Rational design of stapled helical peptides as antidiabetic PPARγ antagonists to target coactivator site by decreasing unfavorable entropy penalty instead of increasing favorable enthalpy contribution.

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated transcription factor belonging to the nuclear hormone receptor and has been exploited as a well-established druggable target for the treatment of diabetes mellitus (DM). Traditionally, small-molecule compounds have been developed to attack at the ligand site and Ser273 phosphorylation site of PPARγ. In this study, we derived helical peptide segments from the LXXLL motif region of coactivator proteins as antidiabetic PPARγ antagonists, which were expected to competitively disrupt the native interaction between PPARγ and its cognate coactivators by rebinding at PPARγ coactivator site. Structural analysis, dynamics simulation and energetics dissection revealed that these peptides cannot be well folded into active helical structure when splitting from the protein context of their parent coactivators and exhibit a large flexibility and intrinsic disorder in the free state, which would, therefore, incur a considerable entropy penalty upon rebinding to PPARγ. Hydrocarbon stapling strategy was employed to constrain these free coactivator peptides into ordered helical conformation, thus largely minimizing unfavorable entropy penalty but having only a moderate effect on favorable enthalpy contribution. The computational findings were further substantiated by fluorescence-based assays; the binding affinity of three potent SRC1, NCoA6 and p300 coactivator peptides to PPARγ was observed to be improved by 7.2-fold, 4.2-fold and 5.7-fold upon the stapling, which were also measured to have an efficient competitive potency with their unstapled counterparts for PPARγ coactivator site, with CC50 = 0.096, 0.12 and 0.18 μM, respectively.