An in silico approach for the discovery of CDK5/p25 interaction inhibitors

The lack of selectivity of all existing ATP competitive inhibitors for a single cyclin-dependent kinase (CDK) has led us to redirect the structure-based molecule design from targeting the classic ATP-binding pocket in CDK5 toward the CDK5/p25 interface. The aim was to seek novel inhibition mechanisms to interrupt protein-protein interactions. A combined strategy of alanine-scanning calculations for locating binding sites, virtual screening for small molecules, molecular dynamics simulations for examining the binding stability of virtual screening hits and bio-assays for testing the level of inhibition was set up and used to explore novel inhibitors capable of interrupting the interactions between the proteins, and consequently of inhibiting the kinase activity. Two compounds were shown to inhibit the complex formation between CDK5 and p25 through p25 binding. They could open avenues for the discovery of new types of structures that prevent interactions between CDK5 and p25 or other CDK and activator proteins, and, more importantly, provide leads in the development of selective inhibitors among CDKs.     

Discovery of thienoquinolone derivatives as selective and ATP non-competitive CDK5/p25 inhibitors by structure-based virtual screening

Calpain mediated cleavage of CDK5 natural precursor p35 causes a stable complex formation of CDK5/p25, which leads to hyperphosphorylation of tau. Thus inhibition of this complex is a viable target for numerous acute and chronic neurodegenerative diseases involving tau protein, including Alzheimer’s disease. Since CDK5 has the highest sequence homology with its mitotic counterpart CDK2, our primary goal was to design selective CDK5/p25 inhibitors targeting neurodegeneration. A novel structure-based virtual screening protocol comprised of e-pharmacophore models and virtual screening workflow was used to identify nine compounds from a commercial database containing 2.84 million compounds. An ATP non-competitive and selective thieno[3,2-c]quinolin-4(5H)-one inhibitor (10) with ligand efficiency (LE) of 0.3 was identified as the lead molecule. Further SAR optimization led to the discovery of several low micromolar inhibitors with good selectivity. The research represents a new class of potent ATP non-competitive CDK5/p25 inhibitors with good CDK2/E selectivity.     

Structure and regulation of the CDK5-p25(nck5a) complex

CDK5 plays an indispensable role in the central nervous system, and its deregulation is involved in neurodegeneration. We report the crystal structure of a complex between CDK5 and p25, a fragment of the p35 activator. Despite its partial structural similarity with the cyclins, p25 displays an unprecedented mechanism for the regulation of a cyclin-dependent kinase. p25 tethers the unphosphorylated T loop of CDK5 in the active conformation. Residue Ser159, equivalent to Thr160 on CDK2, contributes to the specificity of the CDK5-p35 interaction. Its substitution with threonine prevents p35 binding, while the presence of alanine affects neither binding nor kinase activity. Finally, we provide evidence that the CDK5-p25 complex employs a distinct mechanism from the phospho-CDK2-cyclin A complex to establish substrate specificity.     

Design and synthesis of 6-oxo-1,6-dihydropyridines as CDK5 inhibitors

Cyclin-dependent kinase 5 (CDK5) is a serine-threonine protein kinase that plays a significant role in neuronal development. In association with p25, CDK5 abnormally phosphorylates a number of cellular targets involved in neurodegenerative disorders. Using active site homology and previous structure–activity relationships, a new series of potent CDK5 inhibitors was designed. This report describes the optimization of 6-oxo-1,6-dihydropyridines as CDK5 inhibitors.     

Truncation of CDK5 activator p35 induces intensive phosphorylation of Ser202/Thr205 of human tau

Hyperphosphorylated tau is a major component of neurofibrillary tangles, one of the hallmarks of Alzheimer's disease. CDK5 is a kinase that phosphorylates the tau protein, and its endogenous activator, p35, regulates its activity. Recently, calpain was found to digest p35 to its truncated product, p25. Several lines of evidence suggest that p25-CDK5 has much more powerful kinase activity and that it may cause abnormal hyperphosphorylation of tau. In this study, we have examined the kinetic characteristics of in vitro phosphorylation of the longest isoform of human tau by CDK5 and its activators using recombinant proteins. Although the kinase activity of CDK5 in phosphorylating tau was significantly higher in the presence of p25, the affinity of CDK5 for tau was not different. Phosphopeptide mapping revealed enhanced phosphorylation of Ser(202)/Thr(205) residues by p25-CDK5 (amino acid residues of tau are numbered according to the longest isoform of human tau). These results suggest that cleavage of p35 to p25 greatly enhances the kinase activity of CDK5 and increases the phosphorylation of Ser(202)/Thr(205). Considering the fact that phosphorylation of Ser(202)/Thr(205) antagonizes the tau-mediated nucleation of tubulin, p25-CDK5 may play a pivotal role in neuronal cell death in Alzheimer's disease.     

