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.

ClpP Protease, a Promising Antimicrobial Target

“…New pyrrole cores (13i, Figure 7) were recently identified as ClpP1P2 inhibitors by Liu et al after an in silico driven exploration, showing antibacterial properties and room for optimization [104]. Furthermore, a HTS setting for detection of Mtb ClpC1P1P2 complex inhibitors identified two hits [105]. Despite not showing antibacterial properties and having unclear mechanisms of action, both inhibitors showed a new chemical structure for ClpP inhibitors (GSK 17, Figure 7) and forthcoming use of broader libraries envisages a promising future for detection of new inhibitors for this pathogen [105].…”

“…Furthermore, a HTS setting for detection of Mtb ClpC1P1P2 complex inhibitors identified two hits [105]. Despite not showing antibacterial properties and having unclear mechanisms of action, both inhibitors showed a new chemical structure for ClpP inhibitors (GSK 17, Figure 7) and forthcoming use of broader libraries envisages a promising future for detection of new inhibitors for this pathogen [105]. Inhibition of P. falciparum ClpP has also been achieved with a novel class of ClpP inhibitors [106].…”

Control of Toxin-Antitoxin Systems by Proteases in Mycobacterium Tuberculosis

“…This is reminiscent of ClpX interaction with FtsZ that does not lead to altered intracellular levels of FtsZ but rather to an inhibition of Z-ring assembly in M. tuberculosis (Dziedzic et al, 2010). Interestingly, ClpC1 is the target of antimycobacterial peptides such as cyclomarin A or lassomycin, and has emerged as an promising drug target (Lupoli et al, 2018;Fraga et al, 2019;Maurer et al, 2019). More generally, the activation, repression of modification of ClpP mechanism of action has been the focus of many studies to identify new antibiotics (Ye et al, 2016;Moreno-Cinos et al, 2019).…”

The high-throughput screening system for inhibitor Mycobacterium tuberculosis compounds based on ATP hydrolysis activity of recombinant protein ClpC1

“…By the association with ClpP1/ P2 complex, ClpC1 supported ATP-dependent protein degradation. The recombinant ClpC1 was determined for analysis if it had an inherent ATPase activity (Kar et al, 2008;Fraga et al, 2019). We used the radioactive of ATP as a substrate and quantified the radioactive inorganic phosphate generated upon its enzymatic hydrolysis by ClpC1.…”