Poly ADP-ribose Polymerase Activity Research Articles

Converting cell death into senescence by PARP1 inhibition improves recovery from acute oxidative injury.

Excessive amounts of reactive oxygen species (ROS) lead to macromolecular damage and high levels of cell death with consequent pathological sequelae. We hypothesized that switching cell death to a tissue regenerative state could potentially improve the short-term and long-term detrimental effects of ROS-associated acute tissue injury, although the mechanisms regulating oxidative stress-induced cell fate decisions and their manipulation for improving repair are poorly understood. Here, we show that cells exposed to high oxidative stress enter a poly (ADP-ribose) polymerase 1 (PARP1)-mediated regulated cell death, and that blocking PARP1 activation promotes conversion of cell death into senescence (CODIS). We demonstrate that this conversion depends on reducing mitochondrial Ca2+ overload as a consequence of retaining the hexokinase II on mitochondria. In a mouse model of kidney ischemia-reperfusion damage, PARP inhibition reduces necrosis and increases transient senescence at the injury site, alongside improved recovery from damage. Together, these data provide evidence that converting cell death into transient senescence can therapeutically benefit tissue regeneration.

NLRX1 Inhibits LPS-Induced Microglial Death via Inducing p62-Dependent HO-1 Expression, Inhibiting MLKL and Activating PARP-1.

The activation of microglia and the production of cytokines are key factors contributing to progressive neurodegeneration. Despite the well-recognized neuronal programmed cell death regulated by microglial activation, the death of microglia themselves is less investigated. Nucleotide-binding oligomerization domain, leucine-rich repeat-containing X1 (NLRX1) functions as a scaffolding protein and is involved in various central nervous system diseases. In this study, we used the SM826 microglial cells to understand the role of NLRX1 in lipopolysaccharide (LPS)-induced cell death. We found LPS-induced cell death is blocked by necrostatin-1 and zVAD. Meanwhile, LPS can activate poly (ADP-ribose) polymerase-1 (PARP-1) to reduce DNA damage and induce heme oxygenase (HO)-1 expression to counteract cell death. NLRX1 silencing and PARP-1 inhibition by olaparib enhance LPS-induced SM826 microglial cell death in an additive manner. Less PARylation and higher DNA damage are observed in NLRX1-silencing cells. Moreover, LPS-induced HO-1 gene and protein expression through the p62-Keap1-Nrf2 axis are attenuated by NLRX1 silencing. In addition, the Nrf2-mediated positive feedback regulation of p62 is accordingly reduced by NLRX1 silencing. Of note, NLRX1 silencing does not affect LPS-induced cellular reactive oxygen species (ROS) production but increases mixed lineage kinase domain-like pseudokinase (MLKL) activation and cell necroptosis. In addition, NLRX1 silencing blocks bafilomycin A1-induced PARP-1 activation. Taken together, for the first time, we demonstrate the role of NLRX1 in protecting microglia from LPS-induced cell death. The underlying protective mechanisms of NLRX1 include upregulating LPS-induced HO-1 expression via Nrf2-dependent p62 expression and downstream Keap1-Nrf2 axis, mediating PARP-1 activation for DNA repair via ROS- and autophagy-independent pathway, and reducing MLKL activation.

A Novel Inhibitor of Poly(ADP-Ribose) Polymerase-1 Inhibits Proliferation of a BRCA-Deficient Breast Cancer Cell Line via the DNA Damage-Activated cGAS-STING Pathway.

