Member Of Cyclin-dependent Kinase Family Research Articles

Biolayer Interferometry Assay for Cyclin-Dependent Kinase-Cyclin Association Reveals Diverse Effects of Cdk2 Inhibitors on Cyclin Binding Kinetics.

Cyclin-dependent kinases (CDKs) are key mediators of cell proliferation and have been a subject of oncology drug discovery efforts for over two decades. Several CDK and activator cyclin family members have been implicated in regulating the cell division cycle. While it is thought that there are canonical CDK-cyclin pairing preferences, the extent of selectivity is unclear, and increasing evidence suggests that the cell-cycle CDKs can be activated by a pool of available cyclins. The molecular details of CDK-cyclin specificity are not completely understood despite their importance for understanding cancer cell cycles and for pharmacological inhibition of cancer proliferation. We report here a biolayer interferometry assay that allows for facile quantification of CDK binding interactions with their cyclin activators. We applied this assay to measure the impact of Cdk2 inhibitors on Cyclin A (CycA) association and dissociation kinetics. We found that Type I inhibitors increase the affinity between Cdk2 and CycA by virtue of a slowed cyclin dissociation rate. In contrast, Type II inhibitors and other small-molecule Cdk2 binders have distinct effects on the CycA association and dissociation processes to decrease affinity. We propose that the differential impact of small molecules on the cyclin binding kinetics arises from the plasticity of the Cdk2 active site as the kinase transitions between active, intermediate, and inactive states.

Genome-wide identification, evolutionary and expression analysis of the cyclin-dependent kinase gene family in peanut

BackgroundCyclin-dependent kinases (CDKs) are a predominant group of serine/threonine protein kinases that have multi-faceted functions in eukaryotes. The plant CDK members have well-known roles in cell cycle progression, transcriptional regulation, DNA repair, abiotic stress and defense responses, making them promising targets for developing stress adaptable high-yielding crops. There is relatively sparse information available on the CDK family genes of cultivated oilseed crop peanut and its diploid progenitors.ResultsWe have identified 52 putative cyclin-dependent kinases (CDKs) and CDK-like (CDKLs) genes in Arachis hypogaea (cultivated peanut) and total 26 genes in each diploid parent of cultivated peanut (Arachis duranensis and Arachis ipaensis). Both CDK and CDKL genes were classified into eight groups based on their cyclin binding motifs and their phylogenetic relationship with Arabidopsis counterparts. Genes in the same subgroup displayed similar exon–intron structure and conserved motifs. Further, gene duplication analysis suggested that segmental duplication events played major roles in the expansion and evolution of CDK and CDKL genes in cultivated peanuts. Identification of diverse cis-acting response elements in CDK and CDKL genes promoter indicated their potential fundamental roles in multiple biological processes. Various gene expression patterns of CDKs and CDKLs in different peanut tissues suggested their involvement during growth and development. In addition, qRT-PCR analysis demonstrated that most representing CDK and CDKL gene family members were significantly down-regulated under ABA, PEG and mannitol treatments.ConclusionsGenome-wide analysis offers a comprehensive understanding of the classification, evolution, gene structure, and gene expression profiles of CDK and CDKL genes in cultivated peanut and their diploid progenitors. Additionally, it also provides cell cycle regulatory gene resources for further functional characterization to enhance growth, development and abiotic stress tolerance.

Study on the interaction preference between CYCD subclass and CDK family members at the poplar genome level

Cyclin-dependent kinases (CDKs) control the progression of the cell cycle. D-type cyclin (CYCD) is generally believed to form a complex with CDK and control the G1/S transition. In plants, CYCD and CDK gene families can be divided into 6 (D1–D7) and 7 (CDKA–CDKG) subclasses, respectively. Different subclasses in the CYCD and CDK families have different numbers, structures and functions. In some heterologous woody plants, the functions of these subclass family members remain unclear. In this study, 43 CYCD and 27 CDK gene family members were identified in the allodiploid Populus tomentosa Carr. Phylogenetic analysis suggested that these CYCDs and CDKs were divided into 6 and 7 subclasses, respectively, which were the same as other species. The analysis of protein properties, gene structure, motifs, domains, cis-acting elements and tissue-specific expression of all members of these CYCDs and CDKs showed that the differences between members of different subclasses varied widely, but members of the same subclass especially in the CDK gene family were very similar. These findings also demonstrated a strong correlation between CYCD and CDK gene family members in response to hormones and specific expression. The collinear analysis of P. tomentosa, Populus trichocarpa and Arabidopsis thaliana showed that the expansion patterns of CYCD and CDK gene families were predominantly whole genome duplications (WGD). The protein interaction prediction results of different subclasses of CYCD and CDKs showed that the interaction between different subclasses of CYCD and CDKs was significantly different. Our previous study found that transgenic PtoCYCD2;1 and PtoCYCD3;3 poplars exhibited opposite phenotypes. Y2H and BIFC results showed that the interaction between PtoCYCD2;1 and PtoCYCD3;3 was significantly different with CDKs. This finding might suggest that the functional differences of different CYCD subclasses in plant growth and development were closely related to the different interactions between CYCD and CDK. Our results provide a good idea and direction for the functional study of CYCD and CDK proteins in woody plants.

