Metformin inhibits nerve growth factor-induced sympathetic neuron differentiation through p35/CDK5 inhibition.

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.

Cyclin-dependent kinase 5 (CDK5) inhibitors in Parkinson disease.

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.

Design, synthesis, and biological evaluation of dinaciclib and CAN508 hybrids as CDK inhibitors.

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.

Phosphoprotein-based biomarkers as predictors for cancer therapy.

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.

Targeting the cyclin-dependent kinase 5 in metastatic melanoma.

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.

CDK1/2/5 inhibition overcomes IFNG-mediated adaptive immune resistance in pancreatic cancer.

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.

Roscovitine, a Cyclin-Dependent Kinase-5 Inhibitor, Decreases Phosphorylated Tau Formation and Death of Retinal Ganglion Cells of Rats after Optic Nerve Crush.

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.

Three-dimensional structural analysis reveals a Cdk5-mediated kinase cascade regulating hepatic biliary network branching in zebrafish.

The intrahepatic biliary network is a highly branched three-dimensional network lined by biliary epithelial cells, but how its branching patterns are precisely established is not clear. We designed a new computer-based algorithm that quantitatively computes the structural differences of the three-dimensional networks. Utilizing the algorithm, we showed that inhibition of Cyclin-dependent kinase 5 (Cdk5) led to reduced branching in the intrahepatic biliary network in zebrafish. Further, we identified a previously unappreciated downstream kinase cascade regulated by Cdk5. Pharmacological manipulations of this downstream kinase cascade produced a crowded branching defect in the intrahepatic biliary network and influenced actin dynamics in biliary epithelial cells. We generated larvae carrying a mutation in cdk5 regulatory subunit 1a (cdk5r1a), an essential activator of Cdk5. cdk5r1a mutant larvae show similar branching defects as those observed in Cdk5 inhibitor-treated larvae. A small-molecule compound that interferes with the downstream kinase cascade rescued the mutant phenotype. These results provide new insights into branching morphogenesis of the intrahepatic biliary network.

Inhibition of Cyclin-Dependent Kinase 5: A Strategy to Improve Sorafenib Response in Hepatocellular Carcinoma Therapy.

Therapeutic options for patients with advanced-stage hepatocellular carcinoma (HCC) are very limited. The only approved first-line treatment is the multi-tyrosine kinase inhibitor sorafenib, which shows low response rates and severe side effects. In particular, the compensatory activation of growth factor receptors leads to chemoresistance and limits the clinical impact of sorafenib. However, combination approaches to improve sorafenib have failed. Here we investigate the inhibition of cyclin-dependent kinase 5 (Cdk5) as a promising combination strategy to improve sorafenib response in HCC. Combination of sorafenib with Cdk5 inhibition (genetic knockdown by short hairpin RNA or CRISPR/Cas9 and pharmacologic inhibition) synergistically impaired HCC progression in vitro and in vivo by inhibiting both tumor cell proliferation and migration. Importantly, these effects were mediated by a mechanism for Cdk5: A liquid chromatography-tandem mass spectrometry-based proteomic approach revealed that Cdk5 inhibition interferes with intracellular trafficking, a process crucial for cellular homeostasis and growth factor receptor signaling. Cdk5 inhibition resulted in an accumulation of enlarged vesicles and respective cargos in the perinuclear region, considerably impairing the extent and quality of growth factor receptor signaling. Thereby, Cdk5 inhibition offers a comprehensive approach to globally disturb growth factor receptor signaling that is superior to specific inhibition of individual growth factor receptors. Conclusion: Cdk5 inhibition represents an effective approach to improve sorafenib response and to prevent sorafenib treatment escape in HCC. Notably, Cdk5 is an addressable target frequently overexpressed in HCC, and with Dinaciclib, a clinically tested Cdk5 inhibitor is readily available. Thus, our study provides evidence for clinically evaluating the combination of sorafenib and Dinaciclib to improve the therapeutic situation for patients with advanced-stage HCC.

Inhibition of cyclin-dependent kinase 5 activity alleviates diabetes-related cognitive deficits.

