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In order to optimize the isolation procedure of anti-TB lead compounds, a truly targeted isolation approach was developed, which aligns (HP)TLC-bioautography with GUESS1 based countercurrent separation (CCS). (HP)TLC-bioautography was specifically developed for Mycobacterium tuberculosis, using the mc2 7000 lux ABCDE strain, while the GUESS method is used to rapidly determine the optimal CCS solvent system. Optimal CCS conditions target the separation of the inhibition zones within a range of optimal CCS partition coefficients referred to as the “CCS sweet spot”, with a corresponding TLC Rf-value ˜0.5. Hereby, the CCS solvent system is chosen on the basis of the Rf-value of the bioactive component(s) rather than the fraction as a whole. This approach accelerates the isolation process and prevents the isolation of non-active metabolites and byproducts. The new TLC-bioautography-GUESS-CCS workflow is demonstrated with the fractionation of an extract from an anti-TB active actinomycete strain. [Funding: NIH R21 AI093919 – 02] 1Friesen & Pauli, G.U.E.S.S. – A Generally Useful Estimate of Solvent Systems for CCC, Journal of Liquid Chromatography & Related Technologies, 2005, Volume 28, Issue17, 2777 – 2806 *Equal contributors

Our recent efforts at finding new anti-tuberculosis drug leads from actinomycetes have led to the discovery of ecumicin (ECU, MW 1599.22, MIC 0.16 uM) and; more recently, a rufomycin analogue (RUF-I, MW 1041.55, MIC 0.019 uM). Although both of these cyclic peptides target the currently unexploited chaperone protein ClpC1 ATPase complex, there is no observed cross resistance. In this study, the structures of RUF-I and other rufomycin analogues, heptapeptides, were elucidated by 1H, 13C, 2D NMR, MS and HiFSA. To understand the lack of cross resistance, clpC1 from spontaneously generated resistant clones from both leads were sequenced. This revealed several distinct single point mutations, suggesting these compounds bind differently to ClpC1. Checkerboard assay indicated a lack of antagonism between ECU and RUF-I and potential synergistic effects. Preliminary data indicates that ClpC1 could be a multifaceted drug target for multidrug resistant M. tuberculosis. Research pertaining to the peptide drug leads, associated analogues, and their mechanism of action against ClpC1 will be presented.