RESUMEN

Large-scale phase III clinical trials of Olaparib have revealed benefits for ovarian cancer patients with BRCA gene mutations or homologous recombination deficiency (HRD). However, fewer than 50% of ovarian cancer patients have both BRCA mutations and HRD. Therefore, improving the effect of Olaparib in HR-proficient patients is of great clinical value. Here, a combination strategy comprising Olaparib and CDK12-IN-3 effectively inhibited the growth of HR-proficient ovarian cancer in cell line, patient-derived organoid (PDO), and mouse xenograft models. Furthermore, the combination strategy induced severe DNA double-strand break (DSB) formation, increased NHEJ activity in the G2 phase, and reduced HR activity in cancer cells. Mechanistically, the combination treatment impaired Ku80 poly(ADP-ribosyl)ation (PARylation) and phosphorylation, resulting in PARP1-Ku80 complex dissociation. After dissociation, Ku80 occupancy at DSBs and the resulting Ku80-primed NHEJ activity were increased. Owing to Ku80-mediated DNA end protection, MRE11 and Rad51 foci formation was inhibited after the combination treatment, suggesting that this treatment suppressed HR activity. Intriguingly, the combination strategy expedited cGAS nuclear relocalization, further suppressing HR and, conversely, increasing genomic instability. Moreover, the inhibitory effect on cell survival persisted after drug withdrawal. These findings provide a rationale for the clinical application of CDK12-IN-3 in combination with Olaparib.

Asunto(s)

Inestabilidad Genómica , Neoplasias Ováricas , Ftalazinas , Piperazinas , Ftalazinas/farmacología , Ftalazinas/uso terapéutico , Piperazinas/farmacología , Piperazinas/uso terapéutico , Femenino , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/genética , Humanos , Animales , Línea Celular Tumoral , Ratones , Inestabilidad Genómica/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes/metabolismo , Autoantígeno Ku/metabolismo , Roturas del ADN de Doble Cadena/efectos de los fármacos

RESUMEN

Prostate cancer progression is driven by androgen receptor (AR) activity, which is a target for therapeutic approaches. Enzalutamide is an AR inhibitor that prolongs the survival of patients with advanced prostate cancer. However, resistance mechanisms arise and impair its efficacy. One of these mechanisms is the expression of AR-V7, a constitutively active AR splice variant. The Mediator complex is a multisubunit protein that modulates gene expression on a genome-wide scale. MED12 and cyclin-dependent kinase (CDK)8, or its paralog CDK19, are components of the kinase module that regulates the proliferation of prostate cancer cells. In this study, we investigated how MED12 and CDK8/19 influence cancer-driven processes in prostate cancer cell lines, focusing on AR activity and the enzalutamide response. We inhibited MED12 expression and CDK8/19 activity in LNCaP (AR+, enzalutamide-sensitive), 22Rv1 (AR-V7+, enzalutamide-resistant), and PC3 (AR-, enzalutamide-insensitive) cells. Both MED12 and CDK8/19 inhibition reduced cell proliferation in all cell lines, and MED12 inhibition reduced proliferation in the respective 3D spheroids. MED12 knockdown significantly inhibited c-Myc protein expression and signaling pathways. In 22Rv1 cells, it consistently inhibited the AR response, prostate-specific antigen (PSA) secretion, AR target genes, and AR-V7 expression. Combined with enzalutamide, MED12 inhibition additively decreased the AR activity in both LNCaP and 22Rv1 cells. CDK8/19 inhibition significantly decreased PSA secretion in LNCaP and 22Rv1 cells and, when combined with enzalutamide, additively reduced proliferation in 22Rv1 cells. Our study revealed that MED12 and CDK8/19 regulate AR activity and that their inhibition may modulate response to enzalutamide in prostate cancer.

