PARP INHIBITORS

(POLY ADP-RIBOSE POLYMERASE)

Presented By
Mojdeh Yousefian
Ph.D student of Medicinal Chemistry
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Agents that Damage DNA

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Types of DNA repair pathways

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 DNA Repair

• PARP1 is An enzyme involved in various activities

in response to DNA damage.
• Although the structures of the BRCA1 and BRCA2 genes are very different, at least
some functions are interrelated. The proteins made by both genes are essential for
repairing damaged DNA.

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 Breast Cancer

• Breast cancer is one of the most common cancers

in women, accounting for over 20% of all cancer
cases.
• Among them, 5%-10% of breast cancer cases are
ascribed to hereditary predisposition.
• 1 in 8 women (13%) in the United States suffers
from Breast cancer. It is the cancer that forms in
tissues of the breast, usually the ducts (tubes that
carry milk to the nipple) and lobules (glands that
make milk).
• Breast cancer occurs in both men and women,
although male breast cancer is rare.
• A latest study appeared in the Journal of the
American Medical Association shows that
women carrying the BRCA1 and BRCA2 genes
should consider preventive surgery because they
are at a very high risk for breast and ovarian
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cancers.
• Dr. Susan M. Domcheck of the University of Pennsylvania, School of Medicine
in Philadelphia led a study.
• Dr Susan and colleagues studied nearly 2500 genetically at-risk women who
underwent surgeries with those who preferred frequent cancer screenings.
• The screenings consisted of yearly mammograms, MRIS, trans-vaginal
ultrasounds every 6-12 months, along with CA-125 blood testing.

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 Researchers found that:

• Over three years of follow-up, women (10%) who underwent preventive breast
removal didn’t get breast cancer. However, 7% of BRCA-positive women who kept
their breasts developed breast cancer.
• BRCA-positive women (38%) who chose to have their ovaries and fallopian
tubes removed had a significantly lower risk of both breast and ovarian cancer
compared to women who did not opt for the surgery.
• Women who have either of the two BRCA genes have a lifetime risk of 56% to 84%
of developing breast cancer.
• Women with the BRCA1 gene have a 36% to 63% risk of ovarian cancer.
• Women with BRCA2 gene have a 10% to 27% risk.

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 BRCA1 and BRCA2

BRCA1 and BRCA2 are well-established tumor suppressor gene, which is

frequently mutated in familial breast and ovarian cancers.
• The gene product of BRCA1 and BRCA2 functions in a
number of cellular pathways:
1. maintain genomic stability
including DNA damage-induced cell cycle
checkpoint activation
2. DNA damage repair
3. Protein ubiquitination
4. chromatin remodeling
5. Transcriptional regulation
6. apoptosis

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 Protein structure of BRCA1
The human BRCA1 protein consists of four major protein domains.
The BRCA1 amino terminus contains a RING domain that associates with BRCA1-associated
RING domain protein 1 (BARD1) and a nuclear localization sequence (NLS). The central region
of BRCA1 contains a CHK2 phosphorylation site on S988. The carboxyl terminus of BRCA1
contains: a coiled-coil domain that associates with partner and localizer of BRCA2 (PALB2), a
SQ/TQ cluster domain (SCD) that contains approximately ten potential ataxia-telangiectasia
mutated (ATM) phosphorylation sites and spans amino acid residues 1280–1524; and a BRCT
domain that binds ATM-phosphorylated abraxas, CtBP-interacting protein (CtIP) and BRCA1-
interacting protein C-terminal helicase 1 (BRIP1).

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 Protein structure of BRCA2

The N terminus of BRCA2 binds PALB2 at amino acids 21–39. BRCA2 contains
eight BRC repeats between amino acid residues 1009 and 2083 that bind RAD51. The
BRCA2 DNA-binding domain contains a helical domain (H), three oligonucleotide
binding (OB) folds and a tower domain (T), which may facilitate BRCA2 binding to
both single-stranded DNA and double-stranded DNA46. This region also associates
with deleted in split-hand/split-foot syndrome (DSS1)42, 44, 45. The C terminus of
BRCA2 contains an NLS and a cyclin-dependent kinase (CDK) phosphorylation site at
S3291 that also binds RAD51

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Poly-ADP-ribose polymerase (PARP) is a family
of enzymes composed of 17members. PARP is found
in the cell’s nucleus. It is involved in a number of
cellular processes such as cell death, transcriptional
regulation, inflammation, chromatin modification
and DNA repair.
PARPs are one of the important components of base
excision repair pathway for single strand DNA
breaks.
PARP-1 is the best characterized member of this
family. It is the primary enzyme involved in DNA
repair process, whereas PARP2 and PARP3 are
involved to a lesser extent.
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 Structural and functional organization of PARP1