Cognitive deficit is a prevalent and underestimated complication of diabetes, and the underlying cellular and molecular mechanisms are not well understood. Aberrant activity of cyclin-dependent kinase (Cdk)5 is implicated in a number of neurodegenerative diseases. The present study examined the role of Cdk5 in the progression of diabetes-related cognitive deficits. We showed that the Cdk5 protein expression and kinase activity were significantly increased in diabetic mice at 16 wk. In primary cultured hippocampal neurons exposed to 30 mM glucose, Cdk5 protein and kinase activity were also elevated in a time-dependent manner. Moreover, the high glucose exposure led to an aberrant Cdk5 activation due to its activator p25 that was cleaved from p35 by calpain. Both in diabetic mice and in cultured hippocampal neurons exposed to high glucose, inhibition of Cdk5 activity with roscovitine (Ros) or short hairpin RNA (shRNA) decreased the protein levels of cleaved caspase-3 and the ratio of Bax and Bcl-2. The apoptotic rate detected by TUNEL in vivo or Annexin V and propidium iodide staining for flow cytometry in vitro also had obvious reduction. In addition, high glucose exposure resulted in the increase of phosphorylated (phospho)-MAPK kinase (MKK)6, phospho-p38, and c-Jun, which were rescued by Ros or Cdk5 shRNA. It is more important that the cognitive deficits of diabetic mice were also effectively alleviated by Ros. These results indicate that aberrant Cdk5 activity triggered hippocampal neuron apoptosis by activating MKK6/p38 MAPK cascade in hyperglycemia. Inhibition of Cdk5 overactivation attenuates neuronal apoptosis and cognitive deficits and contributes to the relief of diabetic neurotoxicity in the brain.-Liu, W., Zhou, Y., Liang, R., Zhang, Y. Inhibition of cyclin-dependent kinase 5 activity alleviates diabetes-related cognitive deficits.

Rapid Estrogen and Progesterone Signaling to Dendritic Spine Formation via Cortactin/Wave1-Arp2/3 Complex.

BACKGROUND: Synaptic plasticity is the neuronal capacity to modify the function and structure of dendritic spines (DS) in response to neuromodulators. Sex steroids, particularly 17ß-estradiol (E2) and progesterone (P4), are key regulators in the control of DS formation through multiprotein complexes including WAVE1 protein, and are thus fundamental for the development of learning and memory. OBJECTIVES: The aim of this work was to evaluate the molecular switch Cdk5 kinase/protein phosphatase 2A (PP2A) in the control of WAVE1 protein (phosphorylation/dephosphorylation) and the regulation of WAVE1 and cortactin to the Arp2/3 complex, in response to rapid treatments with E2 and P4 in cortical neuronal cells. RESULTS: Rapid treatment with E2 and P4 modified neuronal morphology and significantly increased the number of DS. This effect was reduced by the use of a Cdk5 inhibitor (Roscovitine). In contrast, inhibition of PP2A with PP2A dominant negative construct significantly increased DS formation, evidencing the participation of kinase/phosphatase in the regulation of WAVE1 in DS formation induced by E2 and P4. Cortactin regulates DS formation via Src and PAK1 kinase induced by E2 and P4. Both cortactin and WAVE1 signal to Arp2/3 complex to synergistically promote actin nucleation. CONCLUSION: These results suggest that E2 and P4 dynamically regulate neuron morphology through nongenomic signaling via cortactin/WAVE1-Arp2/3 complex. The control of these proteins is tightly orchestrated by phosphorylation, where kinases and phosphatases are essential for actin nucleation and, finally, DS formation. This work provides a deeper understanding of the biological actions of sex steroids in the regulation of DS turnover and neuronal plasticity processes.

Rapid, quantitative therapeutic screening for Alzheimer's enzymes enabled by optimal signal transduction with transistors.

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.

Phloretin and phloridzin improve insulin sensitivity and enhance glucose uptake by subverting PPARγ/Cdk5 interaction in differentiated adipocytes.

Activators of peroxisome proliferator-activated receptor-γ (PPARγ agonists) are therapeutically promising candidates against insulin resistance and hyperglycemia. Synthetic PPARγ agonists are known to effectively enhance insulin sensitivity, but these are also associated with adverse side-effects and rising cost of treatment. Therefore, natural PPARγ targeting ligands are desirable alternatives for the management of insulin resistance associated with type 2 diabetes. Phloretin (PT) and Phloridzin (PZ) are predominant apple phenolics, which are recognized for their various pharmacological functions. The present study assessed the potential of PT and PZ in enhancing insulin sensitivity and glucose uptake by inhibiting Cdk5 activation and corresponding PPARγ phosphorylation in differentiated 3T3L1 cells. In silico docking and subsequent validation using 3T3L1 cells revealed that PT and PZ not only block the ser273 site of PPARγ but also inhibit the activation of Cdk5 itself, thereby, indicating their potent PPARγ regulatory attributes. Corroborating this, application of PT and PZ significantly enhanced the accumulation of cellular triglycerides as well as expression of insulin-sensitizing genes in adipocytes ultimately resulting in improved glucose uptake. Taken together, the present study reports that PT and PZ inhibit Cdk5 activation, which could be directly influencing the apparent PPARγ inhibition at ser273, ultimately resulting in improved insulin sensitivity and glucose uptake.

