Richard A. Slayden

Biogenesis of the mycobacterial cell wall and the site of action of ethambutol.

The effect of ethambutol (EMB) is primarily on polymerization steps in the biosynthesis of the arabinan component of cell wall arabinogalactan (AG) of Mycobacterium smegmatis. Inhibition of the synthesis of the arabinan of lipoarabinomannan (LAM) occurred later, and thus in the cases of AG and LAM, the polymerization of D-arabinofuranose apparently involves separate pathways. While the synthesis of these arabinans was normal in an EMB-resistant isogeneic strain, the addition of EMB to the resistant strain resulted in partial inhibition of the synthesis of the arabinan of LAM and the emergence of a novel, truncated form of LAM, indicating partial susceptibility of the resistant gene(s) and providing a new intermediate in the LAM biosynthetic sequence. A consequence of inhibition of AG arabinan biosynthesis is the lack of new sites for mycolate attachment and thus the channeling of mycolate residues into a variety of free lipids which then accumulate. The primary biochemical effects of EMB can be explained by postulating separate AG and LAM pathways catalyzed by a variety of extramembranous arabinosyl transferases with various degrees of sensitivity to EMB.

Hypoxic response of Mycobacterium tuberculosis studied by metabolic labeling and proteome analysis of cellular and extracellular proteins.

The events involved in the establishment of a latent infection with Mycobacterium tuberculosis are not fully understood, but hypoxic conditions are generally believed to be the environment encountered by the pathogen in the central part of the granuloma. The present study was undertaken to provide insight into M. tuberculosis protein expression in in vitro latency models where oxygen is depleted. The response of M. tuberculosis to low-oxygen conditions was investigated in both cellular and extracellular proteins by metabolic labeling, two-dimensional electrophoresis, and protein signature peptide analysis by liquid chromatography-mass spectrometry. By peptide mass fingerprinting and immunodetection, five proteins more abundant under low-oxygen conditions were identified from several lysates of M. tuberculosis: Rv0569, Rv2031c (HspX), Rv2623, Rv2626c, and Rv3841 (BfrB). In M. tuberculosis culture filtrates, two additional proteins, Rv0363c (Fba) and Rv2780 (Ald), were found in increased amounts under oxygen limitation. These results extend our understanding of the hypoxic response in M. tuberculosis and potentially provide important insights into the physiology of the latent bacilli.

Unique Mechanism of Action of the Thiourea Drug Isoxyl on Mycobacterium tuberculosis

The thiourea isoxyl (thiocarlide; 4,4′-diisoamyloxydiphenylthiourea) is known to be an effective anti-tuberculosis drug, active against a range of multidrug-resistant strains of Mycobacterium tuberculosis and has been used clinically. Little was known of its mode of action. We now demonstrate that isoxyl results in a dose-dependent decrease in the synthesis of oleic and, consequently, tuberculostearic acid in M. tuberculosis with complete inhibition at 3 μg/ml. Synthesis of mycolic acid was also affected. The anti-bacterial effect of isoxyl was partially reversed by supplementing growth medium with oleic acid. The specificity of this inhibition pointed to a Δ9-stearoyl desaturase as the drug target. Development of a cell-free assay for Δ9-desaturase activity allowed direct demonstration of the inhibition of oleic acid synthesis by isoxyl. Interestingly, sterculic acid, a known inhibitor of Δ9-desaturases, emulated the effect of isoxyl on oleic acid synthesis but did not affect mycolic acid synthesis, demonstrating the lack of a relationship between the two effects of the drug. The three putative fatty acid desaturases in the M. tuberculosis genome, desA1, desA2, and desA3, were cloned and expressed in Mycobacterium bovis BCG. Cell-free assays and whole cell labeling demonstrated increased Δ9-desaturase activity and oleic acid synthesis only in the desA3-overexpressing strain and an increase in the minimal inhibitory concentration for isoxyl, indicating that DesA3 is the target of the drug. These results validate membrane-bound Δ9-desaturase, DesA3, as a new therapeutic target, and the thioureas as anti-tuberculosis drugs worthy of further development.

