Cooperativity basis for small-molecule stabilization of protein-protein interactions

Abstract

A cooperativity framework to describe and interpret small-molecule stabilization of protein-protein interactions (PPI) is presented. The stabilization of PPIs is a versatile and emerging therapeutic strategy to target specific combinations of protein partners within the protein interactome. Currently, the potency of PPI stabilizers is typically expressed by their apparent affinity or EC₅₀. Here, we propose that the effect of a PPI stabilizer be best described involving the cooperativity factor, α, between the stabilizer and binding partners in addition to the intrinsic affinity, K _D ^II, of the stabilizer for one of the apo-proteins. By way of illustration, we combine fluorescence polarization measurements with thermodynamic modeling to determine the α and K _D ^II for the PPI stabilization of 14-3-3 and TASK3 by fusicoccin-A (FC-A) and validate our approach by studying other PPI-partners of 14-3-3 proteins. Finally, we characterize a library of different stabilizer compounds, and perform structure-activity relationship studies in which molecular changes could be attributed to either changes in cooperativity or intrinsic affinity. Such insights should aid in the development of more effective protein-protein stabilizer drugs.

Fig. 1. (A) Examples of natural compounds that stabilize protein–protein interactions. (pdb: 1JFF,; 1FAP,; 2O98. (B) Cooperativity scheme for PPI stabilization involving the sequential addition of PPI partner and stabilizer or vice versa.

Fig. 2. Concentration dependence of fusicoccin-A (FC-A) stabilization of the TASK3/14-3-3σ PPI interaction measured by 2D fluorescence polarization. (A) Titration of 14-3-3 protein to FITC-labelled TASK3 peptide (10 nM, dashed line) against varying fixed concentrations of FC-A, between 0 and 200 μM (left panel). The EC₅₀'s of 14-3-3 to TASK3, for each FC-A concentration is compared to the EC₅₀ values as calculated by the model (red line, right panel). (B) Titration of FC-A to FITC-labelled TASK3 peptide (10 nM, dashed line) against varying fixed concentrations of 14-3-3 between 0 and 164 μM (left panel). The EC₅₀ of the FC-A titration depends on the 14-3-3 concentration in the assay (right panel). Here, the measured EC₅₀ values were not compared to a calculated model fit, as the titration data at both low and high 14-3-3 concentrations prohibit clear determination of the EC₅₀ values.

Fig. 3. (A) Cooperativity scheme of PPI stabilization involving sequential binding of protein (or representative peptide) partners (blue and green) and stabilizer molecule (orange). The binding partner binds to the target protein with K_D^I and in the presence of a stabilizer this affinity is altered to K_D^I/α. Similarly, the stabilizer binds with an intrinsic affinity K_D^II and an enhanced affinity K_D^II/α when the partner is already bound to the target protein. (B) Mass action laws and mass balance equations.

Fig. 4. Fluorescence polarization assays of 14-3-3σ interacting with multiple FITC-labelled binding partners (10 nM, indicated by the dashed vertical line) at various concentration of FC-A between 0 and 500 μM. The cooperativity factor α, is obtained though data-fitting according to the model depicted in Fig. 3. (A) CFTR-sequence (B) RAPTOR-sequence. (C) TASK3-sequence. (D) ExoS-sequence.

Fig. 5. (A) Structure–activity relationship of fusicoccin (FC) analogues as stabilizers for the 14-3-3/TASK3 interaction determined through 2D titrations (Fig. 5B). Variations in overall potency can now be expressed in terms of differences in either the cooperativity factor α or the intrinsic affinity K_D^II or both. (B) 2D-FP titrations with all fusicoccin analogs from this study and their corresponding cooperativity factor α and intrinsic affinity K_D^II for the 14-3-3/TASK3 PPI.

Fig. 6. Expected EC₅₀-enhancement, defined as the ratio of non-stabilized and stabilized EC₅₀, for protein titrations with different stabilizers concentration. The EC₅₀'s are determined via simulated protein titrations depending on α and K_D^II at 10 μM, 100 μM and 1 mM stabilizer. The corresponding position of FC-derivatives are annotated as circles.