Loss-of-function mutations in the Breast Cancer Susceptibility Gene (BRCA1 and BRCA2) are often detected in patients with breast cancer. Poly(ADP-ribose) polymerase-1 (PARP1) plays a key role in the repair of DNA strand breaks, and PARP inhibitors have been shown to induce highly selective killing of BRCA1/2-deficient tumor cells, a mechanism termed synthetic lethality. In our previous study, a novel PARP1 inhibitor─(E)-2-(2,3-dibromo-4,5-dimethoxybenzylidene)-N-(4-fluorophenyl) hydrazine-1-carbothioamide (4F-DDC)─was synthesized, which significantly inhibited PARP1 activity with an IC50 value of 82 ± 9 nM. The current study aimed to explore the mechanism(s) underlying the antitumor activity of 4F-DDC under in vivo and in vitro conditions. 4F-DDC was found to selectively inhibit the proliferation of BRCA mutant cells, with highly potent effects on HCC-1937 (BRCA1-/-) cells. Furthermore, 4F-DDC was found to induce apoptosis and G2/M cell cycle arrest in HCC-1937 cells. Interestingly, immunofluorescence and Western blot results showed that 4F-DDC induced DNA double strand breaks and further activated the cGAS-STING pathway in HCC-1937 cells. In vivo analysis results revealed that 4F-DDC inhibited the growth of HCC-1937-derived tumor xenografts, possibly via the induction of DNA damage and activation of the cGAS-STING pathway. In summary, the current study provides a new perspective on the antitumor mechanism of PARP inhibitors and showcases the therapeutic potential of 4F-DDC in the treatment of breast cancer.

Abstract 7112: Preclinical profile of novel and potent small molecule inhibitors of PARG

Abstract Poly(ADP-ribose) glycohydrolase (PARG) is primary enzyme involved in the dePARylation process of DNA repair, specifically in the single strand break repair (SSBRs). PARG is a macrodomain protein with exo- and endo-glycohydrolase activity that liberates free ADP-ribose and PAR chains, respectively. It interacts with various Poly(ADP-ribose) polymerase (PARP) proteins to facilitate appropriate and timely DNA repair. The balance between PARP and PARG activity is essential for efficient DDR. Inhibition of PARG lead to altered DNA repair in cancer cells. We have identified highly potent PARG inhibitors with picomolar IC50 in. PARG is the primary enzyme for dePARylation and accounts for 90% of dePARylation activity. Treatment of HCC-1806 cells with PARG inhibitors showed dose dependent increase of PARylation signature, with sub micromolar IC50. Compounds also demonstrate significant functional, anti-proliferative activity in ovarian and breast cancer cell lines besides a favourable ADME profile. Further pharmacokinetic and pharmacodynamic characterization of the identified hits is currently in progress. Citation Format: Sukanya Patra, Manoj Pothuganti, Githavani Kummari, Venu Sankeshi, Jyothipriya Kapaka, Navnath Karche, Ramamohan Mekala, Srikant Viswanadha. Preclinical profile of novel and potent small molecule inhibitors of PARG [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7112.

NAMPT/NAD+/PARP1 Pathway Regulates CFA-Induced Inflammatory Pain via NF-κB Signaling in Rodents.

Emerging evidence has implicated nicotinamide adenine dinucleotide (NAD+) metabolism in various inflammatory diseases. In the study, the role of NAD+ metabolism in Complete Freund's Adjuvant (CFA)-evoked inflammatory pain and the underlying mechanisms are investigated. The study demonstrated that CFA induced upregulation of nicotinamide phosphoribosyltransferase (NAMPT) in dorsal root ganglia (DRG) without significant changes in the spinal cord. Inhibition of NAMPT expression by intrathecal injection of NAMPT siRNA alleviated CFA-induced pain-like behavior, decreased NAD+ contents in DRG, and lowered poly-(ADP-ribose) polymerase 1 (PARP1) activity levels. These effects are all reversed by the supplement of nicotinamide mononucleotide (NMN). Inhibition of PARP1 expression by intrathecal injection of PARP1 siRNA alleviated CFA-induced pain-like behavior, while elevated NAD+ levels of DRG. The analgesic effect of inhibiting NAMPT/NAD+/PARP1 axis can be attributed to the downregulation of the NF-κB/IL-1β inflammatory pathway. Double immunofluorescence staining showed that the expression of NAMPT/NAD+/PARP1 axis is restricted to DRG neurons. In conclusion, PARP1 activation in response to CFA stimulation, fueled by NAMPT-derived NAD+, mediates CFA-induced inflammatory pain through NF-κB/IL-1β inflammatory pathway.

Improved QSAR models for PARP-1 inhibition using data balancing, interpretable machine learning, and matched molecular pair analysis.

