RESUMEN
Cyclin-dependent kinase 8 (CDK8) is highly expressed in various cancers and common complex human diseases, and an important therapeutic target for drug discovery and development. The CDK8 inhibitors are actively sought after, especially among natural products. We performed a virtual screening using the ZINC library comprising approximately 90,000 natural compounds. We applied Lipinski's rule of five, absorption, distribution, metabolism, excretion, and toxicity properties, and pan-assay interference compounds filter to eliminate promiscuous binders. Subsequently, the filtered compounds underwent molecular docking to predict their binding affinity and interactions with the CDK8 protein. Interaction analysis were carried out to elucidate the interaction mechanism of the screened hits with binding pockets of the CDK8. The ZINC02152165, ZINC04236005, and ZINC02134595 were selected with appreciable specificity and affinity with CDK8. An all-atom molecular dynamic (MD) simulation followed by essential dynamics was performed for 200 ns. Taken together, the results suggest that ZINC02152165, ZINC04236005, and ZINC02134595 can be harnessed as potential leads in therapeutic development. Moreover, the binding of the molecules brings change in protein conformation in a way that blocks the ATP-binding site of the protein, obstructing its kinase activity. These new findings from natural products offer insights into the molecular mechanisms underlying CDK8 inhibition. CDK8 was previously associated with behavioral and neurological diseases such as autism spectrum disorder, and cancers, for example, colorectal, prostate, breast, and acute myeloid leukemia. Hence, we call for further research and experimental validation, and with an eye to inform future clinical drug discovery and development in these therapeutic fields.
Asunto(s)
Antineoplásicos , Productos Biológicos , Quinasa 8 Dependiente de Ciclina , Inhibidores de Proteínas Quinasas , Humanos , Antineoplásicos/farmacología , Antineoplásicos/química , Sitios de Unión , Productos Biológicos/farmacología , Productos Biológicos/química , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Quinasa 8 Dependiente de Ciclina/metabolismo , Quinasa 8 Dependiente de Ciclina/química , Descubrimiento de Drogas/métodos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/químicaRESUMEN
Macrophages polarize into alternatively activated M2 macrophages through interleukin (IL)-4, and they express high levels of arginase-1, which promotes anti-inflammatory responses. Several studies have confirmed the anti-inflammatory effects of cyclin-dependent kinase (CDK) 8/19 inhibition, and hence, numerous CDK8/19 inhibitors, such as BRD6989, have been developed. However, the effects of CDK8/19 inhibitors on arginase-1 expression in macrophages have not yet been elucidated. This study investigated the effects of CDK8/19 inhibitor on arginase-1 expression in IL-4-activated macrophages. The results showed that BRD6989 increased arginase-1 expression transcriptionally in murine peritoneal macrophages and the murine macrophage cell line RAW264.7 in an IL-4-dependent manner. In addition, the results indicated that BRD6989 enhances signal transducer and activator of transcription (STAT) 6 phosphorylation. Meanwhile, BRD6989 exhibited the capability to activate p38 mitogen-activated protein kinase (MAPKï¼ even in the absence of IL-4 stimulation. Moreover, we observed that a p38 MAPK inhibitor suppressed the BRD6989-induced increase in arginase-1 expression. Besides, BRD6989 increased the surface expression of CD206, an M2 macrophage marker. Thus, this study demonstrated for the first time that CDK8/19 inhibition increases arginase-1 expression, suggesting that this mechanism involves the activation of STAT6 and p38 MAPK. This finding implies that CDK8/19 inhibition may facilitate the production of anti-inflammatory M2 macrophages.
