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Cerebral edema is one of the deadliest complications of ischemic stroke for which there is currently no pharmaceutical treatment. Aquaporin-4 (AQP4), a water-channel polarized at the astrocyte endfoot, is known to be highly implicated in cerebral edema. We previously showed in randomized studies that (S)-roscovitine, a cyclin-dependent kinase inhibitor, reduced cerebral edema 48 h after induction of focal transient ischemia, but its mechanisms of action were unclear. In our recent blind randomized study, we confirmed that (S)-roscovitine was able to reduce cerebral edema by 65% at 24 h post-stroke (t test, p = .006). Immunofluorescence analysis of AQP4 distribution in astrocytes revealed that (S)-roscovitine decreased the non-perivascular pool of AQP4 by 53% and drastically increased AQP4 clusters in astrocyte perivascular end-feet (671%, t test p = .005) compared to vehicle. Non-perivascular and clustered AQP4 compartments were negatively correlated (R = -0.78; p < .0001), suggesting a communicating vessels effect between the two compartments. α1-syntrophin, AQP4 anchoring protein, was colocalized with AQP4 in astrocyte endfeet, and this colocalization was maintained in ischemic area as observed on confocal microscopy. Moreover, (S)-roscovitine increased AQP4/α1-syntrophin interaction (40%, MW p = .0083) as quantified by proximity ligation assay. The quantified interaction was negatively correlated with brain edema in both treated and placebo groups (R = -.57; p = .0074). We showed for the first time, that a kinase inhibitor modulated AQP4/α1-syntrophin interaction, and was implicated in the reduction of cerebral edema. These findings suggest that (S)-roscovitine may hold promise as a potential treatment for cerebral edema in ischemic stroke and as modulator of AQP4 function in other neurological diseases., (© 2023 The Authors. GLIA published by Wiley Periodicals LLC.)

Epithelial-mesenchymal transformation (EMT) plays an important role in the progression of diabetic nephropathy. Dexmedetomidine (DEX) has shown renoprotective effects against ischemic reperfusion injury; however, whether and how DEX prevents high glucose-induced EMT in renal tubular epithelial cells is incompletely known. Here, we conduct in vitro experiments using HK-2 cells, a human tubular epithelial cell line. Our results demonstrate that high glucose increases the expressions of EMT-related proteins, including Vimentin, Slug, Snail and Twist, while decreasing the expression of E-cadherin and increasing Cdk5 expression in HK-2 cells. Both Cdk5 knockdown and inhibition by roscovitine increase the expressions of E-cadherin while decreasing the expressions of other EMT-related markers. DEX inhibits Cdk5 expression without affecting cell viability and changes the expressions of EMT-related markers, similar to effects of Cdk5 inhibition. Furthermore, Cdk5 is found to interact with Drp1 at the protein level and mediate the phosphorylation of Drp1. In addition, Drp1 inhibition with mdivi-1 could also restrain the high glucose-induced EMT process in HK-2 cells. Immunofluorescence results show that roscovitine, Mdivi-1 and DEX inhibit high glucose-induced intracellular ROS accumulation, while the oxidant H 2 O 2 eliminates the protective effect of DEX on the EMT process. These results indicate that DEX mitigates high glucose-induced EMT progression in HK-2 cells via inhibition of the Cdk5/Drp1/ROS pathway.

Synchronization of donor cells is an important step for the success of somatic cell nuclear transfer application and facilitates the development of embryos. Contact inhibition, serum starvation and different chemical agents are used in synchronizing different types of somatic cells. In this study, to synchronize the primary ovine adult (POF) and foetal (POFF) fibroblast cells to G0/G1 phases, the contact inhibition, the serum starvation, roscovitine and trichostatin A (TSA) methods were used. In the first part of the study, roscovitine (10, 15, 20 and 30 μM) and TSA (25, 50, 75 and 100 nM) were applied for 24 h to determine the optimal concentration for POF and POFF cells. In the second part, optimal concentrations of roscovitine and TSA for these cells were compared with contact inhibition and serum starvation methods. Cell cycle distribution and apoptotic activity analysis were performed by flow cytometry to compare this synchronization methods. Serum starvation method resulted in higher cell synchronization rate in both cells compared to other groups. Although contact inhibition and TSA also achieved high success rates of synchronized cell value, it was observed that the difference between serum starvation and these groups was significant (p < .05). When the apoptosis rates of the two cell types were examined, it was observed that the early apoptotic cells in contact inhibition and late apoptotic cells in the serum starvation were higher than the other groups (p < .05). Although the 10 and 15 μM concentrations of roscovitine gave the lowest apoptosis rates, it was observed that it failed to synchronize both the ovine fibroblast cells to G0/G1 phase. As a result, it was concluded that while roscovitine was not successful to synchronize both the POFF and POF cell lines, TSA (50 nM for POF cells and 100 nM for POFF cells) can be used efficiently as an alternative to the contact inhibition and the serum starvation methods., (© 2023 The Authors. Reproduction in Domestic Animals published by Wiley-VCH GmbH.)

