Enzyme Poly ADP-ribose Polymerase Research Articles

Sevoflurane Preconditioning Rescues PKMζ Gene Expression from Broad Hypoxia-Induced mRNA Downregulation Correlating with Improved Neuronal Recovery

Hypoxia due to stroke is a major cause of neuronal damage, leading to loss of cognition and other brain functions. Sevoflurane preconditioning improves recovery after hypoxia. Hypoxia interferes with protein expression at the translational level; however, its effect on mRNA levels for neuronal protein kinase and anti-apoptotic genes is unclear. To investigate the link between sevoflurane preconditioning and gene expression, hippocampal slices were treated with 4% sevoflurane for 15 min, a 5 min washout, 10 min of hypoxia, and 60 min of recovery. We used quantitative PCR to measure mRNA levels in the CA1 region of rat hippocampi. The mRNA levels for specific critical proteins were examined, as follows: Protein kinases, PKCγ (0.22), PKCε (0.38), and PKMζ (0.55) mRNAs, and anti-apoptotic, bcl-2 (0.44) and bcl-xl (0.41), were reduced 60 min after hypoxia relative to their expression in tissue not subjected to hypoxia (set to 1.0). Sevoflurane preconditioning prevented the reduction in PKMζ (0.88 vs. 1.0) mRNA levels after hypoxia. Pro-apoptotic BAD mRNA was not significantly changed after hypoxia, even with sevoflurane preconditioning (hypoxia 0.81, sevo hypoxia 0.84 vs. normoxia 1.0). However, BAD mRNA was increased by sevoflurane in non-hypoxic conditions (1.48 vs. 1.0), which may partially explain the deleterious effects of volatile anesthetics under certain conditions. The DNA repair enzyme poly ADP-ribose polymerase 1 (PARP-1) was increased by sevoflurane in tissue not subjected to hypoxia (1.23). PARP-1 mRNA was reduced in untreated tissue after hypoxia (0.21 vs. 1.0); sevoflurane did not improve PARP-1 after hypoxia (0.27). Interestingly, the mRNA level of the cognitive kinase PKMζ, a kinase essential for learning and memory, was the only one protected against hypoxic downregulation by sevoflurane preconditioning. These findings correlate with previous studies that found that sevoflurane-induced improvement of neuronal survival after hypoxia was dependent on PKMζ. Maintaining mRNA levels for critical proteins may provide an important mechanism for preserving neuronal function after stroke.

Minimizing DNA trapping while maintaining activity inhibition via selective PARP1 degrader

Poly (ADP-ribose) polymerase 1 (PARP1) catalyzes poly (ADP) ribosylation reaction, one of the essential post-translational modifications of proteins in eukaryotic cells. Given that PARP1 inhibition can lead to synthetic lethality in cells with compromised homologous recombination, this enzyme has been identified as a potent target for anti-cancer therapeutics. However, the clinical application of existing PARP1 inhibitors is restrained by side effects associated with DNA trapping and off-target effects, highlighting the need for improved therapeutic strategies. By integrating protein degradation technology, we synthesized a PROTAC molecule 180055 based on the Rucaparib junction and VHL ligand, which efficiently and selectively degraded PARP1 and inhibited PARP1 enzyme activity without a noticeable DNA trapping effect. Furthermore, 180055 kills tumor cells carrying BRCA mutations with a minor impact on the growth of normal cells both in vitro and in vivo. This suggests that 180055 is a PARP1-degrading compound with excellent pharmacological efficacy and extremely high biological safety that deserves further exploration and validation in clinical trials.

Mechanism of Action of PARP Inhibitors

The selective vulnerability of BRCA1/2-mutated (BRCA mut) cancer cells to poly(ADP-ribose) polymerase (PARP) inhibitors provides one of the best examples of synthetic lethality that has been translated into the clinic. The success of this approach has led to a paradigm shift, with four PARP inhibitors now approved by the US Food and Drug Administration (FDA) for the treatment of ovarian, breast, prostate, and/or pancreatic cancers with BRCA mut. Furthermore, recent preclinical and clinical data suggest that many other types of solid tumors might also benefit from PARP inhibitors, regardless of their BRCA mut status. Despite this progress, resistance to PARP inhibitors is frequently observed in the clinic, which is, at least in part, due to the incomplete understanding of the mechanism of action of PARP inhibitors. In this review, we summarize the diverse processes underlying the signaling mechanisms of the PARP enzymes. We also discuss recent progress in utilizing these mechanistic insights for overcoming PARP inhibitor resistance, developing predictive biomarker assays, designing rational combination therapies, and, finally, developing the next-generation PARP1-targeting agents with a more complete and durable therapeutic response.