Abstract 2306: BLU-222, an investigational, potent, and selective CDK2 inhibitor, demonstrated robust antitumor activity in CCNE1-amplified ovarian cancer models

Abstract Background: Cyclin-dependent kinases (CDK) are a class of enzymes that, along with their regulatory cyclin binding partners, drive cell cycle progression. Cell cycle dysregulation is a hallmark of cancer and targeting its genetic drivers can confer therapeutic benefits. Cyclin E1 (CCNE1) gene alterations are common in patients with ovarian cancer and can be found in ~20% of high-grade serous ovarian cancer cases, which tend to be platinum therapy resistant and therefore represent a high medical need. Cyclin E1 is the canonical binding partner of CDK2, which becomes constitutively active when CCNE1 is amplified and overexpressed. Selectively inhibiting CDK2 is an attractive therapeutic option for CCNE1-amplified tumors and may limit off-target CDK-driven toxicities. Here we report preclinical validation studies leading to the development of an orally available CDK2 inhibitor, BLU-222, for the treatment of ovarian cancer harboring a CCNE1 amplification. Methods: BLU-222 selectivity was measured by enzyme assays, cellular target engagement assays (NanoBRET), and proliferation assays in a panel of ovarian cancer cell lines. In vitro cellular potency was assessed by phospho-Rb levels. In vivo antitumor activity of BLU-222 as a single agent or in combination with carboplatin was measured in an OVCAR-3 cell line-derived xenograft (CDX) tumor model harboring a CCNE1 amplification. Results: BLU-222 demonstrated selectivity, both biochemically and in cells, with low nanomolar potency for CDK2 vs other CDK family members (CDK1, -4, -6, -7, and -9). In a panel of ovarian cancer cell lines, those with CCNE1 amplifications were highly sensitive to BLU-222. Consistent with the in vitro profiling, BLU-222 exhibited significant antitumor activity in the OVCAR-3 CDX model. While administration of single-agent carboplatin led to stasis in vivo, the combination of BLU-222 + carboplatin induced durable tumor regression even after treatment cessation. Conclusions: These data provide a strong rationale for advancing BLU-222 towards clinical development in patients with CCNE1-amplified ovarian cancer. Citation Format: Victoria Brown, Phil Ramsden, Nealia House, Richard Vargas, Jian Guo, Ruduan Wang, Riadh Lobbardi, Maxine Chen, Douglas Wilson, Joseph Kim, Neil Bifulco, Michelle Maynard, Emanuele Perola, Dean Zhang, Steve Wenglowsky, Yoon Jong Choi. BLU-222, an investigational, potent, and selective CDK2 inhibitor, demonstrated robust antitumor activity in CCNE1-amplified ovarian cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2306.

CDK Family PROTAC Profiling Reveals Distinct Kinetic Responses and Cell Cycle-Dependent Degradation of CDK2.