Asunto(s)
Arginasa , Quinasa 8 Dependiente de Ciclina , Quinasas Ciclina-Dependientes , Interleucina-4 , Factor de Transcripción STAT6 , Proteínas Quinasas p38 Activadas por Mitógenos , Animales , Arginasa/metabolismo , Arginasa/antagonistas & inhibidores , Factor de Transcripción STAT6/metabolismo , Ratones , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Células RAW 264.7 , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Interleucina-4/metabolismo , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Quinasa 8 Dependiente de Ciclina/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Fosforilación/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Activación Enzimática/efectos de los fármacos , Flavonoides , Piperidinas , Quinasa 9 Dependiente de la CiclinaRESUMEN
Currently, the drugs used in clinical to treat psoriasis mainly broadly suppress cellular immunity. However, these drugs can only provide temporary and partial symptom relief, they do not cure the condition and may lead to recurrence or even serious toxic side effects. In this study, we describe the discovery of a novel potent CDK8 inhibitor as a treatment for psoriasis. Through structure-based design, compound 46 was identified as the most promising candidate, exhibiting a strong inhibitory effect on CDK8 (IC50 value of 57â¯nM) along with favourable inhibition against NF-κB. Additionally, it demonstrated a positive effect in an in vitro psoriasis model induced by TNF-α. Furthermore, this compound enhanced the thermal stability of CDK8 and exerted evident effects on the biological function of CDK8, and it had favourable selectivity across the CDK family and tyrosine kinase. This compound showed no obvious inhibitory effect on CYP450 enzyme. Further studies confirmed that compound 46 exhibited therapeutic effect on IMQ-induced psoriasis, alleviated the inflammatory response in mice, and enhanced the expression of Foxp3 and IL-10 in the dorsal skin in vivo. This discovery provides a new strategy for developing selective CDK8 inhibitors with anti-inflammatory activity for the treatment of psoriasis.
Asunto(s)
Quinasa 8 Dependiente de Ciclina , Inhibidores de Proteínas Quinasas , Psoriasis , Animales , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Quinasa 8 Dependiente de Ciclina/metabolismo , Psoriasis/tratamiento farmacológico , Humanos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Ratones , FN-kappa B/metabolismo , FN-kappa B/antagonistas & inhibidores , Piridinas/farmacología , Piridinas/química , Ratones Endogámicos BALB C , Interleucina-10/metabolismo , Masculino , Pirroles/farmacología , Pirroles/química , Factores de Transcripción Forkhead/metabolismo , Descubrimiento de Drogas/métodos , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Modelos Animales de Enfermedad , Piel/efectos de los fármacos , Piel/patología , Piel/metabolismoRESUMEN
The identification of an effective inhibitor is an important starting step in drug development. Unfortunately, many issues such as the characterization of protein binding sites, the screening library, materials for assays, etc., make drug screening a difficult proposition. As the size of screening libraries increases, more resources will be inefficiently consumed. Thus, new strategies are needed to preprocess and focus a screening library towards a targeted protein. Herein, we report an ensemble machine learning (ML) model to generate a CDK8-focused screening library. The ensemble model consists of six different algorithms optimized for CDK8 inhibitor classification. The models were trained using a CDK8-specific fragment library along with molecules containing CDK8 activity. The optimized ensemble model processed a commercial library containing 1.6 million molecules. This resulted in a CDK8-focused screening library containing 1,672 molecules, a reduction of more than 99.90%. The CDK8-focused library was then subjected to molecular docking, and 25 candidate compounds were selected. Enzymatic assays confirmed six CDK8 inhibitors, with one compound producing an IC50 value of ≤100 nM. Analysis of the ensemble ML model reveals the role of the CDK8 fragment library during training. Structural analysis of molecules reveals the hit compounds to be structurally novel CDK8 inhibitors. Together, the results highlight a pipeline for curating a focused library for a specific protein target, such as CDK8.
Asunto(s)
Quinasa 8 Dependiente de Ciclina , Evaluación Preclínica de Medicamentos , Aprendizaje Automático , Inhibidores de Proteínas Quinasas , Humanos , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Quinasa 8 Dependiente de Ciclina/química , Quinasa 8 Dependiente de Ciclina/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Simulación del Acoplamiento Molecular , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
The mediator kinases CDK8 and CDK19 control the dynamic transcription of selected genes in response to various signals and have been shown to be hijacked to sustain hyperproliferation by various solid and liquid tumors. CDK8/19 is emerging as a promising anticancer therapeutic target. Here, we report the discovery of compound 12, a novel small molecule CDK8/19 inhibitor. This molecule demonstrated not only decent enzymatic and cellular activities but also remarkable selectivity in CDK and kinome panels. Besides, compound 12 also displayed favorable ADME profiles including low CYP1A2 inhibition, acceptable clearance, and high oral bioavailability in multiple preclinical species. Robust in vivo PD and efficacy studies in mice models further demonstrated its potential use as mono- and combination therapy for the treatment of cancers.