Design, synthesis and antitumor activities of phthalazinone derivatives as PARP-1 inhibitors and PARP-1/HDAC-1 inhibitors

In recent years, poly(ADP-ribose)polymerase-1 (PARP-1) and histone deacetylase (HDAC) have emerged as significant targets in tumor therapy, garnering widespread attention. In this study, we designed and synthesized two novel phthalazinone PARP-1 inhibitors and dual PARP-1/HDAC-1 inhibitors, named DLC-1–46 containing dithiocarboxylate fragments and DLC-47–63 containing hydroxamic acid fragments, and evaluated their inhibitory activities on enzymes and cells. Among the PARP-1 inhibitors, most compounds exhibited high inhibitory activity against the PARP-1 enzyme, with DLC-1–6 being particularly notable, showing IC50 values <0.2 nM. Notably, DLC-1 demonstrated significant anti-proliferative activity, with IC50 values for inhibiting the proliferation of MDA-MB-436, MDA-MB-231, and MCF-7 cells reaching 0.08, 26.39, and 1.01 μM, respectively. Further investigation revealed that DLC-1 arrested MDA-MB-231 cells in the G1 phase and induced apoptosis in a dose-dependent manner. Among the designed dual PARP-1/HDAC-1 inhibitors, several compounds exhibited potent dual-target inhibitory activity, with DLC-49 displaying IC50 values of 0.53 nM and 17 nM for PARP-1 and HDAC-1, respectively. DLC-50 demonstrated the most potent anti-proliferative activity, with IC50 values for inhibiting the proliferation of MDA-MB-436, MDA-MB-231, and MCF-7 cells at 0.30, 2.70, and 2.41 μM, respectively. Cell cycle arrest and apoptosis assays indicated that DLC-50 arrested the cell cycle in the G2 phase and induced apoptosis in HCT-116 cells. Our findings present a novel avenue for further exploration of PARP-1 inhibitors and dual PARP-1/HDAC-1 inhibitors.

Abstract LB273: Pharmacological synthetic lethality by co-inhibition of PARP and HDAC enzymes

Abstract Introduction: Inhibition of poly(ADP-ribose) polymerase (PARP) induces synthetic lethality in cells exhibiting defects in homologous repair (HR) pathways, such as BRCA1 and BRCA2-mutated breast or ovarian cancers, and 4 different PARP inhibitors (PARPi) are currently approved by the FDA. While HR-deficient cancers usually respond well to single agent PARPi, HR-proficient cancers are generally less sensitive to the treatment. Acquired resistance to PARPi due to activating mutations in BRCA genes are also not uncommon. In this work, we have examined strategies for enhancing PARPi activity in a HR-proficient context and identified histone deacetylases (HDACs) as targets for pharmacological synthetic lethality in the context of PARPi. Materials and methods: PARP1 and PARP2 activity were measured using Trevigen Universal Colorimetric PARP Assay Kit, BPS Bioscience PARP2 Colorimetric PARP2 Assay Kit, and PARylation assay. HDAC activity was measured using HeLa cell nuclear extracts and a fluorogenic peptide-based biochemical assay. Cell survival EC50 were determined using CellTiter Glo viability assay. Cell cycle analysis was performed by flow cytometry with propidium iodide staining. DNA damage was investigated by immunoblotting and immunofluorescence using gamma-H2AX antibodies, and comet assays for confirmation of double-strand breaks. Spheroid assays were performed using the Incucyte® S3 spheroid analysis module. In vivo metastasis formations were examined using the pulmonary metastasis assay (PuMA). Results and discussion: Combinations of PARP and HDAC inhibitors in HR-proficient tumor cells demonstrated that HDACi could enhance the efficacy of PARPi in vitro. This was associated with increased acute and sustained DNA-damage distinct cell cycle profiles showing activity in both S- and G2/M-phases. Importantly, dual single-molecule PARP-HDAC inhibitors showed potent inhibition of PARP1 and PARP2 activity and PAR synthesis, comparable to olaparib, as well as inhibition of HDAC with IC50 values in the low µM range. Dual PARP-HDAC inhibition decreased survival of HR-proficient cells in both 2D cultures and 3D spheroid assays with lower EC50 values than olaparib or vorinostat alone. Importantly, lung metastasis formation was significantly impaired by dual PARP-HDAC inhibition. Conclusion: Combination of PARP inhibition and HDAC inhibition showed increased efficacy compared to single agent PARP inhibition in HR-proficient cells. Development of bifunctional inhibitors may provide a novel therapeutic opportunity for tumors with limited response rates to single agent PARPi. Citation Format: Sarah Truong, Louise Ramos, Beibei Zhai, Fariba Ghaidi, Mona Marzban, Hans Adomat, Xiaoqi Chen, John Langlands, Dennis Brown, Jeffrey Bacha, Colin Collins, Poul H. Sorensen, Wang Shen, Mads Daugaard. Pharmacological synthetic lethality by co-inhibition of PARP and HDAC enzymes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB273.

