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PARP INHIBITORS(POLY ADP-RIBOSE POLYMERASE)

Presented ByMojdeh Yousefian

Ph.D student of Medicinal Chemistry

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 activitiesin 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 cancers. 5

• 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 repair3. Protein ubiquitination4. chromatin remodeling5. 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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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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Protein structure of BRCA2

PARP 1

PARP2

VPARP (PARP4)

Tankyrase1 (PARP-5a)

Tankyrase2 (PARP-5b)

PARP3

PARP6

TIPARP (PARP7)

PARP8

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 DNArepair process, whereas PARP2 and PARP3 are involved to a lesser extent.

PARP9

PARP10

PARP11

PARP12

PARP14

PARP15

PARP1613

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 importantmolecular 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). 25

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.

26Nicotinamide 5- methyl nicotinamide

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NicotinamideBinding pocket

Adenosine Binding pocket

RiboseBinding pocket

The three binding sites for NAD+

Inhibitors directed to one of the three pockets are expected to behave as NAD+

Competitive PARP inhibitors.

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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.

Second generation inhibitors

QuinazolinoneNicotinamide

1,5-dihydroisoquinolineIC50 = 390 nМ

4-amino-1,8-naphthalimideIC50 = 180 nМ

2-nitro-1,8- phenanthridinoneIC50 = 350 nМ

PD128763IC50 = 420 nМ

NU1025IC50 = 400 nМ

PJ-34IC50 = 20 nM

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Structures of third-generation PARP1 inhibitors

Rucaparib INO-1001 E7016(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

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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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Refferences:• Ames, B. N., & Gold, L. S. (1991). Endogenous mutagens and the causes of aging and

cancer. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 250(1), 3-16.

• Bryant, H. E., Schultz, N., Thomas, H. D., Parker, K. M., Flower, D., Lopez, E., . . . Helleday, T. (2005). Specific killing of BRCA2-deficient tumours with inhibitors of poly (ADP-ribose) polymerase. Nature, 434(7035), 913-917.

• Farmer, H., McCabe, N., Lord, C. J., Tutt, A. N., Johnson, D. A., Richardson, T. B., . . . Knights, C. (2005). Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature, 434(7035), 917-921.

• Fortini, P., Pascucci, B., Parlanti, E., D’errico, M., Simonelli, V., & Dogliotti, E. (2003). The base excision repair: mechanisms and its relevance for cancer susceptibility. Biochimie, 85(11), 1053-1071.

• Hoeijmakers, J. H. (2001). Genome maintenance mechanisms for preventing cancer. Nature, 411(6835), 366-374.

• Liu, J. F., & Matulonis, U. A. (2016). What Is the Place of PARP Inhibitors in Ovarian Cancer Treatment? Current oncology reports, 18(5), 1-9.

• Patel, A. G., Sarkaria, J. N., & Kaufmann, S. H. (2011). Nonhomologous end joining drives poly (ADP-ribose) polymerase (PARP) inhibitor lethality in homologous recombination-deficient cells. Proceedings of the National Academy of Sciences, 108(8), 3406-3411.

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• Wu, J., Lu, L.-Y., & Yu, X. (2010). The role of BRCA1 in DNA damage response. Protein & cell, 1(2), 117-123.

• Zhong, Q., Chen, C.-F., Chen, P.-L., & Lee, W.-H. (2002). BRCA1 facilitates microhomology-mediated end joining of DNA double strand breaks. Journal of Biological Chemistry, 277(32), 28641-28647.

• Zhong, Q., Chen, C.-F., Li, S., Chen, Y., Wang, C.-C., Xiao, J., . . . Lee, W.-H. (1999). Association of BRCA1 with the hRad50-hMre11-p95 complex and the DNA damage response. Science, 285(5428), 747-750.

• Ashworth, A. (2008). A synthetic lethal therapeutic approach: poly (ADP) ribose polymerase inhibitors for the treatment of cancers deficient in DNA double-strand break repair. Journal of Clinical Oncology, 26(22), 3785-3790.

• Dajee, M., Lazarov, M., Zhang, J. Y., Cai, T., Green, C. L., Russell, A. J., . . . Kubo, Y. (2003). NF-κB blockade and oncogenic Ras trigger invasive human epidermal neoplasia. Nature, 421(6923), 639-643.

• de Murcia, G., & de Murcia, J. M. (1994). Poly (ADP-ribose) polymerase: a molecular nick-sensor. Trends in biochemical sciences, 19(4), 172-176.

• Hansen, W. K., & Kelley, M. R. (2000). Review of mammalian DNA repair and translational implications. Journal of Pharmacology and Experimental Therapeutics, 295(1), 1-9.

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• Huber, A., Bai, P., de Murcia, J. M., & de Murcia, G. (2004). PARP-1, PARP-2 and ATM in the DNA damage response: functional synergy in mouse development. DNA repair, 3(8), 1103-1108.

• Malyuchenko, N., Kotova, E. Y., Kulaeva, O., Kirpichnikov, M., & Studitskiy, V. (2015). PARP1 Inhibitors: antitumor drug design. Acta naturae, 7(3), 27.

• Meyer-Ficca, M. L., Meyer, R. G., Jacobson, E. L., & Jacobson, M. K. (2005). Poly (ADP-ribose) polymerases: managing genome stability. The international journal of biochemistry & cell biology, 37(5), 920-926.

