Compounds were profiled for their inhibitory

Compounds – were profiled for their inhibitory activity against dipeptidyl peptidase-IV Activity and/or Structure Homologues (DASH) [e.g. DPP2, DPP8 and DPP9] and non-DASH [eg. post-proline cleaving enzyme (PPCE), neutral endopeptidase (NEP), aminopeptidase P (APP), aminopeptidase N (APN)] enzymes. Compounds – exhibited over one thousand-fold selectivity for DPP4 over DPP2, DPP8 and DPP9 enzymes (), while Compound was approximately one hundred-fold selective. Compounds – exhibited high selectivity (>1000 fold) for non-DASH enzymes PPCE, NEP, APP and APN. While showed >1000 fold selectivity for NEP, APP and APN, it was only∼100 fold selective for PPCE. Compounds – showed that they had no liability for inhibiting common cytochrome P-450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) up to a concentration of 10μM. Furthermore, the compounds were found to be stable to human, mouse, rat and dog liver microsomes. These compounds had very low Caco-2 permeabilities, which is to be expected from the higher values of calculated molecular properties. Despite these compounds showing very low Caco2 permeability, the plasma exposure profiles of compounds – were studied at 30mpk in Wistar rats following oral dosing (). Compound exhibited a very low C and AUC (0.26μg/mL and 0.61μg.h/mL, respectively). Compound exhibited a slightly improved plasma exposure (C=0.55μg/mL; AUC=2.36μg.h/mL), while KN-93 exhibited good plasma exposure (C=2.51μg/mL; AUC=23.49μg.h/mL). Compound also exhibited a good C and AUC values (C=1.33μg/mL; AUC=13.94μg.h/mL); however these concentrations were lower than those for . To conclude, rational design and synthesis has been used to identify compounds having a potent and balanced potency against both human DPP4 and ACE. The compounds have a favourable DMPK profile. While compound exhibited moderate oral plasma exposure, compounds and exhibited high plasma oral exposure in rats. We have thus demonstrated that a merged multiple ligand approach is a feasible strategy for simultaneously inhibiting DPP4 and ACE enzymes. Acknowledgements
Introduction Food-derived peptides with ACE inhibiting properties have been extensively studied in recent years as they may show potential antihypertensive properties in vivo, which could be further developed to health-promoting food products. Most studies have been focused on the production of ACE inhibitory peptides from different sources of food proteins by commercial enzymes as well as the purification and identification of these peptides. However, when peptides are consumed, they are further digested by gastrointestinal (GI) enzymes, leading to structural modification (Vermeirssen, Van Camp, & Verstraete, 2004). Further structural transformation may take place during permeation through intestinal epithelial cells as a result of hydrolysis by brush border proteases (BBPs) (Vermeirssen et al., 2004). It is likely that the peptide transport can result in the fragmentation of their sequences and thus change in the ACE-inhibitory activity. Thus, identified peptides reported in the literature might not be the inhibitor affecting ACE in vivo. It has been reported that tripeptides can be hydrolyzed by BBPs, such as aminotripeptidases or dipeptidases, resulting in the fragmentation of tripeptides and the formation of free amino acids and dipeptides (Ganapathy et al., 1999, Vermeirssen et al., 2004). In addition, dipeptides are absorbed intact across the brush border membrane by a specific peptide transport system (Vermeirssen et al., 2004). The changes of oligopeptides during absorption are varied, depending on the specific peptide sequence. The relationship between peptide structure and the extent of peptide transportation has not been clearly established. It has been recently found that cooked breast of Korat chicken, which is a crossbreed between a male Thai indigenous chicken and a female broiler, released ACE inhibitory peptides upon in vitro GI digestion (Sangsawad, Roytrakul, & Yongsawatdigul, 2017). This implied that chicken breast is a source of health-promoting peptides. The most potent ACE inhibitory peptides were identified to be KPLLCS, KPLL and ELFTT; however, their permeability across intestinal epithelium has not yet been determined. Structural changes of peptides during intestinal transport could inevitably affect ACE inhibitory activity. Therefore, the intact, as well as peptide fragments that exhibit ACE inhibitory activity and are available after intestinal transport can be potential antihypertensive peptides.