Asunto(s)
Antineoplásicos , Quinasa 8 Dependiente de Ciclina , Quinasas Ciclina-Dependientes , Inhibidores de Proteínas Quinasas , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Quinasa 8 Dependiente de Ciclina/metabolismo , Humanos , Animales , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/síntesis química , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Antineoplásicos/síntesis química , Ratones , Descubrimiento de Drogas , Línea Celular Tumoral , Relación Estructura-Actividad , Proliferación Celular/efectos de los fármacos , Neoplasias/tratamiento farmacológico , RatasRESUMEN
Mediator kinases CDK19 and CDK8, pleiotropic regulators of transcriptional reprogramming, are differentially regulated by androgen signaling, but both kinases are upregulated in castration-resistant prostate cancer (CRPC). Genetic or pharmacological inhibition of CDK8 and CDK19 reverses the castration-resistant phenotype and restores the sensitivity of CRPC xenografts to androgen deprivation in vivo. Prolonged CDK8/19 inhibitor treatment combined with castration not only suppressed the growth of CRPC xenografts but also induced tumor regression and cures. Transcriptomic analysis revealed that Mediator kinase inhibition amplified and modulated the effects of castration on gene expression, disrupting CRPC adaptation to androgen deprivation. Mediator kinase inactivation in tumor cells also affected stromal gene expression, indicating that Mediator kinase activity in CRPC molded the tumor microenvironment. The combination of castration and Mediator kinase inhibition downregulated the MYC pathway, and Mediator kinase inhibition suppressed a MYC-driven CRPC tumor model even without castration. CDK8/19 inhibitors showed efficacy in patient-derived xenograft models of CRPC, and a gene signature of Mediator kinase activity correlated with tumor progression and overall survival in clinical samples of metastatic CRPC. These results indicate that Mediator kinases mediated androgen-independent in vivo growth of CRPC, supporting the development of CDK8/19 inhibitors for the treatment of this presently incurable disease.
Asunto(s)
Quinasa 8 Dependiente de Ciclina , Quinasas Ciclina-Dependientes , Neoplasias de la Próstata Resistentes a la Castración , Inhibidores de Proteínas Quinasas , Ensayos Antitumor por Modelo de Xenoinjerto , Masculino , Humanos , Animales , Neoplasias de la Próstata Resistentes a la Castración/patología , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/enzimología , Ratones , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Quinasa 8 Dependiente de Ciclina/genética , Quinasa 8 Dependiente de Ciclina/metabolismo , Línea Celular Tumoral , Inhibidores de Proteínas Quinasas/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacosRESUMEN
Prostate cancer is a prevalent malignancy among men globally, and androgen deprivation therapy is the conventional first-line treatment for metastatic prostate cancer. While androgen deprivation therapy is efficacious in castration-sensitive prostate cancer, it remains less effective in castration-resistant cases. Transcriptional dysregulation is a well-established hallmark of cancer, and targeting proteins involved in transcriptional regulation, such as cyclin-dependent kinase 8 (CDK8), has become an attractive therapeutic strategy. CDK8, a nuclear serine-threonine kinase, is a key component of the mediator complex and plays a critical role in transcriptional regulation. Recent studies have highlighted the promising role of CDK8 as a target in the treatment of metastatic prostate cancer. Our study assessed the efficacy of a novel CDK8 inhibitor, E966-0530-45418, which exhibited potent CDK8 inhibition (IC50 of 129 nM) and high CDK8 selectivity. Treatment with E966-0530-45418 significantly inhibited prostate cancer cell migration and epithelial-to-mesenchymal transition (EMT) at both the RNA and protein levels. Further mechanistic analysis indicated that E966-0530-45418 suppresses prostate cancer metastasis by decreasing CDK8 activity and inhibiting TGF-ß1-mediated Smad3/RNA polymerase II linker phosphorylation and Akt/GSK3ß/ß-catenin signaling. The results in animal model also showed that E966-0530-45418 exhibited anti-metastatic properties in vivo. Our study demonstrated that E966-0530-45418 has great therapeutic potential in the treatment of metastatic prostate cancer.
