RESUMEN
Discovery of potent renin inhibitors which contain a simplified alkylamino Asp-binding group and exhibit improved selectivity for renin over Cyp3A4 is described. Structure-function results in this series are rationalized based on analysis of selected compounds bound to renin, and the contribution of each molecular feature leading to the reduced P450 inhibition is quantified.
Asunto(s)
Ácido Aspártico/metabolismo , Citocromo P-450 CYP3A/metabolismo , Inhibidores de Proteasas/química , Renina/antagonistas & inhibidores , Ácido Aspártico/química , Sitios de Unión , Cristalografía por Rayos X , Citocromo P-450 CYP3A/química , Inhibidores del Citocromo P-450 CYP3A/química , Inhibidores del Citocromo P-450 CYP3A/metabolismo , Evaluación Preclínica de Medicamentos , Humanos , Concentración 50 Inhibidora , Simulación de Dinámica Molecular , Inhibidores de Proteasas/metabolismo , Unión Proteica , Renina/metabolismo , Relación Estructura-ActividadRESUMEN
Epigenetic gene regulation is a critical process controlling differentiation and development, the malfunction of which may underpin a variety of diseases. In this article, we review the current landscape of small-molecule epigenetic modulators including drugs on the market, key compounds in clinical trials, and chemical probes being used in epigenetic mechanistic studies. Hit identification strategies for the discovery of small-molecule epigenetic modulators are summarized with respect to writers, erasers, and readers of histone marks. Perspectives are provided on opportunities for new hit discovery approaches, some of which may define the next generation of therapeutic intervention strategies for epigenetic processes.
Asunto(s)
Descubrimiento de Drogas , Epigénesis Genética , Ensayos Analíticos de Alto Rendimiento , Descubrimiento de Drogas/métodos , Epigénesis Genética/efectos de los fármacos , Epigenómica/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Histonas/metabolismo , Humanos , Unión Proteica/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Large conductance Ca(2+)-activated K(+) (BK) channels are known to be regulated by both intracellular Ca(2+) and voltage. Although BK channel modulators have been identified, there is a paucity of information regarding the molecular entities of this channel that govern interaction with blockers and activators. Using both whole-cell and single-channel electrophysiological studies we have characterized the possible role that a threonine residue in the pore region of the channel has on function and interaction with BK channel modulators. A threonine-to-serine substitution at position 352 (T352S) resulted in a 59-mV leftward shift in the voltage-dependent activation curve. Single-channel conductance was 236 pS for the wild-type channel and 100 pS for the T352S mutant, measured over the range -80 mV to +80 mV. In addition, there was an almost 10-fold reduction in the potency of the BK channel inhibitor 1-[1-hexyl-6-(methyloxy)-1H-indazol-3-yl]-2-methyl-1-propanone (HMIMP), the IC(50) values being 4.3 +/- 0.3 and 38.2 +/- 3.3 nM for wild-type and mutant channel, respectively. There was no significant difference between wild type and the mutant channel in response to inhibition by iberiotoxin. The IC(50) was 8.1 +/- 0.3 nM for the wild type and 7.7 +/- 0.3 nM for the mutant channel. Here, we have identified a residue in the pore region of the BK channel that alters voltage sensitivity and reduces the potency of the blocker HMIMP.
Asunto(s)
Calcio/fisiología , Indazoles/farmacología , Canales de Potasio de Gran Conductancia Activados por el Calcio/antagonistas & inhibidores , Treonina/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Células CHO , Cricetinae , Cricetulus , Conductividad Eléctrica , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Canales de Potasio de Gran Conductancia Activados por el Calcio/fisiología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Técnicas de Placa-Clamp , Homología de Secuencia de AminoácidoRESUMEN
Recent studies using known Rho-associated kinase isoform 1 (ROCK1) inhibitors along with cellular and molecular biology data have revealed a pivotal role of this enzyme in many aspects of cardiovascular function. Here we report a series of ROCK1 inhibitors which were originally derived from a dihydropyrimidinone core 1. Our efforts focused on the optimization of dihydropyrimidine 2, which resulted in the identification of a series of dihydropyrimidines with improved pharmacokinetics and P450 properties.