The poly (ADP-ribose) polymerase-1 (PARP-1) enzyme is an important target in the treatment of breast cancer. Currently, treatment options include the drugs Olaparib, Niraparib, Rucaparib, and Talazoparib; however, these drugs can cause severe side effects including hematological toxicity and cardiotoxicity. Although in silico models for the prediction of PARP-1 activity have been developed, the drawbacks of these models include low specificity, a narrow applicability domain, and a lack of interpretability. To address these issues, a comprehensive machine learning (ML)-based quantitative structure-activity relationship (QSAR) approach for the informed prediction of PARP-1 activity is presented. Classification models built using the Synthetic Minority Oversampling Technique (SMOTE) for data balancing gave robust and predictive models based on the K-nearest neighbor algorithm (accuracy 0.86, sensitivity 0.88, specificity 0.80). Regression models were built on structurally congeneric datasets, with the models for the phthalazinone class and fused cyclic compounds giving the best performance. In accordance with the Organization for Economic Cooperation and Development (OECD) guidelines, a mechanistic interpretation is proposed using the Shapley Additive Explanations (SHAP) to identify the important topological features to differentiate between PARP-1 actives and inactives. Moreover, an analysis of the PARP-1 dataset revealed the prevalence of activity cliffs, which possibly negatively impacts the model's predictive performance. Finally, a set of chemical transformation rules were extracted using the matched molecular pair analysis (MMPA) which provided mechanistic insights and can guide medicinal chemists in the design of novel PARP-1 inhibitors.

Type III secretion system effector YfiD inhibits the activation of host poly(ADP-ribose) polymerase-1 to promote bacterial infection

Modulation of cell death is a powerful strategy employed by pathogenic bacteria to evade host immune clearance and occupy profitable replication niches during infection. Intracellular pathogens employ the type III secretion system (T3SS) to deliver effectors, which interfere with regulated cell death pathways to evade immune defenses. Here, we reveal that poly(ADP-ribose) polymerase-1 (PARP1)-dependent cell death restrains Edwardsiella piscicida’s proliferation in mouse monocyte macrophages J774A.1, of which PARP1 activation results in the accumulation of poly(ADP-ribose) (PAR) and enhanced inflammatory response. Moreover, E. piscicida, an important intracellular pathogen, leverages a T3SS effector YfiD to impair PARP1’s activity and inhibit PAR accumulation. Once translocated into the host nucleus, YfiD binds to the ADP-ribosyl transferase (ART) domain of PARP1 to suppress its PARylation ability as the pharmacological inhibitor of PARP1 behaves. Furthermore, the interaction between YfiD and ART mainly relies on the complete unfolding of the helical domain, which releases the inhibitory effect on ART. In addition, YfiD impairs the inflammatory response and cell death in macrophages and promotes in vivo colonization and virulence of E. piscicida. Collectively, our results establish the functional mechanism of YfiD as a potential PARP1 inhibitor and provide more insights into host defense against bacterial infection.

PARP trapping is governed by the PARP inhibitor dissociation rate constant.

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) are a class of cancer drugs that enzymatically inhibit PARP activity at sites of DNA damage. Yet, PARPi function mainly by trapping PARP1 onto DNA with a wide range of potency among the clinically relevant inhibitors. How PARPi trap and why some are better trappers remain unknown. Here, we show trapping occurs primarily through a kinetic phenomenon at sites of DNA damage that correlates with PARPi koff. Our results suggest PARP trapping is not the physical stalling of PARP1 on DNA, rather the high probability of PARP re-binding damaged DNA in the absence of other DNA-binding protein recruitment. These results clarify how PARPi trap, shed new light on how PARPi function, and describe how PARPi properties correlate to trapping potency.