Targeted protein degradation using heterobifunctional proteolysis-targeting chimera (PROTAC) compounds, which recruit E3 ligase machinery to a target protein, is increasingly becoming an attractive pharmacologic strategy. PROTAC compounds are often developed from existing inhibitors, and assessing selectivity is critical for understanding on-target and off-target degradation. We present here an in-depth kinetic degradation study of the pan-kinase PROTAC, TL12-186, applied to 16 members of the cyclin-dependent kinase (CDK) family. Each CDK family member was endogenously tagged with the 11-amino-acid HiBiT peptide, allowing for live cell luminescent monitoring of degradation. Using this approach, we found striking differences and patterns in kinetic degradation rates, potencies, and Dmax values across the CDK family members. Analysis of the responses revealed that most of the CDKs showed rapid and near complete degradation, yet all cell cycle-associated CDKs (1, 2, 4, and 6) showed multimodal and partial degradation. Further mechanistic investigation of the key cell cycle protein CDK2 was performed and revealed CDK2 PROTAC-dependent degradation in unsynchronized or G1-arrested cells but minimal loss in S or G2/M arrest. The ability of CDK2 to form the PROTAC-mediated ternary complex with CRBN in only G1-arrested cells matched these trends, despite binding of CDK2 to TL12-186 in all phases. These data indicate that target subpopulation degradation can occur, dictated by the formation of the ternary complex. These studies additionally underscore the importance of profiling degradation compounds in cellular systems where complete pathways are intact and target proteins can be characterized in their relevant complexes.

CDK9 attenuation exerts protective effects on catabolism and hypertrophy in chondrocytes and ameliorates osteoarthritis development

Osteoarthritis (OA) is generally considered to be characterized by progressive articular cartilage destruction. Increasing evidence demonstrates that CDK9, which is a member of cyclin-dependent kinase family, plays a significant role in the regulation of acute and chronic inflammatory diseases. IL-1β, a major proinflammatory cytokine, was used to establish a model of OA in vitro after stimulating chondrocytes. We found that CDK9 was highly expressed in in vitro and in vivo models of inflammation. The role of LDC000067 (abbreviated as LDC067), a specific inhibitor of CDK9, in protecting articular cartilage from immune response has not been fully clarified. Intriguingly, in this study, we demonstrated that LDC067 prevented IL-1β-induced production of metalloproteinases (MMPs) and inflammatory cytokines, including MMP3, MMP9, MMP13, IL-6, IL-8 and TNF-ɑ. Furthermore, we revealed that LDC067 inhibited IL-1β-induced NF-κB signaling pathway activation in chondrocytes. The inhibition of CDK9 could also delay cartilage degeneration in an anterior cruciate ligament transection (ACLT) mouse model in vivo. Taken together, these results highlighted the significance of this CDK9 inhibitor in preventing cartilage destruction and indicated that LDC067 might serve as a potential therapeutic agent for OA.

Abstract 4426: Selective inhibition of CDK9 induces apoptosis of TNBC cells independent of Rb status

Abstract The family of cyclin-dependent kinases (CDKs) are serine/threonine protein kinases important for cell cycle control and/or regulation of transcription. CDK9/CyclinT1 is a key regulator of transcription in eukaryotic cells and has been shown to be dysregulated at the level of protein and kinase activity in both hematologic and solid tumors. CDK9/CyclinT1 forms the active P-TEFb complex and phosphorylates Ser2 residues in the carboxy-terminal domain of RNA polymerase II (RNApol II) to initiate elongation of mRNA transcripts. CDK9 activity regulates transcription of a variety of short-lived transcripts that promote survival and directly suppress apoptosis in cancer cells, including MYC,XRN2, MCL-1,and XIAP. MYC-driven tumor types with Rb-loss or high expression levels of cyclin E, such as triple negative breast cancer (TNBC), are difficult to treat and are resistant to existing CDK4/6 inhibitors. Since CDK9 is upstream of these oncogenic drivers, inhibition of CDK9 could potentially bypass innate resistance mechanisms and induce cell death in TNBC through blocking expression of MYC and MCL-1, for example. To target CDK9 in TNBC, we used structure-based drug design and developed a library of novel, potent and selective CDK9 inhibitors (CDK9i). The lead CDK9i exhibit single digit nanomolar potency against CDK9/CyclinT1 complexes and good selectivity against other CDK family members in biochemical assays. In addition, they also display high selectivity over 502 other kinases. Lead CDK9 inhibitors were found to potently inhibit phosphorylation of RNApol II Ser2 in a time and dose dependent manner in TNBC cells and decrease MYC, MCL-1 and XIAP at both mRNA and protein levels. Additionally, treatment of TNBC cells led to a potent G2/M cell cycle arrest, inhibition of cell proliferation and induction of apoptosis within 24 hours, regardless of Rb status of the tumor cells. Primary human bone marrow and normal human fibroblast cells, however, did not undergo apoptosis within 72 hours of treatment with CDK9i, suggesting a potential therapeutic window for CDK9i treatment. Further studies are ongoing to assess the effect of dose and scheduling on tumor efficacy in mouse TNBC tumor models. Selective inhibition of CDK9 presents a novel treatment strategy for difficult-to-treat tumor types, including those resistant to existing targeted therapeutics. Citation Format: Hailey E. Brighton, Claire R. Hall, Kerry A. Dillon, John E. Bisi, Jay C. Strum. Selective inhibition of CDK9 induces apoptosis of TNBC cells independent of Rb status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4426.