Asunto(s)

Benzamidas , Proliferación Celular , Quinasa 8 Dependiente de Ciclina , Quinasas Ciclina-Dependientes , Complejo Mediador , Nitrilos , Feniltiohidantoína , Neoplasias de la Próstata , Receptores Androgénicos , Feniltiohidantoína/farmacología , Masculino , Humanos , Receptores Androgénicos/metabolismo , Receptores Androgénicos/genética , Complejo Mediador/metabolismo , Complejo Mediador/genética , Línea Celular Tumoral , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/genética , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasa 8 Dependiente de Ciclina/metabolismo , Quinasa 8 Dependiente de Ciclina/genética , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos

RESUMEN

Cyclin-dependent kinases (CDKs), including CDK12 and CDK13, play crucial roles in regulating the cell cycle and RNA polymerase II activity, making them vital targets for cancer therapies. SR4835 is a selective inhibitor of CDK12/13, showing significant potential for treating triple-negative breast cancer. To elucidate the selective mechanism of SR4835 among three CDKs (CDK13/12/9), we developed an innovative enhanced sampling method, integrated well-tempered metadynamics-umbrella sampling (IMUS). IMUS synergistically combines the comprehensive pathway exploration capability of well-tempered metadynamics (WT-MetaD) with the precise free energy calculation capability of umbrella sampling, enabling the efficient and accurate characterization of drug-target interactions. The accurate calculation of binding free energy and the detailed analysis of the kinetic mechanism of the drug-target interaction using IMUS successfully elucidate the drug selectivity mechanism targeting the three CDKs, showing that the selectivity is primarily arising from differences in the stability of H-bonds within the Hinge region of the kinases and the interaction patterns during the protein-ligand recognition process. These findings also underscore the utility of IMUS in efficiently and accurately capturing drug-target interaction processes with clear mechanisms.

Asunto(s)

Quinasas Ciclina-Dependientes , Simulación de Dinámica Molecular , Inhibidores de Proteínas Quinasas , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Humanos , Termodinámica , Conformación Proteica , Antineoplásicos/farmacología , Antineoplásicos/química

RESUMEN

Human T-cell leukemia virus type-1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL). The trans-activator protein Tax of HTLV-1 plays crucial roles in leukemogenesis by promoting proliferation of virus-infected cells through activation of growth-promoting genes. However, critical target genes are yet to be elucidated. We show here that Tax activates the gene coding for cyclin-dependent kinase 7 (CDK7), the essential component of both CDK-activating kinase (CAK) and general transcription factor TFIIH. CAK and TFIIH play essential roles in cell cycle progression and transcription by activating CDKs and facilitating transcriptional initiation, respectively. Tax induced CDK7 gene expression not only in human T-cell lines but also in normal peripheral blood lymphocytes (PHA-PBLs) along with increased protein expression. Tax stimulated phosphorylation of CDK2 and RNA polymerase II at sites reported to be mediated by CDK7. Tax activated the CDK7 promoter through the NF-κB pathway, which mainly mediates cell growth promotion by Tax. Knockdown of CDK7 expression reduced Tax-mediated induction of target gene expression and cell cycle progression. These results suggest that the CDK7 gene is a crucial target of Tax-mediated trans-activation to promote cell proliferation by activating CDKs and transcription.

Asunto(s)

Quinasa Activadora de Quinasas Ciclina-Dependientes , Quinasas Ciclina-Dependientes , Productos del Gen tax , Virus Linfotrópico T Tipo 1 Humano , Humanos , Virus Linfotrópico T Tipo 1 Humano/genética , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Productos del Gen tax/genética , Productos del Gen tax/metabolismo , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Factores de Transcripción TFII/genética , Factores de Transcripción TFII/metabolismo , Activación Transcripcional , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Fosforilación