Asunto(s)
Quinasa 8 Dependiente de Ciclina , Neoplasias de la Próstata , Animales , Humanos , Masculino , Antagonistas de Andrógenos , Andrógenos , Línea Celular Tumoral , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Transducción de Señal , Metástasis de la NeoplasiaRESUMEN
As an ideal anti-inflammatory target, cyclin-dependent kinase 8 (CDK8) has gradually attracted the attention of researchers. CDK8 inhibition up-regulates Interleukin-10 (IL-10) expression by enhancing the transcriptional activity of activator protein-1 (AP-1), and augmenting IL-10 abundance is a viable strategy for the treatment of inflammatory bowel disease (IBD). In this research, through structure-based drug design and dominant fragment hybridization, a series of poly-substituted pyridine derivatives were designed and synthesized as CDK8 inhibitors. Ultimately, compound CR16 was identified as the best one, which exhibited good inhibitory activity against CDK8 (IC50 = 74.4 nM). In vitro and in vivo studies indicated that CR16 could enhance the transcriptional activity of AP-1, augment the abundance of IL-10, and affect CDK8-related signaling pathways including TLR7/NF-κB/MAPK and IL-10-JAK1-STAT3 pathways. In addition, CR16 showed potent therapeutic effect in an animal model of IBD.
Asunto(s)
Interleucina-10 , Inhibidores de Proteínas Quinasas , Animales , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Interleucina-10/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Transducción de Señal , Factor de Transcripción AP-1RESUMEN
Acute myeloid leukemia (AML) is the most lethal form of AML due to disease relapse. Cyclin dependent kinase 8 (CDK8) is a serine/threonine kinase that belongs to the family of Cyclin-dependent kinases and is an emerging target for the treatment of AML. MK256, a potent, selective, and orally available CDK8 inhibitor was developed to target AML. We sought to examine the anticancer effect of MK256 on AML. In CD34+/CD38- leukemia stem cells, we found that MK256 induced differentiation and maturation. Treatment of MK256 inhibited proliferation of AML cell lines. Further studies of the inhibitory effect suggested that MK256 not only downregulated phosphorylated STAT1(S727) and STAT5(S726), but also lowered mRNA expressions of MCL-1 and CCL2 in AML cell lines. Efficacy of MK256 was shown in MOLM-14 xenograft models, and the inhibitory effect on phosphorylated STAT1(S727) and STAT5(S726) with treatment of MK256 was observed in vivo. Pharmacologic dynamics study of MK256 in MOLM-14 xenograft models showed dose-dependent inhibition of the STAT pathway. Both in vitro and in vivo studies suggested that MK256 could effectively downregulate the STAT pathway. In vitro ADME, pharmacological kinetics, and toxicity of MK256 were profiled to evaluate the drug properties of MK256. Our results show that MK256 is a novel CDK8 inhibitor with a desirable efficacy and safety profile and has great potential to be a promising drug candidate for AML through regulating the STAT pathway.
