RESUMEN
Castration-resistant prostate cancers (CRPCs) lose sensitivity to androgen-deprivation therapies but frequently remain dependent on oncogenic transcription driven by the androgen receptor (AR) and its splice variants. To discover modulators of AR-variant activity, we used a lysate-based small-molecule microarray assay and identified KI-ARv-03 as an AR-variant complex binder that reduces AR-driven transcription and proliferation in prostate cancer cells. We deduced KI-ARv-03 to be a potent, selective inhibitor of CDK9, an important cofactor for AR, MYC, and other oncogenic transcription factors. Further optimization resulted in KB-0742, an orally bioavailable, selective CDK9 inhibitor with potent anti-tumor activity in CRPC models. In 22Rv1 cells, KB-0742 rapidly downregulates nascent transcription, preferentially depleting short half-life transcripts and AR-driven oncogenic programs. In vivo, oral administration of KB-0742 significantly reduced tumor growth in CRPC, supporting CDK9 inhibition as a promising therapeutic strategy to target AR dependence in CRPC.
Asunto(s)
Antagonistas de Receptores Androgénicos/farmacología , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Receptores Androgénicos/genética , Transcripción Genética/efectos de los fármacos , Antagonistas de Receptores Androgénicos/uso terapéutico , Animales , Línea Celular Tumoral , Quinasa 9 Dependiente de la Ciclina/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Neoplasias de la Próstata Resistentes a la Castración/genética , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
At the onset of a drug discovery program, the goal is to identify novel compounds with appropriate chemical features that can be taken forward as lead series. Here, we describe three prospective case studies, Bruton Tyrosine Kinase (BTK), RAR-Related Orphan Receptor γ t (RORγt), and Human Leukocyte Antigen DR isotype (HLA-DR) to illustrate the positive impact of high throughput virtual screening (HTVS) on the successful identification of novel chemical series. Each case represents a project with a varying degree of difficulty due to the amount of structural and ligand information available internally or in the public domain to utilize in the virtual screens. We show that HTVS can be effectively employed to identify a diverse set of potent hits for each protein system even when the gold standard, high resolution structural data or ligand binding data for benchmarking, is not available.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Agammaglobulinemia Tirosina Quinasa/química , Industria Farmacéutica , Antígenos HLA-DR/química , Antígenos HLA-DR/metabolismo , Humanos , Modelos Moleculares , Receptores Nucleares Huérfanos/química , Receptores Nucleares Huérfanos/metabolismo , Conformación Proteica , Inhibidores de Proteínas Quinasas/farmacología , Factores de Tiempo , Interfaz Usuario-ComputadorRESUMEN
A novel series of benzyl substituted thieno[2,3-d]pyrimidines were identified as potent A2A receptor antagonists. Several five- and six-membered heterocyclic replacements for the optimized methylfuran were explored. Select compounds effectively reverse catalepsy in mice when dosed orally.
Asunto(s)
Antagonistas del Receptor de Adenosina A2/química , Pirimidinas/química , Receptor de Adenosina A2A/metabolismo , Antagonistas del Receptor de Adenosina A2/farmacocinética , Antagonistas del Receptor de Adenosina A2/uso terapéutico , Animales , Encéfalo/metabolismo , Catalepsia/tratamiento farmacológico , Semivida , Humanos , Cinética , Ratones , Unión Proteica , Pirimidinas/farmacocinética , Pirimidinas/uso terapéutico , Ratas , Receptor de Adenosina A2A/químicaRESUMEN
The design and characterization of two, dual adenosine A(2A)/A(1) receptor antagonists in several animal models of Parkinson's disease is described. Compound 1 was previously reported as a potential treatment for Parkinson's disease. Further characterization of 1 revealed that it was metabolized to reactive intermediates that caused the genotoxicity of 1 in the Ames and mouse lymphoma L51784 assays. The identification of the metabolites enabled the preparation of two optimized compounds 13 and 14 that were devoid of the metabolic liabilities associated with 1. Compounds 13 and 14 are potent dual A(2A)/A(1) receptor antagonists that have excellent activity, after oral administration, across a number of animal models of Parkinson's disease including mouse and rat models of haloperidol-induced catalepsy, mouse and rat models of reserpine-induced akinesia, and the rat 6-hydroxydopamine (6-OHDA) lesion model of drug-induced rotation.
