RESUMEN
In seeking novel and potent small molecule hematopoietic prostaglandin D2 synthase (H-PGDS) inhibitors as potential therapies for PGD2-mediated diseases and conditions, we explored a series comprising multiple aryl/heteroaryl rings attached in a linear arrangement. Each compound incorporates an amide or imidazole "linker" between the pyrimidine or pyridine "core" ring and the "tail" ring system. We synthesized and screened twenty analogs by fluorescence polarization binding assay, thermal shift assay, glutathione S-transferase inhibition assay, and a cell-based assay measuring suppression of LPS-induced PGD2 stimulation. Amide analogs show ten-fold greater shift in the thermal shift assay in the presence of glutathione (GSH) versus the same assay run in the absence of GSH. The imidazole analogs did not produce a significant change in thermal shift between the two assay conditions, suggesting a possible stabilization effect of the amide linker in the synthase-GSH-inhibitor complex. Imidazole analog 23, (KMN-010034) demonstrates superior potency across the in vitro assays and good in vitro metabolic stability in both human and guinea pig liver microsomes.
Asunto(s)
Amidas/farmacología , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Lipocalinas/antagonistas & inhibidores , Amidas/síntesis química , Amidas/química , Animales , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Cobayas , Humanos , Imidazoles/síntesis química , Imidazoles/química , Oxidorreductasas Intramoleculares/metabolismo , Lipocalinas/metabolismo , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Estructura Molecular , Relación Estructura-ActividadRESUMEN
We report here on the design, synthesis, and evaluation of small molecule inhibitors of the interaction between a steroid receptor coactivator and estrogen receptor alpha. These inhibitors are based upon an amphipathic benzene scaffold whose hydrophobic face mimics the leucine-rich alpha-helical consensus sequence on the steroid receptor coactivators that interacts with a shallow groove on estrogen receptor alpha. Several of these molecules are among the most potent inhibitors of this interaction described to date and are active at low micromolar concentrations in both in vitro models of estrogen receptor action and in cell-based assays of estrogen receptor-mediated coactivator interaction and transcription.
Asunto(s)
Derivados del Benceno/farmacología , Receptor alfa de Estrógeno/antagonistas & inhibidores , Histona Acetiltransferasas/antagonistas & inhibidores , Pirimidinas/farmacología , Transactivadores/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Derivados del Benceno/síntesis química , Derivados del Benceno/química , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Receptor alfa de Estrógeno/química , Receptor alfa de Estrógeno/genética , Transferencia Resonante de Energía de Fluorescencia , Genes Reporteros , Histona Acetiltransferasas/química , Histona Acetiltransferasas/genética , Humanos , Ligandos , Luciferasas/genética , Coactivador 1 de Receptor Nuclear , Coactivador 3 de Receptor Nuclear , Plásmidos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Pirimidinas/síntesis química , Pirimidinas/química , Transactivadores/química , Transactivadores/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Transcripción Genética/efectos de los fármacos , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
Nuclear hormone receptor (NR) function relies on association of agonist-bound receptors with steroid receptor coactivator (SRC) proteins through a small pentapeptide motif (LXXLL) of the SRC that binds to a hydrophobic groove on the NR. We have synthesized a series of bicyclo[2.2.2]octanes that are close structural mimics of the two key leucine residues of this SRC sequence as bound in the hydrophobic groove of the estrogen receptor. These bicyclic systems block the NR-SRC interaction with modest potency.
Asunto(s)
Compuestos Bicíclicos con Puentes/química , Histona Acetiltransferasas/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Compuestos Bicíclicos con Puentes/farmacología , Transferencia Resonante de Energía de Fluorescencia , Histona Acetiltransferasas/química , Modelos Moleculares , Imitación Molecular , Datos de Secuencia Molecular , Estructura Molecular , Coactivador 1 de Receptor Nuclear , Factores de Transcripción/químicaRESUMEN
Nuclear receptors (NRs) complexed with agonist ligands activate transcription by recruiting coactivator protein complexes. In principle, one should be able to inhibit the transcriptional activity of the NRs by blocking this transcriptionally critical receptor-coactivator interaction directly, using an appropriately designed coactivator binding inhibitor (CBI). To guide our design of various classes of CBIs, we have used the crystal structure of an agonist-bound estrogen receptor (ER) ligand binding domain (LBD) complexed with a coactivator peptide containing the LXXLL signature motif bound to a hydrophobic groove on the surface of the LBD. One set of CBIs, based on an outside-in design approach, has various heterocyclic cores (triazenes, pyrimidines, trithianes, cyclohexanes) that mimic the tether sites of the three leucines on the peptide helix, onto which are appended leucine residue-like substituents. The other set, based on an inside-out approach, has a naphthalene core that mimics the two most deeply buried leucines, with substituents extending outward to mimic other features of the coactivator helical peptide. A fluorescence anisotropy-based coactivator competition assay was developed to measure the specific binding of these CBIs to the groove site on the ER-agonist complex with which coactivators interact; control ligand-binding assays assured that their interaction was not with the ligand binding pocket. The most effective CBIs were those from the pyrimidine family, the best binding with K(i) values of ca. 30 microM. The trithiane- and cyclohexane-based CBIs appear to be poor structural mimics, because of equatorial vs axial conformational constraints, and the triazene-based CBIs are also conformationally constrained by amine-substituent-to-ring resonance overlap, which is not the case with the higher affinity alkyl-substituted pyrimidines. The pyrimidine-based CBIs appear to be the first small molecule inhibitors of NR coactivator binding.