Investigations from the Belly of the Beast: N-Terminally Labeled Incretin Peptides That Are Both Potent Receptor Agonists and Stable to Protease Digestion

Abstract

Type 2 diabetes is an ever more common human disease, which has as its hallmark an inability of the body to properly maintain glucose homeostasis. If left unchecked, it can lead to heart disease, vision loss, and kidney disease; at this particular moment in history, it is also important to note that this condition is correlated with a greater risk for severe illness from COVID-19. Typically, when a healthy person digests a meal, peptide hormone glucagon-like peptide-1 (GLP1) is introduced into the bloodstream and goes on to activate, in several tissues, its cognate receptor GLP1R, initiating numerous events that work to ensure a constant blood sugar level. Thus, when this carefully orchestrated system breaks down, peptides related to GLP1 serve in the clinic as injectable therapeutics to manage the disease. The Achilles’ heel of this class of active compounds, however, is susceptibility to cleavage by proteases and thereby reduced half-lives in patients. Protease sensitivity is frequently a stumbling block for the application of peptide-based drugs. The way this looks on the ground is that the top three clinically applied GLP1R agonists, exenatide, liraglutide, and semaglutide, require twice daily, once daily, and weekly injection schedules, respectively, representing an inconvenience for patients in the best case. Thus, the search for the holy grail of peptide therapeutics must continue, namely, a general strategy for modifying peptides to impart resistance to proteolysis while enabling retention of excellent receptor agonist potency. Or, perhaps, the search is over. In this issue of ACS Central Science, Kumar and coauthors report how an N-terminal trifluoroethyl modification fits the bill, not only in the case of GLP1/GLP1R/DPP4, but also for other related peptides, receptors, and proteases. From the perspective of design, Kumar and coauthors began with the seemingly straightforward task of imparting protease stability to GLP1, a problem that other groups had tackled with success in the past, albeit almost universally at the expense of receptor agonist potency. DPP4, the greatest stumbling block to the therapeutic success of GLP1, is a serine protease that utilizes two active site glutamate residues to bind the positively charged N-terminus of active GLP1(7-37) and then hydrolytically remove the His7Ala8 dipeptide to yield inactive GLP1(9-37). The Kumar paper cites work from 20 years ago by Flatt et al. and Wheeler et al., as well as a more recent patent and paper from 2016 by Sexton et al., all of which explored various types of N-acylation as a successful strategy to confer resistance to DPP4 and also observed disappointing concomitant hits to agonist activity. Operating under the hypothesis that the acylated N-terminus may be refused by GLP1R because it appears to the receptor to be an improperly processed agonist, Kumar and coauthors instead opted for an N-alkylation strategy. The authors then further explored considerations about how N-terminal agonist modifications may impact meaningful binding of the agonist deep within the receptor, the belly of the beast, as the authors figuratively describe. Here the jumping off point was the impressive cryo-EM structure of a GLP1-GLP1R complex (PDB: 5VAI) solved in 2017 by Skiniotis and co-workers. Unfortunately, though this structure clearly reveals the manner in which the receptor makes contact with the N-terminal His7 side chain (namely, a cation-π interaction with R299 and hydrogen bonding to W306 and I309), no obvious direct contact to the N-terminus is observed to help specifically