The atypical cyclin-dependent kinase 5 (CDK5) is considered a neuron-specific kinase that plays important roles in many cellular functions including neuronal migration, neuronal differentiation, synapse development, and synaptic functions. However, the role of CDK5 in microglia under physiological and pathological conditions remains unclear. This study showed that treatment with lipopolysaccharide (LPS) caused the release of pro-inflammatory mediators and increased expression of CDK5 in BV2 microglia in vitro. Moreover, lipopolysaccharide treatment-induced glycolysis by increasing the expression levels of HIF-1α, PFKFB3, and HK2. Application of CDK5 inhibitor roscovitine significantly decreased LPS-induced CDK5 expression and glycolysis, thus suppressing neuroinflammation in the cells. The roscovitine treatment of BV2 cells also significantly blocked the HIF-1 activator, CoCl 2 -mediated HIF-1α, HK2, and PFKFB3 expression. Finally, we demonstrated that roscovitine inhibited microglial activation, metabolic reprogramming, expression of pro-inflammatory markers, cell apoptosis, and alleviated memory impairment in LPS-injected mice. In summary, our results suggest that inhibition of CDK5 can reduce the neuroinflammation of microglia through modulation of metabolic reprogramming., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The accumulation of &beta;‑amyloid peptides (A&beta;) in the brain is a hallmark of Alzheimer's disease (AD). Studies have indicated that ginsenoside Rg1, a primary component of ginseng (Panax&nbsp;ginseng), reduces brain A&beta; levels in an AD model through peroxisome proliferator‑activated receptor &gamma; (PPAR&gamma;), thereby regulating the expression of insulin‑degrading enzyme (Ide) and &beta;‑amyloid cleavage enzyme 1 (Bace1), which are PPAR&gamma; target genes. However, the effects of ginsenoside Rg1 on PPAR&gamma; remain unclear. Since cyclin‑dependent kinase 5 (CDK5) mediates PPAR&gamma; phosphorylation in adipose tissue, this study aimed to investigate whether ginsenoside Rg1 regulates PPAR&gamma; target genes and reduces A&beta; levels by inhibiting PPAR&gamma; phosphorylation through the CDK5 pathway. In the present study, a model of AD was established by treating primary cultured rat hippocampal neurons with A&beta;1‑42. The cells were pretreatment with ginsenoside Rg1 and roscovitine, a CDK5‑inhibitor, prior to the treatment with A&beta;1‑42. Neuronal apoptosis was detected using TUNEL staining. PPAR&gamma; phosphorylation and protein expression levels of PPAR&gamma;, CDK5, IDE, BACE1, amyloid precursor protein (APP) and A&beta;1‑42 were measured by western blotting. The mRNA expression levels of PPAR&gamma;, CDK5, IDE, BACE1 and APP were assessed using reverse transcription‑quantitative PCR. The results of the present study demonstrated that in an AD model induced by A&beta;1‑42, ginsenoside Rg1 significantly decreased CDK5 expression, inhibited PPAR&gamma; phosphorylation at serine 273, elevated IDE expression, downregulated BACE1 and APP expression, decreased A&beta;1‑42 levels and attenuated neuronal apoptosis. The CDK5 inhibitor, roscovitine, demonstrated similar effects. These results suggest that ginsenoside Rg1 has neuroprotective properties and has potential for use in the treatment of AD.

CVT-313 is a potent CDK2 inhibitor that was identified by screening a purine-analogue library and is currently in preclinical studies. Since this molecule has the potential to be developed as a CDK2 inhibitor for cancer therapy, the potency of CVT-313 to bind and stabilize CDK2 was evaluated, together with its ability to inhibit aberrant cell proliferation. CVT-313 increased the melting temperature of CDK2 by 7°C in thermal stabilization studies, thus indicating its protein-stabilizing effect. CVT-313 inhibited the growth of human lung carcinoma cell line A549 in a dose-dependent manner, with an IC 50 of 1.2 µM, which is in line with the reported biochemical potency of 0.5 µM. To support the further chemical modification of CVT-313 and to improve its biochemical and cellular potency, a crystal structure was elucidated in order to understand the molecular interaction of CVT-313 and CDK2. The crystal structure of CDK2 bound to CVT-313 was determined to a resolution of 1.74 Å and clearly demonstrated that CVT-313 binds in the ATP-binding pocket, interacting with Leu83, Asp86 and Asp145 directly, and the binding was further stabilized by a water-mediated interaction with Asn132. Based on the crystal structure, further modifications of CVT-313 are proposed to provide additional interactions with CDK2 in the active site, which may significantly increase the biochemical and cellular potency of CVT-313.

