Ongoing clinical trials are exploring anticancer approaches based on signaling by TRAIL, a ligand for the cell death receptors DR4 and DR5. In this study, we report on the selective apoptotic effects of multivalent DR5 binding peptides (TRAIL mim/DR5 ) on cancer cells in vitro and in vivo. Surface plasmon resonance revealed up to several thousand-fold increased affinities of TRAIL mim/DR5 -receptor complexes on generation of divalent and trivalent molecules, the latter of which was achieved with a conformationally restricted adamantane core. Notably, only multivalent molecules triggered a substantial DR5-dependent apoptotic response in vitro. In tumor models derived from human embryonic kidney cells or primary foreskin fibroblasts, TRAIL mim/DR5peptides exerted a cancer cell-selective action that could synergize with resveratrol in a manner independent of p53. In a xenograft model of human colon cancer, a divalent TRAIL mim/DR5 peptide inhibited tumor growth.Our results offer a proof-of-principle for the development of synthetic small molecules to trigger the TRAIL apoptosis pathway for cancer therapy.

Transglutaminases (TGases) catalyze the intermolecular cross-linking of certain proteins and tissue TGases (TG2) are involved in diverse biological processes. Unregulated, high TGase activities have been implicated in several physiological disorders, but few reversible inhibitors of TG2 have been reported. Herein, we report the synthesis of a series of novel trans-cinammoyl derivatives, discovered to be potent inhibitors of guinea pig liver transglutaminase. The most effective inhibitors evaluated can be sorted into two subclasses: substituted cinnamoyl benzotriazolyl amides and the 3-(substituted cinnamoyl)pyridines, referred to more commonly as azachalcones. Kinetic evaluation of both of these subclasses revealed that they display reversible inhibition and are competitive with acyl donor TGase substrates at IC50 values as low as 18 microM. An analysis of structure-activity relationships within these series of inhibitors permitted the identification of potentially important binding interactions. Further testing of some of the most potent inhibitors demonstrated their selectivity for TG2 and their potential for further development.

Through a multidisciplinary approach comprising organic synthesis, molecular biology, and physical organic kinetic studies, we have studied the mechanism of transglutaminase-mediated transamidation. More recently, we have applied our understanding of the mechanism to the design of reversible inhibitors and affinity labels for biological application. We have also undertaken the engineering of transglutaminase for its application as a “peptide synthase”. Herein, we present a brief overview of previously published work as well as recent results presented at the 2007 Merck–Frosst Centre for Therapeutic Research Award Lecture.Key words: transglutaminase, enzyme mechanism, enzyme inhibition, protein engineering