RESUMEN
Alzheimer's disease (AD) remains one of the most challenging and prevalent neurodegenerative disorders worldwide. Despite extensive research efforts, effective treatments for AD are lacking, emphasising the need for a deeper understanding of its underlying molecular mechanisms. Cyclin-dependent kinase 5 (CDK5), a serine/threonine kinase primarily associated with cell cycle regulation and neuronal development, has emerged as a key player in AD pathology. This review article comprehensively explores the multifaceted roles of CDK5 in the pathogenesis of AD. We begin by elucidating the physiological functions of CDK5 in normal brain development and neuronal maintenance, highlighting its involvement in synaptic plasticity, neurotransmitter release, and cytoskeletal dynamics. Subsequently, we delve into the dysregulation of CDK5 activity observed in AD, encompassing aberrant hyperactivation, and dysregulated protein interactions. Moreover, we discuss the intricate interplay between CDK5 and AD-related proteins, including amyloid-beta precursor protein (APP) and tau protein, elucidating their collective impact on disease progression. Finally, we described various approaches available for the inhibition of CDK-5, which can be explored as future therapeutic intervention for AD. Through synthesizing evidence from in vitro studies, animal models, and clinical investigations, this review provides a comprehensive overview of the intricate relationship between CDK5 dysregulation and AD pathogenesis, offering insights that may inform future therapeutic interventions strategies.
Asunto(s)
Enfermedad de Alzheimer , Quinasa 5 Dependiente de la Ciclina , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Humanos , Quinasa 5 Dependiente de la Ciclina/metabolismo , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Animales , Proteínas tau/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Precursor de Proteína beta-Amiloide/metabolismoRESUMEN
Cyclin-dependent kinase 5 (Cdk5) is a protein expressed in postmitotic neurons in the central nervous system (CNS). Cdk5 is activated by p35 and p39 which are neuron regulatory subunits. Cdk5/p35 complex is activated by calpain protease to form Cdk5/p35 which has a neuroprotective effect by regulating the synaptic plasticity and memory functions. However, exaggerated Cdk5 is implicated in different types of neurodegenerative diseases including Parkinson disease (PD). Therefore, modulation of Cdk5 signalling may mitigate PD neuropathology. Therefore, the aim of the present review was to discuss the critical role of Cdk5 in the pathogenesis of PD, and how Cdk5 inhibitors are effectual in the management of PD. In conclusion, overactivated Cdk5 is involved the development of neurodegeneration, and Cdk5/calpain inhibitors such as statins, metformin, fenofibrates and rosiglitazone can attenuate the progression of PD neuropathology.
Asunto(s)
Quinasa 5 Dependiente de la Ciclina , Enfermedad de Parkinson , Quinasa 5 Dependiente de la Ciclina/metabolismo , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Humanos , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Animales , Calpaína/metabolismo , Calpaína/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
The scaffolds of two known CDK inhibitors (CAN508 and dinaciclib) were the starting point for synthesizing two series of pyarazolo[1,5-a]pyrimidines to obtain potent inhibitors with proper selectivity. The study presented four promising compounds; 10d, 10e, 16a, and 16c based on cytotoxic studies. Compound 16a revealed superior activity in the preliminary anticancer screening with GI % = 79.02-99.13 against 15 cancer cell lines at 10 µM from NCI full panel 60 cancer cell lines and was then selected for further investigation. Furthermore, the four compounds revealed good safety profile toward the normal cell lines WI-38. These four compounds were subjected to CDK inhibitory activity against four different isoforms. All of them showed potent inhibition against CDK5/P25 and CDK9/CYCLINT. Compound 10d revealed the best activity against CDK5/P25 (IC50 = 0.063 µM) with proper selectivity index against CDK1 and CDK2. Compound 16c exhibited the highest inhibitory activity against CDK9/CYCLINT (IC50 = 0.074 µM) with good selectivity index against other isoforms. Finally, docking simulations were performed for compounds 10e and 16c accompanied by molecular dynamic simulations to understand their behavior in the active site of the two CDKs with respect to both CAN508 and dinaciclib.