THU547 Role Of NAD+ Synthesis Pathway In PARP Inhibitor Resistance

Abstract Disclosure: S. Challa: None. ADP-ribosylation is catalyzed by the poly(ADP-ribose) polymerase (PARP) family of enzymes consisting of 17 members that have distinct structural domains, activities, subcellular localizations, and functions. The catalytic activities of PARP enzymes are intimately tied to the synthesis of NAD+, which is consumed during ADPRylation reactions and, thus, must be regenerated. The intracellular salvage pathway utilizing nicotinamide (NAM) is the primary source of NAD+ in the cell. NAM is converted to nicotinamide mononucleotide (NMN) by NAMPT. Nicotinamide mononucleotide adenylyl transferases (NMNATs) catalyze the final step in the NAD+ salvage pathway, combining NMN and ATP to make NAD+. Three different NMNATs, each with a distinct subcellular localization, control the subcellular levels of NAD+ in each compartment: NMNAT-1 in the nucleus, NMNAT-2 is in the outer membrane of golgi, and NMNAT-3 in the mitochondria. We recently demonstrated that such compartment-specific NAD+ synthesis regulates the levels of ADP-ribosylation. Elevated levels of NMNAT-2, the cytosolic NAD+ synthase, reduces nuclear NAD+ levels and PARP1 activity in adipocytes and ovarian cancer cells. In the current study, we aim to delineate the role of compartmentalized synthesis of NAD+ in PARP inhibitor resistance in ovarian cancers. To identify these mechanisms, we generated ovarian cancer cells with acquired resistance to Niraparib, an FDA approved PARP inhibitor (NirR). We found that NirR cells have elevated levels of NAD+ biosynthesis. Moreover, treatment of parental cells with NAD+ precursors reduced the sensitivity to Niraparib while inhibition of NAD+ metabolism in NirR cells increased the sensitivity to Niraparib. Interestingly, the resistant cells have comparable levels of PARP1 activation to the parental cells. These data collectively suggests that NAD+ metabolism confers resistance to Niraparib without altering PARP1 activity. In line with this, NirR cells are sensitive to the effects of depletion of the cytosolic NAD+ biosynthesis, suggesting high NAD+ biosynthesis in NirR cells supports processes regulated by the cytosolic NAD+ which may drive the resistance phenotype. There is an urgent need to identify the mechanisms of resistance to PARP inhibitors to increase their clinical efficacy. This study identifies the NAD+ biosynthesis pathway as a key mechanism of PARP inhibitor resistance. Presentation: Thursday, June 15, 2023

PARP inhibitors for prostate cancer

Poly(ADP-ribose) polymerase (PARP) inhibitors have transformed the treatment landscape for patients with metastatic castration-resistant prostate cancer (mCRPC) and alterations in DNA damage response genes. This has also led to widespread use of genomic testing in all patients with mCRPC. The current review will give an overview of (1) the current understanding of the interplay between DNA damage response and PARP enzymes; (2) the clinical landscape of PARP inhibitors, including the combination of PARP inhibitors with other agents such as androgen-receptor signaling agents; (3) biomarkers related to PARP inhibitor response and resistance; and (4) considerations for interpreting genomic testing results and treating patients with PARP inhibitors.

Poly(ADP-ribose) Polyremase-1 (PARP-1) Inhibition: A Promising Therapeutic Strategy for ETS-Expressing Tumours.