Exposure to fluoride exacerbates the cognitive deficit of diabetic patients living in areas with endemic fluorosis, as well as of rats with type 2 diabetes induced by streptozotocin via a mechanism that may involve excessive activation of the poly(ADP ribose) polymerase-1/P53 pathway

Epidemiology has shown that fluoride exposure is associated with the occurrence of diabetes. However, whether fluoride affects diabetic encephalopathy is unclear. Elderly diabetic patients in areas with endemic (n = 169) or no fluorosis (108) and controls (85) underwent Montreal Cognitive Assessment. Sprague-Dawley rats receiving streptozotocin and/or different fluoride doses were examined for spatial learning and memory, brain morphology, blood-brain barrier, fasting blood glucose and insulin. Cultured SH-SY5Y cells were treated with 50 mM glucose and/or low- or high-dose fluoride, and P53-knockdown or poly-ADP-ribose polymerase-1 (PARP-1) inhibition. The levels of PARP-1, P53, poly-ADP-ribose (PAR), apoptosis-inducing factor (AIF), and phosphorylated-histone H2A.X (ser139) were measured by Western blotting. Reactive oxygen species (ROS), 8-hydroxydeguanosine (8-OHdG), PARP-1 activity, acetyl-P53, nicotinamide adenine dinucleotide (NAD+), activities of mitochondrial hexokinase1 (HK1) and citrate synthase (CS), mitochondrial membrane potential and apoptosis were assessed biochemically. Cognition of diabetic patients in endemic fluorosis areas was poorer than in other regions. In diabetic rats, fasting blood glucose, insulin resistance and blood-brain barrier permeability were elevated, while spatial learning and memory and Nissl body numbers in neurons declined. In these animals, expression and activity of P53 and PARP-1 and levels of NAD+, PAR, ROS, 8-OHdG, p-histone H2A.X (ser139), AIF and apoptosis content increased; whereas mitochondrial HK1 and CS activities and membrane potential decreased. SH-SY5Y cells exposed to glucose exhibited changes identical to diabetic rats. The changes in diabetic rats and cells treated with glucose were aggravated by fluoride. P53-knockout or PARP-1 inhibition mitigated the effects of glucose with/without low-dose fluoride. Elevation of diabetic encephalopathy was induced by exposure to fluoride and the underlying mechanism may involve overactivation of the PARP-1/P53 pathway.

Inhibition of host PARP1 contributes to the anti-inflammatory and antitubercular activity of pyrazinamide

The antibiotic pyrazinamide (PZA) is a cornerstone of tuberculosis (TB) therapy that shortens treatment durations by several months despite being only weakly bactericidal. Intriguingly, PZA is also an anti-inflammatory molecule shown to specifically reduce inflammatory cytokine signaling and lesion activity in TB patients. However, the target and clinical importance of PZA’s host-directed activity during TB therapy remain unclear. Here, we identify the host enzyme Poly(ADP-ribose) Polymerase 1 (PARP1), a pro-inflammatory master regulator strongly activated in TB, as a functionally relevant host target of PZA. We show that PZA inhibits PARP1 enzymatic activity in macrophages and in mice where it reverses TB-induced PARP1 activity in lungs to uninfected levels. Utilizing a PZA-resistant mutant, we demonstrate that PZA’s immune-modulatory effects are PARP1-dependent but independent of its bactericidal activity. Importantly, PZA’s bactericidal efficacy is impaired in PARP1-deficient mice, suggesting that immune modulation may be an integral component of PZA’s antitubercular activity. In addition, adjunctive PARP1 inhibition dramatically reduces inflammation and lesion size in mice and may be a means to reduce lung damage and shorten TB treatment duration. Together, these findings provide insight into PZA’s mechanism of action and the therapeutic potential of PARP1 inhibition in the treatment of TB.

PARP Inhibitor Sensitizes BRCA-mutant Pancreatic Cancer to Oxaliplatin by Suppressing the CDK1/BRCA1 Axis.