RESUMEN

Herpesviral protein kinases, such as the therapy-relevant pUL97 of human cytomegalovirus (HCMV), are important for viral replication efficiency as well as pathogenesis, and represent key antiviral drug targets. HCMV pUL97 is a viral cyclin-dependent kinase (CDK) ortholog, as it shares functional and structural properties with human CDKs. Recently, the formation of vCDK/pUL97-cyclin complexes and the phosphorylation of a variety of viral and cellular substrate proteins has been demonstrated. Genetic mapping and structural modeling approaches helped to define two pUL97 interfaces, IF1 and IF2, responsible for cyclin binding. In particular, the regulatory importance of interactions between vCDK/pUL97 and host cyclins as well as CDKs has been highlighted, both as determinants of virus replication and as a novel drug-targeting option. This aspect was substantiated by the finding that virus replication was impaired upon cyclin type H knock-down, and that such host-directed interference also affected viruses resistant to existing therapies. Beyond the formation of binary interactive complexes, a ternary pUL97-cyclin H-CDK7 complex has also been described, and in light of this, an experimental trans-stimulation of CDK7 activity by pUL97 appeared crucial for virus-host coregulation. In accordance with this understanding, several novel antiviral targeting options have emerged. These include kinase inhibitors directed to pUL97, to host CDKs, and to the pUL97-cyclin H interactive complexes. Importantly, a statistically significant drug synergy has recently been reported for antiviral treatment schemes using combinations of pharmacologically relevant CDK7 and vCDK/pUL97 inhibitors, including maribavir. Combined, such findings provide increased options for anti-HCMV control. This review focuses on regulatory interactions of vCDK/pUL97 with the host cyclin-CDK apparatus, and it addresses the functional relevance of these key effector complexes for viral replication and pathogenesis. On this basis, novel strategies of antiviral drug targeting are defined.

Asunto(s)

Antivirales , Quinasas Ciclina-Dependientes , Citomegalovirus , Proteínas Virales , Humanos , Antivirales/farmacología , Citomegalovirus/efectos de los fármacos , Citomegalovirus/fisiología , Proteínas Virales/metabolismo , Proteínas Virales/química , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Infecciones por Citomegalovirus/tratamiento farmacológico , Infecciones por Citomegalovirus/virología , Animales , Ciclinas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)

RESUMEN

Cyclin-dependent kinase 7 (Cdk7) is required in cell-cycle and transcriptional regulation owing to its function as both a CDK-activating kinase (CAK) and part of transcription factor TFIIH. Cdk7 forms active complexes by associating with Cyclin H and Mat1, and is regulated by two phosphorylations in the activation segment (T loop): the canonical activating modification at T170 and another at S164. Here we report the crystal structure of the human Cdk7/Cyclin H/Mat1 complex containing both T-loop phosphorylations. Whereas pT170 coordinates basic residues conserved in other CDKs, pS164 nucleates an arginine network unique to the ternary Cdk7 complex, involving all three subunits. We identify differential dependencies of kinase activity and substrate recognition on the individual phosphorylations. CAK function is unaffected by T-loop phosphorylation, whereas activity towards non-CDK substrates is increased several-fold by T170 phosphorylation. Moreover, dual T-loop phosphorylation stimulates multisite phosphorylation of the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and SPT5 carboxy-terminal repeat (CTR) region. In human cells, Cdk7 activation is a two-step process wherein S164 phosphorylation precedes, and may prime, T170 phosphorylation. Thus, dual T-loop phosphorylation can regulate Cdk7 through multiple mechanisms, with pS164 supporting tripartite complex formation and possibly influencing processivity, while pT170 enhances activity towards key transcriptional substrates.

Asunto(s)

Quinasa Activadora de Quinasas Ciclina-Dependientes , Quinasas Ciclina-Dependientes , Fosforilación , Humanos , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/genética , Ciclina H/metabolismo , Ciclina H/química , Ciclina H/genética , Cristalografía por Rayos X , ARN Polimerasa II/metabolismo , ARN Polimerasa II/química , Factor de Transcripción TFIIH/metabolismo , Factor de Transcripción TFIIH/química , Factor de Transcripción TFIIH/genética , Modelos Moleculares , Factores de Transcripción/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Dominios Proteicos , Proteínas de Ciclo Celular

RESUMEN

While the structure/function paradigm for folded domains was established decades ago, our understanding of how intrinsically disordered regions (IDRs) contribute to biological function is still evolving. IDRs exist as conformational ensembles that can range from highly compact to highly extended depending on their sequence composition. IDR sequences are less conserved than those of folded domains, but often display short, conserved segments termed short linear motifs (SLiMs), that often mediate protein-protein interactions and are often regulated by posttranslational modifications, giving rise to complex functionality when multiple, differently regulated SLiMs are combined. This combinatorial functionality was associated with signaling and regulation soon after IDRs were first recognized as functional elements within proteins. Here, we discuss roles for disorder in proteins that regulate cyclin-dependent kinases, the master timekeepers of the eukaryotic cell cycle. We illustrate the importance of intrinsic flexibility in the transmission of regulatory signals by these entirely disordered proteins.