• Otto, H., Reche, P. A., Bazan, F., Dittmar, K., Haag, F., & Koch-Nolte, F. (2005). In silico characterization of the family of PARP-like poly (ADP-ribosyl) transferases (pARTs). BMC genomics, 6(1), 139.

• Ratnam, K., & Low, J. A. (2007). Current development of clinical inhibitors of poly (ADP-ribose) polymerase in oncology. Clinical Cancer Research, 13(5), 1383-1388.

• Takata, M., Sasaki, M. S., Sonoda, E., Morrison, C., Hashimoto, M., Utsumi, H., . . . Takeda, S. (1998). Homologous recombination and non homologous end joining ‐ ‐pathways of DNA double strand break repair have overlapping roles in the ‐maintenance of chromosomal integrity in vertebrate cells. The EMBO journal, 17(18), 5497-5508.

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• Banasik, M., Komura, H., Shimoyama, M., & Ueda, K. (1992). Specific inhibitors of poly (ADP-ribose) synthetase and mono (ADP-ribosyl) transferase. Journal of Biological Chemistry, 267(3), 1569-1575.

• Calabrese, C. R., Batey, M. A., Thomas, H. D., Durkacz, B. W., Wang, L.-Z., Kyle, S., . . . Boritzki, T. (2003). Identification of Potent Nontoxic Poly (ADP-Ribose) Polymerase-1 Inhibitors Chemopotentiation and Pharmacological Studies. Clinical Cancer Research, 9(7), 2711-2718.

• Canan Koch, S. S., Thoresen, L. H., Tikhe, J. G., Maegley, K. A., Almassy, R. J., Li, J., . . . Zhang, C. (2002). Novel tricyclic poly (ADP-ribose) polymerase-1 inhibitors with potent anticancer chemopotentiating activity: design, synthesis, and X-ray cocrystal structure. Journal of medicinal chemistry, 45(23), 4961-4974.

• Cazzalini, O., Donà, F., Savio, M., Tillhon, M., Maccario, C., Perucca, P., . . . Prosperi, E. (2010). p21 CDKN1A participates in base excision repair by regulating the activity of poly (ADP-ribose) polymerase-1. DNA repair, 9(6), 627-635.

• Durkacz, B. W., Omidiji, O., Gray, D. A., & Shall, S. (1980). (ADP-ribose) n participates in DNA excision repair.

• Leu, J.-J., Pimkina, J., Frank, A., Murphy, M. E., & George, D. L. (2009). A small molecule inhibitor of inducible heat shock protein 70. Molecular cell, 36(1), 15-27.

• Petesch, S. J., & Lis, J. T. (2008). Rapid, transcription-independent loss of nucleosomes over a large chromatin domain at Hsp70 loci. Cell, 134(1), 74-84.

• Purnell, M. R., & Whish, W. (1980). Novel inhibitors of poly (ADP-ribose) synthetase. Biochemical Journal, 185(3), 775-777.

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• Ruf, A., de Murcia, G., & Schulz, G. E. (1998). Inhibitor and NAD+ binding to poly (ADP-ribose) polymerase as derived from crystal structures and homology modeling. Biochemistry, 37(11), 3893-3900.

• Tulin, A., & Spradling, A. (2003). Chromatin loosening by poly (ADP)-ribose polymerase (PARP) at Drosophila puff loci. Science, 299(5606), 560-562.

• Bryant, H. E., Schultz, N., Thomas, H. D., Parker, K. M., Flower, D., Lopez, E., . . . Helleday, T. (2005). Specific killing of BRCA2-deficient tumours with inhibitors of poly (ADP-ribose) polymerase. Nature, 434(7035), 913-917.

• Chapman, J. R., Taylor, M. R., & Boulton, S. J. (2012). Playing the end game: DNA double-strand break repair pathway choice. Molecular cell, 47(4), 497-510.

• Curtin, N. J., & Szabo, C. (2013). Therapeutic applications of PARP inhibitors: anticancer therapy and beyond. Molecular aspects of medicine, 34(6), 1217-1256.

• De Lorenzo, S., Patel, A., Hurley, R., & Kaufmann, S. H. (2013). The elephant and the blind men: making sense of PARP inhibitors in homologous recombination deficient tumor cells. Frontiers in oncology, 3, 228.

• Deindl, S., Hwang, W. L., Hota, S. K., Blosser, T. R., Prasad, P., Bartholomew, B., & Zhuang, X. (2013). ISWI remodelers slide nucleosomes with coordinated multi-base-pair entry steps and single-base-pair exit steps. Cell, 152(3), 442-452.

• Farmer, H., McCabe, N., Lord, C. J., Tutt, A. N., Johnson, D. A., Richardson, T. B., . . . Knights, C. (2005). Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature, 434(7035), 917-921.

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• Kuzminov, A. (2001). Single-strand interruptions in replicating chromosomes cause double-strand breaks. Proceedings of the National Academy of Sciences, 98(15), 8241-8246.

• Mason, K. A., Raju, U., Buchholz, T. A., Wang, L., Milas, Z. L., & Milas, L. (2014). Poly (ADP-ribose) Polymerase Inhibitors in Cancer Treatment. American journal of clinical oncology, 37(1), 90-100.

• Patel, A. G., Sarkaria, J. N., & Kaufmann, S. H. (2011). Nonhomologous end joining drives poly (ADP-ribose) polymerase (PARP) inhibitor lethality in homologous recombination-deficient cells. Proceedings of the National Academy of Sciences, 108(8), 3406-3411.