NOVEL TARGET SITES

•POLY(ADP-RIBOSE)POLYMERASE(PARP)•PEROXISOME PROLIFERATORS ACTIVATOR RECEPTORS

Physiological functions, pharmacological implications and therapeutic potential

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POLY ADP RIBOSE POLYMERASE

Poly (ADP-ribose) polymerase (PARP) is a family of proteins involved in a number of cellular processes involving mainly DNA repair and programmed cell death.

MEMBERS OF PARP FAMILYFamily comprises 17 members (10 putative). They have all very different structures and functions in the cell.PARP1, PARP2, VPARP (PARP4), Tankyrase-1 and -2 (PARP-5a or TNKS, and PARP-5b or TNKS2) have a confirmed PARP activity.Others include PARP3, PARP6, TIPARP (or "PARP7"), PARP8, PARP9, PARP10, PARP11, PARP12, PARP14, PARP15, and PARP16.

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Poly(ADP-ribose) polymerases (PARP) are a family of enzymes present in eukaryotes, which catalyze the poly(ADP-ribosyl)ation of a limited number of proteins involved in chromatin architecture, DNA repair, or in DNA metabolism, including PARP itself.

Also known as poly(ADP-ribose) synthetase and poly(ADP-ribose) transferase, transfers the ADP-ribose moiety from its substrate, nicotinamide adenine dinucleotide (NAD), to carboxylate groups of aspartic and glutamic residues.

PARP STRUCTUREPARP is composed of four domains of interest: •a DNA-binding domain•a caspase-cleaved domain •an auto-modification domain•a catalytic domain

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FUNCTIONS

The main role is to detect and signal single-strand DNA breaks (SSB) to the enzymatic machinery involved in the SSB repair.

PARP activation is an immediate cellular response to metabolic, chemical, or radiation-induced DNA SSB damage.

Once PARP detects a SSB, it binds to the DNA, and, after a structural change, begins the synthesis of a poly (ADP-ribose) chain (PAR) as a signal for the other DNA-repairing enzymes such as DNA ligase III (LigIII), DNA polymerase beta (polβ), and scaffolding proteins such as X-ray cross-complementing gene 1 (XRCC1).

PARP1 is required for the induction of ICAM-1 gene expression by smooth muscle cells, in response to TNF.

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ACTIVITYThe catalytic domain is responsible for Poly (ADP-ribose) polymerization. This domain has a highly conserved motif that is common to all members of the PARP family.

PAR is synthesized using nicotinamide (NAM) as the leaving group. This leaves a pyrophosphate as the linking group between ribose sugars rather than single phosphate groups.

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PARP INACTIVATIONInactivated by caspase cleavage.

Caspase-3 and caspase-7 are responsible for in vivo cleavage.

Cleavage occurs at aspartic acid 214 and glycine 215, separating PARP into a 24kDA and 89kDA segment.

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PHARMACOLOGICAL IMPLICATIONS AND THERAPEUTIC POTENTIAL

PARP1 is a protein that is important for repairing single-strand breaks ('nicks' in the DNA).

Upon binding to DNA breaks ,activated PARP cleaves NAD(+) into nicotinamide and ADP-ribose and polymerises the latter onto histones,transcription factors ,and PARP itself.

This Poly(ADP-ribosylation)contributes to DNA repair and to the maintenance of genomic stability.

Free radicals ,Reactive Oxygen Species and peroxynitrite causes overactivation of PARP resulting in depletion of NAD(+) and ATP and causing necrosis and organ dysfunction.

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THERAPEUTIC POTENTIAL

PARP becomes rapidly activated in various pathophysiological conditions, & its activation is prolonged & sustained.

Thus PARP inhibitors such as nicotinamide, 3- aminobenzamide, isoquinolones, benzopyrones, phenanthridinones has high benefificial effects in various pathophysiological states.

PARP inhibition plays important role in pathogenesis of several diseases like stroke,MI,circulatory shock,diabetes,neurodegenerative disorders(Parkinsonism and Alzheimers disease)allergy, colitis etc.