Calpain-dependent proteolytic cleavage of the p35 cyclin-dependent kinase 5 activator to p25

Cyclin-dependent kinase 5 (CDK5) is a unique CDK, the activity of which can be detected in postmitotic neurons. To date, CDK5 purified from mammalian brains has always been associated with a truncated form of the 35-kDa major brain specific activator (p35, also known as nck5a) of CDK5, known as p25. In this study, we report that p35 can be cleaved to p25 both in vitro and in vivo by calpain. In a rat brain extract, p35 was cleaved to p25 by incubation with Ca(2+). This cleavage was inhibited by a calpain inhibitor peptide derived from calpastatin and was ablated by separating the p35.CDK5 from calpain by centrifugation. The p35 recovered in the pellet after centrifugation could then be cleaved to p25 by purified calpain. Cleavage of p35 was also induced in primary cultured neurons by treatment with a Ca(2+) ionophore and Ca(2+) and inhibited by calpain inhibitor I. The cleavage changed the solubility of the CDK5 active complex from the particulate fraction to the soluble fraction but did not affect the histone H1 kinase activity. Increased cleavage was detected in cultured neurons undergoing cell death, suggesting a role of the cleavage in neuronal cell death.     

CDK5 and Its Activator P35 in Normal Pituitary and in Pituitary Adenomas: Relationship to VEGF Expression

Pituitary tumors are monoclonal adenomas that account for about 10-15% of intracranial tumors. Cyclin-dependent kinase 5 (CDK5) regulates the activities of various proteins and cellular processes in the nervous system, but its potential roles in pituitary adenomas are poorly understood. The kinase activity of CDK5 requires association with an activating protein, p35 (also known as CDK5 activator 1, p35). Here, we show that functional CDK5, associated with p35, is present in normal human pituitary and in pituitary tumors. Furthermore, p35 mRNA and protein levels were higher in pituitary adenomas than in the normal glands, suggesting that CDK5 activity might be upregulated in pituitary tumors. Inhibition of CDK5 activity in rat pituitary cells, reduced the expression of vascular endothelial growth factor (VEGF), a protein that regulates vasculogenesis and angiogenesis. Our results suggest that increased CDK5-mediated VEGF expression might play a crucial role in the development of pituitary adenomas, and that roscovitine and other CDK5 inhibitors could be useful as anticancer agents.     

Expression and activity of cyclin-dependent kinases and glycogen synthase kinase-3 during NT2 neuronal differentiation

In the presence of retinoic acid undifferentiated NT2 cells turn into terminally differentiated hNT (or NT2N) neurons within 5 weeks. We have used this in vitro cellular model to investigate the changes in expression and activity of cyclin-dependent kinases (CDKs) and glycogen synthase kinase-3 (GSK-3) during this neuronal differentiation process. We here show that CDK1/2 protein level and kinase activity sharply decrease during the NT2-->hNT transition. In contrast, the activity of CDK5/p35 dramatically increases, probably as a result of an enhanced expression of p35 in a stable CDK5 level background. GSK-3 activity increases modestly during the differentiation of hNT cells, and this event correlates with enhanced expression of each of the three GSK-3 isoforms. Pharmacological inhibitors of CDKs and GSK-3 lead to a dose-dependent decrease in cell viability.     

Thiazolo[5,4-f]quinazolin-9-ones, inhibitors of glycogen synthase kinase-3

In an effort to identify new protein kinase inhibitors with increased potency and selectivity, we have developed the microwave-assisted synthesis of thiazolo[5,4-f]quinazolin-9-ones. The effects of eighteen derivatives on CDK1/cyclin B, CDK5/p25, and GSK-3 were investigated. Several turned out to inhibit GSK-3 in the micromolar range. Molecular modeling studies suggest that the most selective GSK-3 inhibitors 7a-d bind into the ATP-binding site through a key hydrogen bond interaction with Val135 and target the specific hydrophobic backpocket of the enzyme.     

