Studies on zinc- and cadmium-bound proteins in bovine kidneys by biochemical and neutron activation techniques

Bovine kidneys were found to contain about 78 ppm Zn and 0.78 ppm Cd. Approximately 45% of Zn and 60% of Cd were present in the cytosol fraction. More than 95% of these two metals were bound to macromolecules. Both Zn- and Cd-protein complexes were observed to be stable between pH 7 and 10.5. Separation and characterization of these proteins were carried out using several chromatographic and electrophoretic techniques in conjunction with instrumental neutron activation analysis (INAA). The results showed the presence of at least four Zn-binding proteins with mol wt>300,000, 260,000, 89,000, and 27,000 and at least three Cd-binding proteins of mol wt>300,000, 32,000, and 13,000.     

Functional characterization of a receptor for vasoactive-intestinal-peptide-related peptides in cultured dermal melanophores from Xenopus laevis

A receptor for vasoactive-intestinal-peptide (VIP)-related peptides was functionally characterized in a cell line derived from Xenopus melanophores using a recently described microtiter-plate-based bioassay. Activation of the melanophore VIP receptor by VIP or the peptides pituitary-adenylate-cyclase-activating polypeptide (PACAP 38), PACAP 27, and helodermin stimulated intracellular 3'-5' cyclic adenosine monophosphate (cAMP) accumulation and pigment dispersion in the cells. Helodermin, with an EC50 (concentration of peptide inducing half-maximal melanosome dispersion) of 46.5 pM, was the most potent activator of pigment dispersion, followed by PACAP 38 > VIP > PACAP 27. A similar order of potencies was observed for the peptides to induce cAMP accumulation. The responses to VIP agonists were selectively inhibited by the VIP antagonists PACAP-(6-27) and (N-Ac-Tyr(1)-D-Phe2)-growth-hormone-releasing factor[GRF](1-29)-NH2. Taken together, the results suggest that the melanophores express a VIP receptor that shares certain characteristics of, but also differs significantly from, other previously identified VIP receptors.     

Identification of potent,nonabsorbable agonists of the calcium-sensing receptor for GI-specific administration

Modulation of gastrointestinal nutrient sensing pathways provides a promising a new approach for the treatment of metabolic diseases including diabetes and obesity. The calcium-sensing receptor has been identified as a key receptor involved in mineral and amino acid nutrient sensing and thus is an attractive target for modulation in the intestine. Herein we describe the optimization of gastrointestinally restricted calcium-sensing receptor agonists starting from a 3-aminopyrrolidine-containing template leading to the identification of GI-restricted agonist 19 (GSK3004774).     

Exploration of phenylpropanoic acids as agonists of the free fatty acid receptor 4 (FFA4): Identification of an orally efficacious FFA4 agonist

The long chain free fatty acid receptor 4 (FFA4/GPR120) has recently been recognized as lipid sensor playing important roles in nutrient sensing and inflammation and thus holds potential as a therapeutic target for type 2 diabetes and metabolic syndrome. To explore the effects of stimulating this receptor in animal models of metabolic disease, we initiated work to identify agonists with appropriate pharmacokinetic properties to support progression into in vivo studies. Extensive SAR studies of a series of phenylpropanoic acids led to the identification of compound 29, a FFA4 agonist which lowers plasma glucose in two preclinical models of type 2 diabetes.     

Purification of a Synaptic Membrane Na+/Ca2+ Antiporter and Immunoextraction with Antibodies to a 36-kDa Protein

The conditions for optimal solubilization and reconstitution of bovine brain synaptic plasma membrane Na+/Ca2+ exchange activity were examined and a series of chromatographic procedures were used for the isolation of a protein involved in this transport activity. The zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate in the presence of 20% (vol/vol) glycerol led to optimal solubilization, and soybean phospholipids in low-pH medium were found to produce optimal reconstitution of activity after dialysis to remove the detergent. Sequential chromatography steps involving the use of gel filtration on Sephacryl S-400 HR, ion exchange on diethylaminoethyl-Sephacel, and metal chelate chromatography on tris-(carboxymethyl)ethylenediamine loaded with LaCl3 led to the isolation of a fraction highly enriched in both Na+/Ca2+ exchange activity and two protein bands identified by denaturing electrophoresis. The estimated molecular masses of the two proteins were 50 and 36 kDa. Development of polyclonal antibodies to the 36-kDa protein permitted immunoextraction of greater than 95% of the antiporter activity from solubilized synaptic plasma membranes. These antibodies cross-reacted with the electroeluted 50-kDa protein on enzyme-linked immunosorbent assays, suggesting a close relationship between the two proteins. These results indicate that the 36-kDa protein is at least a component of the brain membrane Na+/Ca2+ antiporter.     

Synthesis and structure–activity relationships of a series of 3-aryl-4-isoxazolecarboxamides as a new class of TGR5 agonists

A series of 3-aryl-4-isoxazolecarboxamides identified from a high-throughput screening campaign as novel, potent agonists of the human TGR5 G-protein-coupled receptor is described. Many analogues were readily accessible via solution-phase synthesis which resulted in the rapid identification of key structure–activity relationships (SAR), and the discovery of potent exemplars (up to pEC₅₀ = 9). Details of the SAR and optimization of this series are presented herein.     

Going to the well no more: lawn format assays for ultra-high-throughput screening

Screening in a 'well-less' or lawn format provides a means to screen large compound collections against many targets in a fast, versatile and cost effective manner. The development of generic lawn format assays to screen various gene families against large compound collections should facilitate the identification of hits and tools to use in drug discovery and chemogenomic endeavours. Lawn format holds particular promise for screening GPCRs and selected enzyme families with potential use in other gene families.     

A novel approach for oxidation analysis of therapeutic proteins

Measuring and monitoring of protein oxidation modifications is important for biopharmaceutical process development and stability assessment during long-term storage. Currently available methods for biomolecules oxidation analysis use time-consuming peptide mapping analysis. Therefore, it is desirable to develop high-throughput methods for advanced process control of protein oxidation. Here, we present a novel approach by which oxidative protein modifications are monitored by an indirect potentiometric method. The method is based on adding an electron mediator, which enhances electron transfer (ET) between all redox species and the electrode surface. Specifically, the procedure involves measuring the sharp change in the open circuit potential (OCP) for the mediator system (redox couple) as a result of its interaction with the oxidized protein species in the solution. Application of Pt and Ag/AgCl microelectrodes allowed for a high-sensitivity protein oxidation analysis. We found that the Ru(NH₃)₆^2+/3+ redox couple is suitable for measuring the total oxidation of a wide range of therapeutic proteins between 1.1 and 13.6%. Accuracy determined by comparing with the known percentage oxidation of the reference standard showed that percentage oxidation determined for each sample was within ±20% of the expected percentage oxidation determined by mass spectrometry.