (10.Mdluli K. Slayden R.A. Zhu Y. Ramaswamy S. Pan X. Mead D. Crane D.D. Musser J.M. Barry III, C.E. Science. 1998; 280: 1607-1610Crossref PubMed Scopus (373) Google Scholar).Earlier studies have demonstrated that thiolactomycin (TLM) selectively inhibits bacterial and plant type II fatty-acid synthases (FAS-II) through inhibition of β-ketoacyl-ACP synthases (Kas) (13.Hayashi T. Yamamoto O. Sasaki H. Kawaguchi A. Okazaki H. Biochim. Biophys. Res. Commun. 1983; 115: 1108-1113Crossref PubMed Scopus (82) Google Scholar, 14.Sasaki H. Oishi H. Hayashi T. Matsuura I. Ando K. Sawada M. J. Antibiot. (Tokyo). 1982; 35: 396-400Crossref PubMed Scopus (84) Google Scholar, 15.Furukawa H. Tsay J.T. Jackowski S. Takamura Y. Rock C.O. J. Bacteriol. 1993; 175: 3723-3729Crossref PubMed Scopus (72) Google Scholar, 16.Nishida I. Kawaguchi A. Yamada M. J. Biochem. 1986; 99: 1447-1454Crossref PubMed Scopus (86) Google Scholar, 17.Miyakawa S. Suzuki K. Noto T. Harada Y. Okazaki H. J. Antibiot. (Tokyo). 1982; 325: 411-419Crossref Scopus (120) Google Scholar). In addition, we have previously demonstrated that TLM acts as a potent anti-tuberculosis agent by inhibiting both fatty acid and mycolic acid biosynthesis in mycobacteria (18.Slayden R.A. Lee R.E. Armour J.W. Cooper A.M. Orme I.M. Brennan P.J. Besra G.S. Antimicrob. Agents Chemother. 1996; 40: 2813-2819Crossref PubMed Google Scholar). In light of this clinical aspect and of recent genomic information, we have reexamined the mode of action of TLM in mycobacteria. In this study, we have established that KasA and KasB, which both possess a high degree of similarity with other Kas enzymes, are targets for TLM. In addition, we have extended this study in the search of new anti-tuberculosis agents by generating a hypothetical structure of KasA to assist in future drug design and synthesized several lipophilic TLM derivatives. These analogues possess improved activities both in vitro against fatty acid and mycolic acid biosynthesis and in vivo against M. tuberculosis. This opens up new avenues for exploring the development of novel anti-mycobacterial agents based on TLM inhibition of Kas proteins in M. tuberculosis.DISCUSSIONThe antibiotic TLM is a selective inhibitor of type II fatty acid biosynthesis, is not toxic to mice, and affords significant protection against urinary tract and intraperitoneal bacterial infections (13.Hayashi T. Yamamoto O. Sasaki H. Kawaguchi A. Okazaki H. Biochim. Biophys. Res. Commun. 1983; 115: 1108-1113Crossref PubMed Scopus (82) Google Scholar, 14.Sasaki H. Oishi H. Hayashi T. Matsuura I. Ando K. Sawada M. J. Antibiot. (Tokyo). 1982; 35: 396-400Crossref PubMed Scopus (84) Google Scholar, 15.Furukawa H. Tsay J.T. Jackowski S. Takamura Y. Rock C.O. J. Bacteriol. 1993; 175: 3723-3729Crossref PubMed Scopus (72) Google Scholar, 16.Nishida I. Kawaguchi A. Yamada M. J. Biochem. 1986; 99: 1447-1454Crossref PubMed Scopus (86) Google Scholar, 17.Miyakawa S. Suzuki K. Noto T. Harada Y. Okazaki H. J. Antibiot. (Tokyo). 1982; 325: 411-419Crossref Scopus (120) Google Scholar). TLM has moderate in vitro activity against a broad spectrum of pathogens, including Gram-positive cocci and enteric, acid-fast, and anaerobic bacteria (15.Furukawa H. Tsay J.T. Jackowski S. Takamura Y. Rock C.O. J. Bacteriol. 1993; 175: 3723-3729Crossref PubMed Scopus (72) Google Scholar, 17.Miyakawa S. Suzuki K. Noto T. Harada Y. Okazaki H. J. Antibiot. (Tokyo). 1982; 325: 411-419Crossref Scopus (120) Google Scholar). More recently, TLM has shown encouraging anti-malarial activity via inhibition of type-II fatty acid biosynthesis in apicoplasts (41.Waller R.F. Keeling P.J. Donald R.G.K. Striepen B. Handman E. Lang-Unnasch N. Cowman A.F. Besra G.S. Roos D.S. McFadden G.