BIOINFORMATICS ANALYSIS OF CYCLIN-DEPENDENT KINASE 5: INSIGHTS TO DRUG DISCOVERY

Ph.DDOCTOR OF PHILOSOPH

Cdk5 activity in the brain - multiple paths of regulation

Cyclin dependent kinase-5 (Cdk5), a family member of the cyclin-dependent kinases, plays a pivotal role in the central nervous system. During embryogenesis, Cdk5 is indispensable for brain development and, in the adult brain, it is essential for numerous neuronal processes, including higher cognitive functions such as learning and memory formation. However, Cdk5 activity becomes deregulated in several neurological disorders, such as Alzheimer's disease, Parkinson's disease and Huntington's disease, which leads to neurotoxicity. Therefore, precise control over Cdk5 activity is essential for its physiological functions. This Commentary covers the various mechanisms of Cdk5 regulation, including several recently identified protein activators and inhibitors of Cdk5 that control its activity in normal and diseased brains. We also discuss the autoregulatory activity of Cdk5 and its regulation at the transcriptional, post-transcriptional and post-translational levels. We finally highlight physiological and pathological roles of Cdk5 in the brain. Specific modulation of these protein regulators is expected to provide alternative strategies for the development of effective therapeutic interventions that are triggered by deregulation of Cdk5. © 2014. Published by The Company of Biologists Ltd

Targeting cyclin dependent kinase 5 in hepatocellular carcinoma

For a long time cyclin dependent kinase 5 (Cdk5) was thought to be of exclusive importance in neuronal cells. However, recently increasing evidence suggests a function of Cdk5 in cancer progression. In the present study, we examined the role of Cdk5 in hepatocellular carcinoma (HCC), a highly chemoresistant cancer with poor prognosis. Consequently, development of novel targeted therapies for HCC is of paramount clinical importance. Analysis of human HCC patient samples showed an increased expression of Cdk5 as compared to normal liver tissues. Functional ablation of Cdk5 significantly decreases HCC cell proliferation and clonogenic survival, and reduces cell motility and invasion. Of note, genetic as well as pharmacologic inhibition of Cdk5 also shows in vivo efficacy in a HCC xenograft mouse model. Investigating the mechanism behind these functional effects revealed that Cdk5 is most active in the nucleus of cells in G2/M phase. In this cell cycle phase DNA damage response takes place, which is affected by Cdk5 inhibition. Furthermore, Cdk5 leads to phosphorylation of Ataxia Telangiectasia Mutated (ATM) and thereby influence its downstream signaling. Importantly, combination of Cdk5 inhibition with different DNA damage inducing chemotherapeutics or the first-line systemic drug sorafenib inhibits synergistically HCC tumor progression. In conclusion, we introduce: 1. Cdk5 as a novel drugable target for treatment of HCC 2. The combination of Cdk5 inhibition and DNA damage agents as a novel therapeutic approach 3. An increased efficacy of sorafenib treatment by combing with Cdk5 inhibitio

Targeting cyclin dependent kinase 5 in hepatocellular carcinoma

For a long time cyclin dependent kinase 5 (Cdk5) was thought to be of exclusive importance in neuronal cells. However, recently increasing evidence suggests a function of Cdk5 in cancer progression. In the present study, we examined the role of Cdk5 in hepatocellular carcinoma (HCC), a highly chemoresistant cancer with poor prognosis. Consequently, development of novel targeted therapies for HCC is of paramount clinical importance. Analysis of human HCC patient samples showed an increased expression of Cdk5 as compared to normal liver tissues. Functional ablation of Cdk5 significantly decreases HCC cell proliferation and clonogenic survival, and reduces cell motility and invasion. Of note, genetic as well as pharmacologic inhibition of Cdk5 also shows in vivo efficacy in a HCC xenograft mouse model. Investigating the mechanism behind these functional effects revealed that Cdk5 is most active in the nucleus of cells in G2/M phase. In this cell cycle phase DNA damage response takes place, which is affected by Cdk5 inhibition. Furthermore, Cdk5 leads to phosphorylation of Ataxia Telangiectasia Mutated (ATM) and thereby influence its downstream signaling. Importantly, combination of Cdk5 inhibition with different DNA damage inducing chemotherapeutics or the first-line systemic drug sorafenib inhibits synergistically HCC tumor progression. In conclusion, we introduce: 1. Cdk5 as a novel drugable target for treatment of HCC 2. The combination of Cdk5 inhibition and DNA damage agents as a novel therapeutic approach 3. An increased efficacy of sorafenib treatment by combing with Cdk5 inhibitio

Signalling mechanisms associated with cyclin-dependent kinase 5 activities during angiogenesis