ETS transcription factors are a highly conserved family of proteins involved in the progression of many cancers, such as breast and prostate carcinomas, Ewing's sarcoma, and leukaemias. This significant involvement can be explained by their roles at all stages of carcinogenesis progression. Generally, their expression in tumours is associated with a poor prognosis and an aggressive phenotype. Until now, no efficient therapeutic strategy had emerged to specifically target ETS-expressing tumours. Nevertheless, there is evidence that pharmacological inhibition of poly(ADP-ribose) polymerase-1 (PARP-1), a key DNA repair enzyme, specifically sensitises ETS-expressing cancer cells to DNA damage and limits tumour progression by leading some of the cancer cells to death. These effects result from a strong interplay between ETS transcription factors and the PARP-1 enzyme. This review summarises the existing knowledge of this molecular interaction and discusses the promising therapeutic applications.

Design, synthesis, and biological evaluation of a series of benzofuran[3,2-d]pyrimidine-4(3H)-one derivatives containing thiosemicarbazone analogs as novel PARP-1 inhibitors

Poly ADP ribose polymerase-1 (PARP-1), one of the most important members of the PARP protein family, plays a crucial role in DNA damage repair, gene transcription, and apoptosis of cancer cells. In this work, benzofuran[3,2-d]pyrimidine-4(3H)-one was used as a framework to design and synthesize a series of novel PARP-1 inhibitors by introducing thiosemicarbazone or its derivatives into the scafford. Among all the target compounds, 19b and 19c were found to exhibit more potent inhibitory activity and higher selectivity against PARP-1 than Olaparib, especially the latter had an IC50 value of 0.026 μM against PARP-1 enzyme and a PARP-2/PARP-1 selectivity of 85.19-fold over Olapanib. Apart from strong cytotoxicity against the tested cancer cell lines, 19c was most sensitive to SK-OV-3 cells, with an IC50 value of 4.98 μM superior to Olaparib. Anti-cancer mechanism studies revealed that 19c could inhibit DNA single-strand breakage repair and aggravate DNA double-strand breakage by inhibiting PARP-1 activity, and promote the apoptosis of cancer cells through the mitochondrial apoptosis pathway.

Identification of New Potential Prognostic and Predictive Markers in High-Grade Osteosarcoma Using Whole Exome Sequencing.

Conventional high-grade osteosarcoma (OS) is the most common primary cancer of bone and it typically affects the extremities of adolescents. OS has a complex karyotype, and molecular mechanisms related to carcinogenesis, progression and resistance to therapy are still largely unknown. For this reason, the current standard of care is associated with considerable adverse effects. In this study, our aim was to identify gene alterations in OS patients using whole exome sequencing (WES) to find new potential prognostic biomarkers and therapeutic targets. We performed WES on formalin-fixed paraffin-embedded (FFPE) biopsy materials collected from 19 patients affected by conventional high-grade OS. The clinical and genetic data were analyzed according to response to therapy, presence of metastasis and disease status. By comparing good and poor responders to neoadjuvant therapy, we detected a clear prevalence of mutations in the ARID1A, CREBBP, BRCA2 and RAD50 genes in poor responders that negatively influence the progression-free survival time. Moreover, higher tumor mutational burden values correlated with worse prognosis. The identification of mutations in ARID1A, CREBBP, BRCA2 and RAD50 may support the use of a more specific therapy for tumors harboring these alterations. In particular, BRCA2 and RAD50 are involved in homologous recombination repair, and could thus be used as specific therapy targets of inhibitors of the enzyme Poly ADP Ribose Polymerase (PARP). Finally, tumor mutational burden is found to be a potential prognostic marker for OS.

The dynamic process of covalent and non-covalent PARylation in the maintenance of genome integrity: a focus on PARP inhibitors.

Poly(ADP-ribosylation) (PARylation) by poly(ADP-ribose) polymerases (PARPs) is a highly regulated process that consists of the covalent addition of polymers of ADP-ribose (PAR) through post-translational modifications of substrate proteins or non-covalent interactions with PAR via PAR binding domains and motifs, thereby reprogramming their functions. This modification is particularly known for its central role in the maintenance of genomic stability. However, how genomic integrity is controlled by an intricate interplay of covalent PARylation and non-covalent PAR binding remains largely unknown. Of importance, PARylation has caught recent attention for providing a mechanistic basis of synthetic lethality involving PARP inhibitors (PARPi), most notably in homologous recombination (HR)-deficient breast and ovarian tumors. The molecular mechanisms responsible for the anti-cancer effect of PARPi are thought to implicate both catalytic inhibition and trapping of PARP enzymes on DNA. However, the relative contribution of each on tumor-specific cytotoxicity is still unclear. It is paramount to understand these PAR-dependent mechanisms, given that resistance to PARPi is a challenge in the clinic. Deciphering the complex interplay between covalent PARylation and non-covalent PAR binding and defining how PARP trapping and non-trapping events contribute to PARPi anti-tumour activity is essential for developing improved therapeutic strategies. With this perspective, we review the current understanding of PARylation biology in the context of the DNA damage response (DDR) and the mechanisms underlying PARPi activity and resistance.