Ensemble-based modeling and rigidity decomposition of allosteric interaction networks and communication pathways in cyclin-dependent kinases: Differentiating kinase clients of the Hsp90-Cdc37 chaperone.

The overarching goal of delineating molecular principles underlying differentiation of protein kinase clients and chaperone-based modulation of kinase activity is fundamental to understanding activity of many oncogenic kinases that require chaperoning of Hsp70 and Hsp90 systems to attain a functionally competent active form. Despite structural similarities and common activation mechanisms shared by cyclin-dependent kinase (CDK) proteins, members of this family can exhibit vastly different chaperone preferences. The molecular determinants underlying chaperone dependencies of protein kinases are not fully understood as structurally similar kinases may often elicit distinct regulatory responses to the chaperone. The regulatory divergences observed for members of CDK family are of particular interest as functional diversification among these kinases may be related to variations in chaperone dependencies and can be exploited in drug discovery of personalized therapeutic agents. In this work, we report the results of a computational investigation of several members of CDK family (CDK5, CDK6, CDK9) that represented a broad repertoire of chaperone dependencies—from nonclient CDK5, to weak client CDK6, and strong client CDK9. By using molecular simulations of multiple crystal structures we characterized conformational ensembles and collective dynamics of CDK proteins. We found that the elevated dynamics of CDK9 can trigger imbalances in cooperative collective motions and reduce stability of the active fold, thus creating a cascade of favorable conditions for chaperone intervention. The ensemble-based modeling of residue interaction networks and community analysis determined how differences in modularity of allosteric networks and topography of communication pathways can be linked with the client status of CDK proteins. This analysis unveiled depleted modularity of the allosteric network in CDK9 that alters distribution of communication pathways and leads to impaired signaling in the client kinase. According to our results, these network features may uniquely define chaperone dependencies of CDK clients. The perturbation response scanning and rigidity decomposition approaches identified regulatory hotspots that mediate differences in stability and cooperativity of allosteric interaction networks in the CDK structures. By combining these synergistic approaches, our study revealed dynamic and network signatures that can differentiate kinase clients and rationalize subtle divergences in the activation mechanisms of CDK family members. The therapeutic implications of these results are illustrated by identifying structural hotspots of pathogenic mutations that preferentially target regions of the increased flexibility to enable modulation of activation changes. Our study offers a network-based perspective on dynamic kinase mechanisms and drug design by unravelling relationships between protein kinase dynamics, allosteric communications and chaperone dependencies.

Tale of the Good and the Bad Cdk5: Remodeling of the Actin Cytoskeleton in the Brain.

Cdk5 kinase, a cyclin-dependent kinase family member, is a key regulator of cytoskeletal remodeling in the brain. Cdk5 is essential for brain development during embryogenesis. After birth, it is essential for numerous neuronal processes such as learning and memory formation, drug addiction, pain signaling, and long-term behavior changes, all of which rely on rapid alterations in the cytoskeleton. Cdk5 activity is deregulated in various brain disorders including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and ischemic stroke, resulting in profound remodeling of the neuronal cytoskeleton, loss of synapses, and ultimately neurodegeneration. This review focuses on the "good and bad" Cdk5 in the brain and its pleiotropic contribution in regulating neuronal actin cytoskeletal remodeling. A vast majority of physiologicaland pathological Cdk5 substrates are associated with the actin cytoskeleton. Thus, our special emphasis is on the numerous Cdk5 substrates identified in the past two decades such as ephexin1, p27, Mst3, CaMKv, kalirin-7, RasGRF2, Pak1, WAVE1, neurabin-1, TrkB, 5-HT6R, talin, drebrin, synapsin I, synapsin III, CRMP1, GKAP, SPAR, PSD-95, and LRRK2. These substrates have unraveled the molecular mechanisms by which Cdk5 plays divergent roles in regulating neuronal actin cytoskeletal dynamics both in healthy and diseased states.