Mechanism of CDK5 activation revealed by steered molecular dynamics simulations and energy calculations

In the current work, CDK5/p25 complexes were pulled apart by applying external forces with steered molecular dynamics (SMD) simulations. The crucial interactions between the kinase and the activation protein were investigated and the SMD simulations showed that several activation-relevant motifs of CDK5 leave p25 in sequence during the pulling and lead to an apo-CDK2 like CDK5 structure after separation. Based on systematic examination of hydrogen bond breaking and classical MD/molecular mechanics-generalized Born/surface area) (MM-GBSA) calculations, a CDK5 activation mechanism by p25 is suggested. This is the first step towards the systemic development of CDK inhibitors and the mechanism proposed could lead to a better understanding of the protein–protein recognition characteristics between the kinase and its activator.     

The cyclin-dependent kinase inhibitory domain of the yeast Sic1 protein is contained within the C-terminal 70 amino acids

By inhibiting the activity of Cdc28/Clb cyclin-dependent protein kinase (CDK) complexes, Sic1 prevents the premature initiation of S phase in the yeast Saccharomyces cerevisiae. By testing a series of Sic1 truncation mutants, we have mapped the minimal domain necessary for Cdc28/Clb inhibition in vivo to the C-terminal 70 amino acids of Sic1. Site-directed mutagenesis was used to show that a sequence that matches the zRxL motif found in mammalian CDK inhibitors is essential for Sic1 function. This motif is not found in the Schizosaccharomyces CDK inhibitor p25^rum1, which appears to be a structural and functional homolog of Sic1. Based on the mutational data and sequence comparisons, we argue that Sic1 and p25^rum1 are structurally distinct from the known mammalian CDK inhibitors, but may bind CDK complexes in a manner more closely resembling CDK substrates like the retinoblastoma and E2F proteins. Received: 3 February 1999 / Accepted: 23 April 1999     

Cyclin-Dependent Kinase Five Mediates Activation of Lung Xanthine Oxidoreductase in Response to Hypoxia

Background

Xanthine oxidoreductase (XOR) is involved in oxidative metabolism of purines and is a source of reactive oxygen species (ROS). As such, XOR has been implicated in oxidant-mediated injury in multiple cardiopulmonary diseases. XOR enzyme activity is regulated, in part, via a phosphorylation-dependent, post-translational mechanism, although the kinase(s) responsible for such hyperactivation are unknown.

Methods and Results

Using an in silico approach, we identified a cyclin-dependent kinase 5 (CDK5) consensus motif adjacent to the XOR flavin adenine dinucleotide (FAD) binding domain. CDK5 is a proline-directed serine/threonine kinase historically linked to neural development and injury. We tested the hypothesis that CDK5 and its activators are mediators of hypoxia-induced hyperactivation of XOR in pulmonary microvascular endothelial cells (EC) and the intact murine lung. Using complementary molecular and pharmacologic approaches, we demonstrated that hypoxia significantly increased CDK5 activity in EC. This was coincident with increased expression of the CDK5 activators, cyclin-dependent kinase 5 activator 1 (CDK5r1 or p35/p25), and decreased expression of the CDK5 inhibitory peptide, p10. Expression of p35/p25 was necessary for XOR hyperactivation. Further, CDK5 physically associated with XOR and was necessary and sufficient for XOR phosphorylation and hyperactivation both in vitro and in vivo. XOR hyperactivation required the target threonine (T222) within the CDK5-consensus motif.

Conclusions and Significance

These results indicate that p35/CDK5-mediated phosphorylation of T222 is required for hypoxia-induced XOR hyperactivation in the lung. Recognizing the contribution of XOR to oxidative injury in cardiopulmonary disease, these observations identify p35/CDK5 as novel regulators of XOR and potential modifiers of ROS-mediated injury.     

Identification of novel CDK2 inhibitors by a multistage virtual screening method based on SVM,pharmacophore and docking model

Abstract

Cyclin-dependent kinase 2 (CDK2) is the family of Ser/Thr protein kinases that has emerged as a highly selective with low toxic cancer therapy target. A multistage virtual screening method combined by SVM, protein-ligand interaction fingerprints (PLIF) pharmacophore and docking was utilised for screening the CDK2 inhibitors. The evaluation of the validation set indicated that this method can be used to screen large chemical databases because it has a high hit-rate and enrichment factor (80.1% and 332.83 respectively). Six compounds were screened out from NCI, Enamine and Pubchem database. After molecular dynamics and binding free energy calculation, two compounds had great potential as novel CDK2 inhibitors and they also showed selective inhibition against CDK2 in the kinase activity assay.     

