| The most effective strategy towards eradication of tuberculosis relies on effective and powerful chemotherapeutic agents. Nature produces an amazing variety and number of bioactive products. Two of these, thiolactomycin (TLM) and protolichesterinic acid, were used as starter templates to produce a large compound library of synthetic derivatives. More than 100 analogues were screened from this library for efficacy against M. tuberculosis var. bovis BCG. TLM analogues containing aryl hydrazides appending the C-4 hydroxyl displayed marked growth inhibition. However, the kill kinetics for these compounds appeared to involve an intermediary activation step. This activation step provided a new mechanism for designing dual-inhibitors. From TLM, the pyrazolidinone heterocylce core was inspired. Susceptibility testing of these compounds showed primarily weak growth inhibition. For protolichesterinic acid, amide analogues were more active than esters with a clear specificity for long alkyl groups at C-5.; Rational drug design efforts towards inhibitors of the beta-ketoacyl synthase (KAS) condensing enzymes in fatty acid biosynthesis identified a new class of antimycobacterial compounds, the alkylsulfonylacetamides. Three classes of pro-drugs were designed to improve the limited aqueous solubility of these compounds: N-Mannich bases, N-acyloxymethyl esters and hydrazides. Investigations into the mode of action for this compound class were conducted using the most potent analogue N-decanesulfonylacetamide (DSA). Attempts to identify the specific molecular target using standard affinity chromatography based protocols were not successful. However, metabolic studies with radiolabeled DSA identified the presence of intracellular modification enzymes and a strong membrane partitioning of this compound. The DSA-mycobacterium interaction was dynamic involving active efflux systems and global changes in protein expression.; To further understand the drug-pathogen interaction, a new LC-MS proteomic analysis method was validated using isoniazid treated M. tuberculosis var. bovis BCG. In keeping with known effects of isoniazid on the fatty acid synthase II pathway, proteins encoded by the kas operon (AcpM, KasA, KasB, Accd6) were significantly overexpressed, as were those involved in iron metabolism and cell division suggesting a complex interplay of metabolic events leading to cell death. |