Asunto(s)

Quinasas Ciclina-Dependientes , Proteínas Intrínsecamente Desordenadas , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas Intrínsecamente Desordenadas/química , Humanos , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/química , Animales

RESUMEN

The identification of genes involved in replicative stress is key to understanding cancer evolution and to identify therapeutic targets. Here, we show that CDK12 prevents transcription-replication conflicts (TRCs) and the activation of cytotoxic replicative stress upon deregulation of the MYC oncogene. CDK12 was recruited at damaged genes by PARP-dependent DDR-signaling and elongation-competent RNAPII, to repress transcription. Either loss or chemical inhibition of CDK12 led to DDR-resistant transcription of damaged genes. Loss of CDK12 exacerbated TRCs in MYC-overexpressing cells and led to the accumulation of double-strand DNA breaks, occurring between co-directional early-replicating regions and transcribed genes. Overall, our data demonstrate that CDK12 protects genome integrity by repressing transcription of damaged genes, which is required for proper resolution of DSBs at oncogene-induced TRCs. This provides a rationale that explains both how CDK12 deficiency can promote tandem duplications of early-replicated regions during tumor evolution, and how CDK12 targeting can exacerbate replicative-stress in tumors.

Asunto(s)

Quinasas Ciclina-Dependientes , Replicación del ADN , Transcripción Genética , Humanos , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/genética , Roturas del ADN de Doble Cadena , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Línea Celular Tumoral , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Daño del ADN

RESUMEN

MAIN CONCLUSION: Excess of KRP4 in the developing kernels in rice causes poor filling of the grains possibly through inhibition of CDKA;2 and CDKB;1 activity mediated by its interaction with CDKF;3. The potential yield of the rice varieties producing compact and heavy panicles bearing numerous spikelets is compromised because a high percentage of spikelets remain poorly filled, reportedly because of a high expression of KRPs that causes suppression of endosperm cell proliferation. To test the stated negative relationship between KRP expression and grain filling, Orysa;KRP4 was overexpressed under the control of seed-specific glutelin promoter in IR-64 rice variety that shows good grain filling. The transgenic lines showed more than 15-fold increase in expression of KRP4 in the spikelets concomitant with nearly 50% reduction in grain filling compared with the wild type without producing any significant changes on the other yield-related parameters like panicle length and the spikelets numbers that were respectively 30.23 ± 0.89 cm and 229.25 ± 33.72 per panicle in the wild type, suggesting a highly organ-targeted effect of the genetic transformation. Yeast two-hybrid test revealed CDKF;3 as the interacting partner of KRP4, and CDKF;3 was found to interact with CDKA;2, CDKB;1 and CDKD;1. Significant decrease in grain filling in the transgenic lines compared with the wild type due to overexpression of KRP4 could be because of suppression of the activity of CDKB;1 and CDKA;2 by inhibition of their phosphorylation directly by CDKF;3, or mediated through inhibition of phosphorylation of CDKD;1 by CDKF;3. The study thus indicated that suppression of expression of KRP(s) by genetic manipulation of their promoters could be an important way of improving the yield of the rice varieties bearing compact and heavy panicles.