Novel Trisubstituted Benzimidazoles, Targeting Mtb FtsZ, As A New Class of Antitubercular Agents

Libraries of novel trisubstituted benzimidazoles were created through rational drug design. A good number of these benzimidazoles exhibited promising MIC values in the range of 0.5-6 μg/mL (2-15 μM) for their antibacterial activity against Mtb H37Rv strain. Moreover, five of the lead compounds also exhibited excellent activity against clinical Mtb strains with different drug-resistance profiles. All lead compounds did not show appreciable cytotoxicity (IC(50) > 200 μM) against Vero cells, which inhibited Mtb FtsZ assembly in a dose dependent manner. The two lead compounds unexpectedly showed enhancement of the GTPase activity of Mtb FtsZ. The result strongly suggests that the increased GTPase activity destabilizes FtsZ assembly, leading to efficient inhibition of FtsZ polymerization and filament formation. The TEM and SEM analyses of Mtb FtsZ and Mtb cells, respectively, treated with a lead compound strongly suggest that lead benzimidazoles have a novel mechanism of action on the inhibition of Mtb FtsZ assembly and Z-ring formation.

Disease State Differentiation and Identification of Tuberculosis Biomarkers via Native Antigen Array Profiling

A critical element of tuberculosis control is early and sensitive diagnosis of infection and disease. Our laboratories recently showed that different stages of disease were distinguishable via two-dimensional Western blot analyses of Mycobacterium tuberculosis culture filtrate proteins. However, this methodology is not suitable for high throughput testing. Advances in protein microarray technology provide a realistic mechanism to screen a large number of serum samples against thousands of proteins to identify biomarkers of disease states. Techniques were established for separation of native M. tuberculosis cytosol and culture filtrate proteins, resulting in 960 unique protein fractions that were used to generate protein microarrays. Evaluation of serological reactivity from 42 patients in three tuberculosis disease states and healthy purified protein derivative-positive individuals demonstrated that human immunodeficiency virus (HIV)-negative cavitary and noncavitary tuberculosis (TB) patients’ sera recognized 126 and 59 fractions, respectively. Sera from HIV patients coinfected with TB recognized 20 fractions of which five overlapped with those recognized by non-HIV TB patients’ sera and 15 were unique to the HIV+TB+ disease state. Identification of antigens within the reactive fractions yielded 11 products recognized by both cavitary and noncavitary TB patients’ sera and four proteins (HspX, MPT64, PstS1, and TrxC) specific to cavitary TB patients. Moreover four novel B cell antigens (BfrB, LppZ, SodC, and TrxC) of human tuberculosis were identified.

Menaquinone synthesis is critical for maintaining mycobacterial viability during exponential growth and recovery from non-replicating persistence.

Understanding the basis of bacterial persistence in latent infections is critical for eradication of tuberculosis. Analysis of Mycobacterium tuberculosis mRNA expression in an in vitro model of non‐replicating persistence indicated that the bacilli require electron transport chain components and ATP synthesis for survival. Additionally, low μM concentrations of aminoalkoxydiphenylmethane derivatives inhibited both the aerobic growth and survival of non‐replicating, persistent M. tuberculosis. Metabolic labelling studies and quantification of cellular menaquinone levels suggested that menaquinone synthesis, and consequently electron transport, is the target of the aminoalkoxydiphenylmethane derivatives. This hypothesis is strongly supported by the observations that treatment with these compounds inhibits oxygen consumption and that supplementation of growth medium with exogenous menaquinone rescued both growth and oxygen consumption of treated bacilli. In vitro assays indicate that the aminoalkoxydiphenylmethane derivatives specifically inhibit MenA, an enzyme involved in the synthesis of menaquinone. Thus, the results provide insight into the physiology of mycobacterial persistence and a basis for the development of novel drugs that enhance eradication of persistent bacilli and latent tuberculosis.

Slow-onset inhibition of the FabI enoyl reductase from francisella tularensis: residence time and in vivo activity.