While kinases have been attractive targets to combat many diseases, including cancer, selective kinase inhibition has been challenging, because of the high degree of structural homology in the active site, where many kinase inhibitors bind. We have previously discovered that 8-anilino-1-naphthalene sulfonic acid (ANS) binds an allosteric pocket in cyclin-dependent kinase 2 (Cdk2). Here, we detail the positive cooperativity between ANS and orthosteric Cdk2 inhibitors dinaciclib and roscovitine, which increase the affinity of ANS toward Cdk2 5-fold to 10-fold, and the relatively noncooperative effects of ATP. We observe these effects using a fluorescent binding assay and heteronuclear single quantum correlation nuclear magnetic resonance (HSQC NMR), where we noticed a shift from fast exchange to slow exchange upon ANS titration in the presence of roscovitine but not with an ATP mimic. The discovery of cooperative relationships between orthosteric and allosteric kinase inhibitors could further the development of selective kinase inhibitors in general.

Changes in brain energy metabolism in diabetes mellitus, including increased insulin resistance and mitochondrial dysfunction, are critically involved in diabetes-related neurodegeneration, and associate with early cognitive impairment as well. The aim of this study is to detect the specific phosphorylated-Thr485- AMP-activated protein kinase (AMPK-α2), regulated by cyclin-dependent kinase 5 (Cdk5) paly the inhibitory functional role of AMPK-α2, Which is maybe the link to the accelerated diabetic brain damage progression. Here, we used GK rats, the type 2 diabetic animal model for in vivo studies and performed In vitro kinase assay, high glucose treatment, -phosphorylated mutation and protein expression in both HEK-293T and HT-22 cell lines. In vitro, the results show that murine wild-type AMPK-α2 was phosphorylated by Cdk5 at a (S/T)PX(K/H/R) phosphorylation consensus sequence, which was associated with decreased AMPK-α2 activity. Surprisingly, mutation of Thr485 to alanine in AMPK-α2 results in the abolished Cdk5 effects, demonstrating that Thr485-phosphorylation is critical to AMPK-α2 inhibition by Cdk5. In addition, these alterations in AMPK-α2-phosphorylation and -activity induced by Cdk5 is specific at Thr485. Furthermore, in GK rats, the increased phosphorylated- Thr 485 of AMPK-α2 results in the decreased AMPK-α2 activity, which is correlated with the apoptosis of neurons in hippocamps. After high glucose treatment, the decreased survival showed in AMPK-α2 T485A HT-22 cells compared to AMPK-α2 WT . The down-regulated of p-CREB, SNAP25, synaptophysin as well as synapsin-1were shown in both GK rats and HT-22 cell line. Meanwhile, pre-treated with either the specific Cdk5-inhibitor (roscovitine) or the antidiabetic AMPK-α2 - inhibitor (metformin) could restore the alterations in neuronal protein expression. Our results suggest that Cdk5-mediated phosphorylated- Thr485 in AMPK-α2 may be involved in the pathogenesis of diabetic brain damage., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

KHC-4 is a 2-phenyl-4-quinolone analogue that exhibits anticancer activity. Aberrant activation of β-catenin signaling contributes to prostate cancer development and progression. Therefore, targeting β-catenin expression could be a useful approach to treating prostate cancer. We found that KHC-4 can inhibit β-catenin expression and its signaling pathway in DU145 prostate cancer cells. Treatment with KHC-4 decreased total β-catenin expression and concomitantly decreased β-catenin levels in both the cytoplasm and nucleus of cells. KHC-4 treatment also inhibited β-catenin expression and that of its target proteins, PI3K, AKT, GSK3β and TBX3. We monitored the stability of β-catenin with the proteasomal inhibitor, MG132, in DU145 cells and found that MG132 reversed KHC-4-induced proteasomal β-catenin degradation. We verified CDK1/β-catenin expression in KHC-4 treated DU145 cells. We found that roscovitine treatment reversed cell proliferation by arresting the cell cycle at the G2/M phase and β-catenin expression caused by KHC-4 treatment. We suggest that KHC-4 inhibits β-catenin signaling in DU145 prostate cancer cells.

Following the discovery of (R)-roscovitine's beneficial effects in three polycystic kidney disease (PKD) mouse models, cyclin-dependent kinases (CDKs) inhibitors have been investigated as potential treatments. We have used various affinity chromatography approaches to identify the molecular targets of roscovitine and its more potent analog (S)-CR8 in human and murine polycystic kidneys. These methods revealed casein kinases 1 (CK1) as additional targets of the two drugs. CK1ε expression at the mRNA and protein levels is enhanced in polycystic kidneys of 11 different PKD mouse models as well as in human polycystic kidneys. A shift in the pattern of CK1α isoforms is observed in all PKD mouse models. Furthermore, the catalytic activities of both CK1ε and CK1α are increased in mouse polycystic kidneys. Inhibition of CK1ε and CK1α may thus contribute to the long-lasting attenuating effects of roscovitine and (S)-CR8 on cyst development. CDKs and CK1s may constitute a dual therapeutic target to develop kinase inhibitory PKD drug candidates.