Asunto(s)
Enfermedades Cardiovasculares/tratamiento farmacológico , Enfermedades Cardiovasculares/enzimología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/química , Pirimidinas/uso terapéutico , Quinasas Asociadas a rho/antagonistas & inhibidores , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Administración Oral , Aldehídos/química , Animales , Cristalografía por Rayos X , Indazoles/química , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/administración & dosificación , Pirimidinas/administración & dosificación , Ratas , Relación Estructura-Actividad , Quinasas Asociadas a rho/metabolismoRESUMEN
Long and short QT syndromes associated with loss and gain of human ether-a-go-go-related gene (hERG) channel activity, respectively, can cause life-threatening arrhythmias. As such, modulation of hERG channel activity is an important consideration in the development of all new therapeutic agents. In the present study, we investigated the mechanisms of action of 2-[2-(3,4-dichloro-phenyl)-2,3-dihydro-1H-isoindol-5-ylamino]-nicotinic acid (PD-307243), a known hERG channel activator, on hERG channels stably expressed in Chinese hamster ovary (CHO) cells using the patch-clamp technique. In the whole-cell recordings, the extracellular application of PD-307243 concentration-dependently increased the hERG current and markedly slowed hERG channel deactivation and inactivation. PD-307243 had no effect on the selectivity filter of hERG channels. The activity of PD-307243 was use-dependent. PD-307243 (3 and 10 muM) induced instantaneous hERG current with little decay at membrane potentials from -120 to -40 mV. At more positive voltages, PD-307243 induced an I(to)-like upstroke of hERG current. The actions of PD-307243 on the rapid component of delayed rectifier K(+) current (I(Kr)) in rabbit ventricular myocytes were similar to those observed in hERG channel-transfected CHO cells. Inside-out patch experiments revealed that PD-307243 increased hERG tail currents by 2.1 +/- 0.6 (n = 7) and 3.4 +/- 0.3-fold (n = 4) at 3 and 10 muM, respectively, by slowing the channel deactivation but had no effect on channel activation. During a voltage-clamp protocol using a prerecorded cardiac action potential, 3 muM PD-307243 increased the total potassium ions passed through hERG channels by 8.8 +/- 1.0-fold (n = 5). Docking studies suggest that PD-307243 interacts with residues in the S5-P region of the channel.
Asunto(s)
Canales de Potasio Éter-A-Go-Go/efectos de los fármacos , Canales de Potasio Éter-A-Go-Go/fisiología , Isoindoles/farmacología , Niacina/análogos & derivados , Niacina/farmacología , Ácidos Nicotínicos/farmacología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Sitios de Unión , Células CHO , Cricetinae , Cricetulus , Interpretación Estadística de Datos , Relación Dosis-Respuesta a Droga , Conductividad Eléctrica , Canales de Potasio Éter-A-Go-Go/genética , Ventrículos Cardíacos/citología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Isoindoles/química , Cinética , Masculino , Ratones , Microelectrodos , Modelos Moleculares , Estructura Molecular , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Niacina/química , Ácidos Nicotínicos/química , Técnicas de Placa-Clamp , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Conejos , TransfecciónRESUMEN
The discovery, proposed binding mode, and optimization of a novel class of Rho-kinase inhibitors are presented. Appropriate substitution on the 6-position of the azabenzimidazole core provided subnanomolar enzyme potency in vitro while dramatically improving selectivity over a panel of other kinases. Pharmacokinetic data was obtained for the most potent and selective examples and one (6n) has been shown to lower blood pressure in a rat model of hypertension.
Asunto(s)
Antihipertensivos/síntesis química , Bencimidazoles/síntesis química , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Oxadiazoles/síntesis química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Animales , Antihipertensivos/farmacocinética , Antihipertensivos/farmacología , Aorta/efectos de los fármacos , Aorta/fisiología , Bencimidazoles/farmacocinética , Bencimidazoles/farmacología , Presión Sanguínea/efectos de los fármacos , Técnicas In Vitro , Modelos Moleculares , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Oxadiazoles/farmacocinética , Oxadiazoles/farmacología , Ratas , Ratas Endogámicas SHR , Relación Estructura-Actividad , Quinasas Asociadas a rhoRESUMEN
Rho kinase (ROCK1) mediates vascular smooth muscle contraction and is a potential target for the treatment of hypertension and related disorders. Indazole amide 3 was identified as a potent and selective ROCK1 inhibitor but possessed poor oral bioavailability. Optimization of this lead resulted in the discovery of a series of dihydropyridones, exemplified by 13, with improved pharmacokinetic parameters relative to the initial lead. Indazole substitution played a critical role in decreasing clearance and improving oral bioavailability.