Stroke is one of the major causes of death and disability in developing countries. It takes place when the blood supply to a fraction of the brain is abruptly interrupted or severely reduced by, for instance, a blood clot. This is known as an ischaemic stroke. A number of studies indicate that deregulation of a set of cell cycle kinases has been implicated in neural death following an ischaemic insult and in neurodegenerative disorders. Overall the cyclin-dependent kinase 5 (Cdk5) and its two activators; the p35/p25 proteins, have been highlighted as critical players in neural survival, and potential mediators of angiogenesis. Hence, a detailed understanding of the mechanisms involving them in the pathogenesis of stroke can provide a platform for therapeutic intervention and potentially enable adoption of strategies to prevent the disease. The aim of the study was to identify key regulatory factors associated with Cdk5 signalling pathway during angiogenesis in a human brain microvascular endothelial cell line (hBMECs). Two cell clones of hBMECs were generated by stable transfection; overexpression of wild-type human Cdk5 and CIP (Cdk5 inhibitory peptide). Cdk5 and p35 protein co-localisation were detected by Immunofluorescence analysis. The findings suggest that the ratio of p35/p25 is altered in favour of p25 by hypoxia. It was able to cleave p35 into a p25, which reduce the migration. The overexpression of Cdk5 increased the ratios of p35/p25 under hypoxic condition. Activation of p35/Cdk5 signalling occurred at the expense of p25-Cdk5, therefore, may have a protective role against endothelial cells apoptosis in hypoxia and positively contribute to preserving cell motility and the proper spatial and temporal control of cytoskeletal dynamics, which is essential for sprout formation (angiogenesis). Here it is shown that the protective roles of CIP are mediated through the down-regulation of HSP-70 and active caspase-3 and up-regulation of the phospho-extracellular signal-regulated kinase (p-ERK). CIP was able to protect hBMECs against apoptosis, and to allow angiogenesis to continue effectively during hypoxia. Therefore, it may be considered a potential future protector therapeutic after stroke and other brain injury

Mesenchymal Stem Cells Loaded with p5, Derived from CDK5 Activator p35, Inhibit Calcium-Induced CDK5 Activation in Endothelial Cells.

The potential use of stem cells as therapeutics in disease has gained momentum over the last few years and recently phase-I clinical trials have shown favourable results in treatment of a small cohort of acute stroke patients. Similarly, they have been used in preclinical models drug-loaded for the effective treatment of solid tumours. Here we have characterized uptake and release of a novel p5-cyclin-dependent kinase 5 (CDK5) inhibitory peptide by mesenchymal stem cells and showed release levels capable of blocking aberrant cyclin-dependent kinase 5 (CDK5) signaling pathways, through phosphorylation of cyclin-dependent kinase 5 (CDK5) and p53. These pathways represent the major acute mechanism stimulating apoptosis after stroke and hence its modulation could benefit patient recovery. This work indicates a potential use for drug-loaded stem cells as delivery vehicles for stroke therapeutics and in addition as anticancer receptacles particularly, if a targeting and/or holding mechanism can be defined

MOLECULAR MECHANISMS IN P25/CDK5-MEDIATED NEUROINFLAMMATION AND SUBSEQUENT NEURODEGENERATION: THERAPEUTIC IMPLICATIONS FOR ALZHEIMER'S DISEASE

Ph.DDOCTOR OF PHILOSOPH

Cyclin dependent protein kinases as drug targets : potential in cardiovascular diseases