A rapid and sensitive bioanalytical LC-MS/MS method for the quantitation of a novel CDK5 inhibitor 20-223 (CP668863) in plasma: Application to in vitro metabolism and plasma protein-binding studies.

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.

Development of CDK2 and CDK5 Dual Degrader TMX-2172.

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.

CDK5 Inhibitor Downregulates Mcl-1 and Sensitizes Pancreatic Cancer Cell Lines to Navitoclax.

Developing small molecules that indirectly regulate Mcl-1 function has attracted a lot of attention in recent years. Here, we report the discovery of an aminopyrazole, 2-([1,1'-biphenyl]-4-yl)-N-(5-cyclobutyl-1H-pyrazol-3-yl)acetamide (analog 24), which selectively inhibited cyclin-dependent kinase (CDK) 5 over CDK2 in cancer cell lines. We also show that analog 24 reduced Mcl-1 levels in a concentration-dependent manner in cancer cell lines. Using a panel of doxycycline inducible cell lines, we show that CDK5 inhibitor 24 selectively modulates Mcl-1 function while the CDK4/6 inhibitor 6-acetyl-8-cyclopentyl-5-methyl-2-(5-(piperazin-1-yl)pyridin-2-ylamino)pyrido[2,3-day]pyrimidin-7(8H)-one does not. Previous studies using RNA interference and CRISPR showed that concurrent elimination of Bcl-xL and Mcl-1 resulted in induction of apoptosis. In pancreatic cancer cell lines, we show that either CDK5 knockdown or expression of a dominant negative CDK5 results in synergistic induction of apoptosis. Moreover, concurrent pharmacological perturbation of Mcl-1 and Bcl-xL in pancreatic cancer cell lines using a CDK5 inhibitor analog 24 that reduced Mcl-1 levels and 4-(4-{[2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohexen-1-yl]methyl}-1-piperazinyl)-N-[(4-{[(2R)-4-(4-morpholinyl)-1-(phenylsulfanyl)-2-butanyl]amino}-3-[(trifluoromethyl)sulfonyl]phenyl)sulfonyl] benzamide (navitoclax), a Bcl-2/Bcl-xL/Bcl-w inhibitor, resulted in synergistic inhibition of cell growth and induction of apoptosis. In conclusion, we demonstrate targeting CDK5 will sensitize pancreatic cancers to Bcl-2 protein inhibitors. SIGNIFICANCE STATEMENT: Mcl-1 is stabilized by CDK5-mediated phosphorylation in pancreatic ductal adenocarcinoma, resulting in the deregulation of the apoptotic pathway. Thus, genetic or pharmacological targeting of CDK5 sensitizes pancreatic cancers to Bcl-2 inhibitors, such as navitoclax.

Crosstalk between Cdk5/p35 and ERK1/2 signalling mediates spinal astrocyte activity via the PPARγ pathway in a rat model of chronic constriction injury.

The specific mechanisms underlying cyclin-dependent kinase 5 (Cdk5)-mediated neuropathic pain at the spinal cord level remain elusive. The aim of the present study was to explore the role of crosstalk between Cdk5/p35 and extracellular signal-regulated kinase 1/2 (ERK1/2) signalling in mediating spinal astrocyte activity via the PPARγ pathway in a rat model of chronic constriction injury (CCI). Here, we quantified pain behaviour after CCI; detected the localization of p35, Cdk5, phosphorylated ERK1/2 (pERK1/2), phosphorylated peroxisome proliferator-activated receptor γ (pPPARγ), neuronal nuclei (a neuronal marker), glial fibrillary acidic protein (GFAP, an activated astrocyte marker) and ionized calcium binding adaptor molecule 1 (a microglial marker) in the dorsal horn using immunofluorescence; measured the protein levels of Cdk5, p35, pERK1/2, pPPARγ and GFAP using western blot analysis; and gauged the enzyme activity of Cdk5/p35 kinase using a Cdk5/p35 kinase activity assay kit. Tumour necrosis factor-α, interleukin (IL)-1ß and IL-6 levels were measured using enzyme-linked immunosorbent assay (ELISA). Ligation of the right sciatic nerve induced mechanical allodynia; thermal hyperalgesia; and the time-dependent upregulation of p35, pERK1/2 and GFAP and downregulation of pPPARγ. p35 colocalized with Cdk5, pERK1/2, pPPARγ, neurons and astrocytes but not microglia. Meanwhile, intrathecal injection of the Cdk5 inhibitor roscovitine, the mitogen-activated ERK kinase (MEK) inhibitor U0126 and the PPARγ agonist pioglitazone prevented or reversed behavioural allodynia, increased pPPARγ expression, inhibited astrocyte activation and alleviated proinflammatory cytokine (tumour necrosis factor-α, IL-1ß, and IL-6) release from activated astrocytes. Furthermore, crosstalk between the Cdk5/p35 and ERK1/2 pathways was observed with CCI. Blockade of either Cdk5/p35 or ERK1/2 inhibited Cdk5 activity. These findings indicate that spinal crosstalk between the Cdk5/p35 and ERK1/2 pathways mediates astrocyte activity via the PPARγ pathway in CCI rats and that targeting this crosstalk could be an effective strategy to attenuate CCI and astrocyte-derived neuroinflammation.

