Stephen C. McKeown

Pharmacological regulation of insulin secretion in MIN6 cells through the fatty acid receptor GPR40: identification of agonist and antagonist small molecules

1 Long chain fatty acids have recently been identified as agonists for the G protein‐coupled receptors GPR40 and GPR120. Here, we present the first description of GW9508, a small‐molecule agonist of the fatty acid receptors GPR40 and GPR120. In addition, we also describe the pharmacology of GW1100, a selective GPR40 antagonist. These molecules were used to further investigate the role of GPR40 in glucose‐stimulated insulin secretion in the MIN6 mouse pancreatic β‐cell line. 2 GW9508 and linoleic acid both stimulated intracellular Ca2+ mobilization in human embryonic kidney (HEK)293 cells expressing GPR40 (pEC50 values of 7.32±0.03 and 5.65±0.06, respectively) or GPR120 (pEC50 values of 5.46±0.09 and 5.89±0.04, respectively), but not in the parent HEK‐293 cell line. 3 GW1100 dose dependently inhibited GPR40‐mediated Ca2+ elevations stimulated by GW9508 and linoleic acid (pIC50 values of 5.99±0.03 and 5.99±0.06, respectively). GW1100 had no effect on the GPR120‐mediated stimulation of intracellular Ca2+ release produced by either GW9508 or linoleic acid. 4 GW9508 dose dependently potentiated glucose‐stimulated insulin secretion in MIN6 cells, but not in primary rat or mouse islets. Furthermore, GW9508 was able to potentiate the KCl‐mediated increase in insulin secretion in MIN6 cells. The effects of GW9508 on insulin secretion were reversed by GW1100, while linoleic acid‐stimulated insulin secretion was partially attenuated by GW1100. 5 These results add further evidence to a link between GPR40 and the ability of fatty acids to acutely potentiate insulin secretion and demonstrate that small‐molecule GPR40 agonists are glucose‐sensitive insulin secretagogues.

Solid-phase reaction monitoring--chemical derivatization and off-bead analysis.

The aim of this review is to give a compendium of colorimetric assays and spectrophotometric-based quantification methods applicable to solid-phase organic chemistry. Comprehensive experimental details for commonly employed color tests performed on solid support will be documented.

Versatile solid-phase synthesis of secondary amines from alcohols. Development of an N-Boc-(o-nitrobenzene)sulfonamide linker

The development of a versatile amine releasing linker based on the modified o-nitrobenzene sulfonamide protective group is described. This new N-Boc-o-nitrobenzenesulfonamide (Boc-ONBS) linker enables the elaboration on resin of primary and secondary amines by sequential substitution of the sulfonamide moiety using the Mitsunobu reaction. A 16-member array of secondary and Boc protected primary amines was then prepared using this linker.

The application of a thiohydroxamic acid (THA) linker in the solid-phase synthesis of a urea library

A library of mono- and bis-ureas were synthesised on resin derivatised with a thiohydroxamic acid (THA) linker. The ureas were obtained in good yields and the resin was recycled three times with no apparent loss of function.

Application of Differential Isotopic Mass Splitting (DiMaS), a New Mass Spectrometry Methodology for the Analysis of Truncated Peptides, to Amyloid Precursor Protein Cleavage by Human Bleomycin Hydrolase and Neprilysin

A simple method for the analysis of peptides based on mass spectrometry has been applied to the study of interactions between human bleomycin hydrolase (hBH) or neprilysin (NEP; EC 3.4.24.11) and peptides Ac-HHQKLVFFAG-NH2, Ac-SEVNLDAEFG-NH2 and Ac-GGVVIATVIG-NH2, three substrates cleaved by α, β and γ-secretase, respectively. These proteases are involved in the catabolism of amyloid protein precursor (APP), a protein implicated in Alzheimers disease. The results indicate that hBH does not cleave the three substrates tested. Conversely, NEP cleaves Ac-HHQKLVFFAG-NH2 at multiple sites and Ac-SEVNLDAEFG-NH2 at only one position, whereas no cleavage was observed with Ac-GGVVIATVIG-NH2. The fragments generated by cleavage with NEP were clearly identified without any further analysis by tandem mass spectrometry. The implication of the role of NEP in the Alzheimer detoxification process is discussed. This technique provides useful information on peptide cleavage points and could be applied easily to a mixture of peptides for the determination of amino acid sequences preferred by any protease.