PARP-1: a critical regulator in radioprotection and radiotherapy-mechanisms, challenges, and therapeutic opportunities.

Background: Since its discovery, poly (ADP-ribose) polymerase 1 (PARP-1) has been extensively studied due to its regulatory role in numerous biologically crucial pathways. PARP inhibitors have opened new therapeutic avenues for cancer patients and have gained approval as standalone treatments for certain types of cancer. With continued advancements in the research of PARP inhibitors, we can fully realize their potential as therapeutic targets for various diseases. Purpose: To assess the current understanding of PARP-1 mechanisms in radioprotection and radiotherapy based on the literature. Methods: We searched the PubMed database and summarized information on PARP inhibitors, the interaction of PARP-1 with DNA, and the relationships between PARP-1 and p53/ROS, NF-κB/DNA-PK, and caspase3/AIF, respectively. Results: The enzyme PARP-1 plays a crucial role in repairing DNA damage and modifying proteins. Cells exposed to radiation can experience DNA damage, such as single-, intra-, or inter-strand damage. This damage, associated with replication fork stagnation, triggers DNA repair mechanisms, including those involving PARP-1. The activity of PARP-1 increases 500-fold on DNA binding. Studies on PARP-1-knockdown mice have shown that the protein regulates the response to radiation. A lack of PARP-1 also increases the organism's sensitivity to radiation injury. PARP-1 has been found positively or negatively regulate the expression of specific genes through its modulation of key transcription factors and other molecules, including NF-κB, p53, Caspase 3, reactive oxygen species (ROS), and apoptosis-inducing factor (AIF). Conclusion: This review provides a comprehensive analysis of the physiological and pathological roles of PARP-1 and examines the impact of PARP-1 inhibitors under conditions of ionizing radiation exposure. The review also emphasizes the challenges and opportunities for developing PARP-1 inhibitors to improve the clinical outcomes of ionizing radiation damage.

Safety, pharmacokinetics and anti-tumor activity of RP12146, a PARP1/2 inhibitor, in patients with locally advanced or metastatic solid tumors.

3097 Background: RP12146 is an orally bioavailable, potent small-molecule inhibitor of poly (ADP-ribose) polymerase (PARP) 1 and PARP 2. Pre-clinically, RP1246 demonstrated anti-proliferative activity in both BRCA and non- BRCA mutant cancer cell lines of different tumor types as well as in xenograft models. The first-in-human of RP12146 as a single agent is underway to determine the safety, pharmacokinetics and anti-tumor activity. Methods: The phase I/Ib study was designed as two-part study. Phase I was a dose escalation, 3+3 design, MTD determination study and would enroll pts who have tumors which are known to harbour DNA repair deficiencies. Phase Ib is dose expansion at the MTD/ optimal dose and would enroll thirty-six pts (12 pts each of advanced/metastatic ovarian cancer (OC), breast cancer (BC), and castration-resistant Prostate cancer (mCRPC) having a confirmed deleterious HRR mutation. RP12146 was administered orally in a 28-days cycles until disease progression. Safety were the primary outcome and the investigator-assessed ORR, CBR and PFS assessed using RECIST v1.1, are the secondary outcomes. Predictive biomarker analysis included inhibition of PARP enzyme as measured by gH2AX levels in PMBCs. Results: As of 31-Jan-2023, 9 pts in the dose escalation at 3 dose levels (100 mg QD, 200mg BID and 400 mg BID) and 13 pts (with 5 CHEK2, 4 BRCA2, 2 ATM, 1 CDK12, and 1 RAD51C mutations) in dose expansion have been enrolled. No DLTs were reported in dose escalation. The dose of 400mg BID was considered for dose expansion. Out of 22 pts enrolled, 13 pts had PC, 3 pts had OC, and 2 pts had BC. Most of the related AEs reported were mild in severity except one event of Grade 3 anaemia which resolved, and patient continues to be on therapy. None of the pts developed related SAEs or discontinued due to an AE. Eleven patients (50%) in the expansion group continue to be on RP12146 treatment. RP12146 exhibited rapid absorption achieving maximum concentrations in 1 hr, with an elimination half-life of ~ 4 hrs. Out of the 7 efficacy evaluable PC patients, 5pts showed stable disease and 2pts showed progressive disease. Out of 3 OC pts, 2 pts showed stable disease, and one pt had progressive disease. Among two BC pts, one showed stable disease and the other had progressive disease. Overall, out of 16 efficacy evaluable patients, 8 patients (50%) showed stable disease. Conclusions: RP12146 showed a differentiated safety profile so far with limited hematological toxicities reported as compared to the first-generation PARP inhibitors. Updated safety and efficacy data will be provided at the time of presentation. Clinical trial information: NCT05002868 .

