BY

Yogesh K.ChaudhariM Pharm (Pharmacology)

Mumbai University,Mumbai

GUIDED BY

Dr. Vinod GuptaAssistant Manager (Tata Consultancy Services, Mumbai)

Ph. D (Pharmacogenomics) (IITBombay, Mumbai)M. Pharm (Pharmacology) (ICT, Mumbai)

Advanced Pharmacology NOVEL DRUGS TARGET

Pathogenesis

• Greek pathos, "disease", and genesis, "creation".

• It means step by step development of a disease and the chain of events leading to that disease due to a series of changes in the structure and /or function of a cell/tissue/organ being caused by a microbial, chemical or physical agent.

• There are several chemical weapons secreted by pathogens which include enzymes, toxins, growth regulators and polysaccharides.

Metabolic Disease• Metabolism is the sum of the chemical processes and inter-conversions that take place in the cells and the

fluids of the body.

• This includes :• absorption of nutrients and minerals, • breakdown and buildup of large molecules, • production of energy from these chemical reactions.

• Virtually every chemical step of metabolism is catalyzed by an enzyme.

• Disorders of these enzymes that result from abnormalities in their genes are known as inborn errors of metabolism.

• Enzymes are often linked in multistep pathways, such that the product of one reaction becomes the substrate for another.

• When all the enzymes in a pathway are functioning properly, intermediates rarely build up to high concentrations.

Enzyme Defects Cause Metabolic Disorders

• The causes of enzyme defects are genetic mutations that:

• affect the structure or regulation of the enzyme protein or

• create problems with the transport, processing, or binding of cofactors.

Enzyme Defects Cause Metabolic Disorders• Enzymes are proteins that control the rate of chemical reactions in the cell. • Enzymes function by binding to the molecules to be reacted (called substrates or

precursors) and altering their chemical bonds, producing products.

• The binding occurs on the surface of the enzyme, usually in a pocket or groove, called the active site----three-dimensional structure that is required for binding substrates.

• The consequences of an enzyme deficiency is due to cellular chemistry:• either a reduction in the amount of an essential product, the buildup of a toxic intermediate, or • production of a toxic side-product.

• Most metabolic disorders are inherited as autosomal recessive conditions- due to inactivating, or "loss-of-function," mutations.

Approaches to Treatment• Treatment approaches for metabolic disorders include:

• (a) modifying the diet to limit the amount of a precursor that is not metabolized properly;

• (b) using cofactors or vitamins to enhance the residual activity of a defective enzyme system;

• (c) using detoxifying agents to provide alternative pathways for the removal of toxic intermediates;

• (d) enzyme replacement, to provide functional enzymes exogenously (from the outside);

• • (e) organ transplantation, which in principle allows for endogenous (internal) production of

functional enzymes; and

• (f) gene therapy, or replacement of the defective gene.

Why enzymes are target for novel drug??????• The discovery and exploitation of new drug targets is a key focus for both

the pharmaceutical industry and academic biomedical research.

• Enzymes offer unique opportunities for drug design that are not available to:

• cell surface receptors,• nuclear hormone receptors, • ion channels, • transporters, and • DNA

Physiological functions, pharmacological implications and therapeutic potential

• Physiology - science that describes how organisms FUNCTION

• Pharmacological implications - are effects or consequences that may happen in the future.

• Therapeutic potential- known as well under research use

1] ROCK (Rho-associated protein kinase)

Physiological functions, pharmacological implications

and therapeutic potential

ROCK (Rho-associated protein kinase)

• belongs to AGC family of the Serine-threonine kinases (is a kinase enzyme that phosphorylates the OH group of serine or threonine)—Phosphorylation

• Regulates the shape and movement of cells by cytoskeletal

• Present in mammals, invertebrates, zebrafish and chicken

• Subdivided in two isoforms ------ ROCK1 (β) and ROCK2(α)

• ROCK1 - mainly expressed in the lung, liver, spleen, kidney and testis

• ROCK2 - distributed mostly in the brain and heart

Structure of ROCK• Has structure similar to myotonic dystrophy kinase

• Composed of :• N-terminal catalytic domain, • central coiled-coil domain, and • C-terminal domain interrupted by a Cys-rich region

Homologues of ROCKS

• ROCK1 and ROCK2, have high homology (existence of shared ancestry between a pair of structures, or genes, in different species).

• They have 65% amino acid sequences in common and 92% homology within their kinase domains.

• ROCKs are homologous to other metazoan kinases such as myotonic dystrophy kinase (DMPK), DMPK-related cell division control protein 42 (Cdc42)-binding kinases (MRCK) and citron kinase.

