RESUMEN
Accurate ranking of compounds with regards to their binding affinity to a protein using computational methods is of great interest to pharmaceutical research. Physics-based free energy calculations are regarded as the most rigorous way to estimate binding affinity. In recent years, many retrospective studies carried out both in academia and industry have demonstrated its potential. Here, we present the results of large-scale prospective application of the FEP+ method in active drug discovery projects in an industry setting at Merck KGaA, Darmstadt, Germany. We compare these prospective data to results obtained on a new diverse, public benchmark of eight pharmaceutically relevant targets. Our results offer insights into the challenges faced when using free energy calculations in real-life drug discovery projects and identify limitations that could be tackled by future method development. The new public data set we provide to the community can support further method development and comparative benchmarking of free energy calculations.
Asunto(s)
Descubrimiento de Drogas , Ligandos , Estudios Prospectivos , Estudios Retrospectivos , TermodinámicaRESUMEN
There is unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545, a small-molecule inhibitor of the WNT pathway discovered through cell-based screening, is a potent and selective chemical probe for the human Mediator complex-associated protein kinases CDK8 and CDK19 with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates a type 1 binding mode involving insertion of the CDK8 C terminus into the ligand binding site. In contrast to type II inhibitors of CDK8 and CDK19, CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogs alter WNT pathway-regulated gene expression and other on-target effects of modulating CDK8 and CDK19, including expression of genes regulated by STAT1. Consistent with this, we find that phosphorylation of STAT1(SER727) is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally, we demonstrate in vivo activity of CCT251545 in WNT-dependent tumors.
Asunto(s)
Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Quinasa 8 Dependiente de Ciclina/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Sondas Moleculares/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Compuestos de Espiro/farmacología , Línea Celular Tumoral , Quinasa 8 Dependiente de Ciclina/genética , Quinasas Ciclina-Dependientes/genética , Humanos , Modelos Moleculares , Sondas Moleculares/química , Estructura Molecular , Inhibidores de Proteínas Quinasas/química , Piridinas/química , Compuestos de Espiro/químicaRESUMEN
Here we describe the discovery and optimization of 3-benzylindazoles as potent and selective inhibitors of CDK8, also modulating CDK19, discovered from a high-throughput screening (HTS) campaign sampling the Merck compound collection. The primary hits with strong HSP90 affinity were subsequently optimized to potent and selective CDK8 inhibitors which demonstrate inhibition of WNT pathway activity in cell-based assays. X-ray crystallographic data demonstrated that 3-benzylindazoles occupy the ATP binding site of CDK8 and adopt a Type I binding mode. Medicinal chemistry optimization successfully led to improved potency, physicochemical properties and oral pharmacokinetics. Modulation of phospho-STAT1, a pharmacodynamic biomarker of CDK8, was demonstrated in an APC-mutant SW620 human colorectal carcinoma xenograft model following oral administration.
Asunto(s)
Neoplasias Colorrectales/tratamiento farmacológico , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Descubrimiento de Drogas , Proteínas HSP90 de Choque Térmico/metabolismo , Indazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Animales , Neoplasias Colorrectales/metabolismo , Cristalografía por Rayos X , Quinasa 8 Dependiente de Ciclina/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Indazoles/administración & dosificación , Indazoles/química , Ratones , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Ratas , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
While A2A adenosine receptor (AR) was considered as a major contributor to adenosine-mediated immunosuppression, A2B, having the lowest affinity to adenosine, has also emerged as a potential contributor to tumor promotion. Therefore, in adenosine-rich tumor microenvironment (TME), where A2B could be complementary and/or compensatory to A2A, simultaneous targeting of A2A and A2B ARs can provide higher potential for cancer immunotherapy. We developed M1069 - a highly selective dual antagonist of the A2A and A2B AR. In assays with primary human and murine immune cells, M1069 rescued IL 2 production from T cells (A2A dependent) and inhibited VEGF production by myeloid cells (A2B dependent) in adenosine-high settings. M1069 also demonstrated superior suppression of secretion of pro tumorigenic cytokines CXCL1, CXCL5, and rescue of IL 12 secretion from adenosine differentiated dendritic cells compared to an A2A selective antagonist (A2Ai). In a one-way mixed lymphocyte reaction (MLR) assay, adenosine differentiated human and murine dendritic cells treated with M1069 demonstrated superior T cell stimulatory activity compared to dendritic cells differentiated in presence of A2Ai. In vivo, M1069 decreased tumor growth as a monotherapy and enhanced anti-tumor activity of bintrafusp alfa (BA) or cisplatin in syngeneic adenosinehi/CD73hi 4T1 breast tumor model, but not in the CD73 knockout (KO) 4T1 tumor model or in adenosinelow/CD73low MC38 murine colon carcinoma model. In summary, our dual A2A/A2B AR antagonist M1069 may counteract immune-suppressive mechanisms of high concentrations of adenosine in vitro and in vivo and enhance the anti-tumor activity of other agents, including BA and cisplatin.