Structure and inhibitor specificity of the PCTAIRE-family kinase CDK16.

CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that has emerged as a key regulator of neurite outgrowth, vesicle trafficking and cancer cell proliferation. CDK16 is activated through binding to cyclin Y via a phosphorylation-dependent 14-3-3 interaction and has a unique consensus substrate phosphorylation motif compared with conventional CDKs. To elucidate the structure and inhibitor-binding properties of this atypical CDK, we screened the CDK16 kinase domain against different inhibitor libraries and determined the co-structures of identified hits. We discovered that the ATP-binding pocket of CDK16 can accommodate both type I and type II kinase inhibitors. The most potent CDK16 inhibitors revealed by cell-free and cell-based assays were the multitargeted cancer drugs dabrafenib and rebastinib. An inactive DFG-out binding conformation was confirmed by the first crystal structures of CDK16 in separate complexes with the inhibitors indirubin E804 and rebastinib, respectively. The structures revealed considerable conformational plasticity, suggesting that the isolated CDK16 kinase domain was relatively unstable in the absence of a cyclin partner. The unusual structural features and chemical scaffolds identified here hold promise for the development of more selective CDK16 inhibitors and provide opportunity to better characterise the role of CDK16 and its related CDK family members in various physiological and pathological contexts.

Spectrum and Degree of CDK Drug Interactions Predicts Clinical Performance.

Therapeutically targeting aberrant intracellular kinase signaling is attractive from a biological perspective but drug development is often hindered by toxicities and inadequate efficacy. Predicting drug behaviors using cellular and animal models is confounded by redundant kinase activities, a lack of unique substrates, and cell-specific signaling networks. Cyclin-dependent kinase (CDK) drugs exemplify this phenomenon because they are reported to target common processes yet have distinct clinical activities. Tumor cell studies of ATP-competitive CDK drugs (dinaciclib, AG-024322, abemaciclib, palbociclib, ribociclib) indicate similar pharmacology while analyses in untransformed cells illuminates significant differences. To resolve this apparent disconnect, drug behaviors are described at the molecular level. Nonkinase binding studies and kinome interaction analysis (recombinant and endogenous kinases) reveal that proteins outside of the CDK family appear to have little role in dinaciclib/palbociclib/ribociclib pharmacology, may contribute for abemaciclib, and confounds AG-024322 analysis. CDK2 and CDK6 cocrystal structures with the drugs identify the molecular interactions responsible for potency and kinase selectivity. Efficient drug binding to the unique hinge architecture of CDKs enables selectivity toward most of the human kinome. Selectivity between CDK family members is achieved through interactions with nonconserved elements of the ATP-binding pocket. Integrating clinical drug exposures into the analysis predicts that both palbociclib and ribociclib are CDK4/6 inhibitors, abemaciclib inhibits CDK4/6/9, and dinaciclib is a broad-spectrum CDK inhibitor (CDK2/3/4/6/9). Understanding the molecular components of potency and selectivity also facilitates rational design of future generations of kinase-directed drugs. Mol Cancer Ther; 15(10); 2273-81. ©2016 AACR.

Abstract 2823: Identification of highly selective inhibitors of cyclin-dependent kinase 12

