A novel approach to cyclin-dependent kinase 5/p25 inhibitors: A potential treatment for Alzheimer's disease

Based on the earlier results of our in-house database and compound library, a series of novel clubbed thienyl triazoles was designed which may emerge as potential cdk5/p25 inhibitors, for the treatment of Alzheimer's disease. A benign synthesis was planned so as to take an advantage of MAOS (Microwave Assisted Organic Synthesis) method. Evaluation of the SAR of this series has allowed the identification of compounds 4, 5, 7 and 8 from series I while 13, 14, 16 and 17 from series II as significant cdk5/p25 inhibitors and thus have potential as possible treatments for Alzheimer's disease.     

Discovery and SAR of 2-aminothiazole inhibitors of cyclin-dependent kinase 5/p25 as a potential treatment for Alzheimer's disease

High-throughput screening with cyclin-dependent kinase 5 (cdk5)/p25 led to the discovery of N-(5-isopropyl-thiazol-2-yl)isobutyramide (1). This compound is an equipotent inhibitor of cdk5 and cyclin-dependent kinase 2 (cdk2)/cyclin E (IC(50)=ca. 320nM). Parallel and directed synthesis techniques were utilized to explore the SAR of this series. Up to 60-fold improvements in potency at cdk5 and 12-fold selectivity over cdk2 were achieved.     

Docking and 3D-QSAR modeling of cyclin-dependent kinase 5/p25 inhibitors

Structure-based 3D-QSAR approaches (CoMFA and CoMSIA) were applied to understand the structural requirements of the Cyclin-dependent kinase 5/p25 inhibitors. Cyclin-dependent kinase 5 (CDK5) is believed to play an important role in the development of the central nervous system during the process of mammalian embryogenesis. Genetic algorithm based docking program (GOLD) was successfully utilized to orient the compounds inside the binding pocket of the CDK5/p25 structure. The adapted alignment method with the suitable parameters resulted in a reliable model. Furthermore, the final model was robust enough to forecast the activities of test compounds, satisfactorily. The contour maps were produced around the functional groups to understand the SAR requirements. Moreover, we also investigate the structural attributes of the inhibitors which make them selective toward CDK5/p25 over its close counterpart, i.e., CDK2. The study could be helpful to rationalize the new compounds with better inhibition and selectivity profiles against CDK5/p25.     

Clubbed [1,2,3] triazoles by fluorine benzimidazole: a novel approach to H37Rv inhibitors as a potential treatment for tuberculosis

A Novel Clubbed [1,2,3] triazoles with fluorine benzimidazole series of H37Rv strain inhibitors, potentially useful for the treatment of tuberculosis is disclosed on the basis of promising results of preliminary antimicrobial study. Evaluation of the SAR of substitution within these series has followed the identification of a range of compounds. Some of the derivatives are under further evaluation showing better considerable activity compared to rifampin.     

Paullones are potent inhibitors of glycogen synthase kinase-3beta and cyclin-dependent kinase 5/p25.

Paullones constitute a new family of benzazepinones with promising antitumoral properties. They were recently described as potent, ATP-competitive, inhibitors of the cell cycle regulating cyclin-dependent kinases (CDKs). We here report that paullones also act as very potent inhibitors of glycogen synthase kinase-3beta (GSK-3beta) (IC50: 4-80 nM) and the neuronal CDK5/p25 (IC50: 20-200 nM). These two enzymes are responsible for most of the hyperphosphorylation of the microtubule-binding protein tau, a feature observed in the brains of patients with Alzheimer's disease and other neurodegenerative 'taupathies'. Alsterpaullone, the most active paullone, was demonstrated to act by competing with ATP for binding to GSK-3beta. Alsterpaullone inhibits the phosphorylation of tau in vivo at sites which are typically phosphorylated by GSK-3beta in Alzheimer's disease. Alsterpaullone also inhibits the CDK5/p25-dependent phosphorylation of DARPP-32 in mouse striatum slices in vitro. This dual specificity of paullones may turn these compounds into very useful tools for the study and possibly treatment of neurodegenerative and proliferative disorders.     

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.     