Asunto(s)
Antineoplásicos , Compuestos Bicíclicos Heterocíclicos con Puentes , Óxidos N-Cíclicos , Diseño de Fármacos , Indolizinas , Simulación del Acoplamiento Molecular , Inhibidores de Proteínas Quinasas , Compuestos de Piridinio , Humanos , Compuestos de Piridinio/farmacología , Compuestos de Piridinio/química , Indolizinas/farmacología , Indolizinas/química , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Óxidos N-Cíclicos/farmacología , Óxidos N-Cíclicos/química , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Línea Celular Tumoral , Antineoplásicos/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Relación Estructura-Actividad , Pirimidinas/farmacología , Pirimidinas/química , Ensayos de Selección de Medicamentos Antitumorales , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 5 Dependiente de la Ciclina/metabolismo , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 9 Dependiente de la Ciclina/metabolismoRESUMEN
The authors' previous research has shown the pivotal roles of cyclin-dependent kinase 5 (CDK5) and its regulatory protein p35 in nerve growth factor (NGF)-induced differentiation of sympathetic neurons in PC12 cells. During the process of differentiation, neurons are susceptible to environmental influences, including the effects of drugs. Metformin is commonly used in the treatment of diabetes and its associated symptoms, particularly in diabetic neuropathy, which is characterized by dysregulation of the sympathetic neurons. However, the impacts of metformin on sympathetic neuronal differentiation remain unknown. In this study, we investigated the impact of metformin on NGF-induced sympathetic neuronal differentiation using rat pheochromocytoma PC12 cells as a model. We examined the regulation of TrkA-p35/CDK5 signaling in NGF-induced PC12 differentiation. Our results demonstrate that metformin reduces NGF-induced PC12 differentiation by inactivating the TrkA receptor, subsequently inhibiting ERK and EGR1. Inhibition of this cascade ultimately leads to the downregulation of p35/CDK5 in PC12 cells. Furthermore, metformin inhibits the activation of the presynaptic protein Synapsin-I, a substrate of CDK5, in PC12 differentiation. In addition, metformin alters axonal and synaptic bouton formation by inhibiting p35 at both the axons and axon terminals in fully differentiated PC12 cells. In summary, our study elucidates that metformin inhibits sympathetic neuronal differentiation in PC12 cells by disrupting TrkA/ERK/EGR1 and p35/CDK5 signaling. This research contributes to uncovering a novel signaling mechanism in drug response during sympathetic neuronal differentiation, enhancing our understanding of the intricate molecular processes governing this critical aspect of neurodevelopment.NEW & NOTEWORTHY This study unveils a novel mechanism influenced by metformin during sympathetic neuronal differentiation. By elucidating its inhibitory effects from the nerve growth factor (NGF) receptor, TrkA, to the p35/CDK5 signaling pathways, we advance our understanding of metformin's mechanisms of action and emphasize its potential significance in the context of drug responses during sympathetic neuronal differentiation.
Asunto(s)
Diferenciación Celular , Quinasa 5 Dependiente de la Ciclina , Metformina , Factor de Crecimiento Nervioso , Neuronas , Receptor trkA , Animales , Metformina/farmacología , Ratas , Células PC12 , Quinasa 5 Dependiente de la Ciclina/metabolismo , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Factor de Crecimiento Nervioso/metabolismo , Factor de Crecimiento Nervioso/farmacología , Receptor trkA/metabolismo , Receptor trkA/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Diferenciación Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , FosfotransferasasRESUMEN
Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent monogenic human disease, but to date, only one therapy (tolvaptan) is approved to treat kidney cysts in ADPKD patients. Cyclin-dependent kinase 5 (CDK5), an atypical member of the cyclin-dependent kinase family, has been implicated as a target for treating ADPKD. However, no compounds have been disclosed to date that selectively inhibit CDK5 while sparing the broader CDK family members. Herein, we report the discovery of CDK5 inhibitors, including GFB-12811, that are highly selective over the other tested kinases. In cellular assays, our compounds demonstrate CDK5 target engagement while avoiding anti-proliferative effects associated with inhibiting other CDKs. In addition, we show that the compounds in this series exhibit promising in vivo PK profiles, enabling their use as tool compounds for interrogating the role of CDK5 in ADPKD and other diseases.
Asunto(s)
Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Proliferación Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Diseño de Fármacos , Descubrimiento de Drogas , Células HEK293 , Humanos , Modelos Moleculares , Riñón Poliquístico Autosómico Dominante/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Tauopathies are neurodegenerative diseases characterized by abnormal metabolism of misfolded tau proteins and are progressive. Pathological phosphorylation of tau occurs in the retinal ganglion cells (RGCs) after optic nerve injuries. Cyclin-dependent kinase-5 (Cdk5) causes hyperphosphorylation of tau. To determine the roles played by Cdk5 in retinal degeneration, roscovitine, a Cdk5 inhibitor, was injected intravitreally after optic nerve crush (ONC). The neuroprotective effect of roscovitine was determined by the number of Tuj-1-stained RGCs on day 7. The change in the levels of phosphorylated tau, calpain-1, and cleaved α-fodrin was determined by immunoblots on day 3. The expression of P35/P25, a Cdk5 activator, in the RGCs was determined by immunohistochemistry. The results showed that roscovitine reduced the level of phosphorylated tau by 3.5- to 1.6-fold. Calpain-1 (2.1-fold) and cleaved α-fodrin (1.5-fold) were increased on day 3, suggesting that the calpain signaling pathway was activated. P35/P25 was accumulated in the RGCs that were poorly stained by Tuj-1. Calpain inhibition also reduced the increase in phosphorylated tau. The number of RGCs decreased from 2191 ± 178 (sham) to 1216 ± 122 cells/mm2 on day 7, and roscovitine preserved the level at 1622 ± 130 cells/mm2. We conclude that the calpain-mediated activation of Cdk5 is associated with the pathologic phosphorylation of tau.