• All of these kinases are composed of a N-terminal kinase domain, a coiled-coil structure and other functional motifs (structural characteristics) at the C-terminus.

(Rho-associated protein kinase)

Physiological function

1. ROCK is a key regulator of actin organization

• Different substrates can be phosphorylated by ROCKs, including LIM kinase, myosin light chain (MLC) and MLC phosphatase

• These substrates, once phosphorylated, regulate actin filament organization and contractility

A. Effect on Actin Filament

ROCK phosphorylates and activates LIM kinase

phosphorylates ADF/cofilin-

inactivating its actin-depolymerization activity

stabilization of actin filaments and an increase in their numbers

over time actin monomers that are needed to continue actin polymerization for migration become limited

increased stable actin filaments and the loss of actin monomers contribute to a reduction of cell migration

B. Cellular Contractility• ROCK regulates cell migration by promoting cellular contraction and thus cell-substratum contacts.

ROCK increases the activity of the motor protein myosin II by two different mechanisms

Several bundled and active myosins, which are a synchronously active on several actin filaments, move actin filaments against each other

Resulting in the net shortening

of actin fibres.

1)phosphorylation of the

myosin light chain (MLC) increases the myosin

II ATPase activity

ROCK inactivates

MLC phosphatas

e

Leading to increased levels of

phosphorylated MLC

2)

In both above cases, ROCK activation by Rho induces the formation of actin stress fibers, actin filament bundles of opposing polarity, containing myosin II, tropomyosin, and MLC-kinase, and consequently of focal contacts, which are immature integrin-based adhesion points with the extracellular substrate.

Various agonists (neurotransmitters, hormones, etc.) bind to specific receptors to activate contraction in smooth muscle.

Increase phospholipase C activity via coupling through a G protein

Phospholipase C produces two potent second messengers from the membrane: diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP3).

IP3 binds to specific receptors on the sarcoplasmic reticulum, causing release of activator calcium (Ca2+).

DG along with Ca2+ activates PKC, which phosphorylates specific target proteins

PKC has contraction-promoting effects such as phosphorylation of Ca2+ channels or other proteins that regulate cross-bridge cycling.

Activator Ca2+ binds to calmodulin, leading to activation of myosin light chain kinase (MLC kinase).

This Ca2+-sensitizing mechanism is initiated at the same time that phospholipase C is activated, and it involves the activation of the small GTP-binding protein RhoA

Upon activation, RhoA increases Rho kinase activity, leading to inhibition of myosin phosphatase

This promotes the contractile state, since the light chain of myosin cannot be dephosphorylated.Regu

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Physiological function

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RhoA-GTP stimulates the phospholipid phosphatase activity of PTEN (phosphatase and tensin homologue), a human tumor suppressor protein. This stimulation seems to depend on ROCK. In this way, PTEN is important to prevent uncontrolled cell division as is exhibited in cancer cells.

ROCK plays an important role in cell cycle control, it seems to inhibit the premature separation of the two centrioles in G1, and is proposed to be required for contraction of the cleavage furrow, which is necessary for the completion of cytokinesis.

ROCKs also seem to antagonize the insulin signaling pathway, resulting in a reduction of cell size and influence cell fate.

ROCKS play a role in membrane blebbing, a morphological change seen in cells committed to apoptosis.

ROCKs regulate cell-cell adhesion: Loss of ROCK activity seems to lead to loss of tight junction integrity in endothelial cells

Auto-inhibition of ROCKS• ROCK activity is regulated by the disruption of an intramolecular autoinhibition.

• The kinase activity is inhibited by the intramolecular binding between the C-terminal cluster of RBD (Rho-binding domain) domain and the PH domain to the N-terminal kinase domain of ROCK.

• Thus, the kinase activity is off when ROCK is intramolecularly folded.\

• The kinase activity is switched on when Rho-GTP binds to the Rho-binding domain of ROCK, disrupting the auto-inhibitory interaction within ROCK, which liberates the kinase domain.

• ROCK can also be regulated by lipids, in particular arachidonic acid, and protein oligomerization, which induces N-terminal transphosphorylation.

Substrate For ROCKS

ROCK and Diseases• Recent research has shown that ROCK signaling plays an important role in many diseases

including:• diabetes,

• neurodegenerative diseases such as• Parkinson's disease & • amyotrophic lateral sclerosis,

• pulmonary hypertension • cancer

• It has been shown to be involved in causing tissue thickening and stiffening around tumours in a mouse model of skin cancer, principally by increasing the amount of collagen in the tissue around the tumour.