Asunto(s)
Antagonistas del Receptor de Adenosina A1/síntesis química , Antagonistas del Receptor de Adenosina A2/síntesis química , Indenos/síntesis química , Trastornos Parkinsonianos/tratamiento farmacológico , Pirimidinas/síntesis química , Receptor de Adenosina A2A/metabolismo , Antagonistas del Receptor de Adenosina A1/farmacocinética , Antagonistas del Receptor de Adenosina A1/farmacología , Antagonistas del Receptor de Adenosina A2/farmacocinética , Antagonistas del Receptor de Adenosina A2/farmacología , Administración Oral , Animales , Diseño de Fármacos , Femenino , Indenos/farmacocinética , Indenos/farmacología , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos BALB C , Trastornos Parkinsonianos/inducido químicamente , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
The in vivo characterization of a dual adenosine A(2A)/A(1) receptor antagonist in several animal models of Parkinson's disease is described. Discovery and scale-up syntheses of compound 1 are described in detail, highlighting optimization steps that increased the overall yield of 1 from 10.0% to 30.5%. Compound 1 is a potent A(2A)/A(1) receptor antagonist in vitro (A(2A) K(i) = 4.1 nM; A(1) K(i) = 17.0 nM) that has excellent activity, after oral administration, across a number of animal models of Parkinson's disease including mouse and rat models of haloperidol-induced catalepsy, mouse model of reserpine-induced akinesia, rat 6-hydroxydopamine (6-OHDA) lesion model of drug-induced rotation, and MPTP-treated non-human primate model.
Asunto(s)
Antagonistas del Receptor de Adenosina A1/síntesis química , Antagonistas del Receptor de Adenosina A2/síntesis química , Antiparkinsonianos/síntesis química , Indenos/síntesis química , Enfermedad de Parkinson/metabolismo , Pirimidinas/síntesis química , Receptor de Adenosina A2A/fisiología , Antagonistas del Receptor de Adenosina A1/farmacocinética , Antagonistas del Receptor de Adenosina A1/farmacología , Antagonistas del Receptor de Adenosina A2/farmacocinética , Antagonistas del Receptor de Adenosina A2/farmacología , Administración Oral , Animales , Antiparkinsonianos/farmacocinética , Antiparkinsonianos/farmacología , Callithrix , Modelos Animales de Enfermedad , Femenino , Indenos/farmacocinética , Indenos/farmacología , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos BALB C , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
A novel series of arylindenopyrimidines were identified as A(2A) and A(1) receptor antagonists. The series was optimized for in vitro activity by substituting the 8- and 9-positions with methylene amine substituents. The compounds show excellent activity in mouse models of Parkinson's disease when dosed orally.
Asunto(s)
Antagonistas del Receptor de Adenosina A1 , Antagonistas del Receptor de Adenosina A2 , Aminas/química , Neurotransmisores/química , Pirimidinas/química , Aminas/síntesis química , Aminas/uso terapéutico , Animales , Catalepsia/tratamiento farmacológico , Modelos Animales de Enfermedad , Ratones , Neurotransmisores/síntesis química , Neurotransmisores/uso terapéutico , Pirimidinas/síntesis química , Pirimidinas/uso terapéutico , Receptor de Adenosina A1/metabolismo , Receptor de Adenosina A2A/metabolismo , Relación Estructura-ActividadRESUMEN
Two reactive metabolites were identified in vivo for the dual A(2A)/A(1) receptor antagonist 1. Two strategies were implemented to successfully mitigate the metabolic liabilities associated with 1. Optimization of the arylindenopyrimidines led to a number of amide, ether, and amino analogs having comparable in vitro and in vivo activity.