Currently, olaparib, a poly(ADP-ribose) polymerase (PARP) inhibitor, has been approved as maintenance therapy for patients with germline BRCA mutations and metastatic pancreatic cancer. However, platinum-based chemotherapy, which induces synthetic lethality with PARP inhibitor treatment, is still controversial. Hence, we aimed to examine a platinum-based drug in combination with a PARP inhibitor and generate data regarding the use of a PARP inhibitor in the overall treatment of pancreatic cancer. Using the Capan-1 cell line (BRCA2-mutant pancreatic cancer cell line), we evaluated the combinatorial effects of olaparib, a PARP inhibitor, and oxaliplatin by cell viability, combination index, western blotting, immunocytochemistry, flow cytometry, apoptosis assays and in vivo experiments. Capan-1 cells showed high sensitivity to olaparib due to the alteration in PARP activity, which led to cell death through the accumulation of oxaliplatin-induced DNA damage. Beyond DNA damage, oxaliplatin also suppressed the CDK1/BRCA1 signaling axis, which induced defects in homologous recombination repair. Additionally, inhibition of CDK1, a biomarker for oxaliplatin efficacy, induced cell death regardless of the BRCA mutation profile. Oxaliplatin may be used in combination with olaparib in PDAC patients with DNA damage repair mutations. Our findings highlight CDK1 as a potential therapeutic target for pancreatic cancer.

Poly (ADP-ribose) polymerase 1 and neurodegenerative diseases: Past, present, and future.

Poly (ADP-ribose) polymerase 1 (PARP1) is a first responder that recognizes DNA damage and facilitates its repair. Neurodegenerative diseases, characterized by progressive neuron loss driven by various risk factors, including DNA damage, have increasingly shed light on the pivotal involvement of PARP1. During the early phases of neurodegenerative diseases, PARP1 experiences controlled activation to swiftly address mild DNA damage, thereby contributing to maintain brain homeostasis. However, in late stages, exacerbated PARP1 activation precipitated by severe DNA damage exacerbates the disease condition. Consequently, inhibition of PARP1 overactivation emerges as a promising therapeutic approach for neurodegenerative diseases. In this review, we comprehensively synthesize and explore the multifaceted role of PARP1 in neurodegenerative diseases, with a particular emphasis on its over-activation in the aggregation of misfolded proteins, dysfunction of the autophagy-lysosome pathway, mitochondrial dysfunction, neuroinflammation, and blood-brain barrier (BBB) injury. Additionally, we encapsulate the therapeutic applications and limitations intrinsic of PARP1 inhibitors, mainly including limited specificity, intricate pathway dynamics, constrained clinical translation, and the heterogeneity of patient cohorts. We also explore and discuss the potential synergistic implementation of these inhibitors alongside other agents targeting DNA damage cascades within neurodegenerative diseases. Simultaneously, we propose several recommendations for the utilization of PARP1 inhibitors within the realm of neurodegenerative disorders, encompassing factors like the disease-specific roles of PARP1, combinatorial therapeutic strategies, and personalized medical interventions. Lastly, the encompassing review presents a forward-looking perspective along with strategic recommendations that could guide future research endeavors in this field.

The Initial Hepatitis B Virus-Hepatocyte Genomic Integrations and Their Role in Hepatocellular Oncogenesis.

Hepatitis B virus (HBV) remains a dominant cause of hepatocellular carcinoma (HCC). Recently, it was shown that HBV and woodchuck hepatitis virus (WHV) integrate into the hepatocyte genome minutes after invasion. Retrotransposons and transposable sequences were frequent sites of the initial insertions, suggesting a mechanism for spontaneous HBV DNA dispersal throughout the hepatocyte genome. Several somatic genes were also identified as early insertional targets in infected hepatocytes and woodchuck livers. Head-to-tail joints (HTJs) dominated amongst fusions, indicating their creation by non-homologous end-joining (NHEJ). Their formation coincided with the robust oxidative damage of hepatocyte DNA. This was associated with the activation of poly(ADP-ribose) polymerase 1 (PARP1)-mediated dsDNA repair, as reflected by the augmented transcription of PARP1 and XRCC1; the PARP1 binding partner OGG1, a responder to oxidative DNA damage; and increased activity of NAD+, a marker of PARP1 activation, and HO1, an indicator of cell oxidative stress. The engagement of the PARP1-mediated NHEJ repair pathway explains the HTJ format of the initial merges. The findings show that HBV and WHV are immediate inducers of oxidative DNA damage and hijack dsDNA repair to integrate into the hepatocyte genome, and through this mechanism, they may initiate pro-oncogenic processes. Tracking initial integrations may uncover early markers of HCC and help to explain HBV-associated oncogenesis.

