Hytramycins V and I, Anti-<i>Mycobacterium tuberculosis</i> Hexapeptides from a <i>Streptomyces hygroscopicus</i> Strain

Thirty-five thousand actinomycete extracts were screened for anti-<i>Mycobacterium tuberculosis</i> (<i>M. tb</i>) activity, followed by C₁₈ cartridge fractionation of 37 prioritized extracts. Based on MICs against replicating and nonreplicating <i>M. tb</i>, and IC₅₀ values against Vero cells to generate selectivity indices, seven fractions from seven different strains were selected for further examination. When cultured in G.S.S. media and extracted with ethyl acetate, the <i>Streptomyces hygroscopicus</i> strain ECUM 14046 yielded an extract with promising anti-<i>M. tb</i> activity and a well-defined chromatographic profile. Fractionation by preparative HPLC and subsequent structure elucidation of two active fractions using 1D- and 2D-NMR and MS methods revealed the presence of two cyclohexapeptides, hytramycins V and I, each containing three unusual piperazic acid moieties. The use of ¹H iterative full spin analysis (HiFSA) on both hytramycins confirmed that quantum mechanics-simulated spectra match the experimental data, and all <i>J</i>_H,H and δ_H values are consistent with the proposed structures. The absolute configuration of each amino acid moiety was determined by Marfey’s method. The MICs against replicating and, more importantly, nonreplicating <i>M. tb</i> fall into the range of some existing second-line anti-TB drugs, such as streptomycin and capreomycin, respectively. The activities were maintained against <i>M. tb</i> strains that represent the major global clades, as well as H₃₇Rv-isogenic strains that are resistant to individual clinical anti-TB drugs

The Cyclic Peptide Ecumicin Targeting ClpC1 Is Active against Mycobacterium tuberculosis In Vivo

Drug-resistant tuberculosis (TB) has lent urgency to finding new drug leads with novel modes of action. A high-throughput screening campaign of >65,000 actinomycete extracts for inhibition of Mycobacterium tuberculosis viability identified ecumicin, a macrocyclic tridecapeptide that exerts potent, selective bactericidal activity against M. tuberculosis in vitro, including nonreplicating cells. Ecumicin retains activity against isolated multiple-drug-resistant (MDR) and extensively drug-resistant (XDR) strains of M. tuberculosis. The subcutaneous administration to mice of ecumicin in a micellar formulation at 20 mg/kg body weight resulted in plasma and lung exposures exceeding the MIC. Complete inhibition of M. tuberculosis growth in the lungs of mice was achieved following 12 doses at 20 or 32 mg/kg. Genome mining of lab-generated, spontaneous ecumicin-resistant M. tuberculosis strains identified the ClpC1 ATPase complex as the putative target, and this was confirmed by a drug affinity response test. ClpC1 functions in protein breakdown with the ClpP1P2 protease complex. Ecumicin markedly enhanced the ATPase activity of wild-type (WT) ClpC1 but prevented activation of proteolysis by ClpC1. Less stimulation was observed with ClpC1 from ecumicin-resistant mutants. Thus, ClpC1 is a valid drug target against M. tuberculosis, and ecumicin may serve as a lead compound for anti-TB drug development