Asunto(s)

Grano Comestible , Regulación de la Expresión Génica de las Plantas , Oryza , Proteínas de Plantas , Plantas Modificadas Genéticamente , Semillas , Oryza/genética , Oryza/metabolismo , Oryza/crecimiento & desarrollo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Semillas/genética , Semillas/crecimiento & desarrollo , Semillas/metabolismo , Grano Comestible/genética , Grano Comestible/crecimiento & desarrollo , Grano Comestible/metabolismo , Regiones Promotoras Genéticas/genética , Técnicas del Sistema de Dos Híbridos , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/genética

RESUMEN

Protein-protein interactions (PPIs) stabilization with molecular glues plays a crucial role in drug discovery, albeit with significant challenges. In this study, we propose a dual-site approach, targeting the PPI region and its dynamic surroundings. We conduct molecular dynamics simulations to identify critical sites on the PPI that stabilize the cyclin-dependent kinase 12 - DNA damage-binding protein 1 (CDK12-DDB1) complex, resulting in further cyclin K degradation. This exploration leads to the creation of LL-K12-18, a dual-site molecular glue, which enhances the glue properties to augment degradation kinetics and efficiency. Notably, LL-K12-18 demonstrates strong inhibition of gene transcription and anti-proliferative effects in tumor cells, showing significant potency improvements in MDA-MB-231 (88-fold) and MDA-MB-468 cells (307-fold) when compared to its precursor compound SR-4835. These findings underscore the potential of dual-site approaches in disrupting CDK12 function and offer a structural insight-based framework for the design of cyclin K molecular glues.

Asunto(s)

Quinasas Ciclina-Dependientes , Unión Proteica , Humanos , Línea Celular Tumoral , Proliferación Celular , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/química , Simulación de Dinámica Molecular

RESUMEN

Budding yeast, Saccharomyces cerevisiae, is widely used as a model organism to study the genetics underlying eukaryotic cellular processes and growth critical to cancer development, such as cell division and cell cycle progression. The budding yeast cell cycle is also one of the best-studied dynamical systems owing to its thoroughly resolved genetics. However, the dynamics underlying the crucial cell cycle decision point called the START transition, at which the cell commits to a new round of DNA replication and cell division, are under-studied. The START machinery involves a central cyclin-dependent kinase; cyclins responsible for starting the transition, bud formation, and initiating DNA synthesis; and their transcriptional regulators. However, evidence has shown that the mechanism is more complicated than a simple irreversible transition switch. Activating a key transcription regulator SBF requires the phosphorylation of its inhibitor, Whi5, or an SBF/MBF monomeric component, Swi6, but not necessarily both. Also, the timing and mechanism of the inhibitor Whi5's nuclear export, while important, are not critical for the timing and execution of START. Therefore, there is a need for a consolidated model for the budding yeast START transition, reconciling regulatory and spatial dynamics. We built a detailed mathematical model (START-BYCC) for the START transition in the budding yeast cell cycle based on established molecular interactions and experimental phenotypes. START-BYCC recapitulates the underlying dynamics and correctly emulates key phenotypic traits of ~150 known START mutants, including regulation of size control, localization of inhibitor/transcription factor complexes, and the nutritional effects on size control. Such a detailed mechanistic understanding of the underlying dynamics gets us closer towards deconvoluting the aberrant cellular development in cancer.

Asunto(s)

Ciclo Celular , Modelos Biológicos , Saccharomyces cerevisiae , Ciclo Celular/fisiología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Replicación del ADN , Biología Computacional , Saccharomycetales/genética , Saccharomycetales/metabolismo , Saccharomycetales/fisiología , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/genética , Fosforilación , Proteínas Represoras

RESUMEN

The regulation of the cancer cell cycle heavily relies on cyclin-dependent kinases (CDKs). Targeting CDKs has been identified as a promising approach for effective cancer therapy. In recent years, there has been significant attention paid towards developing small-molecule CDK inhibitors in the field of drug discovery. Notably, five such inhibitors have already received regulatory approval for the treatment of different cancers, including breast tumors, lung malignancies, and hematological malignancies. This review provides an overview of the synthetic routes used to produce 17 representative small-molecule CDK inhibitors that have obtained regulatory approval or are currently being evaluated through clinical trials. It also discusses their clinical applications for treating CDK-related diseases and explores the challenges and limitations associated with their use in a clinical setting, which will stimulate the further development of novel CDK inhibitors. By integrating therapeutic applications, synthetic methodologies, and mechanisms of action observed in various clinical trials involving these CDK inhibitors, this review facilitates a comprehensive understanding of the versatile roles and therapeutic potential offered by interventions targeting CDKs.