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12352-12357Crossref PubMed Scopus (635) Google Scholar). In E. coli, all three condensing enzymes (FabB, FabF, and FabH) are inhibited by TLM both in vivo and in vitro (42.Jackowski S. Sutcliffe J. Georgopapadakou N.H. Emerging Targets in Antibacterial and Antifungal Chemotherapy. Routledge, Chapman and Hall, New York1992: 151-162Crossref Google Scholar). In contrast, TLM has no effect on type I fatty acid biosynthesis in Saccharomyces cerevisiae, Candida albicans, or rat liver (17.Miyakawa S. Suzuki K. Noto T. Harada Y. Okazaki H. J. Antibiot. (Tokyo). 1982; 325: 411-419Crossref Scopus (120) Google Scholar).The effect of TLM in mycobacteria has recently been investigated and shown to inhibit specifically the type II but not type I fatty-acid synthases (18.Slayden R.A. Lee R.E. Armour J.W. Cooper A.M. Orme I.M. Brennan P.J. Besra G.S. Antimicrob. Agents Chemother. 1996; 40: 2813-2819Crossref PubMed Google Scholar). In this study, we present evidence that overexpression of KasA or KasB individually or co-expression of both enzymes inM. bovis BCG results in increased resistance levels against TLM. Subsequent MIC determinations using a variety of agents that require penetration through the mycobacterial cell wall (such as rifampicin and other well characterized mycolic acid inhibitors, including INH) suggest that TLM sensitivity is not likely to be mediated by cell wall permeability and that its mechanism of action is dissimilar to other known mycolic acid inhibitors (8.Zhang Y. Heym B. Allen B. Young D. Cole S. Nature. 1992; 358: 591-593Crossref PubMed Scopus (1086) Google Scholar, 9.Banerjee A. Dubnau E. Quémard A. Balasubramanian V. Ulm K.S. Wilson T. Collins D. De Lisle G. Jacobs Jr., W.R. Science. 1994; 263: 227-230Crossref PubMed Scopus (1207) Google Scholar, 10.Mdluli K. Slayden R.A. Zhu Y. Ramaswamy S. Pan X. Mead D. Crane D.D. Musser J.M. Barry III, C.E. Science. 1998; 280: 1607-1610Crossref PubMed Scopus (373) Google Scholar). Overexpression studies provided indirect evidence that KasA and KasB are TLM targets. Further in vivo metabolic labeling and, more importantly, in vitro analyses demonstrated clearly that overexpression of KasA and KasB mediated a protective effect in both a mycobacterial FAS-II assay system (30.Bloch K. Methods Enzymol. 1975; 35: 84-90Crossref PubMed Scopus (47) Google Scholar, 31.Bloch K. Adv. Enzymol. Relat. Areas Mol. Biol. 1977; 45: 1-84PubMed Google Scholar) and a mycolic acid synthesizing extract to TLM (18.Slayden R.A. Lee R.E. Armour J.W. Cooper A.M. Orme I.M. Brennan P.J. Besra G.S. Antimicrob. Agents Chemother. 1996; 40: 2813-2819Crossref PubMed Google Scholar, 27.Wheeler P.R. Besra G.S. Minnikin D.E. Ratledge C. Biochim. Biophys. Acta. 1993; 1167: 182-188Crossref PubMed Scopus (38) Google Scholar). It was also interesting to note that TLM inhibition studies using extracts from M. bovis BCG carrying either pMV261::kasA or pMV261::kasB suggest that KasA may participate in the synthesis of C18-C34 fatty acids, whereas KasB may be involved in later steps of mycolic acid biosynthesis. These results have recently been supported by independent studies conducted by Slayden et al. (43.Slayden, R. A., Ramaswamy, S., Musser, J. M., and Barry, C. E., III (1999) Fourth International Conference on the Pathogenesis of Mycobacterial Infections, Stockholm, Sweden, July 8–11, 1999 p. 156Google Scholar).Mutations in KasA (D66N, G269S, G312S, and F413L), which correlate with low-levels of INH resistance, are thought to inhibit the formation of a trimolecular complex consisting of KasA-INH-AcpM (10.Mdluli K. Slayden R.A. Zhu Y. Ramaswamy S. Pan X. Mead D. Crane D.D. Musser J.M. Barry III, C.E. Science. 