Rational design of cyclic peptides to disrupt TGF-B/SMAD7 signaling in heterotopic ossification

The human TGF-beta/SMAD7 signaling has been recognized as an attractive target of heterotopic ossification (HO). Here, we report a successful rational design of cyclic peptides to disrupt the signaling pathway by targeting TGF-beta-receptor complex. The intermolecular interaction between TGF-beta and its cognate receptor is characterized in detail using molecular dynamics simulation, binding energetic analysis, and alanine scanning. With the computational analysis a binding loop of receptor protein is identified that plays an essential role in the peptide-mediated TGF-beta-receptor interaction. Subsequently, the loop is stripped from the protein context to generate a linear peptide segment, which possesses considerable flexibility and intrinsic disorder, and thus would incur a large entropy penalty upon binding to TGF-beta. In order to minimize the unfavorable entropic effect, the linear peptide is cyclized by adding a disulfide bond between the N- and C-terminal cysteine residues of the peptide, resulting in a cyclic peptide. In vitro fluorescence anisotropy assays substantiate that the cyclic peptide can bind tightly to TGF-beta with determined K-d value of 54 mu M. We also demonstrated that structural optimization can further improve the peptide affinity by site-directed mutagenesis of selected residues based on the computationally modeled complex structure of TGF-beta with the cyclic peptide. (C) 2016 Elsevier Inc. All rights reserved.