Asunto(s)

Antineoplásicos , Quinasas Ciclina-Dependientes , Neoplasias , Inhibidores de Proteínas Quinasas , Bibliotecas de Moléculas Pequeñas , Humanos , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/síntesis química , Neoplasias/tratamiento farmacológico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Antineoplásicos/química , Antineoplásicos/síntesis química , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Bibliotecas de Moléculas Pequeñas/síntesis química , Animales , Descubrimiento de Drogas , Ensayos Clínicos como Asunto

RESUMEN

Fungi have historically been the source of numerous important medicinal compounds, but full exploitation of their genetic potential for drug development has been hampered in traditional discovery paradigms. Here we describe a radically different approach, top-down drug discovery (TD3), starting with a massive digital search through a database of over 100,000 fully genomicized fungi to identify loci encoding molecules with a predetermined human target. We exemplify TD3 by the selection of cyclin-dependent kinases (CDKs) as targets and the discovery of two molecules, 1 and 2, which inhibit therapeutically important human CDKs. 1 and 2 exhibit a remarkable mechanism, forming a site-selective covalent bond to the CDK active site Lys. We explored the structure-activity relationship via semi- and total synthesis, generating an analog, 43, with improved kinase selectivity, bioavailability, and efficacy. This work highlights the power of TD3 to identify mechanistically and structurally novel molecules for the development of new medicines.

Asunto(s)

Quinasas Ciclina-Dependientes , Descubrimiento de Drogas , Inhibidores de Proteínas Quinasas , Humanos , Relación Estructura-Actividad , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/síntesis química , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Animales , Genómica/métodos , Modelos Moleculares

RESUMEN

Cancer immunotherapies are ineffective in nonresponding patients due to absence of immune responses. Here, we identified that dihydroartemisinin (DHA) induced immunogenic cell death (ICD) in hepatocellular carcinoma (HCC), proved by release or surface expose of damage-associated molecular patterns and in vivo protective vaccine activity. Mechanistically, DHA can inhibit cyclin-dependent kinases (CDKs), leading to a buildup of intracellular reactive oxygen species (ROS), which induces immunogenic cell death. In both Hepa1-6 and H22 tumor bearing mice, DHA exerted anti-tumor activity through increasing tumor-infiltrating CD8+ T cells with expression of activation makers (CD25 and CD69), secretion of intracellular cytokines (IFN-γ and TNF-α) and activated dendritic cells expressing MHCⅡ, CD80 and CD86. In hepa1-6 tumor bearing mice, DHA decreased immunosuppressive myeloid-derived suppressor cells. Furthermore, DHA enhanced the anti-PD-1 antibody and chimeric antigen receptor (CAR) T cell-mediated tumor suppression through recruitment and activation of endogenous CD8+ T cells. Overall, we demonstrated that by inhibiting CDKs, DHA can remodel tumor micro-environment to amplify anti-tumor immune responses in HCC. These findings provide a promising therapy option for HCC patients.

Asunto(s)

Artemisininas , Linfocitos T CD8-positivos , Carcinoma Hepatocelular , Quinasas Ciclina-Dependientes , Inmunoterapia , Neoplasias Hepáticas , Ratones Endogámicos C57BL , Microambiente Tumoral , Animales , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología , Artemisininas/farmacología , Artemisininas/uso terapéutico , Ratones , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/terapia , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Línea Celular Tumoral , Humanos , Inmunoterapia/métodos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Células Supresoras de Origen Mieloide/efectos de los fármacos , Células Supresoras de Origen Mieloide/inmunología , Masculino

