Repurposing p97 inhibitors for chemical modulation of the bacterial ClpB–DnaK bichaperone system

Abstract

The ClpB/DnaK bi-chaperone system reactivates aggregated cellular proteins and is essential for survival of bacteria, fungi, protozoa, and plants under stress. AAA+ ATPase ClpB is a promising target for the development of antimicrobials, because a loss of its activity is detrimental for survival of many pathogens and no apparent ClpB orthologs are found in metazoans. We investigated ClpB activity in the presence of several compounds that were previously described as inhibitor leads for the human AAA+ ATPase p97, an anti-tumor target. We discovered that N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ), the least potent among the tested p97 inhibitors, binds to ClpB with a Kd~60 μM and inhibits the casein-activated, but not the basal ATPase activity of ClpB with an IC50~5 μM. The remaining p97 ligands, which displayed a higher affinity towards p97, did not affect the ClpB ATPase. DBeQ also interacted with DnaK with a Kd~100 μM, did not affect the DnaK ATPase, but inhibited the DnaK chaperone activity in vitro. DBeQ inhibited the reactivation of aggregated proteins by the ClpB/DnaK bi-chaperone system in vitro with an IC50~5 μM and suppressed the growth of cultured E. coli. The DBeQ-induced loss of E. coli proliferation was exacerbated by heat shock, but was nearly eliminated in a ClpB-deficient E. coli strain, which demonstrates a significant selectivity of DBeQ towards ClpB in cells. Our results provide chemical validation of ClpB as a target for developing novel antimicrobials. We identified DBeQ as a promising lead compound for structural optimization aimed at selective targeting of ClpB and/or DnaK.

The Role of ClpB in Bacterial Stress Responses and Virulence

“…Currently, only a few ClpB inhibitors have been identified (Grimminger et al, 2004;Martin et al, 2013;Kuczynska-Wisnik et al, 2017;Glaza et al, 2020;Singh et al, 2020). Guanidinium chloride specifically inhibits the ATP hydrolysis by Hsp104 of Saccharomyces cerevisiae and also the ClpB function of Ehrlichia chaffeensis (Grimminger et al, 2004).…”

“…ClpB is highly conserved amongst bacteria, fungi, protozoa, and plants and its role under different stressful conditions has been much studied. It provides protection against, e.g., heat, low pH, osmotic-and oxidative stress, ethanol, and nutrient starvation (Meibom et al, 2008;Krajewska et al, 2017;Glaza et al, 2020;Tripathi et al, 2020). Thus, clpB-deficient mutants demonstrate tremendously decreased survival upon exposure to these stresses.…”

“…For example, the Hsp70 and Hsp90 ATPases have been identified as drug targets for protozoan-derived infectious diseases in humans Shonhai, 2014, 2019). However, due to the high degree of sequence conservation among the Hsps across different domains of life, it is a challenging task (Glaza et al, 2020). ClpB is of special relevance as a drug target, since the homolog of ClpB, Skd3, also known as human ClpB, is conserved in many metazoan lineages, but differs significantly from bacterial and yeast proteins in domain structures.…”

Hsp100 Molecular Chaperone ClpB and Its Role in Virulence of Bacterial Pathogens

“…A recent study explored the hypothesis that known small-molecule ligands of AAA+ ATPases might also interact with ClpB. Among three inhibitors of the human AAA+ ATPase p97, a promising antitumor target, only one affected ClpB [ 73 ]. The identified compound, N 2 ,N 4 -dibenzylquinazoline-2,4-diamine (DBeQ) has been previously used as an antimicrobial [ 74 , 75 ], but without clear identification of its cellular targets.…”

“…The identified compound, N 2 ,N 4 -dibenzylquinazoline-2,4-diamine (DBeQ) has been previously used as an antimicrobial [ 74 , 75 ], but without clear identification of its cellular targets. It has now been shown that ClpB is the main target of DBeQ in E. coli under both permissive conditions and during heat stress [ 73 ]. Thus, ClpB can be selectively targeted with a small-molecule ligand in bacterial cells and such a treatment could produce a loss of bacterial viability.…”