1998; 280: 1607-1610Crossref PubMed Scopus (373) Google Scholar, 43.Slayden, R. A., Ramaswamy, S., Musser, J. M., and Barry, C. E., III (1999) Fourth International Conference on the Pathogenesis of Mycobacterial Infections, Stockholm, Sweden, July 8–11, 1999 p. 156Google Scholar). Interestingly, Lee et al. (44.Lee A.S. Lim I.H. Tang L.L. Telenti A. Wong S.Y. Antimicrob. Agents Chemother. 1999; 43: 2087-2089Crossref PubMed Google Scholar) reported that kasApolymorphisms (R121K, G269S, G312S, and G387D) were identified in only 10% of INH-resistant isolates, with the most frequent substitution (G312S) being associated with INH-susceptible strains. In a recent study, Alland and co-workers (26.Piatek A.S. Telenti A. Murray M.R. El Hajj H. Jacobs Jr., W.R. Kramer F.R. Alland D. Antimicrob. Agents Chemother. 2000; 44: 103-110Crossref PubMed Scopus (194) Google Scholar) did not find mutations inkasA codon 66, 312, or 413 in 165 INH-susceptible and INH-resistant strains. However, 10 strains with mutations in codon 269 were found, 5 among INH-susceptible strains and 5 among INH-resistant strains. Interestingly, these mutations in KasA (Fig. 4 B) do not reside within the catalytic site. Two possible explanations with regard to INH susceptibility/resistance include, first, their influence on the relative degree of acyl-AcpM binding, and second, the stabilization of the dimerization of the KasA protein. As a consequence, these mutations may affect the KasA-INH-AcpM complex, a scenario that remains to be further investigated. We describe two distinct clinical isolates of M. tuberculosis displaying the G269S substitution that are still sensitive to INH, although resistant to TLM. Overall, these results suggest that mutations withinkasA do not confer significant INH resistance. Considering that the serum levels of INH are 35–60 times above the MIC (45.Seth V. Beotra A. Seth S.D. Semwal O.P. Kabra S. Jain Y. Mukhopadhya S. Indian Pediatr. 1993; 30: 1091-1098PubMed Google Scholar), the results suggest that, clinically, point mutations in KasA are not significant in terms of INH resistance. However, because INH has been proven to be one of the most effective anti-tuberculosis agents that targets FAS-II through InhA (9.Banerjee A. Dubnau E. Quémard A. Balasubramanian V. Ulm K.S. Wilson T. Collins D. De Lisle G. Jacobs Jr., W.R. Science. 1994; 263: 227-230Crossref PubMed Scopus (1207) Google Scholar), it would be anticipated that TLM would be of therapeutic value to INH-sensitive and INH-resistant strains through synergistic INH therapy. This is reinforced by the observation that the multidrug-resistant M307 isolate (its resistance is due in part to mutations in KatG) appears to be highly susceptible to TLM, suggesting that TLM may be suitable for the treatment of tuberculosis.During the course of our studies, several elegant structural reports have appeared defining the key catalytic amino acids involved in the elongation and decarboxylation process involved in fatty acid elongation by Kas enzymes (29.Huang W. Jia J. Edwards P. Dehesh K. Schneider G. Lindqvist Y. EMBO J. 1998; 17: 1183-1191Crossref PubMed Scopus (178) Google Scholar, 39.Siggaard-Andersen M. Protein Sequences Data Anal. 1993; 5: 325-335Google Scholar, 40.Siggaard-Andersen M. Bangera G. Olsen J.G. von Wettstein-Knowles P. Sánchez J. Cerdá Olmedo E. Martı́nez-Force E. Advances in Plant Lipid Research. Secretariado de Publicaciones, Universidad de Sevilla, Seville, Spain1998: 67-70Google Scholar). We have taken advantage of this available structural information to predict a model structure for mycobacterial KasA. It is clear that the key catalytic amino acids described for FabB by Siggaard-Anderson and co-workers (39.Siggaard-Andersen M. Protein Sequences Data Anal. 1993; 5: 325-335Google Scholar, 40.Siggaard-Andersen M. Bangera G. Olsen J.G. von Wettstein-Knowles