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Updated: May 27, 2026
System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis
Published on: April 5, 2017
Validation of the essential ClpP protease in Mycobacterium tuberculosis as a novel drug target
Juliane Ollinger1, Theresa O'Malley, Edward A Kesicki
1TB Discovery Research, Infectious Disease Research Institute, Seattle, Washington, USA.
Abstract:
Mycobacterium tuberculosis is a pathogen of major global importance. Validated drug targets are required in order to develop novel therapeutics for drug-resistant strains and to shorten therapy. The Clp protease complexes provide a means for quality control of cellular proteins; the proteolytic activity of ClpP in concert with the ATPase activity of the ClpX/ClpC subunits results in degradation of misfolded or damaged proteins. Thus, the Clp system plays a major role in basic metabolism, as well as in stress responses and pathogenic mechanisms. M. tuberculosis has two ClpP proteolytic subunits. Here we demonstrate that ClpP1 is essential for viability in this organism in culture, since the gene could only be deleted from the chromosome when a second functional copy was provided. Overexpression of clpP1 had no effect on growth in aerobic culture or viability under anaerobic conditions or during nutrient starvation. In contrast, clpP2 overexpression was toxic, suggesting different roles for the two homologs. We synthesized known activators of ClpP protease activity; these acyldepsipeptides (ADEPs) were active against M. tuberculosis. ADEP activity was enhanced by the addition of efflux pump inhibitors, demonstrating that ADEPs gain access to the cell but that export occurs. Taken together, the genetic and chemical validation of ClpP as a drug target leads to new avenues for drug discovery.
Insights
ClpP1 is essential for Mycobacterium tuberculosis survival, while ClpP2 overexpression is toxic. Acyldepsipeptides (ADEPs) show activity against this pathogen, validating ClpP as a potential drug target for tuberculosis.
Area of Science:
- Microbiology
- Molecular Biology
- Drug Discovery
Background:
- Mycobacterium tuberculosis poses a significant global health challenge, necessitating new drugs against resistant strains.
- Clp protease complexes (ClpP, ClpX, ClpC) are crucial for protein quality control, metabolism, and pathogenicity.
- M. tuberculosis possesses two ClpP homologs, ClpP1 and ClpP2, with potentially distinct functions.
Purpose of the Study:
- To investigate the essentiality and roles of ClpP1 and ClpP2 in M. tuberculosis.
- To evaluate the potential of ClpP as a drug target using acyldepsipeptides (ADEPs).
Main Methods:
- Gene deletion and overexpression studies to assess ClpP1 and ClpP2 essentiality.
- Synthesis and testing of acyldepsipeptides (ADEPs) as ClpP activators.
- Assessment of ADEP activity in combination with efflux pump inhibitors.
Main Results:
- ClpP1 is essential for M. tuberculosis viability in culture.
- Overexpression of clpP1 did not affect growth under various conditions.
- Overexpression of clpP2 was toxic, indicating differential roles.
- Synthesized ADEPs demonstrated activity against M. tuberculosis.
- ADEP efficacy was enhanced by efflux pump inhibitors, suggesting cellular export.
Conclusions:
- ClpP1 is a validated essential gene and potential drug target in M. tuberculosis.
- ClpP2 exhibits distinct functions and toxicity upon overexpression.
- ADEPs represent a promising class of compounds for developing novel tuberculosis therapeutics by targeting ClpP.
