Discovery and structure–activity relationships of a novel class of quinazoline glucokinase activators

We describe design, syntheses and structure–activity relationships of a novel class of 4,6-disubstituted quinazoline glucokinase activators. Prototype quinazoline leads (4 and 5) were designed based on the X-ray analyses of the previous 2-aminobenzamide lead classes. Modifications of the quinazoline leads led to the identification of a potent GK activator (21d).     

A Novel Method to Quantify Contribution of Channels and Transporters to Membrane Potential Dynamics

The action potential, once triggered in ventricular or atrial myocytes, automatically proceeds on its time course or is generated spontaneously in sinoatrial node pacemaker cells. It is induced by complex interactions among such cellular components as ion channels, transporters, intracellular ion concentrations, and signaling molecules. We have developed what is, to our knowledge, a new method using a mathematical model to quantify the contribution of each cellular component to the automatic time courses of the action potential. In this method, an equilibrium value, which the membrane potential is approaching at a given moment, is calculated along the time course of the membrane potential. The calculation itself is based on the time-varying conductance and the reversal potentials of individual ion channels and electrogenic ion transporters. Since the equilibrium potential moves in advance of the membrane potential change, we refer to it as the lead potential, V_L. The contribution of an individual current was successfully quantified by comparing dV_L/dt before and after fixing the time-dependent change of a component of interest, such as the variations in the open probability of a channel or the turnover rate of an ion transporter. In addition to the action potential, the lead-potential analysis should also be applicable in all types of membrane excitation in many different kinds of cells.     

Metabolic activation of N-thiazol-2-yl benzamide as glucokinase activators: Impacts of glutathione trapping on covalent binding

Glucokinase activators (GKAs) are currently under investigation as potential antidiabetic agents by many pharmaceutical companies. Most of GKAs reported previously possess N-aminothiazol-2-yl amide moiety in their structures because the aminothiazole moiety interacts with glucokinase (GK) and shows strong GK activation. During the development of N-aminothiazol-2-yl amide derivatives, we identified a bioactivation and metabolic liability of 2-aminothizole substructure of GKA 3 by assessing covalent binding, metabolites in liver microsomes and glutathione (GSH) trap assay.     

Amino Acid Residues Critical for Endoplasmic Reticulum Export and Trafficking of Platelet-activating Factor Receptor

Several residues are conserved in the transmembrane domains (TMs) of G-protein coupled receptors. Here we demonstrate that a conserved proline, Pro²⁴⁷, in TM6 of platelet-activating factor receptor (PAFR) is required for endoplasmic reticulum (ER) export and trafficking after agonist-induced internalization. Alanine-substituted mutants of the conserved residues of PAFRs, including P247A, were retained in the ER. Because a PAFR antagonist, Y-24180, acted as a pharmacological chaperone to rescue ER retention, this retention is due to misfolding of PAFR. Methylcarbamyl (mc)-PAF, a PAFR agonist, did not increase the cell surface expression of P247A, even though another ER-retained mutant, D63A, was effectively trafficked. Signaling and accumulation of the receptors in the early endosomes were observed in the mc-PAF-treated P247A-expressing cells, suggesting that P247A was trafficked to the cell surface by mc-PAF, and thereafter disappeared from the surface due to aberrant trafficking, e.g. enhanced internalization, deficiency in recycling, and/or accelerated degradation. The aberrant trafficking was confirmed with a sortase-A-mediated method for labeling cell surface proteins. These results demonstrate that the conserved proline in TM6 is crucial for intracellular trafficking of PAFR.     

Expression of heat shock protein 70a mRNA in Bombyx mori diapause eggs

In an effort to understand whether heat shock protein 70 (Hsp70) participates in the environmental 5 °C signal reception/transduction toward breaking embryonic diapause of the silkworm Bombyx mori, we isolated a cDNA for Hsp70a and examined the expression of Hsp70a mRNA in B. mori diapause and nondiapause eggs by quantitative real-time PCR. Hsp70a mRNA gradually increased in diapause eggs continuously kept at 25 °C after oviposition to maintain diapause. When diapause eggs were exposed to the diapause-terminating condition of 5 °C beginning at 2 days post-oviposition, Hsp70a mRNA increased beginning at 5 days post-cold treatment. Even in nondiapause eggs, Hsp70a mRNA increased slightly with exposure to 5 °C. These results suggest that Hsp70a is involved in reception/transduction of the diapause-terminating (5 °C) signal via gene activation. The expression patterns of Hsp70a mRNA are discussed in relation to those of the cold-response gene Samui.     

Homogeneous sandwich immunoassay based on the enzymatic complementation induced by single-chain Fv fragments

We describe a novel homogeneous sandwich immunoassay based on beta-galactosidase (beta-gal) complementation (the crab-claw sandwich enzymatic complementation immunoassay, CS-ECIA). We chose a high-molecular-weight antigen human serum albumin (HSA) as a model and constructed two chimeric proteins, in which a pair of single-chain Fvs (scFvs) recognizing distant epitopes of HSA was fused to either an N-terminal deletion mutant of beta-gal (deltaalpha) or a C-terminal deletion mutant of beta-gal (deltaomega). Upon simple mixing of the reagents with the sample, the two chimeric proteins became associated through binding separate epitopes on HSA that allowed reassociation of the two mutant enzymes. The resulting enzymatic complementation was measured as an increase in beta-gal activity using a luminescent substrate. With this CS-ECIA, a HSA concentration of 10-1000 pg/mL could be determined. In addition, the assay was easy to operate and required less time, handling, and sample volume than conventional sandwich enzyme-linked immunoassays. The assay will have general utility by substituting scFvs with other pairs of scFvs recognizing any polyvalent antigens.