[Ser]- and [Ser(P)]Incretin Analogs COMPARISON OF DIPEPTIDYL PEPTIDASE IV RESISTANCE AND BIOLOGICAL ACTIVITIES IN VITRO AND IN VIVO*

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP; also known as gastric inhibitory polypeptide) are incretin hormones that reduce postprandial glycemic excursions via enhancing insulin release but are rapidly inactivated by enzymatic N-terminal truncation. As such, efforts have been made to improve their plasma stability by synthetic modification or by inhibition of the responsible protease, dipeptidyl peptidase (DP) IV. Here we report a parallel comparison of synthetic GIP and GLP-1 with their Serand Ser(P)substituted analogs, examining receptor binding and activation, metabolic stability, and biological effects in vivo. Both incretins and their Ser-substituted analogs showed similar EC50s (0.16–0.52 nM) and IC50s (4.3–8.1 nM) at their respective cloned receptors. Although both phosphoserine 2-modified (Ser(PO3H2); Ser(P)) peptides were able to stimulate maximal cAMP production and fully displace receptor-bound tracer, they showed significantly rightshifted concentration-response curves and binding affinities. Ser-substituted analogs were moderately resistant to DP IV cleavage, whereas [Ser(P)]GIP and [Ser(P)] GLP-1 showed complete resistance to purified DP IV. It was shown that the Ser(P) forms were dephosphorylated in serum and thus in vivo act as precursor forms of Sersubstituted analogs. When injected subcutaneously into conscious Wistar rats, all peptides reduced glycemic excursions (rank potency: [Ser(P)]incretins > [Ser] incretins > native hormones). Insulin determinations indicated that the reductions in postprandial glycemia were at least in part insulin-mediated. Thus it has been shown that despite having low in vitro bioactivity using receptor-transfected cells, in vivo potency of [Ser(P)] incretins was comparable with or greater than that of native or [Ser]peptides. Hence, Ser(P)-modified incretins present as novel glucose-lowering agents.