P. Sánchez J. Cerdá Olmedo E. Martı́nez-Force E. Advances in Plant Lipid Research. Secretariado de Publicaciones, Universidad de Sevilla, Seville, Spain1998: 67-70Google Scholar) are highly conserved and that a deep hydrophobic pocket surrounds the catalytic Cys-163 residue (Fig. 4). It should also be pointed out that the mycobacterial FAS-II and meromycolate system elongate C16 fatty acid primers, initially through Kas enzymes, whereas E. coli FAS-II performs de novo synthesis from acetyl-CoA/ACP and malonyl-ACP (42.Jackowski S. Sutcliffe J. Georgopapadakou N.H. Emerging Targets in Antibacterial and Antifungal Chemotherapy. Routledge, Chapman and Hall, New York1992: 151-162Crossref Google Scholar). As a consequence, we decided to investigate whether a range of TLM inhibitors differing in hydrophobicity, and possibly resembling more the nature of the condensing enzyme substrates, would be more potent than TLM itself. Clearly, there was a strict requirement for a C-4 side chain as the parent thiolactone moiety was inactive both in vivo andin vitro. Analogues 2 and 3 were very similar in relation to the overall structure of TLM and possessed similar in vitro properties to TLM but were very poorin vivo inhibitors. Analogues 3-7 were influenced by the overall chain length and degree of saturation of the C-4 side chain in terms of in vivo activity, with6 and 7 possessing a 4-fold increase in potency. It was interesting to observe that there was a lack of inhibition against mycobacterial FAS-II by analogues 4-8 but excellent inhibitory properties against the mycolic-acid synthase extract. However, it should be pointed out that further studies are required to determine true structure-activity relationships for TLM analogues and FAS-II inhibition. For instance, studies using chloroplasts from peas demonstrated that analogue 4(inactive in the mycobacterial FAS-II system) was a potent inhibitor of FAS-II within the pea FAS-II assay system (46.Jones A.L. Dancer J.E. Harwood J.L. Biochem. Soc. Trans. 1994; 22: 258SCrossref PubMed Scopus (7) Google Scholar). Overall, the results suggest that TLM targets both KasA and KasB, whereas the more hydrophobic derivatives may target KasB and meromycolate synthesis. Studies examining recombinant E. coli expression of soluble Kas proteins (i.e. KasA and KasB) and site-directed mutants and studies of structural and binding/co-crystallization using TLM are currently in progress. In this regard, although it is a weak inhibitor, the benzophenone analogue 8 may provide a useful photoprobe for labeling investigations and covalent modification of the Kas enzymes.TLM remains an interesting but rather unexploited antibiotic sharing little or no cross-resistance with other classes of antibiotics. The TLM family are confirmed as members of a group of mycobacterial mycolic acid inhibitors (INH, ethionamide, and isoxyl) that target various fatty acid biosynthetic genes. The selective enhanced activity of a number of analogues of TLM demonstrates the potential for antibiotic development. A key feature of TLM (and its analogues) is its selectivity for type-II FAS systems, which promotes its effectiveness as a broad spectrum antibiotic. The added value in the context ofM. tuberculosis is the presence of multiple Kas enzymes. It can be envisaged that a suitable TLM derivative would block multiple condensing enzymes, i.e. both KasA and KasB, and thus reduce the frequency of appearance of TLM-resistance in M. tuberculosis. In this regard, several attempts to generate a laboratory-cultured M. bovis BCG strain resistant to TLM has failed (data not shown), supporting this notion. In summary, further characterization of genes involved in the biosynthesis of mycolic acids, such as KasA and KasB, may lead to the development of