Cardiovascular Diseases• Dysfunction in the regulation of vascular tone is a prominent cause of the

cardiovascular pathologies hypertension, hypertrophy and fibrosis.

• Hypertension is characterized by an increase in systemic blood pressure that results from increased vasculature resistance, desensitization of vessels to vasoactive molecules and fibrosis.

• Hypertensive mouse models manifest enhanced RhoA-ROCK signaling whereas inhibition of ROCK activity in these mouse models as well as in spontaneous hypertensive rats normalizes their blood pressure.

• ROCK activity is also implicated in the regulation of vasoactive molecules.

• ROCK inhibited mice have reduced sensitivity to angiotensin II, a peptide hormone that promotes vasoconstriction .

• ROCK activation reduces endothelial nitric oxide (NO) synthase expression which is an important enzyme in the catalysis of L-arginine to produce NO, a potent vasodilator.

• Deregulated vaso-tone, as in hypertension, results in blood vessel stenosis and ultimately ischemic episodes that, in severe cases, cause tissue fibrosis.

• ROCK1 inhibitor in mice showed decreased myocardial fibrosis as well as decreased expression of the fibrotic markers transforming growth factor-β and type III collagen.• • Inhibition of ROCK activity with small-molecule inhibitors prevents

morphological changes in endothelial cells that accompany ischemic injury .

Cardiovascular Diseases– contd…..

Cancer• Cancer encompasses a group of diseases, each characterized by key phenotypes.

• Transformation of benign tumours to metastatic cancers results from several stochastic events in hyperplasic cells leading to:• loss of tissue cohesion,• increased cell motility and • invasion as well as the growth of metastatic cell colonies at locations foreign to the cells origin.

• Amplification of ROCK signaling enhances both:• cell migration and • Proliferation

• Therefore, it is conceivable that abberrant ROCK expression and activation contribute to cancer development.

• ROCK levels are elevated in testicular, bladder, esophageal cancers and invasive sarcoma cell lines.

Cancer• Several of the ROCK substrates are prominent players in the development of

cancer and its associated phenotypes.

• For example: • tumor suppressor phosphatase and • tensin homologue (PTEN), which is frequently inactivated in melanoma by

ROCK phosphorylation.

• ROCK signaling may fuel JNK (c-Jun N-terminal Kinase) signaling to promote melanoma.

• Signalling in skin cancers recently demonstrated that ROCK expression in mouse epidermis induces hyperplasia and tumor formation.

• Further evaluation of the role of ROCK signalling in cancer is required.

ROCK Inhibitors• Researchers are developing ROCK inhibitors for treating disease.

• For example, such drugs have potential to prevent cancer from spreading by blocking cell migration, stopping cancer cells from spreading into neighbouring tissue.

• To elucidate the physiological roles of Rho-kinase, small molecule inhibitors have been developed and investigated in various cell types and animal models.

• Fasudil (HA-1077) and Y-27632 have been broadly used as Rho-kinase selective inhibitors and function in an ATP-competitive manner.

• Fasudil, composed of the isoquinoline ring and the pendant ring of the seven-membered homopiperazine, is used clinically for cerebral vasospasm after subarachnoid hemorrhage in Japan.

• Y-27632 was identified by its ability to inhibit phenylephrine induced contraction of a rabbit aortic strip and contains a 4- aminopyridine ring.

• Rho-kinase (ROCK2)-Fasudil complex revealed the inhibitors cause an induced-fit conformational change to increase contacts with Rho-kinase phosphate loop, which may account for their specificity.

• Several pharmaceutical companies are investing in the development of Rho-kinase inhibitors for the treatment of certain diseases, such as glaucoma.

• ROCK2-selective inhibitor SLx-2119 is now in clinical trials (Kadmon Corporation).

ROCK Inhibitors

ROCK inhibitors in Glaucoma• Modulating ROCK activity within the aqueous humor outflow pathway using

selective inhibitors could achieve very significant benefits for the treatment of increased intraocular pressure in patients with glaucoma.

• ROCK and Rho GTPase inhibitors can increase aqueous humor drainage through the trabecular meshwork, leading to a decrease in intraocular pressure.

• Inhibitors of both ROCK and Rho GTPase have been also shown to enhance ocular blood flow, retinal ganglion cell survival and axon regeneration.

• These properties of the ROCK and Rho GTPase inhibitors indicate that targeting the Rho GTPase/ROCK pathway with selective inhibitors represents a novel therapeutic approach aimed at lowering increased intraocular pressure in glaucoma patients.

