Exenatide: NMR/CD evaluation of the medium dependence of conformation and aggregation state

Exenatide, synthetic exendin‐4, is the first member of the incretin mimetic class of potential therapeutic agents. It has been the subject of extensive clinical trials in people with Type 2 diabetes. Results to date indicate that exenatide decreases postmeal blood glucose concentrations and that this effect is associated with weight loss. Prior NMR studies of exendin‐4 utilized 30% trifluoroethanol because this medium affords sharp, high‐resolution NMR spectra. These studies defined its three‐dimensional structure in this medium. The NMR‐derived ensemble included a novel tertiary structure motif that has subsequently been optimized, yielding water‐soluble Trp‐cage miniproteins. Prior to the present study, the structuring propensities (and aggregation/association state) of exendin‐4 in strictly aqueous media had not been established. Studies of exendin‐4 and N‐terminally truncated analogs of exendin have established that the structuring propensities of these species are highly medium dependent. This study extends knowledge of the medium dependence of exendin structure to DMSO–water mixtures and to aqueous media mimicking the formulation conditions for this investigational drug. Exenatide retains a substantial helical propensity from residues 9–27 even in 98% DMSO. The addition of water leads to the appearance of NMR diagnostics of the Trp‐cage formation. In strictly aqueous media (pH 4–4.4), exenatide is monomeric only at <10 μM peptide concentrations. Under these conditions the Trp cage is partially formed. NMR and CD data indicate that higher concentrations lead to helix bundle formation and that the helix/helix interactions involve residues 11–26. Both the N‐ and C‐termini of the helix bundle state display rapid segmental motion. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004