Structure-activity relationship study of 2,4-diaminothiazoles as Cdk5/p25 kinase inhibitors

Cdk5/p25 has emerged as a principle therapeutic target for numerous acute and chronic neurodegenerative diseases, including Alzheimer’s disease. A structure-activity relationship study of 2,4-diaminothiazole inhibitors revealed that increased Cdk5/p25 inhibitory activity could be accomplished by incorporating pyridines on the 2-amino group and addition of substituents to the 2- or 3-position of the phenyl ketone moiety. Interpretation of the SAR results for many of the analogs was aided through in silico docking with Cdk5/p25 and calculating protein hydrations sites using WaterMap. Finally, improved in vitro mouse microsomal stability was also achieved.     

Cleavage of the cyclin-dependent kinase 5 activator p35 to p25 does not induce tau hyperphosphorylation

Hyperphosphorylated tau protein is the primary component of neurofibrillary tangles observed in several neurodegenerative disorders. It has been hypothesized that in certain pathological conditions, the calcium activated protease, calpain, would cleave the cyclin-dependent kinase 5 (cdk5) activator p35 to a p25 fragment, which would lead to augmented cdk5 activity, and cdk5-mediated tau hyperphosphorylation. To test this hypothesis, we induced calpain-mediated p35 cleavage in rat hippocampal neuronal cultures and studied the relationship between p25 production, cdk5 activity, and tau phosphorylation. In glutamate-treated cells p35 was cleaved to p25 and this was associated with elevated cdk5 activity. However, tau phosphorylation was concomitantly decreased at multiple sites. The calpain inhibitor MDL28170 prevented the cleavage of p35 but had no effect on tau phosphorylation, suggesting that calpain-mediated processes, i.e., the cleavage of p35 to p25 and cdk5 activation, do not contribute to tau phosphorylation in these conditions. Treatment of the neuronal cultures with N-methyl-D-aspartic acid or with calcium ionophores resulted in an outcome highly similar to that of glutamate. We conclude that, in neuronal cells, the cleavage of p35 to p25 is associated with increased activity of cdk5 but not with tau hyperphosphorylation.     

Design of a novel class of peptide inhibitors of cyclin-dependent kinase/cyclin activation

Cyclin dependent kinases (CDKs) are key regulators of the cell cycle progression and therefore constitute excellent targets for the design of anticancer agents. Most of the inhibitors identified to date inhibit kinase activity by interfering with the ATP-binding site of CDKs. We recently proposed that the protein/protein interface and conformational changes required in the molecular mechanism of CDK2-cyclin A activation were potential targets for the design of specific inhibitors of cell cycle progression. To this aim, we have designed and characterized a small peptide, termed C4, derived from amino acids 285-306 in the alpha5 helix of cyclin A. We demonstrate that this peptide does not interfere with complex formation but forms stable complexes with CDK2-cyclin A. The C4 peptide significantly inhibits kinase activity of complexes harboring CDK2 in a competitive fashion with respect to substrates but does not behave as an ATP antagonist. Moreover, when coupled with the protein transduction domain of Tat, the C4 peptide blocks the proliferation of tumor cell lines, thereby constituting a potent lead for the development of specific CDK-cyclin inhibitors.     

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.     

A novel series of p38 MAP kinase inhibitors for the potential treatment of rheumatoid arthritis

A novel p38 MAP kinase inhibitor structural class was discovered through selectivity screening. The rational analogue design, synthesis and structure-activity relationship of this series of bis-amide inhibitors is reported. The inhibition in vitro of human p38alpha enzyme activity and lipopolysaccharide-induced tumour necrosis factor-alpha release is described for the series. The activity in vivo and pharmacokinetic properties are exemplified for the more potent analogues.     

Phosphorylation of FTDP-17 mutant tau by cyclin-dependent kinase 5 complexed with p35, p25, or p39

One of the major pathological hallmarks of Alzheimer disease is neurofibrillary tangles. Neurofibrillary tangles are bundles of paired helical filaments composed of hyperphosphorylated tau. Cyclin-dependent kinase 5 (Cdk5) is one of the tau protein kinases that increase paired helical filament epitopes in tau by phosphorylation. Recently, various mutations of tau have been identified in frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Here, we investigated the phosphorylation of FTDP-17 mutant tau proteins, K257T, P301L, P301S, and R406W, by Cdk5 complexed with p35, p25, or p39 in vitro and in cultured cells. The extent of phosphorylation by all Cdk5 species was slightly lower in mutant tau than in wild-type tau. Major phosphorylation sites, including Ser202, Ser235, and Ser404, were the same among the wild-type, K257T, P301L, and P301S tau proteins phosphorylated by any Cdk5. On the other hand, R406W tau was less phosphorylated at Ser404 than were the other variants. This was not due to the simple replacement of amino acid Arg406 with Trp close to the phosphorylation site, because Ser404 in a R406W peptide was equally phosphorylated in a wild-type peptide. The decreased phosphorylation of mutant tau by Cdk5s was canceled when tau protein bound to microtubules was phosphorylated. These results indicate that FTDP-17 mutations do not affect the phosphorylatability of tau by Cdk5 complexed with p35, p25, or p39 and may explain part of the discrepancy reported previously between in vivo and in vitro phosphorylation of FTDP-17 tau mutants.     

