Figure 3. Characterization of selective binding and identification of Tropane 1.
(A) The structural overlay of HsHsp90 bound to XL888 (PDB: 4AWO, black ribbons) and BX-2819 (PDB: 3HHU, white ribbons), displaying a section of the lid subdomain (Thr99-Gln123) and N-terminal helix (Phe22-Thr36). The residues Lys112 and Glu25 form a salt bridge in the XL888-bound HsHsp90 structure. These residues are substituted by Arg98 and Asp11 in PfHsp90, respectively. (B-C) The dose-dependent displacement of FITC-GA by XL888 (B) or BX-2819 (C) from purified wild-type (WT) or salt bridge double mutant (E25D/K112R) HsHsp90 nucleotide-binding domain (NBD). XL888 exhibits a higher affinity to the E25D/K112R mutant compared to the wild-type NBD (B), with apparent Ki = 9.6 ± 1.6 nM and Ki = 68 ± 23 nM, respectively (p = 0.0463). BX-2819 does not exhibit a change in affinity to the E25D/K112R mutant compared to the wild-type NBD (C), with apparent Ki = 13 ± 2.5 nM and Ki = 19 ± 4.0 nM, respectively (p = 0.2103). Data shown as means ± SEM, n = 4; unpaired t-test. (D) Heatmap (light blue to black) displays the amount of FITC-GA bound to PfHsp90 or HsHsp90 after displacement by 4 tropane-derived compounds at 100 nM. FITC-GA bound to PfHsp90 is competitively displaced (<50%) by compounds 1-3. A higher ratio of bound FITC-GA to HsHsp90 compared to PfHsp90 (Hs/Pf; color coded orange) for 1 suggests improved selectivity. (E) Structure of Tropane 1. (F) Table summarizing Hsp90 binding affinity and Plasmodium inhibition potency for Tropane 1. The compound exhibits 9.6-fold selectivity for PfHsp90 over HsHsp90 and retains activity against the P. falciparum blood and P. berghei liver stages without host HuH7 cell cytotoxicity. Apparent Ki values reported as means ± SEM, n = 4; *<0.05; unpaired t-test. Parasite inhibition EC50 and host viability values reported as means ± SEM, n = 2.