Asunto(s)
Antagonistas del Receptor de Adenosina A1 , Antagonistas del Receptor de Adenosina A2 , Neurotransmisores/química , Pirimidinas/química , Animales , Catalepsia/tratamiento farmacológico , Modelos Animales de Enfermedad , Ratones , Neurotransmisores/síntesis química , Neurotransmisores/uso terapéutico , Pirimidinas/síntesis química , Pirimidinas/uso terapéutico , Receptor de Adenosina A1/metabolismo , Receptor de Adenosina A2A/metabolismo , Relación Estructura-ActividadRESUMEN
A novel series of acyclic 3-(7-azaindolyl)-4-(aryl/heteroaryl)maleimides was synthesized and evaluated for activity against GSK-3beta and selectivity versus PKC-betaII, as well as a broad panel of protein kinases. Compounds 14 and 17c potently inhibited GSK-3beta (IC(50)=7 and 26 nM, respectively) and exhibited excellent selectivity over PKC-betaII (325 and >385-fold, respectively). Compound 17c was also highly selective against 68 other protein kinases. In a cell-based functional assay, both 14 and 17c effectively increased glycogen synthase activity by inhibiting GSK-3beta.
Asunto(s)
Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Maleimidas/química , Inhibidores de Proteínas Quinasas/química , Línea Celular , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Maleimidas/farmacología , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Two approaches were developed to synthesize the novel 7-azaindolyl-heteroarylmaleimides. The first approach was based upon the palladium-catalyzed Suzuki cross-coupling or Stille cross-coupling of 2-chloro-maleimide 5 with various arylboronic acids or arylstannanes. The second approach was based upon the condensation of ethyl 7-azaindolyl-3-glyoxylate 12 with various acetamides. The hydroxypropyl-substituted 7-azaindolylmaleimide template was first used to screen different heteroaryls attached to the maleimide. Replacement of hydroxypropyl with different chain lengths and different functional groups were studied next. Many compounds synthesized were demonstrated to have high potency at GSK-3beta, good GS activity in HEK293 cells and good to excellent metabolic stability in human liver microsomes. Three representative compounds (21, 33, and 34) were demonstrated to have good selectivity against a panel of 80 kinase assays. Among them, compound 33 exhibited very weak inhibitions at the other 79 kinase assays, and behaved as a highly selective GSK-3beta inhibitor.
Asunto(s)
Compuestos Aza/síntesis química , Compuestos Aza/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Maleimidas/química , Maleimidas/farmacología , Animales , Compuestos Aza/química , Línea Celular , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Maleimidas/síntesis química , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Estructura Molecular , Conejos , Especificidad por SustratoRESUMEN
Palladium catalyzed cross-coupling reactions were used to synthesize two key intermediates 3 and 5 that resulted in the synthesis of novel series of macrocyclic bis-7-azaindolylmaleimides. Among the three series of macrocycles, the oxygen atom and thiophene containing linkers yielded molecules with higher inhibitory potency at GSK-3 beta (K(i)=0.011-0.079 microM) while the nitrogen atom containing linkers yielded molecules with lower potency (K(i)=0.150->1 microM). Compound 33 and 36 displayed 1-2 orders of magnitude selectivity at GSK-3 beta against CDK2, PKC beta II, Rsk3 and little or no inhibitions to the other 62 protein kinases. Compound 46 was at least 100-fold more selective towards GSK-3 beta than PKC beta II, and it had little or no activity against a panel of 65 protein kinases, almost behaved as a GSK-3 beta 'specific inhibitor'. All three compounds showed good potency in GS assay. Molecular docking studies were conducted in an attempt to rationalize the GSK-3 beta selectivity of azaindolylmaleimides. The high selectivity, inhibitory potency and cellular activities of these non-crown-ether typed molecules may provide them as a valuable pharmacological tools in elucidating the complex roles of GSK-3 beta in cell signaling pathways and the potential usage for the treatment of elevated level of GSK-3 beta involved diseases.