Abstract CDK12 and CDK13 belong to the cyclin-dependent kinase (CDK) family, which includes at least 20 different human CDKs and CDK-like enzymes. One subclass of CDKs (e.g. CDK7, CDK8, CDK9, CDK12 and CDK13) preferentially regulates transcription by phosphorylation of the C-terminal domain of RNA polymerase II (RNAPII), whereas another subgroup of CDKs plays a pivotal role in controlling cell cycle progression (e.g. CDK1, CDK2, CDK4, CDK6 and CDK7). Both groups - cell cycle as well as transcriptional CDKs - have been reported to be involved in cancer development and progression. Recent reports indicated CDK12 to be involved in DNA damage repair mechanisms and linked its mutation to high-grade serous ovarian carcinoma rendering CDK12 a promising target for drug development. However, the pivotal role of individual CDKs and their complex regulation requests for highly selective inhibitors to avoid unwanted adverse effects. Non-specific first-generation CDK-inhibitors cause toxicity in vivo issues. They showed little to no therapeutic window in clinical trials and thus shifted the focus to the optimization of selective small molecule CDK inhibitors. To identify selective small molecular weight inhibitors of CDK12 and CDK13 we employed a rationale drug discovery approach based on a focused kinase library screen. Screening of slightly over 16.000 compounds identified several hit compound classes. Medicinal chemistry based optimization yielded nanomolar CDK12 inhibitors. The selectivity of these improved CDK12 inhibitors is excellent, when tested against 12 CDK family members. Such selective CDK12 inhibitors have also shown promising physicochemical properties, indicating lead- and drug-likeness. Although, potency on the target still needs to be improved, the frontrunners from our selective CDK12 inhibitor family have shown cellular inhibition of CDK12 substrate phosphorylation such as those of RNA polymerase II. Currently, potency of these CDK12 inhibitors is being improved by using structure-guided medicinal chemistry-based optimization. The resulting lead candidates will then be tested in in vivo cancer models. Citation Format: Axel Choidas, Carsten Schultz-Fademrecht, Carsten Degeenhardt, Peter Habenberger, Uwe Koch, Jan E. Eickhoff, Ann Kathrin Greifenberg, Peter Nussbaumer, Matthias Geyer, Bert M. Klebl. Identification of highly selective inhibitors of cyclin-dependent kinase 12. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2823.

P21 and p27 Immunoexpression in Upper Urinary Tract Urothelial Carcinomas.

p21 and p27 are members of cyclin-dependent kinase family, which function as tumor suppressors and they are involved in development and progression of several malignancies. We investigated their expression in upper urinary tract urothelial carcinoma (UUTUC). Radical nephroureterectomy materials of 34 patients were assessed by immunohistochemistry to evaluate expression of p21 and p27 in UUTUC. Results were correlated with various clinicopathological variables as age, gender, tumor grade and stage, tumor architecture, multifocality, subsequent bladder carcinoma development and clinical outcome.p21 and p27 expression was observed in 52.9% (n=18) and 88.2% (n=30), respectively. A total of 21 tumors (61.7%) showed either total loss of p21 expression (n=16, 47%) or lower expression (n=5, 14.7%). No correlation was found between p21 expression and clinicopathologic variables. Cases showing total loss or lower p27 expression (11.7% and <25.6%, respectively) (n=19, 55.8%) constituted 67.6% (n=23) of the cases totally. This loss or lower p27 expression correlated with a shorter overall survival in both univariate and multivariate analysis (p=0.039 and p=0.037, respectively). None of the noninvasive tumors (papillary and nodular tumors) showed loss of p27 (p=0.016) while 33.3% of invasive ones showed p27 loss. Noninvasive tumor architecture also correlated with subsequent bladder carcinoma development (p=0.032) while invasive tumor architecture correlated with advanced stage (T3 and T4) (p=0.003). p27 is widely expressed in UUTUC, while p21 expression is observed in half of the cases. Loss of p27 expression correlated with tumor architecture and overall survival in UUTUC. However, further research is needed to assess their role in UUTUC.

A Tale of the Good and Bad: Remodeling of the Microtubule Network in the Brain by Cdk5.

Cdk5, a cyclin-dependent kinase family member, is a global orchestrator of neuronal cytoskeletal dynamics. During embryogenesis, Cdk5 is indispensable for brain development. In adults, it is essential for numerous neuronal processes, including higher cognitive functions such as learning and memory formation, drug addiction, pain signaling, and long-term behavior changes through long-term potentiation and long-term depression, all of which rely on rapid alterations in the cytoskeleton. Cdk5 activity becomes deregulated in various brain disorders, including Alzheimer's disease, Parkinson's disease, Huntington's disease, attention-deficit hyperactivity disorder, epilepsy, schizophrenia, and ischemic stroke; these all result in profound remodeling of the neuronal cytoskeleton. This Commentary specifically focuses on the pleiotropic contribution of Cdk5 in regulating neuronal microtubule remodeling. Because the vast majority of the physiological substrates of Cdk5 are associated with the neuronal cytoskeleton, our emphasis is on the Cdk5 substrates, such as CRMP2, stathmin, drebrin, dixdc1, axin,MAP2, MAP1B, doublecortin, kinesin-5, and tau, that have allowed to unravel the molecular mechanisms through which Cdk5 exerts its divergent roles in regulating neuronal microtubule dynamics, both in healthy and disease states.