Asunto(s)
Quinasa 5 Dependiente de la Ciclina/fisiología , Traumatismos del Nervio Óptico , Células Ganglionares de la Retina , Tauopatías , Proteínas tau/metabolismo , Animales , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Traumatismos del Nervio Óptico/metabolismo , Traumatismos del Nervio Óptico/patología , Fosforilación , Ratas , Ratas Wistar , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patología , Roscovitina/farmacología , Tauopatías/metabolismo , Tauopatías/patologíaRESUMEN
Mesial temporal lobe epilepsy with hippocamapal sclerosis (MTLE-HS) is the most common form of drug resistant epilepsy (DRE). MTLE-HS is a distributed network disorder comprising of not only the hippocampus, but other anatomically related extrahippocampal regions. Excitatory synaptic transmission is differentially regulated in the hippocampal and extra-hippocampal regions of patients with MTLE-HS, but its mechanism not understood. Cyclin-dependent kinase 5 (Cdk5) is known to regulate synaptic transmission and plasticity through up-regulation of NMDA receptors by phosphorylating NR2Asubunits. The present study is designed to investigate whether Cdk5 differentially regulates the excitatory synaptic transmission in the hippocampus and anterior temporal lobe (ATL) samples obtained from patients of MTLE-HS. We have measured the Cdk5 kinase activity and the protein levels of Cdk5, p-Cdk5, p35/p25, NR2A, pNR2A in the hippocampal and ATL samples obtained from patients with MTLE-HS. We have also determined the effect of roscovitine, a Cdk5 antagonist, on spontaneous excitatory postsynaptic currents (EPSCs) recorded from the hippocampal and ATL using patch-clamp technique. We observed significant increase in the expression of Cdk5, p-Cdk5, p35/p25, NR2A, pNR2A in the ATL samples as compared to the hippocampal samples. Cdk5 activity was significantly higher in ATL samples as compared to the hippocampal samples. Magnitude of reduction in the frequency of EPSCs by roscovitine in the ATL samples was higher than that in the hippocampal samples. Our studies suggest that Cdk5 differentially regulates excitatory synaptic activity in the hippocampal and ATL region of patients with MTLE-HS.
Asunto(s)
Quinasa 5 Dependiente de la Ciclina/metabolismo , Epilepsia del Lóbulo Temporal/metabolismo , Potenciales Postsinápticos Excitadores , Hipocampo/metabolismo , Lóbulo Temporal/metabolismo , Adolescente , Adulto , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Epilepsia del Lóbulo Temporal/fisiopatología , Femenino , Hipocampo/patología , Hipocampo/fisiopatología , Humanos , Masculino , Persona de Mediana Edad , Inhibidores de Proteínas Quinasas/farmacología , Receptores de N-Metil-D-Aspartato/metabolismo , Roscovitina/farmacología , Esclerosis , Lóbulo Temporal/fisiopatologíaRESUMEN
The marked overexpression of cyclin-dependent kinase 5 (CDK5) or Notch1 receptor, which plays critical roles in pancreatic ductal adenocarcinoma (PDAC) development, has been detected in numerous PDAC cell lines and tissues. Although, a previous study has demonstrated that CDK5 inhibition disrupts Notch1 functions in human umbilical vein endothelial cells, the mechanism underlying Notch1 activation regulated by CDK5 remains unclear. Herein, we identified a physical interaction between CDK5 and Notch1 in PDAC cells, with the Notch1 peptide phosphorylated by CDK5/p25 kinase. CDK5 blockade resulted in the profound inhibition of Notch signaling. Accordingly, CDK5 inhibition sensitized PDAC cell proliferation and migration following Notch inhibition. In conclusion, CDK5 positively regulates Notch1 function via phosphorylation, which in turn promotes cell proliferation and migration. The combinational inhibition of CDK5 and Notch signaling may be an effective strategy in the treatment of PDAC.
