RESUMEN
A cell-based assay for the chemokine G-protein-coupled receptor CCR4 was developed, and used to screen a small-molecule compound collection in a multiplex format. A series of bipiperidinyl carboxylic acid amides amenable to parallel chemistry were derived that were potent and selective antagonists of CCR4. One prototype compound was shown to be active in a functional model of chemotaxis, making it a useful chemical tool to explore the role of CCR4 in asthma, allergy, diabetes, and cancer.
Asunto(s)
Amidas/química , Amidas/farmacología , Biperideno/química , Ácidos Carboxílicos/química , Receptores de Quimiocina/antagonistas & inhibidores , Concentración 50 Inhibidora , Estructura Molecular , Receptores CCR4 , Receptores de Quimiocina/metabolismo , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
An early drug discovery approach focusing on gene families can benefit from strategies that exploit common signaling mechanisms to more effectively identify and characterize novel chemical lead structures. Multiplexing, defined as the screening of multiple targets within the same experiment, is an example of this strategy. Here, the authors describe a technique that allows multiplexing of a common assay type used to study G-protein-coupled receptors: changes in intracellular Ca2+ levels as measured by Molecular Device's fluorometric imaging plate reader (FLIPR). The multiplexed FLIPR assays showed the expected pharmacological properties of single assays, with good reproducibility and Z* factors. The authors used them to screen large compound libraries in 2 multiplexed assay designs. The 1st used a single-cell line expressing 2 different receptors and the 2nd a mixture of 2 cell lines of the same type each expressing distinct receptors. Screening using these multiplexed assays produced significant savings in reagents, time, and human resources and allowed the authors to quickly identify specific and selective hits.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Fluorometría , Receptores Acoplados a Proteínas G/metabolismo , Bioensayo , Calcio/metabolismo , Línea Celular , Receptores Acoplados a Proteínas G/genéticaRESUMEN
3-(2-Carboxyethyl)-4,6-dichloro-1H-indole-2-carboxylic acid (MDL-29951), an antagonist of the glycine site of the NMDA receptor, has been found to be an allosteric inhibitor of the enzyme fructose 1,6-bisphosphatase. The compound binds at the AMP regulatory site by X-ray crystallography. This represents a new approach to inhibition of fructose 1,6-bisphosphatase and serves as a lead for further drug design.
Asunto(s)
Adenosina Monofosfato/metabolismo , Fructosa-Bifosfatasa/antagonistas & inhibidores , Indoles/metabolismo , Indoles/farmacología , Propionatos/metabolismo , Propionatos/farmacología , Sitio Alostérico , Animales , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Fructosa-Bifosfatasa/metabolismo , Humanos , Indoles/química , Modelos Moleculares , Propionatos/química , Conejos , Ratas , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , PorcinosRESUMEN
The synthesis and in vitro structure-activity relationships (SAR) of a novel series of anilinoquinazolines as allosteric inhibitors of fructose-1,6-bisphosphatase (F16Bpase) are reported. The compounds have a different SAR as inhibitors of F16Bpase than anilinoquinazolines previously reported. Selective inhibition of F16Bpase can be attained through the addition of appropriate polar functional groups at the quinazoline 2-position, thus separating the F16Bpase inhibitory activity from the epidermal growth factor receptor tyrosine kinase inhibitory activity previously observed with similar structures. The compounds have been found to bind at a symmetry-repeated novel allosteric site at the subunit interface of the enzyme. Inhibition is brought about by binding to a loop comprised of residues 52-72, preventing the necessary participation of these residues in the assembly of the catalytic site. Mutagenesis studies have identified the key amino acid residues in the loop that are required for inhibitor recognition and binding.