Asunto(s)
Quinasa 8 Dependiente de Ciclina , Leucemia Mieloide Aguda , Inhibidores de Proteínas Quinasas , Factores de Transcripción STAT , Humanos , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Regulación hacia Abajo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Factores de Transcripción STAT/genética , AnimalesRESUMEN
Senexins are potent and selective quinazoline inhibitors of CDK8/19 Mediator kinases. To improve their potency and metabolic stability, quinoline-based derivatives were designed through a structure-guided strategy based on the simulated drug-target docking model of Senexin A and Senexin B. A library of quinoline-Senexin derivatives was synthesized to explore the structure-activity relationship (SAR). An optimized compound 20a (Senexin C) exhibits potent CDK8/19 inhibitory activity with high selectivity. Senexin C is more metabolically stable and provides a more sustained inhibition of CDK8/19-dependent cellular gene expression when compared with the prototype inhibitor Senexin B. In vivo pharmacokinetic (PK) and pharmacodynamic (PD) evaluation using a novel tumor-based PD assay showed good oral bioavailability of Senexin C with a strong tumor-enrichment PK profile and tumor-PD marker responses. Senexin C inhibits MV4-11 leukemia growth in a systemic in vivo model with good tolerability.
Asunto(s)
Antineoplásicos/farmacología , Antineoplásicos/farmacocinética , Neoplasias del Colon/tratamiento farmacológico , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/farmacocinética , Animales , Antineoplásicos/uso terapéutico , Disponibilidad Biológica , Línea Celular Tumoral , Humanos , Leucemia/tratamiento farmacológico , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Simulación del Acoplamiento Molecular , Estructura Molecular , Inhibidores de Proteínas Quinasas/toxicidad , Quinolinas , Relación Estructura-Actividad , Especificidad por Sustrato , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chronic inflammation is an underlying cause in a number of diseases. Cyclin-dependent kinase 8 (CDK8) has been implicated as an inflammatory mediator, indicating its potential as an anti-inflammatory target. Herein, we performed structure-based virtual screening (SBVS) to identify novel CDK8 inhibitors. The pharmacological interactions for CDK8 were identified and incorporated into a SBVS protocol. Selected compounds were tested in enzymatic assays, and one compound was confirmed to be a CDK8 inhibitor with a 50% inhibitory concentration (IC50) value of 1684.4 nM. Comparing structural analogs identified a compound, F059-1017, with greater potency (IC50 558.1 nM). When tested in cell lines, the compounds displayed low cytotoxicity. Cellular assays revealed that the identified CDK8 inhibitors can reduce phosphorylation and expression of signaling mediators associated with inflammation. In addition, results of kinase profiling showed that compound F059-1017 is selective towards CDK8. These findings suggest that the new inhibitors have great potential as lead compounds for developing novel anti-inflammatory therapeutics.
Asunto(s)
Antiinflamatorios/farmacología , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Quinasa 8 Dependiente de Ciclina/metabolismo , Ciclooxigenasa 2/metabolismo , Citocinas/metabolismo , Humanos , Lipopolisacáridos/farmacología , Ratones , Modelos Moleculares , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , OxindolesRESUMEN
Immune health requires innate and adaptive immune cells to engage precisely balanced pro- and anti-inflammatory forces. We employ the concept of chemical immunophenotypes to classify small molecules functionally or mechanistically according to their patterns of effects on primary innate and adaptive immune cells. The high-specificity, low-toxicity cyclin-dependent kinase 8 (CDK8) inhibitor 16-didehydro-cortistatin A (DCA) exerts a distinct tolerogenic profile in both innate and adaptive immune cells. DCA promotes regulatory T cells (Treg) and Th2 differentiation while inhibiting Th1 and Th17 differentiation in both murine and human cells. This unique chemical immunophenotype led to mechanistic studies showing that DCA promotes Treg differentiation in part by regulating a previously undescribed CDK8-GATA3-FOXP3 pathway that regulates early pathways of Foxp3 expression. These results highlight previously unappreciated links between Treg and Th2 differentiation and extend our understanding of the transcription factors that regulate Treg differentiation and their temporal sequencing. These findings have significant implications for future mechanistic and translational studies of CDK8 and CDK8 inhibitors.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Factores de Transcripción Forkhead/metabolismo , Factor de Transcripción GATA3/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Tolerancia Inmunológica/efectos de los fármacos , Inmunofenotipificación , Isoquinolinas/farmacología , Adolescente , Adulto , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Quinasa 8 Dependiente de Ciclina/metabolismo , Humanos , Inmunidad Innata/efectos de los fármacos , Ratones Endogámicos BALB C , Persona de Mediana Edad , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal/efectos de los fármacos , Adulto JovenRESUMEN