Asunto(s)
Carcinoma Ductal Pancreático/metabolismo , Quinasa 5 Dependiente de la Ciclina/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptor Notch1/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 5 Dependiente de la Ciclina/genética , Dipéptidos/farmacología , Silenciador del Gen , Humanos , Inmunoprecipitación , Neoplasias Pancreáticas/patología , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/metabolismo , Receptor Notch1/antagonistas & inhibidores , Receptor Notch1/genética , Roscovitina/farmacología , Transducción de SeñalRESUMEN
BACKGROUND: General anesthetics such as sevoflurane interfere with dendritic development and synaptogenesis, resulting in cognitive impairment. The collapsin response mediator protein2 (CRMP2) plays important roles in dendritic development and synaptic plasticity and its phosphorylation is regulated by cycline dependent kinase-5 (Cdk5) and glycogen synthase kinase-3ß (GSK-3ß). Here we investigated whether Cdk5/CRMP2 or GSK-3ß/CRMP2 pathway is involved in sevoflurane-induced developmental neurotoxicity. METHODS: Rats at postnatal day 7 (PND7) were i.p. injected with Cdk5 inhibitor roscovitine, GSK-3ß inhibitor SB415286 or saline 20 min. before exposure to 2.8% sevoflurane for 4 h. Western-blotting was applied to measure the expression of Cdk5/CRMP2 and GSK-3ß/CRMP2 pathway proteins in the hippocampus 6 h after the sevoflurane exposure. When rats grew to adolescence (from PND25), they were tested for open-field and contextual fear conditioning, and then long term potentiation (LTP) from hippocampal slices was recorded, and morphology of pyramidal neuron was examined by Golgi staining and synaptic plasticity-related proteins expression in hippocampus were measured by western-blotting. In another batch of experiment, siRNA-CRMP2 or vehicle control was injected into hippocampus on PND5. RESULTS: Sevoflurane activated Cdk5/CRMP2 and GSK-3ß/CRMP2 pathways in the hippocampus of neonatal rats, reduced dendritic length, branches and the density of dendritic spine in pyramidal neurons. It also reduced the expressions of PSD-95, drebrin and synaptophysin in hippocampus, impaired memory ability of rats and inhibited LTP in hippocampal slices. All the impairment effects by sevoflurane were attenuated by pretreatment with inhibitor of Cdk5 or GSK-3ß. Furthermore, rat transfected with siRNA-CRMP2 eliminated the neuroprotective effects of Cdk5 or GSK-3ß blocker in neurobehavioral and LTP tests. CONCLUSION: Cdk5/CRMP2 and GSK-3ß/CRMP2 pathways participate in sevoflurane-induced dendritic development abnormalities and cognitive dysfunction in developing rats.
Asunto(s)
Disfunción Cognitiva/inducido químicamente , Quinasa 5 Dependiente de la Ciclina/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Sevoflurano/toxicidad , Aminofenoles/farmacología , Animales , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 5 Dependiente de la Ciclina/genética , Dendritas/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3 beta/genética , Hipocampo/citología , Hipocampo/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular/genética , Maleimidas/farmacología , Proteínas del Tejido Nervioso/genética , Inhibidores de Proteínas Quinasas/farmacología , Células Piramidales/efectos de los fármacos , Ratas , Roscovitina/farmacologíaRESUMEN
AIMS: Cyclin-dependent kinase 5 (CDK5) is a potential target for the treatment of cerebral ischemia. CDK5 is one of the upstream regulators for Dynamin-related protein 1 (Drp1) phosphorylation. This study intends to discuss whether CDK5 inhibition conferring neuroprotection in cerebral ischemia through regulating Drp1 phosphorylation. MATERIALS AND METHODS: Mouse neuroblastoma N2a cells and N1E-115 cells were cultured and subjected to oxygen-glucose deprivation/reperfusion (OGDR). N2a cells and N1E-115 cells were treated with Roscovitine, a pharmacological inhibitor of CDK5, or transfected with CDK5 siRNA to knock down CDK5 expression. N2a cells were transfected with different plasmids (Drp1-Myc, the dephosphorylation-mimic mutant Drp1S616A-Myc and the phosphorylation-mimic mutant Drp1S616D-Myc). The expression of CDK5 and its activator p35, Drp1 and phosphorylated Drp1 on S616 was determined by western blot. The morphology of mitochondria was detected by immunofluorescence staining and the proportion of N2a cells with apoptosis was detected by flow cytometry analysis. KEY FINDINGS: Expression of CDK5, p35 and phosphorylated Drp1 on S616 was strongly upregulated after 4 h and 12 h reperfusion following 4 h oxygen-glucose deprivation (OGD) at protein level. CDK5 inhibition by pre-treated with Roscovitine or transfection with CDK5 siRNA significantly ameliorated OGDR induced mitochondrial fragmentation and apoptosis. Overexpression of the phosphorylation-mimic mutant Drp1S616D abrogated the protective effect of CDK5 inhibition against OGDR induced mitochondrial fragmentation and apoptosis. SIGNIFICANCE: Our data indicate that the neuroprotective effect of CDK5 inhibition against OGDR induced neuronal damage is Drp1S616 phosphorylation dependent. A better understanding of the neuroprotective mechanisms of CDK5 inhibition in cerebral ischemia will help to develop safe and efficacious drugs targeting CDK5 signaling for clinical use.