MiR-16 induction after CDK4 knockdown is mediated by c-Myc suppression and inhibits cell growth as well as sensitizes nasopharyngeal carcinoma cells to chemotherapy.

Cyclin-dependent kinase 4 (CDK4) is a member of cyclin-dependent kinase family which regulates G1 to S cell cycle transition. CDK4 activity is increased in many tumor types. Here, we report a negative automodulatory feedback loop between CDK4 and miR-16 that regulates cell cycle progression in nasopharyngeal carcinoma (NPC). By miRNA array and real-time PCR, we identified upregulation of tumor suppressor miR-16a, which inhibited cell cycle progression and sensitized NPC cells to chemotherapy. CDK4 knockdown reduced the expression of c-Myc, the latter of which directly suppresses the miR-16 expression by directly binding to the miR-16 promoter. Moreover, we found that miR-16 upregulation could reduce CDK4 expression by repressing CCND1 and thus forms a feedback loop via the CDK4/c-Myc/miR-16/CCND1 pathway. Finally, miR-16 was negatively correlated with CDK4 expression in NPC biopsies. In summary, our results define a double-negative feedback loop involving CDK4 and miR-16 mediated by c-Myc that modulates NPC cell growth and chemotherapy sensitivity.

An Inhibitor’s-Eye View of the ATP-Binding Site of CDKs in Different Regulatory States

We have used a chemically diverse panel of kinase inhibitors to assess the chemical similarity of the ATP-binding sites of cyclin-dependent kinase (CDK) subfamily members in a range of activation states. Using this approach, we find that different activation states of a particular CDK may differ from each other as much as different CDKs in the same activation state. We also find that inhibitors discriminate more effectively among CDK family members in their monomeric state than in their cyclin-bound state, providing direct evidence for the belief that selective binding to inactive kinase states might be more readily achieved than selective binding to active states.

Exploring the selectivity of a ligand complex with CDK2/CDK1: a molecular dynamics simulation approach

Cyclin-dependent kinases (CDKs) are core components of the cell cycle machinery that govern the transition between phases during cell cycle progression. Abnormalities in CDKs activity and regulation are common features of cancer, making CDK family members attractive targets for the development of anticancer drugs. Their inhibitors have entered in clinical trials to treat cancer. Very recently, Heathcote et al. (J. Med. Chem. 2010, 53:8508-8522) have found a ligand BS194 that has a high affinity with CDK2 (IC(50) = 3 nM) but shows low affinity with CDK1 (IC(50) = 30 nM). To understand the selectivity, we used homology modeling, molecular docking, molecular dynamics, and free-energy calculation to analyze the interactions. A rational three-dimensional model of the CDK1/BS194 complex is built. We found that Leu83 is a key residue that recognizes BS194 more effectively with CDK2 with good binding free energies rather than CDK1. Energetic analysis reveals that van der Waals interaction and non-polar contributions to solvent are favorable in the formation of complexes and amine group of the ligand, which plays a crucial role for binding selectivity between CDK2 and CDK1.