Selective inhibition of cyclin-dependent kinase 8 (CDK8) has been recently regarded as a potential approach for cancer therapy. A series of novel CDK8 inhibitors with the pyridine core was identified via scaffold hopping from the known CDK8 inhibitor A-7. The new inhibitors were designed to improve the ligand efficiency so as to enhance drug-likeness. Most of the compounds showed significant inhibition against CDK8/cyclin C, and the most active compounds (5d, 5e and 7') displayed IC50 values of 2.4 nM, 5.0 nM and 7.7 nM, respectively. Preliminary kinase profiling of selected compounds against a panel of kinases from different families indicated that this compound class might selectively inhibit CDK8 as well as its paralog CDK19. Some compounds exhibited cellular activity in both MTT and SRB assays against a variety of tumor cells, including HCT-116, A549, MDA-MB-231, KB, KB-VIN and MCF-7. Further flow cytometry analysis revealed a dose-dependent G2/M phase arrest in MDA-MB-231 cells treated with compounds 6'a, 6'b, 6'j and 6'k. In addition, compound 6'k demonstrated moderate antitumor efficacy in HCT-116 mouse models, although unfavorable pharmacokinetic profiles were suggested by preliminary study in mice. The results provided a new structural prototype for the search of selective CDK8 inhibitors as antitumor agents.
Asunto(s)
Antineoplásicos/uso terapéutico , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/uso terapéutico , Animales , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa 8 Dependiente de Ciclina/metabolismo , Diseño de Fármacos , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Humanos , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Simulación del Acoplamiento Molecular , Estructura Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacocinética , Piridinas/síntesis química , Piridinas/metabolismo , Piridinas/farmacocinética , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
CDK8 is deregulated in multiple types of human cancer and is viewed as a therapeutic target for the treatment of the disease. Accordingly, the search for small-molecule inhibitors of CDK8 is being intensified. Capitalising on our initial discovery of AU1-100, a potent CDK8 inhibitor yet with a limited degree of kinase selectivity, a structure-based optimisation was carried out, with a series of new multi-substituted pyridines rationally designed, chemically prepared and biologically evaluated. Such endeavour has culminated in the identification of 42, a more potent CDK8 inhibitor with superior kinomic selectivity and oral bioavailability. The mechanism underlying the anti-proliferative effect of 42 on MV4-11 cells was studied, revealing that the compound arrested the G1 cell cycle and triggered apoptosis. The low risk of hepato- and cardio-toxicity of 42 was estimated. These findings merit further investigation of 42 as a targeted cancer therapeutic.
Asunto(s)
Antineoplásicos/farmacología , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Descubrimiento de Drogas , Inhibidores de Proteínas Quinasas/farmacología , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Disponibilidad Biológica , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa 8 Dependiente de Ciclina/metabolismo , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Pluripotent stem cells can be stabilized in vitro at different developmental states by the use of specific chemicals and soluble factors. The naïve and primed states are the best characterized pluripotency states. Naïve pluripotent stem cells (PSCs) correspond to the early pre-implantation blastocyst and, in mice, constitute the optimal starting state for subsequent developmental applications. However, the stabilization of human naïve PSCs remains challenging because, after short-term culture, most current methods result in karyotypic abnormalities, aberrant DNA methylation patterns, loss of imprinting and severely compromised developmental potency. We have recently developed a novel method to induce and stabilize naïve human PSCs that consists in the simple addition of a chemical inhibitor for the closely related CDK8 and CDK19 kinases (CDK8/19i). Long-term cultured CDK8/19i-naïve human PSCs preserve their normal karyotype and do not show widespread DNA demethylation. Here, we investigate the long-term stability of allele-specific methylation at imprinted loci and the differentiation potency of CDK8/19i-naïve human PSCs. We report that long-term cultured CDK8/19i-naïve human PSCs retain the imprinting profile of their parental primed cells, and imprints are further retained upon differentiation in the context of teratoma formation. We have also tested the capacity of long-term cultured CDK8/19i-naïve human PSCs to differentiate into primordial germ cell (PGC)-like cells (PGCLCs) and trophoblast stem cells (TSCs), two cell types that are accessible from the naïve state. Interestingly, long-term cultured CDK8/19i-naïve human PSCs differentiated into PGCLCs with a similar efficiency to their primed counterparts. Also, long-term cultured CDK8/19i-naïve human PSCs were able to differentiate into TSCs, a transition that was not possible for primed PSCs. We conclude that inhibition of CDK8/19 stabilizes human PSCs in a functional naïve state that preserves imprinting and potency over long-term culture.