new therapeutic anti-tubercular agents, especially in the context of more potent TLM mimics. Tuberculosis, in terms of infectious diseases, is the leading cause of morbidity and mortality worldwide, infecting 8 million and killing 3 million people annually (1.Snider D.E. Raviglione M. Kochi A. Bloom B.R. Tuberculosis: Pathogenesis, Protection and Control. American Society for Microbiology, Washington, D. C.1994: 3-11Google Scholar). The situation has recently been exacerbated by the human immunodeficiency virus pandemic and the increased prevalence of multidrug-resistant strains ofMycobacterium tuberculosis (2.Smith P.G. Moss A.R. Bloom B.R. Tuberculosis: Pathogenesis, Protection and Control. American Society for Microbiology, Washington, D. C.1994: 47-59Google Scholar). Vaccine prophylaxis, usingMycobacterium bovis BCG, has proven unsatisfactory in many parts of the world (3.Colditz G.A. Brewer T.F. Berkey C.S. Wilson M.E. Burdick E. Fineberg H.V. Mosteller F. J. Am. Med. Assoc. 1994; 271: 698-702Crossref PubMed Scopus (1695) Google Scholar). Recent research has focused on understanding the molecular basis of drug resistance in M. tuberculosis, and a great deal of progress has been made in this regard in relation to several major anti-tuberculous drugs, including rifampicin (4.Telenti A. Imboden P. Marchesi F. Lowrie D. Cole S.T. Colston M.J. Matter L. Schopfer K. Bodmer T. Lancet. 1993; 341: 647-650Abstract PubMed Scopus (1085) Google Scholar), streptomycin (5.Finken M. Kirschner P. Meier A. Wrede A. Bottger E.C. Mol. Microbiol. 1993; 9: 1239-1246Crossref PubMed Scopus (301) Google Scholar), pyrazinamide (6.Scorpio A. Zhang Y. Nat. Med. 1996; 2: 662-667Crossref PubMed Scopus (599) Google Scholar), ethambutol (7.Belanger A.E. Besra G.S. Ford M.E. Mikusova K. Belisle J.T. Brennan P. Inamine J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11919-11924Crossref PubMed Scopus (390) Google Scholar), and isoniazid (INH)1 (8.Zhang Y. Heym B. Allen B. Young D. Cole S. Nature. 1992; 358: 591-593Crossref PubMed Scopus (1086) Google Scholar, 9.Banerjee A. Dubnau E. Quémard A. Balasubramanian V. Ulm K.S. Wilson T. Collins D. De Lisle G. Jacobs Jr., W.R. Science. 1994; 263: 227-230Crossref PubMed Scopus (1207) Google Scholar, 10.Mdluli K. Slayden R.A. Zhu Y. Ramaswamy S. Pan X. Mead D. Crane D.D. Musser J.M. Barry III, C.E. Science. 1998; 280: 1607-1610Crossref PubMed Scopus (373) Google Scholar). Mycolic acids are high molecular weight α-alkyl, β-hydroxy fatty acids with the general structure R-CH(OH)-CH(R′)-COOH, where R is a “meromycolate” chain consisting of 50–56 carbons and R′ is a shorter aliphatic branch possessing 22–26 carbons (11.Brennan P.J. Nikaido H. Annu. Rev. Biochem. 1995; 64: 29-63Crossref PubMed Scopus (1545) Google Scholar). Mycolic acids are key components of the mycobacterial cell wall and may play a role as an effective lipophilic barrier to the penetration of some antibiotics (11.Brennan P.J. Nikaido H. Annu. Rev. Biochem. 1995; 64: 29-63Crossref PubMed Scopus (1545) Google Scholar). Considering the importance of mycolic acids in bacterial survival, enzymes involved in the metabolism of these specific molecules represent attractive targets for the design of new anti-mycobacterial agents. Although there has been controversy about the mechanism of action of INH, it is clear that disruption of mycolic acid biosynthesis is one of its earliest detectable effects. This is apparently achieved through inhibition of inhA, an enoyl-acyl carrier protein (ACP) reductase, a key enzyme involved in the biosynthesis of fatty acids and mycolic acids (9.Banerjee A. Dubnau E. Quémard A. Balasubramanian V. Ulm K.S. Wilson T. Collins D. De Lisle G. Jacobs Jr., W.R. Science. 