Synthesis and structure-activity relationship of 4-(1,3-benzothiazol-2-yl)-thiophene-2-sulfonamides as cyclin-dependent kinase 5 (cdk5)/p25 inhibitors

4-(1,3-Benzothiazol-2-yl)thiophene-2-sulfonamide (4a) was found to be a moderately potent inhibitor of cyclin-dependent kinase 5 (cdk5) from a HTS screen. The synthesis and SAR around this hit is described. The X-ray coordinates of ligand 4a with cdk5 are also reported, showing an unusual binding mode to the hinge region via a water molecule.     

Stem cell-based therapy as a promising approach in Alzheimer's disease: current perspectives on novel treatment

Alzheimer's disease (AD) is a neuronal disorder with insidious onset and slow progression, leading to growing global concern with huge implications for individuals and society. The occurrence of AD has been increased and has become an important health issue throughout the world. In recent years, the care of more than 35 million patients with AD costs over $ 600 billion per year, it is approximately 1 percent of the global Gross Domestic Product. Currently, the therapeutic approach is not effective for neurological deficits especially after the development of these major neurological disorders. The discovery of the technique called cell-based therapy has shown promising results and made important conclusions beyond AD using the stem cells approach. Here we review recent progress on stem cell-based therapy in the context of AD.

     

p25/cyclin-dependent kinase 5 promotes the progression of cell death in nucleus of endoplasmic reticulum-stressed neurons

Dysregulation of cyclin-dependent kinase 5 (Cdk5) by cleavage of its activator p35 to p25 by calpain is involved in the neuronal cell death observed in neurodegenerative disorders, including Alzheimer's disease. However, it is not yet clear how p25/Cdk5 induces cell death, although its cytosolic localization or extended half life are thought to be involved. We show here that endoplasmic reticulum (ER) stress causes the calpain-dependent cleavage of p35 to p25 in primary cultured cortical neurons. Generation of p25 occurred at a cell death execution step in ER-stressed neurons. p25 translocated to the nucleus in ER-stressed neurons, whereas p35/Cdk5 was perinuclear in control neurons. Cdk5 inhibitors or dominant-negative Cdk5 suppressed ER stress-induced neuronal cell death. These findings indicate that p25/Cdk5 is a proapoptotic factor that promotes ER stress-induced neuronal cell death in nuclei.     

Mutant superoxide dismutase 1 causes motor neuron degeneration independent of cyclin-dependent kinase 5 activation by p35 or p25

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective loss of motor neurons in the brain and spinal cord. Neurotoxicity mediated by glutamate is thought to play a role in the neuronal death through intracellular calcium-dependent signaling cascades. Cyclin-dependent kinase 5 (Cdk5) has been proposed as one of the calcium-dependent mediators that may cause neuronal death observed in this disease. Cdk5 is activated in neurons by the association with its activators, p35 or p39. The calcium-activated protease calpain cleaves p35 to its truncated product, p25, which eventually causes the cellular mislocalization and prolonged activation of Cdk5. This deregulated Cdk5 induces cytoskeletal disruption and apoptosis. To examine whether inhibition of the calpain-mediated conversion of p35 to p25 can delay the disease progression of ALS, we generated double transgenic mice in which ALS-linked mutant copper/zinc superoxide dismutase 1 (SOD1G93A) was expressed in a p35-null background. The absence of p35 neither affected the onset and progression of motor neuron disease in the mutant SOD1 mice nor ameliorated the pathological lesions in these mice. Our results provide direct evidence that the pathogenesis of motor neuron disease in the mutant SOD1 mice is independent of the Cdk5 activation by p35 or p25.     