The cdc2Ms Kinase Is Differently Regulated in the Cytoplasm and in the Nucleus

To study a cyclin-dependent kinase (CDK) from alfalfa (Medicago sativa L.), an antibody was raised against the C-terminal 16 amino acids of the protein cdc2aMs. The cdc2Ms protein was immunopurified with this antibody and its histone kinase activity was measured. The cdc2Ms kinase is activated at the G1/S transition when phosphate-starved cells from the G0 phase re-enter the cell cycle and remain active as cells transit the S, G2, and M phases, indicating that the same CDK regulates all of these phases in alfalfa. In contrast, when cdc2Ms kinase was purified by binding to p13suc1, it was active only in the G2 and M phases. In immunoblots the C-terminal antibody detected an equal amount of the cdc2Ms protein in the cytoplasm and in the nucleus. By indirect immunofluorescence, however, the cytoplasmic form of cdc2Ms could not be found in the S phase of the cells, indicating that the epitope for the cdc2 antibody is not accessible. Binding of putative inhibitor proteins to cdc2 was shown by inactivation of purified plant CDK when cell extracts were added. Furthermore, purified CDK inhibitors, such as the mouse p27kip1 and the yeast p40sic1, blocked the purified plant CDK activity.     

Investigating the neuroprotective mechanism of action of a CDK5 inhibitor by phosphoproteome analysis

Small molecule inhibitors of cyclin-dependent kinase 5 (CDK5) protect neurons from cell death following various insults. To elucidate the cellular mechanism of action we investigated changes in protein phosphorylation in cultured rat cerebellar granule neurons after administration of the CDK5 inhibitor Indolinone A. By immunoblot analysis we detected enhanced phosphorylation of the extracellular signal-regulated kinase1/2 (ERK1/2) and the Jun N-terminal kinase (JNK) substrate c-Jun. Co-administration of U0126, an inhibitor of ERK1/2, or SP600125, an inhibitor of JNK, blocked phosphorylation of ERK1/2 or c-Jun, but did not affect neuroprotection by the CDK5 inhibitor. By metal affinity chromatography, two-dimensional (2D) gel electrophoresis, and MALDI-TOF mass spectrometry we identified several phosphoproteins that accumulated in neurons treated with Indolinone A. Among them were proteins involved in neurotransmitter release, which is consistent with a physiological function of CDK5 in synaptic signaling. Moreover, we identified proteins acting in energy metabolism, protein folding, and oxidative stress response. Similar findings have been reported in yeast following inhibition of Pho85 kinase, which is homologous to mammalian CDK5 and acts in environmental stress signaling. These results suggest that inhibition of CDK5 activates stress responsive proteins that may protect neurons against subsequent injurious stimuli.     

The p34cdc2-related cyclin-dependent kinase 11 interacts with the p47 subunit of eukaryotic initiation factor 3 during apoptosis

Cyclin-dependent kinase 11 (CDK11; also named PITSLRE) is part of the large family of p34(cdc2)-related kinases whose functions appear to be linked with cell cycle progression, tumorigenesis, and apoptotic signaling. However, substrates of CDK11 during apoptosis have not been identified. We used a yeast two-hybrid screening strategy and identified eukaryotic initiation factor 3 p47 protein (eIF3 p47) as an interacting partner of caspase-processed C-terminal kinase domain of CDK11 (CDK11(p46)). We demonstrate that the eIF3 p47 can interact with CDK11 in vitro and in vivo, and the interaction can be strengthened by stimulation of apoptosis. EIF3 p47 contains a Mov34/JAB domain and appears to interact with CDK11(p46) through this motif. We show in vitro that the caspase-processed CDK11(p46) can phosphorylate eIF3 p47 at a specific serine residue (Ser(46)) and that eIF3 p47 is phosphorylated in vivo during apoptosis. Purified recombinant CDK11(p46) inhibited translation of a reporter gene in vitro in a dose-dependent manner. In contrast, a kinase-defective mutant CDK11(p46M) did not inhibit translation of the reporter gene. Stable expression of CDK11(p46) in vivo inhibited the synthesis of a transfected luciferase reporter protein and overall cellular protein synthesis. These data provide insight into the cellular function of CDK11 during apoptosis.     