Multiple Roles of Cyclin-Dependent Kinase 4/6 Inhibitors in Cancer Therapy

Cyclin-dependent kinases (CDKs) regulate cell proliferation and coordinate the cell cycle checkpoint response to DNA damage. Although inhibitors with varying selectivity to specific CDK family members have been developed, selective CDK4/6 inhibitors have emerged as the most attractive antineoplastic agents because of the importance of CDK4/6 activity in regulating cell proliferation and the toxic effects associated with inhibition of other CDKs (eg, CDK1 and CDK2). FVB/N wild-type mice (n = 13) were used to evaluate carboplatin-induced myelosuppression in bone marrow by complete blood cell counts after treatment with the CDK4/6 inhibitor PD0332991. Genetically engineered murine models of retinoblastoma (Rb)-competent (MMTV-c-neu) and Rb-incompetent (C3-TAg) breast cancer (n = 16 MMTV-c-neu mice in the carboplatin plus vehicle control group, n = 17 MMTV-c-neu mice in the carboplatin plus PD0332991 group, n = 17 C3-TAg mice in the carboplatin plus vehicle control group, and n = 14 C3-TAg mice in the carboplatin plus PD0332991 group) were used to investigate the antitumor activity of PD0332991 alone or in combination with chemotherapy. All statistical tests were two-sided. Coadministration of PD0332991 with carboplatin compared with carboplatin alone in FVB/N wild-type mice increased hematocrit (51.2% vs 33.5%, difference = 17.7%, 95% confidence interval [CI] = -26.7% to -8.6%, P < .001), platelet counts (1321 vs 758.5 thousand cells per μL, difference = 562.5 thousand cells per μL, 95% CI = -902.8 to -222.6, P = .002), myeloid cells (granulocytes and monocytes; 3.1 vs 1.6 thousand cells per μL, difference = 1.5 thousand cells per μL, 95% CI = -2.23 to -0.67, P < .001), and lymphocytes (7.9 vs 5.4 thousand cells per μL, difference = 2.5 thousand cells per μL, 95% CI = -4.75 to -0.18, P = .02). Daily administration of PD0332991 exhibited antitumor activity in MMTV-c-neu mice as a single agent. However, the combination of carboplatin plus PD0332991 decreased antitumor activity compared with carboplatin alone in Rb-competent mice (mean percent change in tumor volume at day 21 = -52.6% vs 3.7% for carboplatin and carboplatin plus PD0332991, respectively, difference = 56.3%, 95% CI = -109.0% to -3.6%, P = .04). In contrast, Rb-deficient tumors in C3-Tag mice were resistant to PD0332991, and coadministration of PD0332991 plus carboplatin had no effect on in vivo tumor growth (mean percent change in tumor volume at day 21 = 118.8% and 109.1% for carboplatin and carboplatin plus PD0332991, respectively, difference = 9.7%, 95% CI = -183.5% to 202.9%, P = .92). Finally, in tumor-bearing mice, coadministration of PD0332991 with carboplatin provided statistically significant protection of platelets (P = .04). We believe that the present data support a possible role for CDK4/6 inhibitors in a majority of patients with advanced cancer: to either inhibit tumor growth in CDK4/6-dependent tumors or ameliorate the dose-limiting toxicities of chemotherapy in CDK4/6-indepdendent tumors. Our data also suggest CDK4/6 inhibitors should not be combined with DNA-damaging therapies, such as carboplatin, to treat tumors that require CDK4/6 activity for proliferation.

The Versatile Roles of Cyclin-Dependent Kinase 5 in Human Diseases

Cyclin-dependent kinase 5 (Cdk5) is a member of cyclindependent kinase family without the ability of cell cycle control. In the initial stage of research history, Cdk5 was believed to respond for the development of nervous system. However, more and more evidence identifies the expressions and physiological functions of Cdk5 in extra-neuronal tissues. This article introduces readers some recent findings which demonstrate what roles that Cdk5 plays in different tissues and how Cdk5 changes to involve the formation of diseases. In addition to the physiological and pathological roles of Cdk5, the possibility of Cdk5 which becomes a drug target for treating diseases is also discussed in this article.

The Cyclin-Dependent Kinase (CDK) Family Member PNQALRE/CCRK Supports Cell Proliferation but has no Intrinsic CDK-Activating Kinase (CAK) Activity

The cyclin-dependent kinases (CDKs) that drive the eukaryotic cell cycle must be phosphorylated within the activation segment (T-loop) by a CDK-activating kinase (CAK) to achieve full activity. Although a requirement for CDK-activating phosphorylation is conserved throughout eukaryotic evolution, CAK itself has diverged between metazoans and budding yeast, and fission yeast has two CAKs, raising the possibility that additional mammalian enzymes remain to be identified. We report here the characterization of PNQALRE (also known as CCRK or p42), a member of the mammalian CDK family most similar to the cell-cycle effectors Cdk1 and Cdk2 and to the CAK, Cdk7. Although PNQALRE/CCRK was recently proposed to activate Cdk2, we show that the monomeric protein has no intrinsic CAK activity. Depletion of PNQALRE by >80% due to RNA interference (RNAi) impairs cell proliferation, but fails to arrest the cell cycle at a discrete point. Instead, both the fraction of cells with a sub-G1 DNA content and cleavage of poly(ADP-ribose) polymerase (PARP) increase. PNQALRE knockdown did not diminish Cdk2 T-loop phosphorylation in vivo or decrease CAK activity of a cell extract. In contrast, depletion of Cdk7 by RNAi causes a proportional decrease in the ability of an extract to activate recombinant Cdk2. Our data do not support the proposed function of PNQALRE/CCRK in activating CDKs, butinstead reinforce the notion of Cdk7 as the major, and to date the only, CAK in mammalian cells.