1994; 263: 227-230Crossref PubMed Scopus (1207) Google Scholar). However, mutations within katG, which encodes a catalase-peroxidase enzyme, lead to the majority of INH-resistant isolates (8.Zhang Y. Heym B. Allen B. Young D. Cole S. Nature. 1992; 358: 591-593Crossref PubMed Scopus (1086) Google Scholar), demonstrating that INH is a prodrug and that an activated metabolite is responsible for its mode of action (12.Blanchard J.S. Annu. Rev. Biochem. 1996; 65: 215-239Crossref PubMed Scopus (227) Google Scholar). Presumably, inhA is the primary target for the activated form of INH, and indeed, mutations in the inhA gene account for some cases of INH resistance inM. tuberculosis. More recently, mutations have also been observed within clinical M. tuberculosis isolates resistant to INH, traceable to kasA, which encodes a β-ketoacyl-ACP synthase, another key enzyme involved in mycolic acid biosynthesis (10.Mdluli K. Slayden R.A. Zhu Y. Ramaswamy S. Pan X. Mead D. Crane D.D. Musser J.M. Barry III, C.E. Science. 1998; 280: 1607-1610Crossref PubMed Scopus (373) Google Scholar). Earlier studies have demonstrated that thiolactomycin (TLM) selectively inhibits bacterial and plant type II fatty-acid synthases (FAS-II) through inhibition of β-ketoacyl-ACP synthases (Kas) (13.Hayashi T. Yamamoto O. Sasaki H. Kawaguchi A. Okazaki H. Biochim. Biophys. Res. Commun. 1983; 115: 1108-1113Crossref PubMed Scopus (82) Google Scholar, 14.Sasaki H. Oishi H. Hayashi T. Matsuura I. Ando K. Sawada M. J. Antibiot. (Tokyo). 1982; 35: 396-400Crossref PubMed Scopus (84) Google Scholar, 15.Furukawa H. Tsay J.T. Jackowski S. Takamura Y. Rock C.O. J. Bacteriol. 1993; 175: 3723-3729Crossref PubMed Scopus (72) Google Scholar, 16.Nishida I. Kawaguchi A. Yamada M. J. Biochem. 1986; 99: 1447-1454Crossref PubMed Scopus (86) Google Scholar, 17.Miyakawa S. Suzuki K. Noto T. Harada Y. Okazaki H. J. Antibiot. (Tokyo). 1982; 325: 411-419Crossref Scopus (120) Google Scholar). In addition, we have previously demonstrated that TLM acts as a potent anti-tuberculosis agent by inhibiting both fatty acid and mycolic acid biosynthesis in mycobacteria (18.Slayden R.A. Lee R.E. Armour J.W. Cooper A.M. Orme I.M. Brennan P.J. Besra G.S. Antimicrob. Agents Chemother. 1996; 40: 2813-2819Crossref PubMed Google Scholar). In light of this clinical aspect and of recent genomic information, we have reexamined the mode of action of TLM in mycobacteria. In this study, we have established that KasA and KasB, which both possess a high degree of similarity with other Kas enzymes, are targets for TLM. In addition, we have extended this study in the search of new anti-tuberculosis agents by generating a hypothetical structure of KasA to assist in future drug design and synthesized several lipophilic TLM derivatives. These analogues possess improved activities both in vitro against fatty acid and mycolic acid biosynthesis and in vivo against M. tuberculosis. This opens up new avenues for exploring the development of novel anti-mycobacterial agents based on TLM inhibition of Kas proteins in M. tuberculosis. DISCUSSIONThe antibiotic TLM is a selective inhibitor of type II fatty acid biosynthesis, is not toxic to mice, and affords significant protection against urinary tract and intraperitoneal bacterial infections (13.Hayashi T. Yamamoto O. Sasaki H. Kawaguchi A. Okazaki H. Biochim. Biophys. Res. Commun. 1983; 115: 1108-1113Crossref PubMed Scopus (82) Google Scholar, 14.Sasaki H. Oishi H. Hayashi T. Matsuura I. Ando K. Sawada M. J. Antibiot. (Tokyo). 1982; 35: 39" @default.
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• W2143144919 title "Thiolactomycin and Related Analogues as Novel Anti-mycobacterial Agents Targeting KasA and KasB Condensing Enzymes inMycobacterium tuberculosis" @default.
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