Asunto(s)
Apoptosis , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Dinaminas/metabolismo , Glucosa/deficiencia , Mitocondrias/patología , Neuroblastoma/prevención & control , Oxígeno/metabolismo , Animales , Quinasa 5 Dependiente de la Ciclina/genética , Quinasa 5 Dependiente de la Ciclina/metabolismo , Dinaminas/genética , Ratones , Mitocondrias/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patología , Fármacos Neuroprotectores , Fosforilación , Daño por Reperfusión/complicaciones , Transducción de Señal , Células Tumorales CultivadasRESUMEN
The neurovascular unit (NVU) is responsible for synchronizing the energetic demand, vasodynamic changes, and neurochemical and electrical function of the brain through a closed and interdependent interaction of cell components conforming to brain tissue. In this review, we will focus on cyclin-dependent kinase 5 (CDK5) as a molecular pivot, which plays a crucial role in the healthy function of neurons, astrocytes, and the endothelium and is implicated in the cross-talk of cellular adhesion signaling, ion transmission, and cytoskeletal remodeling, thus allowing the individual and interconnected homeostasis of cerebral parenchyma. Then, we discuss how CDK5 overactivation affects the integrity of the NVU in Alzheimer's disease (AD) and cognitive impairment; we emphasize how CDK5 is involved in the excitotoxicity spreading of glutamate and Ca2+ imbalance under acute and chronic injury. Additionally, we present pharmacological and gene therapy strategies for producing partial depletion of CDK5 activity on neurons, astrocytes, or endothelium to recover neuroplasticity and neurotransmission, suggesting that the NVU should be the targeted tissue unit in protective strategies. Finally, we conclude that CDK5 could be effective due to its intervention on astrocytes by its end feet on the endothelium and neurons, acting as an intermediary cell between systemic and central communication in the brain. This review provides integrated guidance regarding the pathogenesis of and potential repair strategies for AD.
Asunto(s)
Astrocitos/metabolismo , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 5 Dependiente de la Ciclina/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Silenciador del Gen/fisiología , Acoplamiento Neurovascular/fisiología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/patología , Ensayos Clínicos como Asunto/métodos , Silenciador del Gen/efectos de los fármacos , Humanos , Acoplamiento Neurovascular/efectos de los fármacos , Inhibidores de Proteínas Quinasas/administración & dosificaciónRESUMEN
OBJECTIVE: Adaptive immune resistance mediated by the cytokine interferon gamma (IFNG) still constitutes a major problem in cancer immunotherapy. We develop strategies for overcoming IFNG-mediated adaptive immune resistance in pancreatic ductal adenocarcinoma cancer (PDAC). DESIGN: We screened 429 kinase inhibitors for blocking IFNG-induced immune checkpoint (indoleamine 2,3-dioxygenase 1 (IDO1) and CD274) expression in a human PDAC cell line. We evaluated the ability of the cyclin-dependent kinase (CDK) inhibitor dinaciclib to block IFNG-induced IDO1 and CD274 expression in 24 human and mouse cancer cell lines as well as in primary cancer cells from patients with PDAC or ovarian carcinoma. We tested the effects of dinaciclib on IFNG-induced signal transducer and activator of transcription 1 activation and immunological cell death, and investigated the potential utility of dinaciclib in combination with IFNG for pancreatic cancer therapy in vivo, and compared gene expression levels between human cancer tissues with patient survival times using the Cancer Genome Atlas datasets. RESULTS: Pharmacological (using dinaciclib) or genetic (using shRNA or siRNA) inactivation of CDK1/2/5 not only blocks JUN-dependent immune checkpoint expression, but also triggers histone-dependent immunogenic cell death in immortalised or primary cancer cells in response to IFNG. This dual mechanism turns an immunologically 'cold' tumour microenvironment into a 'hot' one, dramatically improving overall survival rates in mouse pancreatic tumour models (subcutaneous, orthotopic and transgenic models). The abnormal expression of CDK1/2/5 and IDO1 was associated with poor patient survival in several cancer types, including PDAC. CONCLUSION: CDK1/2/5 kinase activity is essential for IFNG-mediated cancer immunoevasion. CDK1/2/5 inhibition by dinaciclib provides a novel strategy to overcome IFNG-triggered acquired resistance in pancreatic tumour immunity.