RESUMEN
Here we describe the discovery and optimization of hexahydro-2H-pyrano[3,2-c]quinolines (HHPQs) as potent and selective inhibitors of the mitotic kinesin-5 originally found during a high-throughput screening (HTS) campaign sampling our in-house compound collection. The compounds optimized subsequently and characterized herein were potently inhibiting the ATPase activity of Kinesin-5 and also exhibited consistent cellular activity, in that cells arrested in mitosis and apoptosis induction could be observed. X-ray crystallographic data demonstrated that these inhibitors bind in an allosteric pocket of Kinesin-5 distant from the nucleotide and microtubule binding sites. The selected clinical candidate EMD 534085 caused strong growth inhibition in human tumor xenograft models using Colo 205 colon carcinoma cells at doses below 30mg/kg administered twice weekly without showing severe toxicity as determined by loss of body weight.
Asunto(s)
Antineoplásicos/química , Inhibidores Enzimáticos/química , Cinesinas/antagonistas & inhibidores , Mitosis , Quinolinas/química , Regulación Alostérica , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Sitios de Unión , Línea Celular Tumoral , Cristalografía por Rayos X , Descubrimiento de Drogas , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , Cinesinas/metabolismo , Ratones , Quinolinas/síntesis química , Quinolinas/farmacología , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The mediator complex-associated cyclin dependent kinase CDK8 regulates ß-catenin-dependent transcription following activation of WNT signaling. Multiple lines of evidence suggest CDK8 may act as an oncogene in the development of colorectal cancer. Here we describe the successful optimization of an imidazo-thiadiazole series of CDK8 inhibitors that was identified in a high-throughput screening campaign and further progressed by structure-based design. In several optimization cycles, we improved the microsomal stability, potency, and kinase selectivity. The initial imidazo-thiadiazole scaffold was replaced by a 3-methyl-1H-pyrazolo[3,4-b]-pyridine which resulted in compound 25 (MSC2530818) that displayed excellent kinase selectivity, biochemical and cellular potency, microsomal stability, and is orally bioavailable. Furthermore, we demonstrated modulation of phospho-STAT1, a pharmacodynamic biomarker of CDK8 activity, and tumor growth inhibition in an APC mutant SW620 human colorectal carcinoma xenograft model after oral administration. Compound 25 demonstrated suitable potency and selectivity to progress into preclinical in vivo efficacy and safety studies.
Asunto(s)
Antineoplásicos/farmacología , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento , Imidazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Tiadiazoles/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Biomarcadores de Tumor/antagonistas & inhibidores , Biomarcadores de Tumor/metabolismo , Proliferación Celular/efectos de los fármacos , Quinasa 8 Dependiente de Ciclina/metabolismo , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Imidazoles/síntesis química , Imidazoles/química , Ratones , Ratones Desnudos , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad , Tiadiazoles/síntesis química , Tiadiazoles/químicaRESUMEN
We demonstrate a designed scaffold-hop approach to the discovery of 2,8-disubstituted-1,6-naphthyridine- and 4,6-disubstituted-isoquinoline-based dual CDK8/19 ligands. Optimized compounds in both series exhibited rapid aldehyde oxidase-mediated metabolism, which could be abrogated by introduction of an amino substituent at C5 of the 1,6-naphthyridine scaffold or at C1 of the isoquinoline scaffold. Compounds 51 and 59 were progressed to in vivo pharmacokinetic studies, and 51 also demonstrated sustained inhibition of STAT1(SER727) phosphorylation, a biomarker of CDK8 inhibition, in an SW620 colorectal carcinoma human tumor xenograft model following oral dosing.
RESUMEN
The Mediator complex-associated cyclin-dependent kinase CDK8 has been implicated in human disease, particularly in colorectal cancer where it has been reported as a putative oncogene. Here we report the discovery of 109 (CCT251921), a potent, selective, and orally bioavailable inhibitor of CDK8 with equipotent affinity for CDK19. We describe a structure-based design approach leading to the discovery of a 3,4,5-trisubstituted-2-aminopyridine series and present the application of physicochemical property analyses to successfully reduce in vivo metabolic clearance, minimize transporter-mediated biliary elimination while maintaining acceptable aqueous solubility. Compound 109 affords the optimal compromise of in vitro biochemical, pharmacokinetic, and physicochemical properties and is suitable for progression to animal models of cancer.