PARP activation can also act as a signal that initiates cell death programs.

PARP inhibition is important in inhibiting expression of inflammatory mediators.

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PARP IN PATHOGENESIS OF DIABETESThe primary DNA damage and subsequent decrease in cellular NAD+

levels may activate PARP. The decrease in NAD+ levels is responsible for the loss of cellular ATP and leads to the inhibition of proinsulin biosynthesis, ultimately resulting in the loss of β-cell viability and cell death.

PARP IN INFLAMMATORY BOWEL DISEASEGenetic ablation of PARP gene or pharmacological inhibition of PARP with 3- aminobenzamide may provide resistance to damage induced by mucosal erosion & ulceration associated with increased neutrophil infiltration, lipid peroxidation & progressive weight loss.

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PARP IN ADJUVANT TREATMENT OF CANCER The ionizing radiation, alkylating agents, and topoisomerase inhibitors cause DNA damage and PARP activation. PARP inhibitors have two therapeutic applications in cancer:•As chemo/radiopotentiator.•As a stand-alone therapy for tumour types that are already deficient in certain types of DNA repair mechanisms.

ANTIRETROVIRAL EFFECT OF PARP INHIBITORSDuring HIV infection nicking of DNA strands occur thereby activation of PARP, which may regulate HIV infection at 2 levels: integration & transcription. Thus PARP inhibitors are necessary for the antiviral effect.

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PARP INHIBITORS IN ARTHRITISMercaptoethylguanidine, an anti-inflammatory agent with a combined mechanism of action (inhibition of the inducible isoform of NO synthase, scavenging peroxynitrite, and inhibition of cyclooxygenase) also provided marked beneficial effects in collagen-induced arthritis inhibition of PARP not only prevented the development of arthritis, it also inhibited the progress of established collagen-induced arthritis. GPI 6150, a novel potent PARP inhibitor was found to be effective in attenuating joint swelling, various parameters of inflammation & also effective in adjuvant arthritis. PJ34 another potent PARP inhibitor effective against collagen induced arthritis.

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ADDITIONAL MODE OF ACTION FOR PARP INHIBITORS(RECENT FINDINGS)PARP inhibitors were thought to work primarily by blocking PARP enzyme activity, thus preventing the repair of DNA damage and ultimately causing cell death.

But now, scientists established that PARP inhibitors have an additional mode of action: localizing PARP proteins at sites of DNA damage.

The trapped PARP protein–DNA complexes are highly toxic to cells because they block DNA replication.trapped PARP–DNA complexes are more toxic to cells than the unrepaired single-strand DNA breaks that accumulate in the absence of PARP activity, indicating that PARP inhibitors act as PARP poisons.Two classes of PARP inhibitors:• catalytic inhibitors that act mainly to inhibit PARP enzyme activity and do not trap PARP proteins on DNA• dual inhibitors that both block PARP enzyme activity and act as PARP poison.

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PARP Inhibitors in clinical trials

Drug Phase Use

Iniparib (BSI 201)

III Breast cancer and squamous cell lung cancer.

Olaparib (AZD-2281)

II breast, ovarian and colorectal cancer

Rucaparib(AG014699, PF-01367338)

II metastatic breast and ovarian cancer

Veliparib (ABT-888)

II metastatic melanoma and breast cancer.

MK 4827 I Inhibitor of PARP1 and PARP2

PEROXISOME PROLIFERATOR –ACTIVATED RECEPTORS

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Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear hormone receptors that mediate critical transcriptional regulation of genes associated with lipid homeostasis.

They also play a role in regulation of cellular differentiation,development and metabolism(carbohydrate,lipid,protein) and tumorigenesis of higher organism.

THREE TYPES OF PPARS ARE DESIGNATED AS: PPARα (alpha), PPARβ/δ (delta/beta) PPARγ (gamma)

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STRUCTURE OF PPAR

Like all other nuclear receptors PPARs also have an N- terminal having DNA binding domain (DBD) with zinc fingers and the C terminal with the ligand binding domain (LBD). The PPAR LBD consists of 12 α- helices that form the characteristic 3- layer antiparallel α- helical sandwich with a small 4 stranded sheet. This structure delineates a large Y- shape hydrophobic pocket, the ligand- binding domain.