2] Phosphoinositide 3-kinase

Physiological functions, pharmacological implications and therapeutic potential

Phosphoinositide 3-kinase (PI3K)• Also called as phosphatidylinositide 3-kinases, phosphatidylinositol-3-kinases, PI 3-

kinases, PI(3)Ks, PI-3Ks.

• PI3K(s) are a family of enzymes involved in cellular functions such as:• cell growth,• proliferation, • differentiation,• motility, • survival and • intracellular trafficking

• PI3Ks are a family of related intracellular signal transducer enzymes capable of phosphorylating the 3 position hydroxyl group of the inositol ring of phosphatidylinositol (PtdIns).

Classification of PI3K• The phosphoinositol-3-kinase family is divided into four different

classes: •Class I, •Class II, •Class III, and •Class IV

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

Class I• Class I PI3Ks are responsible for the production of phosphatidylinositol 3-

phosphate (PI(3)P), phosphatidylinositol (3,4)-bisphosphate(PI(3,4)P2), and phosphatidylinosito (3,4,5)-trisphosphate (PI(3,4,5)P3).

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

Class II• Class II is differentiated from the Class I by their structure and function.

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

• This domain lacks critical Asp residues to coordinate binding of Ca2+, which suggests class II PI3Ks bind lipids in a Ca2+-independent manner.

• Class II catalyse the production of PI(3)P from PI and PI(3,4)P2 from PIP; however, little is known about their role in immune cells.

Class III• Class III produces only PI(3)P from PI but are more similar to Class I in structure.

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

• Evidences are there to show that they are able to contribute to the effectiveness of several process important to immune cells.

Class IV• A group of more distantly related enzymes are sometimes referred to as class IV PI 3-

kinases.

• It 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).

• They are protein serine/threonine kinases.

Structure of PI3K

PhysiologyAKT binds directly to 1] PtdIns(3,4,5)P3 and PtdIns(3,4)P2 and 2] phosphoinositide-dependent

kinase-1 (PDK1), which are produced by activated PI 3-kinase

Results in translocation of AKT/PKD1 to the plasma membrane, as they are restricted to the plasma membrane

Interaction of activated PDK1 and AKT allows AKT to become phosphorylated by PDK1 on threonine 308, leading to partial activation of AKT

Full activation of AKT occurs upon phosphorylation of serine 473 by the TORC2 complex of the mTOR protein kinase

The "PI3-k/AKT" signaling pathway has been shown to be required for an extremely diverse array of cellular activities - most notably cellular proliferation and survival.

Metabolism of PI3K

Functions of PI3K

Functions of PI3K• PI3-kinases have been linked to an extraordinarily diverse group of cellular

functions, including 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.

• Hence there is great interest in the role of PI 3-kinase signaling in diabetes mellitus.

Therapeutic potential

• PI3Ks interact with the insulin receptor substrate (IRS) to regulate glucose uptake through a series of phosphorylation events.

• Absence of these interaction will lead to diabetes.

• The class IA PI 3-kinase p110α is mutated in many cancers.

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

Inhibitors of PI3K• First Generation:• The inhibitors being studied inhibit one or more isoforms of the class I PI3Ks.

• They are also being considered for inflammatory respiratory disease.

• Wortmannin (steroidal metabolite of fungi): The concentration of wortmannin required to inhibit PI3Ks ranges from 1–100 nM.

• Irreversible inhibition of lipid and serine kinase activity of class 1 and class III PI 3-kinases occurs by covalent modification of the catalytic sites.

• Nucleophilic residues equivalent to Lys802 are present in all PI3Ks and protein kinases, such as myosin light chain kinase, protein kinase A, and DNA-dependent protein kinase catalytic subunit as well as target of rapamycin (TOR)-related proteins, and probably account for the poor selectivity of wortmannin.

• Bioflavanoid-Related Compounds

• The bioflavanoid quercetin (structure 2) was initially shown to effectively inhibit PI3K with an IC50 of 3.8 μM but has poor selectivity, inhibiting related enzymes such as PI 4-kinase and several tyrosine and serine/threonine kinases.

• LY294002 (structure 3) completely inhibits PI3K at 100 μM.

• Both of these compounds are competitive inhibitors at the ATP binding site of PI3K, but unlike quercetin, LY294002 has no detectable effect on other ATP-requiring enzymes such as protein kinases and PI 4-kinase. • • Neither wortmannin nor LY294002 exhibit any degree of selectivity for individual PI3K

isoforms

Inhibitors of PI3K

Structure of First Generation inhibitors

Isoform Selective Inhibitors: The Second Generation

• SF1126 is the first PI-3 kinase inhibitor to enter paediatrics cancer clinical trials via the (New Approaches to Neuroblastoma Therapy) NANT consortium.

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