Complications of atherosclerosis such as myocardial infarction and stroke are the primary cause of death in Western societies. The development of atherosclerotic lesions is a complex process, including endothelial cell dysfunction, inflammation, extracellular matrix alteration and vascular smooth muscle cell (VSMC) proliferation and migration. Various cell cycle regulatory proteins control VSMC proliferation. Protein kinases called cyclin dependent kinases (CDKs) play a major role in regulation of cell cycle progression. At specific phases of the cell cycle, CDKs pair with cyclins to become catalytically active and phosphorylate numerous substrates contributing to cell cycle progression. CDKs are also regulated by cyclin dependent kinase inhibitors, activating and inhibitory phosphorylation, proteolysis and transcription factors. This tight regulation of cell cycle is essential; thus its deregulation is connected to the development of cancer and other proliferative disorders such as atherosclerosis and restenosis as well as neurodegenerative diseases. Proteins of the cell cycle provide potential and attractive targets for drug development. Consequently, various low molecular weight CDK inhibitors have been identified and are in clinical development. Tylophorine is a phenanthroindolizidine alkaloid, which has been shown to inhibit the growth of several human cancer cell lines. It was used in Ayurvedic medicine to treat inflammatory disorders. The aim of this study was to investigate the effect of tylophorine on human umbilical vein smooth muscle cell (HUVSMC) proliferation, cell cycle progression and the expression of various cell cycle regulatory proteins in order to confirm the findings made with tylophorine in rat cells. We used several methods to determine our hypothesis, including cell proliferation assay, western blot and flow cytometric cell cycle distribution analysis. We demonstrated by cell proliferation assay that tylophorine inhibits HUVSMC proliferation dose-dependently with an IC50 value of 164 nM ± 50. Western blot analysis was used to determine the effect of tylophorine on expression of cell cycle regulatory proteins. Tylophorine downregulates cyclin D1 and p21 expression levels. The results of tylophorine's effect on phosphorylation sites of p53 were not consistent. More sensitive methods are required in order to completely determine this effect. We used flow cytometric cell cycle analysis to investigate whether tylophorine interferes with cell cycle progression and arrests cells in a specific cell cycle phase. Tylophorine was shown to induce the accumulation of asynchronized HUVSMCs in S phase. Tylophorine has a significant effect on cell cycle, but its role as cell cycle regulator in treatment of vascular proliferative diseases and cancer requires more experiments in vitro and in vivo.Ateroskleroosin erilaiset komplikaatiot, kuten sydäninfarkti ja aivohalvaus, kuuluvat yleisimpiin kuolinsyyn aiheuttajiin länsimaissa. Ateroskleroottisten plakkien kehittymistä edistävät monet eri prosessit kuten LDL-kolesterolin kertyminen verisuonten seinämiin, tulehdustila, endoteelisolujen toimintahäiriö, soluväliaineen muutokset ja verisuonten sileiden lihassolujen proliferaatio, jota säätelee useat solusyklin säätelyproteiinit. Sykliinistä riippuvaiset kinaasit (cyclin dependent kinase, CDK) ovat tärkeitä solusyklin säätelijöitä. Ne muodostavat aktiivisen kompleksin sykliinien kanssa ja fosforyloivat kohdeproteiinejaan, jotka osaltaan edistävät solusykliä. Sykliinien lisäksi CDK:n toimintaa säätelevät CDK:n inhibiittoriproteiinit, aktivoiva ja estävä fosforylaatio, proteolyysi ja monet transkriptiotekijät. Solusyklin tarkka säätely on välttämätöntä. Solusyklin häiriintynyt säätely onkin monien sairauksien, kuten syövän, ateroskleroosin, restenoosin ja aivojen rappeumasairauksien, taustalla. Lääkehoidon kohdistaminen solusyklin säätelyproteiineihin on noussut mielen-kiintoiseksi lähtökohdaksi näiden sairauksien hoidossa. Kliinisissä kokeissa tutkitaankin useiden pienimolekyylisten CDK:n estäjien vaikutuksia. Tyloforiini on fenantroindolitsidiinialkaloidi, jonka on osoitettu estävän useiden syöpä-solulinjojen kasvua. Sitä on käytetty perinteisessä intialaisessa lääkinnässä tulehduksellisten sairauksien hoidossa. Tutkimuksen tavoitteena oli selvittää tyloforiinin vaikutus ihmisen napanuoran suonen sileiden lihassolujen (HUVSMC) proliferaatioon, solusykliin ja solusyklin säätelyproteiinien ilmentymiseen. Tavoitteena oli osoittaa rotan soluilla tehtyjen kokeiden tulokset myös ihmisen soluilla. Käytettyjä menetelmiä olivat soluproliferaatiokoe, western blot -analyysi ja virtaussytometria. Soluproliferaatiokoe osoitti, että tyloforiini estää solujen proliferaatiota pitoisuudesta riippuen (IC50 164 nM ± 50). Western blot -analyysia käytettiin tutkittaessa tyloforiinin vaikutusta säätelyproteiinien ilmentymiseen. Tyloforiini vähentää sykliini D1:n ja CDK:n inhibiittori-proteiini p21:n ilmentymistä. Tulokset tyloforiinin vaikutuksesta p53 proteiinin fosforylaatioon eivät oleet yhtenäisiä. Tämän vaikutuksen tutkimiseen tarvitaan lisäkokeita herkemmillä menetelmillä. Virtaussytometriaa käytettiin määrittämään tyloforiinin vaikutus solujen jakautumiseen eri solusyklin vaiheisiin. Tyloforiini aiheutti synkronoimattomien solujen akkumuloitumisen solusyklin S-vaiheeseen. Tyloforiinin vaikutus solusykliin on ilmeinen. Lisätutkimuksia kuitenkin tarvitaan selvittämään sen todellinen merkitys solusyklin säätelyssä sekä syövän ja verisuonten proliferatiivisten sairauksien hoidossa