RESUMEN

Colorectal cancer (CRC) is the third most common cancer worldwide. Recent experiments suggest that CDK12 can be a good therapeutic target in CRC, and therefore, novel inhibitors targeting this protein are currently in preclinical development. Lipid-based formulations of chemical entities have demonstrated the ability to enhance activity while improving the safety profile. In the present work, we explore the antitumor activity of a new CDK12 inhibitor (CDK12-IN-E9, CDK12i) and its lipid-based formulation (LP-CDK12i) in CRC models, to increase efficacy. SW620, SW480 and HCT116 CRC cell lines were used to evaluate the inhibitor and the liposomal formulation using MTT proliferation assay, 3D invasion cultures, flow cytometry, Western blotting and immunofluorescence experiments. Free-cholesterol liposomal formulations of CDK12i (LP-CDK12i) were obtained by solvent injection method and fully characterized by size, shape, polydispersity, encapsulation efficiency, and release profile and stability assessments. LP-CDK12i induced a higher antiproliferative effect compared with CDK12i as a free agent. The IC50 value was lower across all cell lines tested, leading to a reduction in cell proliferation and the formation of 3D structures. Evaluation of apoptosis revealed an increase in cell death, while biochemical studies demonstrated modifications of apoptosis and DNA damage components. In conclusion, we confirm the role of targeting CDK12 for the treatment of CRC and describe, for the first time, a liposomal formulation of a CDK12i with higher antiproliferative activity compared with the free compound.

Asunto(s)

Antineoplásicos , Apoptosis , Proliferación Celular , Neoplasias Colorrectales , Quinasas Ciclina-Dependientes , Liposomas , Humanos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Proliferación Celular/efectos de los fármacos , Antineoplásicos/farmacología , Línea Celular Tumoral , Apoptosis/efectos de los fármacos , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Células HCT116 , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química

RESUMEN

The biology of the cyclin-dependent kinase-like (CDKL) kinase family remains enigmatic. Contrary to their nomenclature, CDKLs do not rely on cyclins for activation and are not involved in cell cycle regulation. Instead, they share structural similarities with mitogen-activated protein kinases and glycogen synthase kinase-3, although their specific functions and associated signaling pathways are still unknown. Previous studies have shown that the activation of CDKL5 kinase contributes to the development of acute kidney injury (AKI) by suppressing the protective SOX9-dependent transcriptional program in tubular epithelial cells. In the current study, we measured the functional activity of all five CDKL kinases and discovered that, in addition to CDKL5, CDKL1 is also activated in tubular epithelial cells during AKI. To explore the role of CDKL1, we generated a germline knockout mouse that exhibited no abnormalities under normal conditions. Notably, when these mice were challenged with bilateral ischemia-reperfusion and rhabdomyolysis, they were found to be protected from AKI. Further mechanistic investigations revealed that CDKL1 phosphorylates and destabilizes SOX11, contributing to tubular dysfunction. In summary, this study has unveiled a previously unknown CDKL1-SOX11 axis that drives tubular dysfunction during AKI.NEW & NOTEWORTHY Identifying and targeting pathogenic protein kinases holds potential for drug discovery in treating acute kidney injury. Our study, using novel germline knockout mice, revealed that Cdkl1 kinase deficiency does not affect mouse viability but provides protection against acute kidney injury. This underscores the importance of Cdkl1 kinase in kidney injury and supports the development of targeted small-molecule inhibitors as potential therapeutics.

Asunto(s)

Lesión Renal Aguda , Quinasas Ciclina-Dependientes , Factores de Transcripción SOXC , Transducción de Señal , Animales , Masculino , Ratones , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Lesión Renal Aguda/genética , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/genética , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Rabdomiólisis/metabolismo , Factores de Transcripción SOXC/metabolismo , Factores de Transcripción SOXC/genética

RESUMEN

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 (MAPK） 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 Ciclina

RESUMEN

Cell cycle regulation is largely abnormal in cancers. Molecular understanding and therapeutic targeting of the aberrant cell cycle are essential. Here, we identified that an underappreciated serine/threonine kinase, cyclin-dependent kinase-like 3 (CDKL3), crucially drives rapid cell cycle progression and cell growth in cancers. With regard to mechanism, CDKL3 localizes in the nucleus and associates with specific cyclin to directly phosphorylate retinoblastoma (Rb) for quiescence exit. In parallel, CDKL3 prevents the ubiquitin-proteasomal degradation of cyclin-dependent kinase 4 (CDK4) by direct phosphorylation on T172 to sustain G1 phase advancement. The crucial function of CDKL3 in cancers was demonstrated both in vitro and in vivo. We also designed, synthesized, and characterized a first-in-class CDKL3-specific inhibitor, HZ1. HZ1 exhibits greater potency than CDK4/6 inhibitor in pan-cancer treatment by causing cell cycle arrest and overcomes acquired resistance to CDK4/6 inhibitor. In particular, CDKL3 has significant clinical relevance in colon cancer, and the effectiveness of HZ1 was demonstrated by murine and patient-derived cancer models. Collectively, this work presents an integrated paradigm of cancer cell cycle regulation and suggests CDKL3 targeting as a feasible approach in cancer treatment.

