novel target sites

1. NOVEL TARGET SITES
•POLY(ADP-RIBOSE)POLYMERASE(PARP)
•PEROXISOME PROLIFERATORS ACTIVATOR
RECEPTORS
Physiological functions, pharmacological implications
and therapeutic potential

2. 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 FAMILY
Family 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.
2

3. 3
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.

4. PARP STRUCTURE
PARP 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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5. 5
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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ACTIVITY
The 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 INACTIVATION
Inactivated 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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9. 9
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 DIABETES
The 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 DISEASE
Genetic 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 INHIBITORS
During 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 ARTHRITIS
Mercaptoethylguanidine, 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(AG014
699, 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

16. 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 dyslipidemias
These 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/Synt
hetic)
Known physiologic
actions
Known associated
metabolic disease
or pathogenesis
PPARα Fatty
acids/Fibrate(gemfi
brozil,fenofibrate,cl
ofibrate)
Fatty acid oxidation Dyslipidemia,
Atherosclerosis,
Diabetic
cardiomyopathy
PPARγ Fatty
acid,Eicosanoids/th
iazolidine
dione(pioglitazone,r
osiglitazone),FMO
C-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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REFERENCE
1. Wikipedia.org
2. http://pharmrev.aspetjournals.org/content/54/3/375.full
3. http://intl.pharmrev.org/content/58/4/726.full