Asunto(s)
Diferenciación Celular , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Impresión Genómica , Inhibidores de Proteínas Quinasas/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular , Quinasa 8 Dependiente de Ciclina/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Células Germinativas/citología , Células Germinativas/efectos de los fármacos , Células Germinativas/metabolismo , Humanos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Trofoblastos/citología , Trofoblastos/efectos de los fármacosRESUMEN
Embryonic stem cells (ESCs) can be maintained in the naïve state through inhibition of Mek1/2 and Gsk3 (2i). A relevant effect of 2i is the inhibition of Cdk8/19, which are negative regulators of the Mediator complex, responsible for the activity of enhancers. Inhibition of Cdk8/19 (Cdk8/19i) stimulates enhancers and, similar to 2i, stabilizes ESCs in the naïve state. Here, we use mass spectrometry to describe the molecular events (phosphoproteome, proteome, and metabolome) triggered by 2i and Cdk8/19i on ESCs. Our data reveal widespread commonalities between these two treatments, suggesting overlapping processes. We find that post-transcriptional de-repression by both 2i and Cdk8/19i might support the mitochondrial capacity of naive cells. However, proteome reprogramming in each treatment is achieved by different mechanisms. Cdk8/19i acts directly on the transcriptional machinery, activating key identity genes to promote the naïve program. In contrast, 2i stabilizes the naïve circuitry through, in part, de-phosphorylation of downstream transcriptional effectors.
Asunto(s)
Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Células Madre Embrionarias de Ratones/citología , Células Madre Pluripotentes/citología , Animales , Benzamidas/farmacología , Línea Celular , Difenilamina/análogos & derivados , Difenilamina/farmacología , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Fosforilación/fisiología , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genéticaRESUMEN
CDK8 regulates transcription either by phosphorylation of transcription factors or, as part of a four-subunit kinase module, through a reversible association of the kinase module with the Mediator complex, a highly conserved transcriptional coactivator. Deregulation of CDK8 has been found in various types of human cancer, while the role of CDK8 in supressing anti-cancer response of natural killer cells is being understood. Currently, CDK8-targeting cancer drugs are highly sought-after. Herein we detail the discovery of a series of novel pyridine-derived CDK8 inhibitors. Medicinal chemistry optimisation gave rise to 38 (AU1-100), a potent CDK8 inhibitor with oral bioavailability. The compound inhibited the proliferation of MV4-11 acute myeloid leukaemia cells with the kinase activity of cellular CDK8 dampened. No systemic toxicology was observed in the mice treated with 38. These results warrant further pre-clinical studies of 38 as an anti-cancer agent.