24 Correlation of clinical outcomes and poly ADP ribose polymerase activity in high grade serous ovarian cancer

24 Correlation of clinical outcomes and poly ADP ribose polymerase activity in high grade serous ovarian cancer

PARP-1 improves leukemia outcomes by inducing parthanatos during chemotherapy

Previous chemotherapy research has focused almost exclusively on apoptosis. Here, a standard frontlinedrug combination of cytarabine and idarubicin induces distinct features of caspase-independent, poly(ADP-ribose)polymerase 1 (PARP-1)-mediated programmed cell death "parthanatos" in acute myeloid leukemia (AML) cell lines (n= 3/10 tested), peripheral blood mononuclear cells from healthy human donors (n= 10/10 tested), and primary cell samples from patients with AML (n= 18/39 tested, French-American-British subtypes M4 and M5). A 3-fold improvement in survival rates is observed in the parthanatos-positive versus -negative patient groups (hazard ratio [HR]= 0.28-0.37, p= 0.002-0.046). Manipulation of PARP-1 activity in parthanatos-competent cells reveals higher drug sensitivity in cells that have basal PARP-1 levels as compared with those subjected to PARP-1 overexpression or suppression. The same trends are observed in RNA expression databases and support the conclusion that PARP-1 can have optimal levels for favorable chemotherapeutic responses.

Inhibiting PRMT5 induces DNA damage and increases anti-proliferative activity of Niraparib, a PARP inhibitor, in models of breast and ovarian cancer

BackgroundInhibitors of Poly (ADP-Ribose) Polymerases (PARP) provide clinical benefit to patients with breast and ovarian cancers, by compromising the DNA repair activity of cancer cells. Although these agents extend progression-free survival in many patients, responses can be short lived with many patients ultimately progressing. Identification of combination partners that increase dependence of cancer cells to the DNA repair activity of PARPs may represent a strategy to increase the utility of PARP inhibitors. Protein arginine methyltransferase 5 (PRMT5) regulates DNA damage response pathways through splicing and protein modification, and inhibitors of PRMT5 have recently entered clinical trials.MethodsThe effect of PRMT5 inhibition on the levels of DNA damage and repair markers including γH2AX, RAD51, and 53BP1 was determined using high content immunofluorescent imaging. The anti-proliferative activity of the combination of PRMT5 and PARP inhibitors was evaluated using in vitro models of breast and ovarian cancers using both cell lines and ex vivo patient derived xenografts. Finally, the combinations of PRMT5 and PARP inhibitors were evaluated in cell line xenograft models in vivo.ResultsInhibition of PRMT5 by GSK3326595 led to increased levels of markers of DNA damage. The addition of GSK3326595 to the PARP inhibitor, niraparib, resulted in increased growth inhibition of breast and ovarian cancer cell lines and patient derived spheroids. In vivo, the combination improved the partial effects on tumor growth inhibition achieved by either single agent, producing complete tumor stasis and regression.ConclusionThese data demonstrate that inhibition of PRMT5 induced signatures of DNA damage in models of breast and ovarian cancer. Furthermore, combination with the PARP inhibitor, Niraparib, resulted in increased anti-tumor activity in vitro and in vivo. Overall, these data suggest inhibition of PRMT5 as a mechanism to broaden and enhance the clinical application of PARP inhibitors.

Poly(ADP-Ribose) Polymerase-1 Lacking Enzymatic Activity Is Not Compatible with Mouse Development.

