Development of high throughput screening methods for inhibitors of ClpC1P1P2 from Mycobacteria tuberculosis

Abstract

Tuberculosis affects about 100 million people worldwide and causes nearly 2 million deaths annually. It has been estimated that one third of all humans is infected with latent Mycobacterium tuberculosis (Mtb). Moreover, Mtb has become increasingly resistant to available antibiotics. Consequently, it is important to identify and characterize new therapeutic targets in Mtb and to synthesize selective inhibitors. ClpP1, ClpP2 and their associated regulatory ATPases, ClpX and ClpC1 are required for the growth of Mtb and for its virulence during murine infection and are highly attractive drug targets, especially since they are not present in the cytosol of mammalian cells, and they differ markedly from the mitochondrial ClpP complex. The importance of these proteins in Mtb is emphasized by the existence of several natural antibiotics targeting this system. In order to find new inhibitors of ClpC1P1P2 system, we developed an assay based on the ATP-dependent degradation of a fluorescent protein substrate. The hits obtained were further characterized with a set of secondary assays to identify precise targets within a complex. A large library of compounds was screened and led to the identification of a ClpC1 ATPase inhibitor demonstrating that this approach can be used in future searches for anti-TB agents.

Keywords: ClpC1; ClpP; ClpX; Mycobacterium tuberculosis; Screening.