Kinetic studies of Cdk5/p25 kinase: phosphorylation of tau and complex inhibition by two prototype inhibitors

Cdk5/p25 is a member of the family of cyclin-dependent, Ser/Thr kinases and is thought to play a causal role in Alzheimer's disease (AD) due to its ability to phosphorylate the protein tau, and thus promote the latter's aggregation into intraneuronal tangles. Given this, we and others are seeking inhibitors of cdk5/p25 as possible disease-modifying therapeutics for AD. In this paper, we first report the kinetic mechanism for the cdk5/p25-catalyzed phosphorylation of tau and histone H-1-derived peptide (H1P). These studies served as a necessary kinetic backdrop for investigations of the mechanism of inhibition by prototype inhibitors N4-(6-aminopyrimidin-4-yl)-sulfanilamide (APS) and 1-(5-cyclobutyl-thiazol-2-yl)-3-isoquinolin-5-yl-urea (CTIU). We found that the cdk5/p25-catalyzed phosphorylation of tau follows a rapid equilibrium, random kinetic mechanism, as evidenced by initial velocity analysis indicating sequential addition of tau and ATP, and studies of the mechanism of inhibition by substrate analogue AMP, product ADP, and analogues of peptide substrate H1P. Identical mechanistic conclusions were drawn when H1P was the phosphoryl acceptor. Subsequent studies of inhibition by APS and CTIU revealed that both compounds can bind to all four steady-state forms of the enzyme, to form the complexes E:I, E:I:tau, E:I:ATP, and E:I:tau:ATP. These results contrast with reported claims that APS and CTIU are competitive inhibitors of the binding of ATP.     

AMP-activated protein kinase: a potential player in Alzheimer's disease

AMP-activated protein kinase (AMPK) stimulates energy production via glucose and lipid metabolism, whereas it inhibits energy consuming functions, such as protein and cholesterol synthesis. Increased cytoplasmic AMP and Ca(2+) levels are the major activators of neuronal AMPK signaling. Interestingly, Alzheimer's disease (AD) is associated with several abnormalities in neuronal energy metabolism, for example, decline in glucose uptake, mitochondrial dysfunctions and defects in cholesterol metabolism, and in addition, with problems in maintaining Ca(2+) homeostasis. Epidemiological studies have also revealed that many metabolic and cardiovascular diseases are risk factors for cognitive impairment and sporadic AD. Emerging studies indicate that AMPK signaling can regulate tau protein phosphorylation and amyloidogenesis, the major hallmarks of AD. AMPK is also a potent activator of autophagic degradation which seems to be suppressed in AD. All these observations imply that AMPK is involved in the pathogenesis of AD. However, the responses of AMPK activation are dependent on stimulation and the extent of activating stress. Evidently, AMPK signaling can repress and delay the appearance of AD pathology but later on, with increasing neuronal stress, it can trigger detrimental effects that augment AD pathogenesis. We will outline the potential role of AMPK function in respect to various aspects affecting AD pathogenesis.     

Tau phosphorylation by cyclin-dependent kinase 5/p39 during brain development reduces its affinity for microtubules

The microtubule-associated protein tau is a developmentally regulated neuronal phosphoprotein. The phosphorylation of tau reduces its ability to bind and stabilize axonal microtubules during axonal growth. Although tau is phosphorylated by cyclin-dependent kinase 5 (Cdk5) in vitro, its in vivo roles remain unclear. Here, we show that tau is phosphorylated by Cdk5/p39 during brain development, resulting in a reduction of its affinity for microtubules. The activity of Cdk5 is tightly regulated by association with its neuronal activators, p35 or p39. The p35 and p39 expression levels were investigated in the developing mouse brain; the p39 expression level was higher in embryonic hind brain and spinal cord and in postnatal cerebral cortex, whereas that of p35 was most prominent in cerebral cortex at earlier stages of development. The ability of Cdk5 to phosphorylate tau was higher when in association with p39 than in association with p35. Tau phosphorylation at Ser-202 and Thr-205 was decreased in Cdk5-/- mouse brain but not in p35-/- mouse brain, suggesting that Cdk5/p39 is responsible for the in vivo phosphorylation of tau at these sites. Our data suggest that tau phosphorylation by Cdk5 may provide the neuronal microtubules with dynamic properties in a region-specific and developmentally regulated manner.