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Óxidos N-Cíclicos/farmacología , Inhibidores de Puntos de Control Inmunológico/farmacología , Indolizinas/farmacología , Interferón gamma/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Fragmentos de Péptidos/farmacología , Compuestos de Piridinio/farmacología , Inmunidad Adaptativa , Adenocarcinoma/genética , Adenocarcinoma/inmunología , Animales , Antígeno B7-H1/antagonistas & inhibidores , Proteína Quinasa CDC2/antagonistas & inhibidores , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/inmunología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Expresión Génica , Humanos , Ratones , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/inmunología , Transducción de Señal , Tasa de Supervivencia , Microambiente Tumoral/efectos de los fármacosRESUMEN
Radiation-induced optic neuropathy (RION) is a devastating complication following external beam radiation therapy (EBRT) that leads to acute vision loss. To date, no efficient, available treatment for this complication, due partly to the lack of understanding regarding the developmental processes behind RION. Here, we report radiation caused changes in mitochondrial dynamics by regulating the mitochondrial fission proteins dynamin-related protein 1 (Drp1) and fission-1 (Fis1). Concurrent with an excessive production of reactive oxygen species (ROS), both neuronal injury and visual dysfunction resulted. Further, our findings delineate an important mechanism by which cyclin-dependent kinase 5 (Cdk5)-mediated phosphorylation of Drp1 (Ser616) regulates defects in mitochondrial dynamics associated with neuronal injury in the development of RION. Both the pharmacological inhibition of Cdk5 by roscovitine and the inhibition of Drp1 by mdivi-1 inhibited mitochondrial fission and the production of ROS associated with radiation-induced neuronal loss. Taken together, these findings may have clinical significance in preventing the development of RION.
Asunto(s)
Quinasa 5 Dependiente de la Ciclina/metabolismo , Dinaminas/metabolismo , Mitocondrias/efectos de la radiación , Enfermedades del Nervio Óptico/etiología , Animales , Apoptosis/efectos de la radiación , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Dinaminas/antagonistas & inhibidores , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Mitocondrias/metabolismo , Dinámicas Mitocondriales/efectos de la radiación , Neuronas/metabolismo , Neuronas/patología , Neuronas/efectos de la radiación , Enfermedades del Nervio Óptico/sangre , Enfermedades del Nervio Óptico/metabolismo , Enfermedades del Nervio Óptico/patología , Fosforilación , Quinazolinonas/farmacología , Traumatismos Experimentales por Radiación/metabolismo , Radioterapia/efectos adversos , Ratas , Roscovitina/farmacologíaRESUMEN
Disparities in cancer patient responses have prompted widespread searches to identify differences in sensitive vs. nonsensitive populations and form the basis of personalized medicine. This customized approach is dependent upon the development of pathway-specific therapeutics in conjunction with biomarkers that predict patient responses. Here, we show that Cdk5 drives growth in subgroups of patients with multiple types of neuroendocrine neoplasms. Phosphoproteomics and high throughput screening identified phosphorylation sites downstream of Cdk5. These phosphorylation events serve as biomarkers and effectively pinpoint Cdk5-driven tumors. Toward achieving targeted therapy, we demonstrate that mouse models of neuroendocrine cancer are responsive to selective Cdk5 inhibitors and biomimetic nanoparticles are effective vehicles for enhanced tumor targeting and reduction of drug toxicity. Finally, we show that biomarkers of Cdk5-dependent tumors effectively predict response to anti-Cdk5 therapy in patient-derived xenografts. Thus, a phosphoprotein-based diagnostic assay combined with Cdk5-targeted therapy is a rational treatment approach for neuroendocrine malignancies.
