Mutation in clpC1 encoding an ATP-dependent ATPase involved in protein degradation is associated with pyrazinamide resistance in Mycobacterium tuberculosis

Pyrazinamide (PZA) is an important sterilising tuberculosis drug that helps to shorten the duration of current chemotherapy regimens for tuberculosis. When first discovered, it had activity in murine tuberculosis but no apparent in vitro activity, and its subsequent use in treatment depended largely on classic experiments at Cornell University, which showed its requirement for an acid pH for activity and its sterilising activity in the mouse. Recent studies have shown that PZA enters Mycobacterium tuberculosis by passive diffusion, is converted to pyrazinoic acid (POA) by nicotinamidase/pyrazinamidase (PZase) and is then excreted by a weak efflux pump. Protonated POA (HPOA) is reabsorbed into the bacilli under acid conditions and accumulates because the efflux pump is inefficient, causing cellular damage. Unlike other antibacterials, PZA has no defined target of action. PZA is more active against old than against actively growing cultures, probably because the energy production and efflux pump would be slowed down by low bacterial metabolism. This review deals with the activity of PZA in vitro, in macrophages and in animal models. It describes the evidence from clinical trials that it is an effective sterilising drug that acts synergistically with rifampicin. The highly diverse mutations in the PZase gene (pncA) that lead to loss of PZase activity cause PZA resistance. Methods for susceptibility determination either as tests against PZA or nicotinamide in liquid and solid media, as tests for PZase activity or for mutations in pncA, are reviewed.

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.