Bacterial DnaK belongs to the ATP-dependent heat shock protein 70 (Hsp70) family of chaperones, which catalyze nascent protein folding and salvage misfolded proteins. DnaK collaborates with cofactors, DnaJ and GrpE, which regulate its catalytic cycle and conformational state. In the bacterial pathogen <i>Mycobacterium tuberculosis</i>, DnaK is assisted by two DnaJs, DnaJ1 and DnaJ2. Functional protein-protein interactions between DnaK and at least one DnaJ is essential for survival of mycobacteria; hence, chaperone-cofactor interactions may represent untapped antibacterial targets. Some Hsp70 inhibitors show cross-reactivity with bacterial homologs, but we lack selective probes for bacterial DnaK.  Here, we describe unbiased and rational approaches to modulate bacterial DnaK ATPase and chaperone activity, with the goal of designing probes with improved specificity that may be used to assess DnaK as an antibiotic target.  First, we detail the discovery of small molecule inhibitors of mycobacterial DnaK from a high through-put screen, most notably a peptidomimetic called telaprevir, which is able to inhibit chaperone function through interactions with the peptide-binding cleft of DnaK.  Binding of telaprevir leads to conformational changes that prevent ATP hydrolysis in a distal domain.  Using in vitro and in vivo chaperone assays, we demonstrate that telaprevir allosterically modulates the function of mycobacterial DnaK and its cofactor protein DnaJ2.  As part of an orthogonal approach, we synthesize protein fragments that mimic a crucial N-terminal domain of mycobacterial DnaJ proteins in order to disrupt DnaK-DnaJ interactions.  We find that proteomimetics of DnaJ1 are able to inhibit DnaK activation by cofactors in vitro, and cause cell death when mycobacterial cells are exposed to heat, a common proteotoxic stress.  Similarly, while telaprevir alone is not toxic to mycobacteria, co-treatment of cells with telaprevir and aminoglycosides, which further stress the proteome, enhances the potency of these antibiotics. In addition, telaprevir combats mycobacterial resistance to the frontline TB drug rifampin, as DnaK-DnaJ2 function is required for stabilization of protein mutants that confer drug tolerance.  Collectively, our work sets the stage for the design of peptide-based inhibitors with improved selectivity for bacterial chaperones to probe chaperone-protein interactions and explore chaperone inhibitors as adjuvants for different classes of antibiotics.

Selective probes for targeting bacterial Hsp70 chaperones to potentiate antibiotics

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ACS Fall 2022

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