Asunto(s)
Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Tumores Neuroectodérmicos/tratamiento farmacológico , Fosfoproteínas/metabolismo , Inhibidores de Proteínas Quinasas/administración & dosificación , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 5 Dependiente de la Ciclina/genética , Quinasa 5 Dependiente de la Ciclina/metabolismo , Xenoinjertos , Humanos , Ratones , Neoplasias/genética , Tumores Neuroectodérmicos/genética , Tumores Neuroectodérmicos/metabolismo , Fosfoproteínas/análisis , Fosfoproteínas/genética , FosforilaciónRESUMEN
Mild hypothermia has promising effects in the treatment of acute brain insults and also affects cell cycle progression. Mitochondrial dynamics, fusion and fission, are changed along with the cell cycle and disrupted in neurodegenerative diseases, including Parkinson's disease (PD). However, the effects of hypothermia on aberrant mitochondrial dynamics in PD remain unknown. We hypothesized that mild hypothermia protects neurons by regulating cell cycle-dependent protein expression and mitochondrial dynamics in a 1-methyl-4-phenylpyridinium (MPP+)-induced cell model of PD. We found that the hypothermia treatment at 32 °C prevented MPP+-induced neuron death; however, 32 °C treatment itself also reduced cell viability. This reduction was associated with cell cycle arrest and downregulation of cyclin-dependent kinase 4 (CDK4) in proliferating human SK-N-SH neuroblastoma cells but upregulation in well-differentiated primary rat cortical neurons. In both types of neurons, hypothermia upregulated p27 (an endogenous inhibitor of CDKs) and p35 (CDK5 activator) protein expression. Treatment with hypothermia, or a selective CDK4 inhibitor, or roscovitine (CDK5 inhibitor) prevented MPP+-induced mitochondrial fission, upregulation of mitochondrial fission protein dynamin-related protein 1 (Drp1), and neuron death. In addition, overexpression of dominant negative mutant Drp1K38A improved MPP+-induced mitochondrial fission while overexpression of wild-type Drp1 blunted the prevention of mitochondrial fission by hypothermia as well as CDK4 inhibitor and roscovitine. These results elucidate that hypothermia may inhibit CDK4 and CDK5 activation by upregulating p27 and p35 expression to prevent Drp1-dependent mitochondrial fission and neuron loss after MPP+ treatment. CDK4 and CDK5 inhibition imitates the neuroprotective functions of hypothermia as a potential therapy for PD.
Asunto(s)
Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Dinaminas/metabolismo , Hipotermia Inducida , Dinámicas Mitocondriales , Neuronas/patología , 1-Metil-4-fenilpiridinio , Animales , Proteínas de Ciclo Celular/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Corteza Cerebral/patología , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 5 Dependiente de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Dinámicas Mitocondriales/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Ratas Sprague-Dawley , Roscovitina/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Cyclin-dependent kinase 2 (CDK2) is a potential therapeutic target for the treatment of cancer. Development of CDK2 inhibitors has been extremely challenging as its ATP-binding site shares high similarity with CDK1, a related kinase whose inhibition causes toxic effects. Here, we report the development of TMX-2172, a heterobifunctional CDK2 degrader with degradation selectivity for CDK2 and CDK5 over not only CDK1, but transcriptional CDKs (CDK7 and CDK9) and cell cycle CDKs (CDK4 and CDK6) as well. In addition, we demonstrate that antiproliferative activity in ovarian cancer cells (OVCAR8) depends on CDK2 degradation and correlates with high expression of cyclin E1 (CCNE1), which functions as a regulatory subunit of CDK2. Collectively, our work provides evidence that TMX-2172 represents a lead for further development and that CDK2 degradation is a potentially valuable therapeutic strategy in ovarian and other cancers that overexpress CCNE1.
Asunto(s)
Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , División Celular/efectos de los fármacos , Línea Celular Tumoral , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 5 Dependiente de la Ciclina/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/patología , FosforilaciónRESUMEN
The emergence of new drugs is a major feature of the treatment history of multiple myeloma (MM), which also reflects the current incurability of MM. As a unique member of cyclin dependent kinase (CDK) family, CDK5 participates in numerous tumorigenic or nontumorigenic processes. The aim of this study is to investigate the effects of CDK5 on the viability of MM cells and bortezomib resistance using western blotting, immunohistochemistry, transient transfection, MTT assays, cell cycle analysis, apoptosis assays and a myeloma xenograft mouse model. The present study found that MM patients with high CDK5 expression in the bone marrow do not respond well to bortezomib, have higher DS stage and worse prognosis. Genetic and pharmacological (dinaciclib) inhibition of CDK5 triggers MM cell viability inhibition. Dinaciclib induces G2/M arrest and apoptosis of MM cells. In vivo experiments with myeloma xenograft mice indicate that dinaciclib significantly reduces the volume of tumors with good tolerance. Dinaciclib combined with bortezomib exerts a synergistic antimyeloma activity accompanied by inhibiting the activation of the nuclear factorκB pathway. This study demonstrates the important role of CDK5 in the pathogenesis, viability, prognosis and resistance to bortezomib of MM, laying a solid theoretical foundation for further clinical use of CDK5 inhibitors.