Asunto(s)
Aminopiridinas/farmacología , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Descubrimiento de Drogas , Bibliotecas de Moléculas Pequeñas/farmacología , Administración Oral , Aminopiridinas/administración & dosificación , Aminopiridinas/química , Animales , Disponibilidad Biológica , Células CACO-2 , Quinasa 8 Dependiente de Ciclina/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Perros , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Masculino , Ratones , Modelos Moleculares , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Ratas , Ratas Wistar , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Bibliotecas de Moléculas Pequeñas/química , Solubilidad , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mediator-associated kinases CDK8/19 are context-dependent drivers or suppressors of tumorigenesis. Their inhibition is predicted to have pleiotropic effects, but it is unclear whether this will impact on the clinical utility of CDK8/19 inhibitors. We discovered two series of potent chemical probes with high selectivity for CDK8/19. Despite pharmacodynamic evidence for robust on-target activity, the compounds exhibited modest, though significant, efficacy against human tumor lines and patient-derived xenografts. Altered gene expression was consistent with CDK8/19 inhibition, including profiles associated with super-enhancers, immune and inflammatory responses and stem cell function. In a mouse model expressing oncogenic beta-catenin, treatment shifted cells within hyperplastic intestinal crypts from a stem cell to a transit amplifying phenotype. In two species, neither probe was tolerated at therapeutically-relevant exposures. The complex nature of the toxicity observed with two structurally-differentiated chemical series is consistent with on-target effects posing significant challenges to the clinical development of CDK8/19 inhibitors.
Asunto(s)
Antiinflamatorios/administración & dosificación , Antineoplásicos/administración & dosificación , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Complejo Mediador/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/administración & dosificación , Animales , Antiinflamatorios/efectos adversos , Antiinflamatorios/toxicidad , Antineoplásicos/efectos adversos , Antineoplásicos/toxicidad , Modelos Animales de Enfermedad , Xenoinjertos , Humanos , Hiperplasia/tratamiento farmacológico , Ratones , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/efectos adversos , Inhibidores de Proteínas Quinasas/toxicidad , Resultado del TratamientoRESUMEN
WNT signaling is frequently deregulated in malignancy, particularly in colon cancer, and plays a key role in the generation and maintenance of cancer stem cells. We report the discovery and optimization of a 3,4,5-trisubstituted pyridine 9 using a high-throughput cell-based reporter assay of WNT pathway activity. We demonstrate a twisted conformation about the pyridine-piperidine bond of 9 by small-molecule X-ray crystallography. Medicinal chemistry optimization to maintain this twisted conformation, cognisant of physicochemical properties likely to maintain good cell permeability, led to 74 (CCT251545), a potent small-molecule inhibitor of WNT signaling with good oral pharmacokinetics. We demonstrate inhibition of WNT pathway activity in a solid human tumor xenograft model with evidence for tumor growth inhibition following oral dosing. This work provides a successful example of hypothesis-driven medicinal chemistry optimization from a singleton hit against a cell-based pathway assay without knowledge of the biochemical target.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Evaluación Preclínica de Medicamentos/métodos , Luciferasas/antagonistas & inhibidores , Piridinas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Compuestos de Espiro/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Bioensayo/métodos , Disponibilidad Biológica , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Luciferasas/metabolismo , Ratones , Modelos Moleculares , Estructura Molecular , Piridinas/administración & dosificación , Piridinas/química , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Bibliotecas de Moléculas Pequeñas/química , Compuestos de Espiro/administración & dosificación , Compuestos de Espiro/química , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The dual serotonin (5-HT) re-uptake inhibitor and 5-HT(1A) receptor agonist vilazodone was found to increase central serotonin levels in rat brain. In the course of structural modifications of vilazodone 3-[4-[4-(2-oxo-2H-1-benzopyran-6-yl)-1-piperazinyl]-butyl]-1H-indole-5-carbonitrile 8i and its fluorine analogue 6-[4-[4-(5-fluor-3-indolyl)-butyl]-1-piperazinyl]-2H-1-benzopyran-2-one have been identified. These unsubstituted chromenones are equally potent at the 5-HT(1A) receptor and 5-HT transporter. The implementation of nitrogen functionalities in position 3 of the chromenones resulted in compounds acting as agonists at the 5-HT(1A) receptor and as 5-HT re-uptake inhibitors like vilazodone. Ex vivo 5-HT re-uptake inhibition and in vitro 5-HT agonism were determined in the PCA- and GTPgammaS-assay, respectively. The potential of these chromenones to increase central 5-HT levels was measured in microdialysis studies and especially the derivatives 3-[4-[4-(3-amino-2-oxo-2H-chromen-6-yl)-piperazin-1-yl]-butyl]-1H-indole-5-carbonitrile 8f, ethyl (6-[4-[4-(5-cyano-1H-indol-3-yl)-butyl]-piperazin-1-yl]-2-oxo-2H-chromen-3-yl)-carbamate 8h and N-(6-[4-[4-(5-cyano-1H-indol-3-yl)-butyl]-piperazin-1-yl]-2-oxo-2H-chromen-3-yl)-acetamide 8k give rise to rapid development of increased serotonin levels in rat brain cortex, lasting longer than 3h.