The HU177 Collagen Epitope Controls Melanoma Cell Migration and Experimental Metastasis by a CDK5/YAP-Dependent Mechanism.

Stromal components not only help form the structure of neoplasms such as melanomas, but they also functionally contribute to their malignant phenotype. Thus, uncovering signaling pathways that integrate the behavior of both tumor and stromal cells may provide unique opportunities for the development of more effective strategies to control tumor progression. In this regard, extracellular matrix-mediated signaling plays a role in coordinating the behavior of both tumor and stromal cells. Here, evidence is provided that targeting a cryptic region of the extracellular matrix protein collagen (HU177 epitope) inhibits melanoma tumor growth and metastasis and reduces angiogenesis and the accumulation of α-SMA-expressing stromal cell in these tumors. The current study suggests that the ability of the HU177 epitope to control melanoma cell migration and metastasis depends on the transcriptional coactivator Yes-associated protein (YAP). Melanoma cell interactions with the HU177 epitope promoted nuclear accumulation of YAP by a cyclin-dependent kinase-5-associated mechanism. These findings provide new insights into the mechanism by which the anti-HU177 antibody inhibits metastasis, and uncovers an unknown signaling pathway by which the HU177 epitope selectively reprograms melanoma cells by regulating nuclear localization of YAP. This study helps to define a potential new therapeutic strategy to control melanoma tumor growth and metastasis that might be used alone or in combination with other therapeutics.

Systemic Administration of the Cyclin-Dependent Kinase Inhibitor (S)-CR8 Selectively Reduces Escalated Ethanol Intake in Dependent Rats.

BACKGROUND: Chronic exposure to ethanol (EtOH) and other drugs of abuse can alter the expression and activity of cyclin-dependent kinase 5 (CDK5) and its cofactor p35, but the functional implication of CDK5 signaling in the regulation of EtOH-related behaviors remains unknown. In the present study, we sought to determine whether CDK5 activity plays a role in the escalation of EtOH self-administration triggered by dependence. METHODS: We tested the effect of systemically administered (S)-CR8, a nonselective CDK inhibitor, on operant responding for EtOH or saccharin, a highly palatable reinforcer, in adult male Wistar rats. Half of the rats were made EtOH-dependent via chronic intermittent EtOH inhalation (CIE). We then sought to identify a possible neuroanatomical locus for the behavioral effect of (S)-CR8 by quantifying protein levels of CDK5 and p35 in subregions of the extended amygdala and prefrontal cortex from EtOH-naïve, nondependent, and dependent rats at the expected time of EtOH self-administration. We also analyzed the phosphorylation of 4 CDK5 substrates and of the CDK substrate consensus motif. RESULTS: (S)-CR8 dose-dependently reduced EtOH self-administration in dependent rats. It had no effect on water or saccharin self-administration, nor in nondependent rats. The abundance of CDK5 or p35 was not altered in any of the brain regions analyzed. In the bed nucleus of the stria terminalis, CDK5 abundance was negatively correlated with intoxication levels during EtOH vapor exposure but there was no effect of dependence on the phosphorylation ratio of CDK5 substrates. In contrast, EtOH dependence increased the phosphorylation of low-molecular-weight CDK substrates in the basolateral amygdala (BLA). CONCLUSIONS: The selective effect of (S)-CR8 on excessive EtOH intake has potential therapeutic value for the treatment of alcohol use disorders. Our data do not support the hypothesis that this effect would be mediated by the inhibition of up-regulated CDK5 activity in the extended amygdala nor prefrontal cortex. However, increased activity of CDKs other than CDK5 in the BLA may contribute to excessive EtOH consumption in alcohol dependence. Other (S)-CR8 targets may also be implicated.

Kinase inhibitions in pyrido[4,3-h] and [3,4-g]quinazolines: Synthesis, SAR and molecular modeling studies.

New pyrido[3,4-g]quinazoline derivatives were prepared and evaluated for their inhibitory potency toward 5 protein kinases (CLK1, DYRK1A, GSK3, CDK5, CK1). A related pyrido[4,3-h]quinazoline scaffold with an angular structure was also synthesized and its potency against the same protein kinase panel was compared to the analogous pyrido[3,4-g]quinazoline. Best results were obtained for 10-nitropyrido[3,4-g]quinazoline 4 toward CLK1 with nanomolar activities.