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Phosphoinositide 3-kinase&

Rho kinase

Presented by Shijina N 1ST yr M Pharm

Pharmacology

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Phosphoinositide 3 kinase

IntroductionDiscoveryClassesMechanismFunctionTherapeutical role

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Phosphoinositide 3-kinase

orPI 3-Kinases

orPI3Ks

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Introduction • PI3-Kinases (phosphoinositide 3-kinases, PI3Ks) are family of

enzymes capable of phosphorylating the 3'OH of the inositol ring of phosphoinositides. (PtdIns).

• They are responsible for coordinating a diverse range of cell functions including proliferation, cell survival, degranulation, vesicular trafficking and cell migration, which in turn are involved in cancer.

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Discovery

• Lewis Cantley & colleagues - phosphoinositide kinase associated with the polyoma middle T protein.

• Cantley and colleagues demonstrated that in vivo the enzyme prefers PtdIns(4,5)P2 as a substrate, producing the novel phosphoinositide PtdIns(3,4,5)P3.

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Classes

• Classifications are based on primary structure, regulation, and in vitro lipid substrate specificity.

• Divided into three different classes:•  Class I,Class II, and Class III

• Class I :• Responsible for the production of Phosphatidylinositol

3-phosphate(PI(3)P), Phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P2), and  Phosphatidylinositol(3,4,5)-triphosphate(PI(3,4,5P3)

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• The PI3K is activated by G protein-coupled receptors and tyrosine kinase receptors.

• Class I PI3K are heterodimeric molecules composed of a regulatory and a catalytic subunit.

• They are further divided between IA and IB subsets on sequence similarity.

• Class IA PI3K is composed of a heterodimer between a p110 catalytic subunit and a p85 regulatory subunit.

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• Five variants of the p85 regulatory subunit, designated p85α, p55α, p50α, p85β, and p85γ

• There are also three variants of the p110 catalytic subunit designated p110α, β, or δ catalytic subunit.

• The p85 subunits contain SH2 and SH3 domains

• Class II :• Class II and III PI3K are differentiated from the Class

I by their structure and function.

• Class II comprises three catalytic isoforms (C2α, C2β, and C2γ)

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• Unlike Classes I and III, no regulatory proteins.

• Class II catalyse the production of PI(3)P and PI(3,4)P2 from PI.

• C2α and C2β are expressed through the body, however expression of C2γ is limited to hepatocytes

• The distinct feature of Class II PI3Ks is the C-terminal C2 domain.

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• Class III:• Class III produces only PI(3)P from PI  

• More similar to Class I in structure, as they exist as a heterodimers of a catalytic (Vps34) and a regulatory (Vps15/p150) subunits

• Class III seems to be primarily involved in the trafficking of proteins and vesicles

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Mechanism

• An assorted group of signalling proteins, containing PX domain, pleckstrin homology domains(PH domains), FYVE domains and other phosphoinositide-binding domains, are

recruited to various cellular membranes.

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Function

• Cell growth, proliferation, differentiation, motility, survival and intracellular trafficking.

• Many of these functions relate to the ability of class I PI 3-kinases to activate protein kinase B (PKB, aka Akt) as in the PI3K/AKT/mTOR pathway.

• The p110δ and p110γ isoforms regulate different aspects of immune responses.

• PI 3-kinases are also a key component of the insulin signaling pathway.

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• Cancer :• The class IA PI 3-kinase p110α is mutated in many

cancers.

• Many of the mutations cause the kinase to be more active.

• The PtdIns(3,4,5)P3 phosphatase PTEN that antagonises PI 3-kinase signaling is absent from many tumours.

• PI 3-kinase activity contributes significantly to cellular transformation and the development of cancer

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• Learning & Memory :

• PI3K has also been implicated in Long-term potentiation (LTP). • Whether it is required for the expression or

the induction of LTP is still debated.

• PI3K inhibitors suppressed the induction, but not the expression, of LTP in mouse hippocampal CA1.

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PI 3-Kinases as Protein Kinases

• Many of the PI 3-kinases appear to have a serine/threonine kinase activity in vitro; however, it is unclear whether this has any role in vivo.

• In addition to the class I – class III PI 3-kinases there is a group of more distantly related enzymes that are sometimes referred to as class IV PI 3-kinases.

• The class IV PI 3-kinases family is composed of ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3 related (ATR), DNA-dependent protein kinase(DNA-PK) and mammalian Target Of Rapamycin (mTOR).

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Inhibitors as Therapeutics

• Wortmannin

• LY294002

• As therapeutics :• Too toxic• P110δ isoform specific

inhibitors, IC486068andIC87114, ICOS Corporation.

• GDC-0941 is a highly selective inhibitor of p110α with little activity against mTOR.

 

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Rho kinase

• Introduction• Experimental tools for Rho kinase• Studies at molecular level• Studies at cellular level• Animal studies• Therapeutic targets• Clinical studies

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Introduction

• Rho-kinase has been identified as one of the effectors of the small GTP-binding protein Rho.

• Vascular smooth muscle cell (VSMC) contraction ,actin cytoskeleton organization, cell adhesion and motility, cytokinesis, and gene expressions.

• Rho-kinase upregulates various molecules that accelerate inflammation/oxidative stress, thrombus formation, and fibrosis, whereas it downregulates endothelial nitric oxide synthase.

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• VSMC hypercontraction, stimulates VSMC proliferation and migration, and enhances inflammatory cell motility.

• Pathogenesis of vasospasm, arteriosclerosis, ischemia/ reperfusion injury, hypertension, pulmonary hypertension, stroke and heart failure, and to enhance central sympathetic nerve activity.

• Rho-kinase is an important therapeutic target in cardiovascular medicine.

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• Rho family - Rho (isoforms A to E, and G), Rac (isoforms 1 to 3), Cdc42, and TC10. 

• Rho-kinase α3/ROKα4/ROCK2

• Rho kinase substrates-Myosin-binding subunit (MBS) of myosin phosphatase (MLCPh), ERM (ezrin, radixin, moesin) family, adducin, intermediate filament (vimentin), Na+-H+ exchanger, and LIM-kinase.

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Eeperimental Tools for Rho-Kinase

• 3 major domains

• Catalytic domain• Coiled coil domain• Putative pleckstrin – homology domain

• Rho-kinase activity is enhanced by binding GTP-Rho.

• Fasudil and Y-27632 have been developed and they inhibit Rho-kinase activity in a competitive manner with ATP.

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Studies at Molecular Level

• Rho-kinase plays an important role in mediating various cellular functions, not only VSMC contraction but also actin cytoskeleton organization,cell adhesion and motility,cytokinesis, and, gene expression, all of which may be involved in the pathogenesis of arteriosclerosis/atherosclerosis.

• Arteriosclerosis is a slowly progressing inflammatory process of arterial wall that involves all 3 layers.

• NO antagonizes the vasoconstrictor effect of Rho-kinase through activation of myosin phosphatase.

• Rho-kinase also upregulates tissue factor in the intima and is involved in endothelial contraction that increases endothelial permeability and hence enhances atherosclerosis.

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Animal Studies

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Clinical Studies

• Fasudil is the only clinically available Rho-kinase inhibitor at present, several other Rho-kinase inhibitors are currently undergoing investigation.

• Intravenous form of fasudil is used for the treatment of cerebral vasospasm only in Japan,its oral form is undergoing clinical trials for angina pectoris in Japan and Northern America.

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Thank You