Closing the In Vitro/In Vivo Gap in Tuberculosis Drug Discovery

This study establishes a new anti-M. tuberculosis (M. tb) drug lead screening scheme that integrates in vitro and in vivo methods for early detection of bioactive constituents during the purification of nature-derived extracts. Two in vivo M. tb quantification methods were established: 1) the determination of a Mycobacterium genus-specific cell wall fatty acid, tuberculostearic acid, with GC-MS/MS and 2) determination of M. tb marker RNAs with real-time PCR. Both are efficient, accurate and relatively inexpensive, and adaptable to in vitro and in vivo M. tb growth and inhibition monitoring in anti-M. tb drug discovery programs. The conventional in vitro high-throughput phenotypic screening, the newly designed M. tb bioautography on thin layer chromatography plates, as well as the determination of quantitative purity-activity relationship study all aid in exploring and screening for bioactive principles in a crude state. Two classes of cyclic peptides, hytramycins and a xylamycin, were isolated from the extracts of two different actinomycete strains through bioassay-guided fractionation. The structures were elucidated mainly with LC-MS and 1D/2D-NMR. Both peptides contain unusual amino acid residues in the structural cores, and also exhibit strong anti-M. tb activity in vitro with unique antimicrobial mechanisms of action.