Roles of ADP-Ribosylation during Infection Establishment by Trypanosomatidae Parasites.

ADP-ribosylation is a reversible post-translational protein modification, which is evolutionarily conserved in prokaryotic and eukaryotic organisms. It governs critical cellular functions, including, but not limited to cellular proliferation, differentiation, RNA translation, and genomic repair. The addition of one or multiple ADP-ribose moieties can be catalysed by poly(ADP-ribose) polymerase (PARP) enzymes, while in eukaryotic organisms, ADP-ribosylation can be reversed through the action of specific enzymes capable of ADP-ribose signalling regulation. In several lower eukaryotic organisms, including Trypanosomatidae parasites, ADP-ribosylation is thought to be important for infection establishment. Trypanosomatidae encompasses several human disease-causing pathogens, including Trypanosoma cruzi, T. brucei, and the Leishmania genus. These parasites are the etiological agents of Chagas disease, African trypanosomiasis (sleeping sickness), and leishmaniasis, respectively. Currently, licenced medications for these infections are outdated and often result in harmful side effects, and can be inaccessible to those carrying infections, due to them being classified as neglected tropical diseases (NTDs), meaning that many infected individuals will belong to already marginalised communities in countries already facing socioeconomic challenges. Consequently, funding to develop novel therapeutics for these infections is overlooked. As such, understanding the molecular mechanisms of infection, and how ADP-ribosylation facilitates infection establishment by these organisms may allow the identification of potential molecular interventions that would disrupt infection. In contrast to the complex ADP-ribosylation pathways in eukaryotes, the process of Trypanosomatidae is more linear, with the parasites only expressing one PARP enzyme, compared to the, at least, 17 genes that encode human PARP enzymes. If this simplified pathway can be understood and exploited, it may reveal new avenues for combatting Trypanosomatidae infection. This review will focus on the current state of knowledge on the importance of ADP-ribosylation in Trypanosomatidae during infection establishment in human hosts, and the potential therapeutic options that disrupting ADP-ribosylation may offer to combat Trypanosomatidae.

Abstract 6194: A bifunctional inhibitor of PARP and HDAC enzymes with activity in Ewing sarcoma 3D spheroid and metastasis models