Regulation of cyclin-dependent kinase 2 activity by ceramide

Cyclin-dependent kinases have been implicated in the inactivation of retinoblastoma (Rb) protein and cell cycle progression. Recent studies have demonstrated that the lipid molecule ceramide is able to induce Rb hypophosphorylation leading to growth arrest and cellular senescence. In this study, we examined the underlying mechanisms of Rb hypophosphorylation and cell cycle progression utilizing the antiproliferative molecule ceramide. C6-Ceramide induced a G0/G1 arrest of the cell cycle in WI38 human diploid fibroblasts. Employing immunoprecipitation kinase assays, we found that ceramide specifically inhibited cyclin-dependent kinase CDK2, with a mild effect on CDC2 and significantly less effect on CDK4. The effect of ceramide was specific such that C6-dihydroceramide was not effective. Ceramide did not directly inhibit CDK2 in vitro but caused activation of p21, a major class of CDK-inhibitory proteins, and led to a greater association of p21 to CDK2. Using purified protein phosphatases, we showed that ceramide activated both protein phosphatase 1 and protein phosphatase 2A activities specific for CDK2 in vitro. Further, calyculin A and okadaic acid, both potent protein phosphatase inhibitors, together almost completely reversed the effects of ceramide on CDK2 inhibition. Taken together, these results demonstrate a dual mechanism by which ceramide inhibits the cell cycle. Ceramide causes an increase in p21 association with CDK2 and through activation of protein phosphatases selectively regulates CDK2. These events may lead to activation of Rb protein and subsequent cell cycle arrest.     

A triple exon-skipping luciferase reporter assay identifies a new CLK inhibitor pharmacophore

The splicing of pre-mRNA is a critical process in normal cells and is deregulated in cancer. Compounds that modulate this process have recently been shown to target a specific vulnerability in tumors. We have developed a novel cell-based assay that specifically activates luciferase in cells exposed to SF3B1 targeted compounds, such as sudemycin D6. This assay was used to screen a combined collection of approved drugs and bioactive compounds. This screening approach identified several active hits, the most potent of which were CGP-74514A and aminopurvalanol A, both have been reported to be cyclin-dependent kinases (CDKs) inhibitors. We found that these compounds, and their analogs, show significant cdc2-like kinase (CLK) inhibition and clear structure-activity relationships (SAR) at CLKs. We prepared a set of analogs and were able to ‘dial out’ the CDK activity and simultaneously developed CLK inhibitors with low nanomolar activity. Thus, we have demonstrated the utility of our exon-skipping assay and identified new molecules that exhibit potency and selectivity for CLK, as well as some structurally related dual CLK/CDK inhibitors.     

Explaining the inhibition of cyclin-dependent kinase 5 by peptides derived from p25 with molecular dynamics simulations and MM-PBSA

A cyclin-dependent kinase (CDK) 5 inhibitory peptide (CIP) from p25 was recently reported to inhibit CDK5/p25 activity in vitro but had no effect on endogenous cdc2 kinase activity. This may lead to a specific CDK5 inhibition strategy in the treatment of neurodegeneration. However, the mechanism of the inhibition remains unclear. In this work, molecular dynamics simulations and energy decomposition calculation models were set up to investigate the deregulation mechanisms of CIP on CDK5 activity. The results show that truncation of the N, and C terminals of p25 introduces important conformational changes into a hydrophobic pocket that is crucial for accommodating Ile153 on the activation loop of CDK5. In addition, such truncations lead to distortion and displacement of the activation loop and consequently affect binding of the substrate peptide. New inhibition sites for selectively inhibiting the activity of CDK5 are also suggested.     

Identification of a neuronal Cdk5 activator-binding protein as Cdk5 inhibitor

Neuronal Cdc2-like kinase (Nclk) plays an important role in a variety of cellular processes, including neuronal cell differentiation, apoptosis, neuron migration, and formation of neuromuscular junction. The active kinase consists of a catalytic subunit, Cdk5, and an essential regulatory subunit, neuronal Cdk5 activator (p35(nck5a) or p25(nck5a)), which is expressed primarily in neurons of central nervous tissue. In our previous study using the yeast two-hybrid screening method, three novel p35(nck5a)-associated proteins were isolated. Here we show that one of these proteins, called C42, specifically inhibits the activation of Cdk5 by Nck5a. Co-immunoprecipitation data suggested that C42 and p35(nck5a) could form a complex within cultured mammalian cells. Deletion analysis has mapped the inhibitory domain of C42 to a region of 135 amino acids, which is conserved in Pho81, a yeast protein that inhibits the yeast cyclin-dependent protein kinase Pho85. The Pho85.Pho80 kinase complex has been shown to be the yeast functional homologue of the mammalian Cdk5/p35(nck5a) kinase.