Asunto(s)

Quinasa 4 Dependiente de la Ciclina , Humanos , Animales , Ratones , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/genética , Línea Celular Tumoral , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Neoplasias del Colon/patología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/genética , Neoplasias del Colon/tratamiento farmacológico , Ciclo Celular

RESUMEN

Cyclin-dependent kinase 7, along with cyclin H and MAT1, forms the CDK-activating complex (CAK), which directs cell cycle progression via T-loop phosphorylation of cell cycle CDKs. Pharmacological inhibition of CDK7 leads to selective anti-cancer effects in cellular and in vivo models, motivating several ongoing clinical investigations of this target. Current CDK7 inhibitors are either reversible or covalent inhibitors of its catalytic activity. We hypothesized that small molecule targeted protein degradation (TPD) might result in differentiated pharmacology due to the loss of scaffolding functions. Here, we report the design and characterization of a potent CDK7 degrader that is comprised of an ATP-competitive CDK7 binder linked to a CRL2VHL recruiter. JWZ-5-13 effectively degrades CDK7 in multiple cancer cells and leads to a potent inhibition of cell proliferation. Additionally, compound JWZ-5-13 displayed bioavailability in a pharmacokinetic study conducted in mice. Therefore, JWZ-5-13 is a useful chemical probe to investigate the pharmacological consequences of CDK7 degradation.

Asunto(s)

Proliferación Celular , Quinasas Ciclina-Dependientes , Inhibidores de Proteínas Quinasas , Humanos , Animales , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/síntesis química , Proliferación Celular/efectos de los fármacos , Ratones , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Relación Estructura-Actividad , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/síntesis química , Descubrimiento de Drogas , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Relación Dosis-Respuesta a Droga , Quinasa Activadora de Quinasas Ciclina-Dependientes , Proteolisis/efectos de los fármacos , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales

RESUMEN

In cells, signal transduction heavily relies on the intricate regulation of protein kinases, which provide the fundamental framework for modulating most signaling pathways. Dysregulation of kinase activity has been implicated in numerous pathological conditions, particularly in cancer. The druggable nature of most kinases positions them into a focal point during the process of drug development. However, a significant challenge persists, as the role and biological function of nearly one third of human kinases remains largely unknown.Within this diverse landscape, cyclin-dependent kinases (CDKs) emerge as an intriguing molecular subgroup. In human, this kinase family encompasses 21 members, involved in several key biological processes. Remarkably, 13 of these CDKs belong to the category of understudied kinases, and only 5 having undergone broad investigation to date. This knowledge gap underscores the pressing need to delve into the study of these kinases, starting with a comprehensive review of the less-explored ones.Here, we will focus on the PCTAIRE subfamily of CDKs, which includes CDK16, CDK17, and CDK18, arguably among the most understudied CDKs members. To contextualize PCTAIREs within the spectrum of human pathophysiology, we conducted an exhaustive review of the existing literature and examined available databases. This approach resulted in an articulate depiction of these PCTAIREs, encompassing their expression patterns, 3D configurations, mechanisms of activation, and potential functions in normal tissues and in cancer.We propose that this effort offers the possibility of identifying promising areas of future research that extend from basic research to potential clinical and therapeutic applications.

Asunto(s)

Quinasas Ciclina-Dependientes , Humanos , Quinasas Ciclina-Dependientes/metabolismo , Animales , Neoplasias/metabolismo , Neoplasias/patología , Neoplasias/genética , Transducción de Señal , Relación Estructura-Actividad , Conformación Proteica