Poly(ADP-ribose) polymerase-1 (PARP1) binds DNA lesions to catalyse poly(ADP-ribosyl)ation (PARylation) using NAD+ as a substrate. PARP1 plays multiple roles in cellular activities, including DNA repair, transcription, cell death, and chromatin remodelling. However, whether these functions are governed by the enzymatic activity or scaffolding function of PARP1 remains elusive. In this study, we inactivated in mice the enzymatic activity of PARP1 by truncating its C-terminus that is essential for ART catalysis (PARP1ΔC/ΔC, designated as PARP1-ΔC). The mutation caused embryonic lethality between embryonic day E8.5 and E13.5, in stark contrast to PARP1 complete knockout (PARP1-/-) mice, which are viable. Embryonic stem (ES) cell lines can be derived from PARP1ΔC/ΔC blastocysts, and these mutant ES cells can differentiate into all three germ layers, yet, with a high degree of cystic structures, indicating defects in epithelial cells. Intriguingly, PARP1-ΔC protein is expressed at very low levels compared to its full-length counterpart, suggesting a selective advantage for cell survival. Noticeably, PARP2 is particularly elevated and permanently present at the chromatin in PARP1-ΔC cells, indicating an engagement of PARP2 by non-enzymatic PARP1 protein at the chromatin. Surprisingly, the introduction of PARP1-ΔC mutation in adult mice did not impair their viability; yet, these mutant mice are hypersensitive to alkylating agents, similar to PARP1-/- mutant mice. Our study demonstrates that the catalytically inactive mutant of PARP1 causes the developmental block, plausibly involving PARP2 trapping.

The kynurenine pathway of tryptophan metabolism: a neglected therapeutic target of COVID-19 pathophysiologyand immunotherapy.

SARS-CoV-2 (COVID-19) exerts profound changes in the kynurenine (Kyn) pathway (KP) of tryptophan (Trp) metabolism that may underpin its pathophysiology. The KP is the main source of the vital cellular effector NAD+ and intermediate metabolites that modulate immune and neuronal functions. Trp metabolism is the top pathway influenced by COVID-19. Sixteenstudies established virus-induced activation of the KPmediated mainly by induction of indoleamine 2,3-dioxygenase(IDO1) in most affected tissues and of IDO2 in lung by the increased release of proinflammatory cytokines, but could additionally involve increased flux of plasma free Trp and induction of Trp 2,3-dioxygenase (TDO) by cortisol. The major Kyn metabolite targeted by COVID-19 is kynurenic acid (KA), the Kyn metabolite with the greatest affinity for the aryl hydrocarbon receptor (AhR), which is also activated by COVID-19. AhR activation initiates two important series of events: a vicious circle involving IDO1induction, KA accumulation and further AhR activation, and activation of poly (ADP-ribose)polymerase (PARP) leading to NAD+ depletion and cell death. The virus further deprives the host of NAD+ by inhibiting its main biosynthetic pathway from quinolinic acid, while simultaneously acquiring NAD+ by promoting its synthesis from nicotinamide in the salvage pathway. Additionally, the protective effects of sirtuin 1 are minimised by the PARP activation. KP dysfunction may also underpin the mood and neurological disorders acutely and during "long COVID". More studies of potential effects of vaccination therapy on the KP are required andexploration of therapeutic strategies involving modulation of the KP changes are proposed.

5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) induces apoptosis in breast cancer cells through inhibiting of Mcl-1 expression

The effective treatment of breast cancer remains a profound clinical challenge, especially due to drug resistance and metastasis which unfortunately arise in many patients. The transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB), as a selective inhibitor of cyclin-dependent kinase 9, was shown to be effective in inducing apoptosis in various hematopoietic malignancies. However, the anticancer efficacy of DRB against breast cancer is still unclear. Herein, we demonstrated that administration of DRB to the breast cancer cell line led to the inhibition of cellular proliferation and induction of the typical signs of apoptotic cells, including the increases in Annexin V-positive cells, DNA fragmentation, and activation of caspase-7, caspase-9, and poly (ADP ribose) polymerase (PARP). Treatment of DRB resulted in a rapid decline in the myeloid cell leukemia 1 (Mcl-1) protein, whereas levels of other antiapoptotic proteins did not change. Overexpression of Mcl-1 decreased the DRB-induced PARP cleavage, whereas knockdown of Mcl-1 enhanced the effects of DRB on PARP activation, indicating that loss of Mcl-1 accounts for the DRB-mediated apoptosis in MCF-7 cells, but not in T-47D. Furthermore, we found that co-treatment of MCF-7 cells with an inhibitor of AKT (LY294002) or an inhibitor of the proteasome (MG-132) significantly augmented the DRB-induced apoptosis. These data suggested that DRB in combination with LY294002 or MG-132 may have a greater therapeutic potency against breast cancer cells.

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.