Asunto(s)
Bortezomib/farmacología , Muerte Celular/efectos de los fármacos , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Resistencia a Antineoplásicos/efectos de los fármacos , Mieloma Múltiple/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Animales , Apoptosis/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Tumoral , Óxidos N-Cíclicos/farmacología , Sinergismo Farmacológico , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Indolizinas/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Mieloma Múltiple/metabolismo , FN-kappa B/metabolismo , Pronóstico , Compuestos de Piridinio/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A rapid, selective, and sensitive liquid chromatography coupled with tandem mass spectrometry (MS/MS) method was developed and validated for the quantitation of the novel CDK5 inhibitor '20-223' in mouse plasma. Separation of analytes was achieved by a reverse-phase ACE Excel C18 column (1.7 µm, 100 × 2.1 mm) with gradient elution using 0.1% formic acid (FA) in methanol and 0.1% FA as the mobile phase. Analytes were monitored by MS/MS with an electrospray ionization source in the positive multiple reaction monitoring mode. The MS/MS response was linear over the concentration range 0.2-500 ng/mL for 20-223. The within- and between-batch precision were within the acceptable limits as per Food and Drug Administration guidelines. The validated method was successfully applied to plasma protein binding and in vitro metabolism studies. Compound 20-223 was highly bound to mouse plasma proteins (>98% bound). Utilizing mouse S9 fractions, in vitro intrinsic clearance (CLint ) was 24.68 ± 0.99 µL/min/mg protein. A total of 12 phase I and II metabolites were identified with hydroxylation found to be the major metabolic pathway. The validate method required a low sample volume, was linear from 0.2 to 500 ng/mL, and had acceptable accuracy and precision.
Asunto(s)
Cromatografía Liquida/métodos , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/sangre , Inhibidores de Proteínas Quinasas/farmacocinética , Espectrometría de Masas en Tándem/métodos , Animales , Proteínas Sanguíneas/metabolismo , Límite de Detección , Modelos Lineales , Ratones , Unión Proteica , Inhibidores de Proteínas Quinasas/química , Reproducibilidad de los ResultadosRESUMEN
We show that commercially sourced n-channel silicon field-effect transistors (nFETs) operating above their threshold voltage with closed loop feedback to maintain a constant channel current allow a pH readout resolution of (7.2 ± 0.3) × 10-3 at a bandwidth of 10 Hz, or ≈3-fold better than the open loop operation commonly employed by integrated ion-sensitive field-effect transistors (ISFETs). We leveraged the improved nFET performance to measure the change in solution pH arising from the activity of a pathological form of the kinase Cdk5, an enzyme implicated in Alzheimer's disease, and showed quantitative agreement with previous measurements. The improved pH resolution was realized while the devices were operated in a remote sensing configuration with the pH sensing element off-chip and connected electrically to the FET gate terminal. We compared these results with those measured by using a custom-built dual-gate 2D field-effect transistor (dg2DFET) fabricated with 2D semi-conducting MoS2 channels and a signal amplification of 8. Under identical solution conditions the nFET performance approached the dg2DFETs pH resolution of (3.9 ± 0.7) × 10-3. Finally, using the nFETs, we demonstrated the effectiveness of a custom polypeptide, p5, as a therapeutic agent in restoring the function of Cdk5. We expect that the straight-forward modifications to commercially sourced nFETs demonstrated here will lower the barrier to widespread adoption of these remote-gate devices and enable sensitive bioanalytical measurements for high throughput screening in drug discovery and precision medicine applications.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Quinasa 5 Dependiente de la Ciclina/análisis , Transistores Electrónicos , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Técnicas Electroquímicas/instrumentación , Técnicas Electroquímicas/métodos , Humanos , Concentración de Iones de Hidrógeno , Fármacos Neuroprotectores/química , Péptidos/química , Silicio/químicaRESUMEN
The cyclin-dependent kinase 5 (CDK5), originally described as a neuronal-specific kinase, is also frequently activated in human cancers. Using conditional CDK5 knockout mice and a mouse model of highly metastatic melanoma, we found that CDK5 is dispensable for the growth of primary tumors. However, we observed that ablation of CDK5 completely abrogated the metastasis, revealing that CDK5 is essential for the metastatic spread. In mouse and human melanoma cells CDK5 promotes cell invasiveness by directly phosphorylating an intermediate filament protein, vimentin, thereby inhibiting assembly of vimentin filaments. Chemical inhibition of CDK5 blocks the metastatic spread of patient-derived melanomas in patient-derived xenograft (PDX) mouse models. Hence, inhibition of CDK5 might represent a very potent therapeutic strategy to impede the metastatic dissemination of malignant cells.