Francisella tularensis is a highly virulent and contagious Gram-negative intracellular bacterium that causes the disease tularemia in mammals. The high infectivity and the ability of the bacterium to survive for weeks in a cool, moist environment have raised the possibility that this organism could be exploited deliberately as a potential biological weapon. Fatty acid biosynthesis (FAS-II) is essential for bacterial viability and has been validated as a target for the discovery of novel antibacterials. The FAS-II enoyl reductase ftuFabI has been cloned and expressed, and a series of diphenyl ethers have been identified that are subnanomolar inhibitors of the enzyme with MIC90 values as low as 0.00018 microg mL(-1). The existence of a linear correlation between the Ki and MIC values strongly suggests that the antibacterial activity of the diphenyl ethers results from direct inhibition of ftuFabI within the cell. The compounds are slow-onset inhibitors of ftuFabI, and the residence time of the inhibitors on the enzyme correlates with their in vivo activity in a mouse model of tularemia infection. Significantly, the rate of breakdown of the enzyme-inhibitor complex is a better predictor of in vivo activity than the overall thermodynamic stability of the complex, a concept that has important implications for the discovery of novel chemotherapeutics that normally rely on equilibrium measurements of potency.

A Point Mutation in the mma3 Gene Is Responsible for Impaired Methoxymycolic Acid Production in Mycobacterium bovis BCG Strains Obtained after 1927

BCG vaccines are substrains of Mycobacterium bovis derived by attenuation in vitro. After the original attenuation (1908 to 1921), BCG strains were maintained by serial propagation in different BCG laboratories (1921 to 1961). As a result, various BCG substrains developed which are now known to differ in a number of genetic and phenotypic properties. However, to date, none of these differences has permitted a direct phenotype-genotype link. Since BCG strains differ in their abilities to synthesize methoxymycolic acids and since recent work has shown that the mma3 gene is responsible for O-methylation of hydroxymycolate precursors to form methoxymycolic acids, we analyzed methoxymycolate production and mma3 gene sequences for a genetically defined collection of BCG strains. We found that BCG strains obtained from the Pasteur Institute in 1927 and earlier produced methoxymycolates in vitro but that those obtained from the Pasteur Institute in 1931 and later all failed to synthesize methoxymycolates, and furthermore, the mma3 sequence of the latter strains differs from that of Mycobacterium tuberculosis H37Rv by a point mutation at bp 293. Site-specific introduction of this guanine-to-adenine mutation into wild-type mma3 (resulting in the replacement of glycine 98 with aspartic acid) eliminated the ability of this enzyme to produce O-methylated mycolic acids when the mutant was cloned in tandem with mma4 into Mycobacterium smegmatis. These findings indicate that a point mutation in mma3 occurred between 1927 and 1931, and that this mutant population became the dominant clone of BCG at the Pasteur Institute.

Synthesis and in vitro antimycobacterial activity of B-ring modified diaryl ether InhA inhibitors

Previous structure-based design studies resulted in the discovery of alkyl substituted diphenyl ether inhibitors of InhA, the enoyl reductase from Mycobacterium tuberculosis. Compounds such as 5-hexyl-2-phenoxyphenol 19 are nM inhibitors of InhA and inhibit the growth of both sensitive and isoniazid-resistant strains of Mycobacterium tuberculosis with MIC(90) values of 1-2 microg/mL. However, despite their promising in vitro activity, these compounds have ClogP values of over 5. In efforts to reduce the lipophilicity of the compounds, and potentially enhance compound bioavailability, a series of B ring analogues of 19 were synthesized that contained either heterocylic nitrogen rings or phenyl rings having amino, nitro, amide, or piperazine functionalities. Compounds 3c, 3e, and 14a show comparable MIC(90) values to that of 19, but have improved ClogP values.

FtsZ: A Novel Target for Tuberculosis Drug Discovery

The emergence of multi-drug resistant Mycobacterium tuberculosis (Mtb) strains has made many of the currently available anti-TB drugs ineffective. Accordingly there is a pressing need to identify new drug targets. FtsZ, a bacterial tubulin homologue, is an essential cell division protein that polymerizes in a GTP-dependent manner, forming a highly dynamic cytokinetic ring, designated as the Z ring, at the septum site. Following recruitment of other cell division proteins, the Z ring contracts, resulting in closure of the septum and then formation of two daughter cells. Since inactivation of FtsZ or alteration of FtsZ assembly results in the inhibition of Z ring and septum formation, FtsZ is a very promising target for new antimicrobial drug development. This review describes the function and dynamic behaviors of FtsZ, its homology to tubulin, and recent development of FtsZ inhibitors as potential anti-TB agents.