Abstract Introduction: Poly(ADP-ribose) polymerase (PARP) plays a major role in DNA repair and PARP inhibitors (PARPi) have shown promise in pre-clinical studies for the treatment of Ewing sarcoma (ES). While a clinical trial using olaparib as a single agent failed to show significant response against ES, combination therapies with PARPi have emerged as an area of interest. Deacetylation of histones, controlled by histone deacetylases (HDACs) is a key regulatory event in DNA repair and inhibition of HDACs has been shown to reduce ES tumor growth in vitro and in vivo. PARP inhibition combined with HDAC inhibition has demonstrated enhanced efficacy in pre-clinical studies in various tumor indications, and a clinical trial of olaparib and vorinostat combination therapy against metastatic breast cancer is currently ongoing. However, combination therapies can be limited in clinical utility due to overlapping toxicities and different pharmacokinetic profiles. Here, we report the efficacy of a novel bifunctional small-molecule compound, kt-3283, designed to have both PARP and HDAC inhibitory activities. Materials and methods: PARP1 and PARP2 activity were measured using Trevigen Universal Colorimetric PARP Assay Kit, BPS Bioscience PARP2 Colorimetric PARP2 Assay Kit, and PARylation assay. HDAC activity was measured using HeLa cell nuclear extracts and a fluorogenic peptide-based biochemical assay. Cell survival EC50s were determined using live cell imaging with an Incucyte® S3 system and CellTiter Glo viability assay. Cell cycle analysis was performed by flow cytometry with propidium iodide staining. DNA damage was investigated by western blot, immunofluorescence, and comet assay. Spheroid assays were performed using the Incucyte® S3 spheroid analysis module and inhibition of metastases was assessed in a PUMA ES mouse model. Results and discussion: Kt-3283 showed potent inhibition of PARP1/2 activity and PAR synthesis with IC50 values comparable to olaparib. Kt-3283 also showed inhibition of HDACs with an IC50 value in the low µM range. Cell survival EC50 values for the compound were also superior to those of olaparib and vorinostat in ES cell lines. Cell cycle and DNA damage analyses indicated S/G2/M cell cycle arrest and strong DNA damage upon treatment with kt-3283 at lower concentration range compared to olaparib and vorinostat. This compound also exhibited potent inhibition of 3D spheroid growth of ES cells with low µM EC50 values, and inhibited metastatic growth in a PUMA mouse model. Conclusion: Kt-3283 shows potent inhibition of PARP1/2 and HDAC activities. It induces S and G2/M cell cycle arrest and DNA damage, and inhibits 3D spheroid growth and metastatic potential of ES cells. Further investigation of this bifunctional single-molecule inhibitor may offer a novel treatment opportunity for ES and other solid tumors with limited responses to PARPi. Citation Format: Sarah Truong, Louise Ramos, Beibei Zhai, Jay Joshi, Fariba Ghaidi, Michael M. Lizardo, Taras Shyp, John Langlands, Dennis Brown, Jeffrey Bacha, Poul Sorensen, Wang Shen, Mads Daugaard. A bifunctional inhibitor of PARP and HDAC enzymes with activity in Ewing sarcoma 3D spheroid and metastasis models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6194.

Poly ADP-Ribose Polymerase-1 inhibition by 3-aminobenzamide recuperates HEI-OC1 auditory hair cells from blast overpressure-induced cell death.

Introduction: Poly ADP-Ribose Polymerase-1 (PARP1), a DNA repair enzyme is implicated as a key molecule in the pathogenesis of several neurodegenerative disorders. Traumatic insults inducing oxidative stress results in its over-activation causing inflammation and cell death (Parthanatos). As PARP1 inhibition is known to reduce oxidative stress, we hypothesized that PARP1 inhibition by a known inhibitor 3-aminobenzamide (3AB) might recuperate the damage in an in vitro model of blast injury using HEI-OC1 cells (mouse auditory hair cells). Methods: Here, we evaluated the protective effect of 3AB on HEI-OC1 cells following single and repetitive blast overpressures (BOPs). Results: We found that inhibition of PARP1 b 3AB inhibits the PARP1 enzyme and its action of a post-translational modification i.e. formation of Poly ADP-Ribose Polymers which leads to massive ATP depletion. PARP inhibition (3AB treatment) reduced the oxidative stress (4HNE, a marker of lipid peroxidation, and 8OHdG, a marker of oxidative DNA damage) in cells exposed to single/repetitive BOPS through up-regulation of Nrf2, a transcriptional regulator of antioxidant defense and the GCLC, a rate limiting enzyme in the synthesis of glutathione. Discussion: Overall, we found that PARP inhibition by 3AB helps to maintain the viability of BOP-exposed auditory hair cells by recuperating the ATP pool from both mitochondrial and glycolytic sources.

Design, synthesis, and evaluation of 1H-benzo[d]imidazole-4-carboxamide PARP-1 inhibitors using different saturated nitrogen-contained heterocycle as linker group.

Poly (ADP-ribose) polymerase-1 (PARP-1) inhibitors have been successfully applied in the clinical treatment of various cancer. Side effects and drug resistant cases were reported, and more effective PARP-1 inhibitors were required. However, studies on the AD site of PARP-1 inhibitors are currently incomplete. Therefore, to synthesize more potential candidate PARP-1 inhibitors and disclose some AD site SAR of the PARP-1 inhibitors, herein, a series of 2-phenyl-benzimidazole-4-carboxamide derivatives using different saturated nitrogen-contained heterocycles as linker group (6a-6t) have been designed, synthesized, and evaluated PARP-1 inhibitory activity and proliferation inhibitory against BRCA-1 mutant MDA-MB-436 cell line in vitro. The results showed 6b (IC50=8.65 nM) exhibited the most PARP-1 enzyme inhibitory activity comparable with Veliparib (IC50=15.54 nM) and Olaparib (IC50=2.77 nM); 6m exhibited the strongest MDA-MB-436 cell anti-proliferation activity (IC50=25.36 ± 6.06 μM) comparable with Olaparib (IC50=23.89 ± 3.81 μM). The compounds 6b, 6r, and 6m could be potential candidates for effective PARP-1 inhibitors and valuable for further optimization. The analysis of activity data also showed that the holistically anti-proliferation activity of the 1,4-diazepane group was about~twofold than that of the piperazine group. Meanwhile, the terminal 3-methyl-furanyl group exhibited the most robust PARP-1 inhibitory and anti-proliferation activity. It is hoped that the results could benefitable for further optimization of PARP-1 inhibitors. Furthermore, we note that some compounds (6d,6g,6n,6p,6s) showed poor PARP-1 inhibitory (>500 nM) but relatively good anti-proliferation activity, which indicates the proliferation inhibitory mechanism against MDA-MB-436 cell line was worth investigating in-depth.