Asunto(s)
Antineoplásicos/farmacología , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Diseño de Fármacos , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Disponibilidad Biológica , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quinasa 8 Dependiente de Ciclina/metabolismo , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Estructura Molecular , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Piridinas/administración & dosificación , Piridinas/química , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Alkylating drugs are among the most often used chemotherapeutics. While cancer cells frequently develop resistance to alkylation treatments, detailed understanding of mechanisms that lead to the resistance is limited. Here, by using genome-wide CRISPR-Cas9 based screen, we identify transcriptional Mediator complex subunit 13 (MED13) as a novel modulator of alkylation response. The alkylation exposure causes significant MED13 downregulation, while complete loss of MED13 results in reduced apoptosis and resistance to alkylating agents. Transcriptome analysis identified cyclin D1 (CCND1) as one of the highly overexpressed genes in MED13 knock-out (KO) cells, characterized by shorter G1 phase. MED13 is able to bind to CCND1 regulatory elements thus influencing the expression. The resistance of MED13 KO cells is directly dependent on the cyclin D1 overexpression, and its down-regulation is sufficient to re-sensitize the cells to alkylating agents. We further demonstrate the therapeutic potential of MED13-mediated response, by applying combinatory treatment with CDK8/19 inhibitor Senexin A. Importantly, the treatment with Senexin A stabilizes MED13, and in combination with alkylating agents significantly reduces viability of cancer cells. In summary, our findings identify novel alkylation stress response mechanism dependent on MED13 and cyclin D1 that can serve as basis for development of innovative therapeutic strategies.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Ciclina D1/genética , Complejo Mediador/fisiología , Sistemas CRISPR-Cas , Línea Celular , Línea Celular Tumoral , Ciclina D1/metabolismo , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Daño del ADN , Resistencia a Antineoplásicos , Regulación de la Expresión Génica , Humanos , Complejo Mediador/metabolismo , Regulación hacia ArribaRESUMEN
Drug resistance is the main obstacle to achieving cures with both conventional and targeted anticancer drugs. The emergence of acquired drug resistance is initially mediated by non-genetic transcriptional changes, which occur at a much higher frequency than mutations and may involve population-scale transcriptomic adaptation. CDK8/19 kinases, through association with transcriptional Mediator complex, regulate transcriptional reprogramming by co-operating with different signal-responsive transcription factors. Here we tested if CDK8/19 inhibition could prevent adaptation to drugs acting on epidermal growth factor receptor (EGFR/ERBB1/HER1). The development of resistance was analyzed following long-term exposure of BT474 and SKBR3 breast cancer cells to EGFR-targeting small molecules (gefitinib, erlotinib) and of SW48 colon cancer cells to an anti-EGFR monoclonal antibody cetuximab. In all cases, treatment of small cell populations (~105 cells) with a single dose of the drug initially led to growth inhibition that was followed by the resumption of proliferation and development of drug resistance in the adapted populations. However, this adaptation was always prevented by the addition of selective CDK8/19 inhibitors, even though such inhibitors alone had only moderate or no effect on cell growth. These results indicate that combining EGFR-targeting drugs with CDK8/19 inhibitors may delay or prevent the development of tumor resistance to therapy.
Asunto(s)
Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Resistencia a Antineoplásicos , Receptores ErbB/metabolismo , Terapia Molecular Dirigida , Inhibidores de Proteínas Quinasas/farmacología , Línea Celular Tumoral , Cetuximab/farmacología , Quinasa 8 Dependiente de Ciclina/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Clorhidrato de Erlotinib/farmacología , Gefitinib/farmacología , Humanos , Concentración 50 InhibidoraRESUMEN
Cyclin-dependent kinase 8 (CDK8) plays an momentous role in transcription regulation by forming kinase module or transcription factor phosphorylation. A large number of evidences have identified CDK8 as an important factor in cancer occurrence and development. In addition, CDK8 also participates in the regulation of cancer cell stress response to radiotherapy and chemotherapy, assists tumor cell invasion, metastasis, and drug resistance. Therefore, CDK8 is regarded as a promising target for cancer therapy. Most studies in recent years supported the role of CDK8 as a carcinogen, however, under certain conditions, CDK8 exists as a tumor suppressor. The functional diversity of CDK8 and its exceptional role in different types of cancer have aroused great interest from scientists but even more controversy during the discovery of CDK8 inhibitors. In addition, CDK8 appears to be an effective target for inflammation diseases and immune system disorders. Therefore, we summarized the research results of CDK8, involving physiological/pathogenic mechanisms and the development status of compounds targeting CDK8, provide a reference for the feasibility evaluation of CDK8 as a therapeutic target, and guidance for researchers who are involved in this field for the first time.