The PARP1 structure is composed of three main functional domains:

• N-terminal DNA-binding domain
• internal automodification domain
• C-terminal catalytic domain

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Mechanism of action of PARP1

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Mechanism of action of BRCA1 and BRCA2

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Mechanism of tumor-specific synthetic lethality in homologous
recombination deficient tumors treated with PARP inhibitors

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Regulating PARP-1 through post-translational modifications
• ADP-ribosylation: Auto-ADP-ribosylation of PARP-1, especially extensive
autopoly(ADP-ribosyl)ation in response to DNA damage.
• Phosphorylation:PARP-1 is phosphorylated by ERK1/2 at Ser 372 and Thr
373 and these modifications are required for maximal PARP-1 activation after
DNA damage.
• Acetylation: PARP-1 are modified by acetylation, and evidence is
accumulating for the specific roles for these acetylation events in stress-
related responses.
• Ubiquitylation and SUMOylation: Recent studies have begun to elucidate
roles for the polypeptide protein modifiers ubiquitin and SUMO as well as the
E3 ligases that promote their covalent attachment, in the stress-related
function of PARP-1.

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 Why is PARP1 regarded as an important
molecular target for designed antitumor agents?
• Carcinogenesis can be caused by PARP1-dependent deregulation of the factors
involved in the cell cycle and mitosis, as well as the factors regulating the
expression of the genes associated with the initiation and development of tumors.
• The relationship between PARP1 and the NF-kB factor has been revealed. PARP1
was found to co-regulate the NF-kB activity and lead to increased secretion of pro-
metastatic cytokines. The NF-kB signaling cascade is known to be essential for
tumor growth.
• PARP1 is known to control the expression of heat shock protein 70 (HSP70),
which significantly contributes to the survival of tumor cells and their resistance to
antitumor agents.
• PARP1 interacts with the p21 protein, which controls the cell cycle. The p21
protein directly interacts with PARP1 during DNA repair, and p21 knockdown
leads to an increased enzymatic activity of PARP1.
• In prostate cancer cells expressing the androgen receptor (AR), PARP1 is recruited
to the sites of AR localization and stimulates AR activity.
• PARP1 are involved in the estrogen-dependent regulation of gene expression in
breast cancer (BC).
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 EARLY DEVELOPMENT OF PARP INHIBITORS

• PARP inhibitors were initially developed for the purposes of elucidating

PARP function.
• In 1971, Clark et al. first described nicotinamide itself and its 5-methyl
derivative as inhibitors of poly ADP-ribose formation after DNA damage in
cells.
• The first generation of typical PARP1 inhibitors, nicotinamide analogues,
was developed about 30 years ago based on observations that nicotinamide,
a second product of the PARP1-catalyzed reaction, causes moderate
inhibition of the reaction.

Nicotinamide 5- methyl nicotinamide

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 Inhibitors directed to one of the three pockets are expected to behave as NAD+
Competitive PARP inhibitors.

Nicotinamide
Binding pocket

Ribose
Binding pocket Adenosine
Binding pocket

The three binding sites for NAD+

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• A pivotal study in the 1980s established that the PARP inhibitor, 3-
aminobenzamide (3-AB) a nicotinamide analogue was able to enhance the
cytotoxic effects of DNA methylating agents thus supporting for the first time a
chemopotentiating role for PARP inhibition.
• These early nicotanimide analogue-based PARP inhibitors however lacked
antitumor activity and have been replaced by more potent and specific 3rd
generation lead compounds through the process of empirical compound
screening and structural refining.

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 Second generation inhibitors

Nicotinamide Quinazolinone

1,5-dihydroisoquinoline 4-amino-1,8-naphthalimide 2-nitro-1,8- PD128763 NU1025

IC50 = 390 nМ IC50 = 180 nМ phenanthridinone IC50 = 420 nМ IC50 = 400 nМ
IC50 = 350 nМ

PJ-34
IC50 = 20 nM

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 Structures of third-generation PARP1 inhibitors

Rucaparib E7016
INO-1001
(GPI21016)

