HSP60/10 chaperonin systems are inhibited by a variety of approved drugs, natural products, and known bioactive molecules

Abstract

All living organisms contain a unique class of molecular chaperones called 60 kDa heat shock proteins (HSP60 - also known as GroEL in bacteria). While some organisms contain more than one HSP60 or GroEL isoform, at least one isoform has always proven to be essential. Because of this, we have been investigating targeting HSP60 and GroEL chaperonin systems as an antibiotic strategy. Our initial studies focused on applying this antibiotic strategy for treating African sleeping sickness (caused by Trypanosoma brucei parasites) and drug-resistant bacterial infections (in particular Methicillin-resistant Staphylococcus aureus - MRSA). Intriguingly, during our studies we found that three known antibiotics - suramin, closantel, and rafoxanide - were potent inhibitors of bacterial GroEL and human HSP60 chaperonin systems. These findings prompted us to explore what other approved drugs, natural products, and known bioactive molecules might also inhibit HSP60 and GroEL chaperonin systems. Initial high-throughput screening of 3680 approved drugs, natural products, and known bioactives identified 161 hit inhibitors of the Escherichia coli GroEL chaperonin system (4.3% hit rate). From a purchased subset of 60 hits, 29 compounds (48%) re-confirmed as selective GroEL inhibitors in our assays, all of which were nearly equipotent against human HSP60. These findings illuminate the notion that targeting chaperonin systems might be a more common occurrence than we previously appreciated. Future studies are needed to determine if the in vivo modes of action of these approved drugs, natural products, and known bioactive molecules are related to GroEL and HSP60 inhibition.

Keywords: Chaperonin; GroEL; GroES; HSP10; HSP60; Molecular chaperone; Natural products; Proteostasis; Small molecule inhibitors.

Figure 1.

Structures of compounds previously found to inhibit E. coli GroEL/ES and/or human HSP60/10 chaperonin systems.

Figure 2.

Representative analysis of the binding of suramin (28) to E. coli GroEL measured by Isothermal Titration Calorimetry (ITC). The top panel shows a representative binding isotherm obtained by titrating suramin (2 mM) into a solution of GroEL (150 μM monomer concentration) in the ITC cell. The lower panel shows the integrated data (solid squares) fit to a single-site binding model (solid line). The molar ratio refers to the binding stoichiometry of suramin to monomeric GroEL. Average results for the various binding parameters (K_d, n, ΔH, ΔS, and ΔG) obtained from triplicate analyses are presented in Table 1.

Figure 3.. Protocol for the primary multiplexed high-throughput screening assay.

For primary screening of the LOPAC and MicroSource Spectrum libraries, we employed a new assay where we combined our individual GroEL/ES-dMDH refolding and GroEL/ES-dMDH ATPase assays into one multiplexed format. In this assay, a solution containing GroES and a binary complex of denatured malate dehydrogenase (dMDH) bound to GroEL was dispensed into the wells of a 384-well microplate. Compounds from the LOPAC and MicroSource Spectrum libraries (single concentrations) were then pin-transferred into the wells. The chaperonin-mediated refolding cycle was initiated by addition of ATP, the plates were incubated at 37°C for ~30 minutes (t₁ – until ~90% of the dMDH would have been refolded in the absence of inhibitors), and EDTA was then added to quench the refolding cycle. The substrates for the refolded, native MDH (nMDH) were added (sodium mesoxalate and NADH) and the enzymatic reporter reaction was monitored over time by reading well absorbance at 340 nm (t₂ – until the DMSO control wells had reached ~90% conversion of NADH to NAD⁺). In this coupled assay, the extent of chaperonin inhibition is proportional to the amount of enzymatic activity, and thus refolded MDH, present. In the same plate, we then added the malachite green phosphate reporter reagents to evaluate chaperonin-mediated hydrolysis of ATP.

Figure 4.

Correlation plots of IC₅₀ values for compounds tested in the respective biochemical assays. Each data point represents results for individual compounds tested in the respective assays, with color coding of points corresponding to the selectivity classifications of compound results presented in Table 2. Compounds inhibited nearly equipotently in both the GroEL/ES-dMDH and GroEL/ES-dRho refolding assays (panel A, Spearman correlation coefficients presented in Table 2), with few that inhibited both the native MDH and Rho reporter counter-screens (panel B), supporting on-target effects against the chaperonin-mediated refolding cycle. Compounds inhibited the human HSP60/10 and E. coli GroEL/ES chaperonin systems nearly equipotently, suggesting binding sites may be highly conserved between the two (panel C, Spearman correlation coefficients presented in Table 2). As indicated in panel A, For the purposes of categorizing inhibitor potencies in the various biochemical assays, we consider compounds with IC₅₀ values plotted in the grey zones to be inactive (i.e. greater than the maximum concentrations tested), >30 μM to be weak inhibitors, 10–30 μM moderate inhibitors, 1–10 μM potent inhibitors, and <1 μM very potent and acting near stoichiometrically since the concentration of GroEL tetradecamer is 50 nM during the refolding cycle (i.e. 700 nM GroEL monomeric subunits).