Asunto(s)
Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Maleimidas/síntesis química , Maleimidas/farmacología , Secuencia de Aminoácidos , Línea Celular , Simulación por Computador , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Glucógeno Sintasa Quinasa 3 beta , Humanos , Maleimidas/química , Proteínas Quinasas/química , Proteínas Quinasas/clasificación , Proteínas Quinasas/efectos de los fármacos , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
The use of a multiplex mimetic assay led us to identify 1,4-dihydropyrimidines with potent and selective calcitonin receptor mimetic activity. Subsequent modification of the dihydropyrimidine scaffold led to a series of molecules that were efficacious in a neonatal mouse calvaria in vitro model. Dihydropyrimidine 5h, in particular, was identified as a calcitonin mimetic (EC(50)=6 microM), active in-vivo in the Weanling rat model when administered subcutaneously.
Asunto(s)
Calcitonina/química , Calcitonina/fisiología , Dihidropiridinas/química , Imitación Molecular/fisiología , Pirimidinas/química , Animales , Calcitonina/orina , Línea Celular , Dihidropiridinas/farmacología , Dihidropiridinas/orina , Humanos , Pirimidinas/farmacología , Pirimidinas/orina , RatasRESUMEN
Stimulation of a cell with insulin initiates a signal transduction cascade that results in cellular activities that include phosphorylation of the receptor itself. Measurement of the degree of phosphorylation can serve as a marker for receptor activation. Receptor phosphorylation has been measured using Western blot analysis, which is very low throughput and not easily quantifiable. The goal of this project was to develop a cell-based assay to measure receptor phosphorylation in high throughput. This report describes a cell-based assay for insulin receptor phosphorylation that is robust and amenable to high-volume screening in a microwell format.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Insulina/metabolismo , Receptor de Insulina/metabolismo , Animales , Células CHO , Cricetinae , Indicadores y Reactivos , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de SeñalRESUMEN
Attempts to design the macrocyclic maleimides as selective protein kinase C gamma inhibitors led to the unexpected discovery of a novel series of potent and highly selective glycogen synthase kinase-3beta (GSK-3beta) inhibitors. Palladium-catalyzed cross-coupling reactions were used to synthesize the key intermediates 17 and 22 that resulted in the synthesis of novel macrocycles. All three macrocyclic series (bisindolyl-, mixed 7-azaindoleindolyl-, and bis-7-azaindolylmaleimides) were found to have submicromolar inhibitory potency at GSK-3beta with various degrees of selectivity toward other protein kinases. To gain the inhibitory potency at GSK-3beta, the ring sizes of these macrocycles may play a major role. To achieve the selectivity at GSK-3beta, the additional nitrogen atoms in the indole rings may contribute to a significant degree. Overall, the bis-7-azaindolylmaleimides 28 and 29 exhibited little or no inhibitions to a panel of 50 protein kinases. Compound 29 almost behaved as a GSK-3beta specific inhibitor. Both 28 and 29 displayed good potency in GS cell-based assay. Molecular docking studies were conducted in an attempt to rationalize the GSK-3beta selectivity of azaindolylmaleimides.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Éteres Cíclicos/síntesis química , Éteres Cíclicos/farmacología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Maleimidas/síntesis química , Maleimidas/farmacología , Adipocitos/citología , Adipocitos/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Células Cultivadas , Diseño de Fármacos , Éteres Cíclicos/química , Glucógeno Sintasa Quinasa 3/metabolismo , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Maleimidas/química , Modelos Moleculares , Datos de Secuencia Molecular , Inhibidores de Proteínas Quinasas , Proteínas Quinasas/metabolismo , Ratas , Alineación de Secuencia , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Efficient methods were developed to synthesize a novel series of macrocyclic bisindolylmaleimides containing linkers with multiple heteroatoms. Potent inhibitors (single digit nanomolar IC(50)) for PKC-beta and GSK-3beta were identified, and compounds showed good selectivity over PKC-alpha, -gamma, -delta, -epsilon, and -zeta. Representative compound 5a also had high selectivity in a screening panel of 10 other protein kinases. In cell-based functional assays, several compounds effectively blocked interleukin-8 release induced by PKC-betaII and increased glycogen synthase activity by inhibiting GSK-3beta.