Mechanisms of HSP60/10 Chaperonin Inhibitors in Trypanosoma brucei
Stevens, McKayla, Department of Biochemistry and Molecular Biology, Indiana University School of Medicine; Emma Doud, Department of Biochemistry and Molecular Biology, Indiana University School of Medicine;Anne-Marie Ray, Department of Biochemistry and Molecular Biology, Indiana University School of Medicine; Jared Sivinski, Dept of Pharmacology and Toxicology, College of Pharmacy, University of Arizona; Eli Chapman, Dept of Pharmacology and Toxicology, College of Pharmacy, University of Arizona; Steven Johnson, Department of Biochemistry and Molecular Biology, Indiana University School of Medicine
Background/Significance/Rationale: Trypanosoma brucei is a trypanosomatid parasite that causes Human African Trypanosomiasis (African Sleeping Sickness), one of several Neglected Tropical Diseases (NTDs). Current therapeutics have significant problems including intrinsically low efficacy, high toxicity, poor oral and CNS bioavailability, complex dosing regimens, and rising incidences of drug-resistance. To address the need for more effective therapeutics, we are investigating the potential of targeting HSP60/10 chaperonin systems as a mechanistically unique antibiotic strategy. Trypanosomes have multiple HSP60 isoforms, one of which is essential for survival; thus, trypanosomes may be particularly susceptible to HSP60 inhibition.
Methods: Here, I screened a diverse library of known HSP60 inhibitors in an Alamar Blue cell viability assay against T. brucei parasites and counter-screened in human FHS74Int and FHC cells to establish selectivity profiles. Compound series with greatest efficacy and selectivity were selected for mechanistic studies including biotin-streptavidin pulldown assays and LC-MS/MS analyses for binding site identification
Results/Findings: The most potent hits contained nitrofuran (NF) and α,β-unsaturated ketone (ABK) moieties. Pulldown analyses support target engagement in cells, while LC-MS/MS analyses indicate ABK binding to the C411 residue on HSP60. Nifurtimox, a standard of care drug, functions as a pro-drug whose active metabolite, a cyano-α,β-unsaturated ketone, likewise binds to the C411 residue. The C411 residue lies in a cleft beneath the apical domains wherein inhibitor binding may perturb the apical domain movements necessary for engagement with the HSP10 co-chaperone during the refolding cycle.
Conclusions/Discussion: Ultimately, compounds with promising potential for trypanocidal development were identified, many surpassing efficacy of standard of care drugs. Preliminary mechanisms of action were also established. Significantly, MOA of Nifurtimox are currently unknown: these studies suggest HSP60 inhibition may contribute to its trypanocidal properties.
Translational/Human Health Impact: The cysteine residue identified is conserved across all trypanosomatids: optimizing inhibitors to exploit this mechanism could afford an effective class of broad-spectrum agents for treating trypanosomatid NTDs.