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PHARMACOLOGICAL FUNCTIONS OF PPAR PPARα Regulation of energy homeostasis.

Activates fatty acid catabolism, stimulates gluconeogenesis and ketone body synthesis, and is involved in the control of lipoprotein assembly(in liver) Stimulates heme synthesis & cholesterol catabolism.

Attenuates inflammatory responses & participates in the control of amino acid metabolism & urea synthesis.

Increased fatty acid oxidation by activated PPAR α lowers circulating triglyceride levels, liver & muscle steatosis, & reduces adiposity which improves insulin sensitivity.

Gemfibrozil,Clofibrate and Fenofibrate(fibrate drugs)that are used to treat hypertriglyceridemia are activators of PPAR α.

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PPAR β/δ necessary for placental & gut development & is also involved in the control of energy homeostasis by stimulating genes involved in fatty acid catabolism & adaptive thermogenesis.

It also has an important role in control of cell proliferation, differentiation & survival & is involved in tissue repair.

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PPAR γ Is a pivotal actor in adipose tissue differentiation & in maintaining adipocyte specific functions such as lipid storage in white adipose tissue & energy dissipation in brown adipose tissue.It is also involved in glucose metabolism by improving insulin sensitivity.Like PPARα, PPARγ activation seems to limit inflammation, also has a role in limiting atherosclerosis and/ or diabetes.

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THERAPEUTIC POTENTIALS OF PPAR

PPAR agonists in the treatment of dyslipedmia

Fibrates were introduced for treatment of hyperlipidemia. Trials with fibrates have shown a reduction in coronary heart disease (CHD) risk through modification of atherogenic dislipidemia. PPARα potentiates fatty acid oxidation in the liver, heart, kidney, and skeletal muscle. Activation of PPARα leads to an increase in expression of lipoprotein lipase and apolipoprotein A-V (apoA-V) and to a decrease in hepatic apoC-III. Eg: bezafibrate, gemfibrozil or combination of fibrates with statins.

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PPAR agonists in the management insulin resistance & type 2 diabetes In patients with diabetes, PPAR agonists restore insulin sensitivity and glucose homeostasis. PPARγ agonists promote adipocyte differentiation, and they promote free fatty acid uptake & storage in subcutaneous adipose rather than visceral adipose tissue. This reduce free fatty acid levels, with associated reductions in insulin resistance.Eg: pioglitazone, rosiglitazone

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PPAR agonists in vascular & metabolic disorders

“Metabolic syndrome: The metabolic syndrome describes a complex of metabolic abnormalities, including obesity, diabetes, hypertension, and dyslipidemia, to which insulin resistance is central. “PPAR α and γ agonists as ideal agents for managing the metabolic syndrome. Eg:Thiazolidinediones improve glycemic control Fibrates improve a range of atherogenic dyslipidemiasThese agents also influence many other components of metabolic syndrome, including hypertension, inflammation, and vascular dysfunction and remodeling.

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Nuclear Receptor Ligands(Physiologic/Synthetic)

Known physiologic actions

Known associated metabolic disease or pathogenesis

PPARα Fatty acids/Fibrate(gemfibrozil,fenofibrate,clofibrate)

Fatty acid oxidation Dyslipidemia, Atherosclerosis, Diabetic cardiomyopathy

PPARγ Fatty acid,Eicosanoids/thiazolidine dione(pioglitazone,rosiglitazone),FMOC-L-Leucine

Adipogenesis,Lipid storage

Insulin resistance, Obesity,Metabolic Syndrome

PPAR β/δ Fatty acid Fatty acid oxidation, Energy expenditure

Dyslipidemia, Atherosclerosis, Obesity

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REFERENCE1. Wikipedia.org

2. http://pharmrev.aspetjournals.org/content/54/3/375.full

3. http://intl.pharmrev.org/content/58/4/726.full