CEP8983 BMN673 Niraparib

Veliparib Olaparib

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• Olaparib (AZD-2281, trade name Lynparza) is an FDA-approved targeted therapy for
cancer, developed by KuDOS Pharmaceuticals and later by AstraZeneca.
• It is a PARP inhibitor, inhibiting poly ADP ribose polymerase (PARP), an enzyme
involved in DNA repair.
• It acts against cancers in people with hereditary BRCA1 or BRCA2 mutations, which
include some ovarian, breast, and prostate cancers.
• In December 2014 , olaparib was approved for use as a single agent by the EMA and
the FDA at a recommended dose of 400 mg taken twice per day.
• Side effects Side effects include gastrointestinal effects such as nausea, vomiting,
and loss of appetite; fatigue; muscle and joint pain; and low blood counts such as
anemia, with occasional leukemia. 33
 OLAPARIB as Single Therapy

• PARPis has been their relatively low side effect profile, especially in comparison to
traditional chemotherapies.
• This low side effect profile has also made them appealing agents to consider as single
therapy.
• Inhibiting PARP1 alone may be sufficient to cause tumor cell death and avoid the toxic
effects of chemotherapy and radiation. PARP inhibitors killed BRCA2 deficient cells at doses
that were nontoxic to normal cells.

• Olaparib has few of the adverse effects of

conventional chemotherapy, inhibits PARP, and
has antitumor activity in cancer associated with
the BRCA1 or BRCA2 mutation.

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• A phase I study of single agent olaparib was Conducted.A total of 50 ovarian
cancer patients with BRCA mutations were enrolled.
• 20 patients had CR or PR by response evaluation criteria in solid tumors
(RECIST) and 3 patients had been SD for longer than 4 months, resulting
in a clinical benefit rate of 46% (23/50).
• Olaparib also showed activity in BRCA mutation positive breast cancer.
• In a phase II trial of a single agent olaparib, 54 patients with BRCA mutation
positive breast cancer were randomized to receive either 100 mg or 400 mg of
olaparib twice per day. The overall response rate (ORR) was 41%.

• This study provided further evidence of PARP inhibitor

activity in BRCA associated tumor.
• The potential of selectively targeting tumor cells without
affecting the normal cells seemed possible in BRCAassociated
tumors using single agent PARP inhibitor.
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 Olaparib in combination
with cytotoxic therapy

• Preclinically, PARP inhibitors have enhanced the effects of various

chemotherapies. PARP1 elicited resistance to the effect of methylating agents,
which is negated by the presence of a PARP inhibitor.
• Olaparib in combination with paclitaxel was also investigated in a phase I/II trial
in TNBC. 19 patients, most of whom had received prior taxane therapy, were
treated with daily 200 mg of olaparib given orally in combination with paclitaxel
90 mg/m intravenous weekly for 3 out of 4 weeks. 37% of patients had
confirmed PRs.
• Olaparib is also being combined with DNA damaging agents, such as topotecan,
doxorubicin, carboplatin,, irinotecan, dacarbazine, and gemcitabine and cisplatin
as well as with antiangiogenesis agents.
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• PARP1, one of the key components of the enzymatic machinery involved in
DNA repair, is activated by DNA strand breaks and participates in BER
pathway.
• At the same time, increased PARP1 expression is observed in melanomas and
lung and breast tumors.
• PARP inhibitors are one of the most exciting new class of targeted treatments to
have entered the clinic in recent years.
• PARPi have shown significant activity in BRCA-associated breast, ovarian, and
other cancers.
• Almost all existing PARP1 inhibitors are nicotinamide mimetics, i.e. aimed at
binding to the catalytic domain of PARP1 and competition with NAD+.

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• The third generation inhibotors are more potent, because the Crystallization
of PARP1 inhibitors showed that the carboxamide group forms several
important hydrogen bonds with Ser904-OG and Gly863-N in the catalytic
domain of PARP1, which improves the interaction between the heterocycle of
these inhibitors and the protein.
• Olaparib (AZD-2281, trade name Lynparza) is an FDA-approved targeted
therapy for cancer In December 2014, developed by KuDOS Pharmaceuticals
and later by AstraZeneca.
• PARP1 inhibitors in some cases increase the efficacy of DNA-alkylating
agents (e.g., Temozolomide) and topoisomerase I inhibitors (e.g., topotecan), as
well as ionizing radiation. PARP1inhibitors are also effective in
radiosensitization of tumor cells.
• Along with the synergistic effect of PARP inhibitors and other DNA-damaging
antineoplastic agents, a direct toxic effect of PAPR1 inhibitors is observed in
some tumor cells.

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108(8), 3406-3411.

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