Engineering Novel VPAC2-Selective Agonists with Improved Stability and Glucose-Lowering Activity in Vivo

Abstract

A previously described VPAC2-selective agonist, BAY 55-9837 (peptide HSDAVFTDNYTRLRKQVAAKKYLQSIKNKRY), had several limitations with respect to its potential as an insulin secretagogue for the treatment of type 2 diabetes. These limitations were primarily poor stability in aqueous buffer and short duration of action in vivo. In this report, we describe a series of novel analogs of BAY 55-9837 that were designed around the likely degradation mechanisms and structure-activity relationship of this peptide with a view to overcoming its limitations. These analogs were tested for improved liquid stability and retention of VPAC2-selective binding and activation, as well as prolonged activity in vivo. Although several degradation mechanisms were possible based on the degradation pattern, it was determined that deamidation at the two asparagines (N9 and N28) was the major instability determinant. Changing these two asparagines to glutamines did not negatively affect VPAC2-selective binding and activation. The double glutamine mutein analog, BAY(Q9Q28), retained full VPAC2 activity and selectivity while displaying no significant degradation when stored at 40°C for 4 weeks. This is in contrast to BAY 55-9837, which showed greater than 80% degradation when stored at 40°C for 2 weeks. A cysteine was added to the C terminus of BAY(Q9Q28), followed by site-specific cysteine conjugation with a 22-or 43-kDa polyethylene glycol (PEG) to yield BAY(Q9Q28C32)PEG22 or BAY(Q9Q28C32)PEG43, respectively. These PEGylated peptides retain the ability to selectively bind and activate the VPAC2 receptor and have prolonged glucose-lowering activity in vivo.

Therapeutic potential of VIP vs PACAP in diabetes

“…VPAC2 selective agonists were very effective in augmenting insulin secretion without causing hypoglycemia (Tsutsumi et al 2002). However, as the first generation of VPAC2 selective agonists manifested stability problems in vivo, new DPP4-resistant VPAC2 analogs were generated (Pan et al 2007) and studies are underway to determine the efficacy of these new therapeutic compounds. Because VPAC2 selective agonists are considered to be new therapeutic agents for the treatment of T2D, and VIP is the natural ligand for VPAC2, VIP-mediated gene transfer strategies also represent an experimental treatment modality for diabetes.…”

A novel selective VPAC2 agonist peptide-conjugated chitosan modified selenium nanoparticles with enhanced anti-type 2 diabetes synergy effects

“…12,18,19 Pan et al explored the coupling of dipeptidyl peptidase IV-resistant VPAC2 analog peptides with 22 or 44 kDa polyethylene glycol (PEG) through specific cysteine moieties to improve the pharmacokinetics of these peptides in vivo. 13 But the large polymers formed by PEG or other modifications often decrease the biological activity of the peptides or interfere with the biological function of the peptides. Therefore, to maintain the activity and function of the peptide drugs in vivo, appropriate slow-release carriers may be used to liberate the active peptides in order.…”

“…17 Mainly, dipeptidyl peptidase IV-mediated enzymolysis leads to the H-S lack of BAY55-9837 at the N-terminus, which may cause it cannot activate VPAC2. 13 Moreover, deamidation occurring in the ninth and 28th amino acid Asn (N) results in destabilization and its small molecular size causes rapid renal clearance.…”

“…Thus, PACAP derivatives as VPAC2-specific agonists can effectively promote glucose-dependent insulin secretion, control circulating glucose and protect islet beta cells without causing glucagon secretion and glycogenolysis, and have been proposed as potential T2D therapeutics. [12][13][14] BAY55-9837 is a reported specific VPAC2 agonist constructed through site-directed mutagenesis, and it can increase plasma insulin levels in a dose-dependent manner, but not leading to any hypoglycemia in rats. 15,16 Nevertheless, its therapeutic application has been hampered by its short half-life (~5 min) and limited bioavailability in vivo.…”

Chitosan-decorated selenium nanoparticles as protein carriers to improve the in vivo half-life of the peptide therapeutic BAY 55-9837 for type 2 diabetes mellitus

“…Recent attempts have been made to overcome these limitations, eg, amino acid substitutions and polyethylene glycolation were used to prolong the half-life of BAY 55-9837 in vivo. 13 The in vivo half-life of protein-based products significantly affects both dosing requirements and therapeutic efficacy; therefore, strategies to prolong the half-life of such drugs are highly significant. One such strategy, which utilizes the polyethylene glycolation of a given therapeutic protein, works to prolong the protein half-life by increasing the apparent size of the polypeptide.…”