Thermodynamics of Aryl-Dihydroxyphenyl-Thiadiazole Binding to Human Hsp90

Abstract

The design of specific inhibitors against the Hsp90 chaperone and other enzyme relies on the detailed and correct understanding of both the thermodynamics of inhibitor binding and the structural features of the protein-inhibitor complex. Here we present a detailed thermodynamic study of binding of aryl-dihydroxyphenyl-thiadiazole inhibitor series to recombinant human Hsp90 alpha isozyme. The inhibitors are highly potent, with the intrinsic Kd approximately equal to 1 nM as determined by isothermal titration calorimetry (ITC) and thermal shift assay (TSA). Dissection of protonation contributions yielded the intrinsic thermodynamic parameters of binding, such as enthalpy, entropy, Gibbs free energy, and the heat capacity. The differences in binding thermodynamic parameters between the series of inhibitors revealed contributions of the functional groups, thus providing insight into molecular reasons for improved or diminished binding efficiency. The inhibitor binding to Hsp90 alpha primarily depended on a large favorable enthalpic contribution combined with the smaller favorable entropic contribution, thus suggesting that their binding was both enthalpically and entropically optimized. The enthalpy-entropy compensation phenomenon was highly evident when comparing the inhibitor binding enthalpies and entropies. This study illustrates how detailed thermodynamic analysis helps to understand energetic reasons for the binding efficiency and develop more potent inhibitors that could be applied for therapeutic use as Hsp90 inhibitors.

Protein polymer nanoparticles engineered as chaperones protect against apoptosis in human retinal pigment epithelial cells

“…For protein systems, either a decrease in enthalpy decrease or an increase in entropy is beneficial for maintaining stability. Although no direct studies have focused on the thermodynamic mechanism underlying sHSPs’ chaperone activity towards ADH, several studies suggest that stabilization of client proteins by sHSPs is dominated by enthalpic changes [37, 40]. The enthalpy increase of both plain ELPs [41] and ADH when heated indicated that both the phase transition and aggregation are endothermic processes (data not shown).…”

Intrinsic binding of 4‐substituted‐2,3,5,6‐tetrafluorobenezenesulfonamides to native and recombinant human carbonic anhydrase VI

“…In order to determine the pH dependence of the observed binding enthalpy, experiments were performed at 25 °C in 50 m m phosphate or 50 m m Tris buffer containing 100 m m NaCl at pH 5.5–9.5 with a final DMSO concentration of 1%, equal in the syringe and the cell. Data were integrated, fitted and analyzed as previously described . All experiments were repeated at least twice.…”

SEDPHAT – A platform for global ITC analysis and global multi-method analysis of molecular interactions

“…Global strategies combine multiple experiments in global analyses of ITC (gITC) [9,21–28] and in global multi-method analysis (GMMA), the combination of ITC data with data from complementary biophysical disciplines [29–32,82]. Beyond merely organizing separate complementary experiments into a hierarchical, multi-stage interpretation, global analysis takes full advantage of all possible constraints of the model and the full statistics of the data by simultaneously and directly fitting all experimental data with one explicit global model.…”

“…Beyond merely organizing separate complementary experiments into a hierarchical, multi-stage interpretation, global analysis takes full advantage of all possible constraints of the model and the full statistics of the data by simultaneously and directly fitting all experimental data with one explicit global model. Natural applications of gITC analysis are multi-site binding processes of homo- and hetero-oligomerizing macromolecules [9–11,13,14,15 ( this volume ),23,33–37], displacement experiments [22,38,39], and protonation-linked and other linked binding analyses varying temperature to determine heat capacity changes or buffer composition to determine salt or other co-factor linkage [24,26,28]. …”