Dihydrokaempferol attenuates CCl4-induced hepatic fibrosis by inhibiting PARP-1 to affect multiple downstream pathways and cytokines

The pathophysiological mechanism of hepatic fibrosis (HF) is related to the excessive activation of the DNA repair enzyme poly ADP-ribose polymerase-1 (PARP-1). The drugs, targeting PARP-1, are scarce. Therefore, the lead compound, moderately inhibiting PARP-1, with anti-HF properties should be identified. This study screened dihydrokaempferol (DHK) from herbs based on preliminary studies to intervene in a CCl4-induced liver injury and HF model in mice. In vitro, the expression levels of PARP-1-regulated related proteins and phosphorylation were examined. The binding pattern of DHK and PARP-1 was analyzed using molecular docking and molecular dynamics platforms. The results showed that DHK could significantly attenuate CCl4-induced liver injury and HF in mice. Moreover, it could also attenuate the toxic effects of CCl4 on HepG2 and inhibit α-SMA and Collagen 1/3 synthesis of LX-2 cells in-vitro. Molecular docking revealed that DHK could competitively bind to the Glu-988 and His-862 residues of the upstream DNA repair enzyme PARP-1, moderately inhibiting its overactivation. This led to maintaining NAD+ levels and energy metabolism in hepatocytes and inhibiting the activation of PARP-1-regulated downstream signaling pathways (TGF-β1, etc.), related proteins (p-Smd2/3, etc.), and inflammatory mediators while acting indirectly. Thus, DHK could attenuate CCl4-induced liver injury and HF in mice in a different mechanism from those of the existing reported flavonoids. It was associated with inhibiting the expression of downstream pathways and related cytokines by competitively binding to PARP-1. This study might provide a basis and direction for the design and exploration of anti-HF lead compounds.

330 Poly(ADP-ribose) polymerase (PARP)-2 promotes inflammation in psoriasis by regulating estradiol biosynthesis in keratinocytes

Activation of PARP enzymes is generally a proinflammatory process in humans. PARP2 has inflammatory role in the nervous system, however, its effect in skin inflammation is not characterized. Therefore, we set out to investigate the role of PARP2 in psoriasis. PARP2 mRNA expression is induced in psoriatic skin. In mice, PARP2 deletion reduces the severity of imiquimod (IMQ)-induced dermatitis. PARP2 is involved in regulating steroid synthesis and signalling, hence we asked if PARP2 regulates cutaneous lipid synthesis.

Dose Adjustment of Poly (ADP‑Ribose) Polymerase Inhibitors in Patients with Hepatic or Renal Impairment.

Poly (ADP-ribose) polymerase (PARP) inhibitors are small-molecule inhibitors of PARP enzymes (including PARP1, PARP2, and PARP3) that exhibit activity against tumor cells with defects in DNA repair. In recent years, five PARP inhibitors, olaparib, niraparib, rucaparib, talazoparib and veliparib, have been developed for the treatment of solid tumors, particularly in patients with breast-related cancer antigen (BRCA) 1/2 mutations, or those without a functional homologous recombination repair pathway. These novel treatments exhibit improved efficacy and toxicity when compared to conventional chemotherapy agents. The five PARP inhibitors are eliminated primarily via the liver and kidneys, hepatic or renal impairment may significantly affect their pharmacokinetics (PK). Therefore, it is important to know the effects of hepatic or renal impairment on the PK and safety of PARP inhibitors. In this review, we characterize and summarize the effects of hepatic and renal function on the PK of PARP inhibitors and provide specific recommendations for clinicians when prescribing PARP inhibitors in patients with hepatic or renal impairment.