Asunto(s)
Amidas/síntesis química , Antihipertensivos/síntesis química , Indazoles/síntesis química , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Piridonas/síntesis química , Amidas/farmacocinética , Amidas/farmacología , Animales , Antihipertensivos/farmacocinética , Antihipertensivos/farmacología , Aorta/efectos de los fármacos , Aorta/fisiología , Presión Sanguínea/efectos de los fármacos , Técnicas In Vitro , Indazoles/farmacocinética , Indazoles/farmacología , Péptidos y Proteínas de Señalización Intracelular/química , Modelos Moleculares , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Proteínas Serina-Treonina Quinasas/química , Piridonas/farmacocinética , Piridonas/farmacología , Pirimidinas/síntesis química , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Ratas , Ratas Endogámicas SHR , Relación Estructura-Actividad , Quinasas Asociadas a rhoRESUMEN
Docking is a computational technique that samples conformations of small molecules in protein binding sites; scoring functions are used to assess which of these conformations best complements the protein binding site. An evaluation of 10 docking programs and 37 scoring functions was conducted against eight proteins of seven protein types for three tasks: binding mode prediction, virtual screening for lead identification, and rank-ordering by affinity for lead optimization. All of the docking programs were able to generate ligand conformations similar to crystallographically determined protein/ligand complex structures for at least one of the targets. However, scoring functions were less successful at distinguishing the crystallographic conformation from the set of docked poses. Docking programs identified active compounds from a pharmaceutically relevant pool of decoy compounds; however, no single program performed well for all of the targets. For prediction of compound affinity, none of the docking programs or scoring functions made a useful prediction of ligand binding affinity.
Asunto(s)
Ligandos , Proteínas/química , Relación Estructura-Actividad Cuantitativa , Algoritmos , Sitios de Unión , Diseño de Fármacos , Modelos Moleculares , Conformación Molecular , Unión Proteica , Programas InformáticosAsunto(s)
Diseño de Fármacos , Relación Estructura-Actividad Cuantitativa , Algoritmos , Biología Computacional , Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/farmacología , Humanos , Modelos Moleculares , Preparaciones Farmacéuticas/química , Receptores de Droga/químicaRESUMEN
We have used alanine scanning mutagenesis to identify residues in transmembrane domain 5 of the histamine H3 receptor that are important for agonist binding. All of the mutants generated were functionally expressed as demonstrated by their ability to bind [(125)I]iodoproxyfan with comparable affinity to the wild-type receptor and their ability to inhibit forskolin-stimulated cAMP formation when activated by histamine. Many mutations produced small changes in the potency of histamine, but the most pronounced reduction in potency and affinity of the agonists, histamine, R-alpha-methylhistamine, imetit, and impentamine, was seen with mutation of glutamate 206. Our modeling suggests that this residue plays a key role in ligand binding by interacting with the imidazole ring of histamine. Interestingly, L199A greatly reduced agonist potency in functional assays but had only minor effects on agonist affinity, implicating a role for this residue in the mechanism of receptor activation. We also studied the functional effects of the mutations by linking the receptor to calcium signaling using a chimeric G protein. A comparison of the two functional assays demonstrated contrasting effects on agonist activity. Histamine, imetit, and impentamine were full agonists in the cAMP assay, but imetit exhibited only partial agonist activity through the chimeric G protein. Furthermore, impentamine, another potent agonist in the cAMP assay, was only able to activate the E206A mutant in the calcium assay despite being inactive at the wild-type receptor. These observations suggest that the agonist receptor complexes formed by these three different H3 agonists are not conformationally equivalent.