A PARP1-TIMELESS alliance in cancer therapy.

In a recent paper in Nature, Petropoulos et al.1 report that PARP1 acts together with the replisome components TIMELESS and TIPIN to protect the genome from transcription-replication conflicts, which has important implications for the clinical use of PARP inhibitors.

Achillea fragrantissima (Forssk.) Sch.Bip instigates the ROS/FADD/c-PARP expression that triggers apoptosis in breast cancer cell (MCF-7).

Achillea fragrantissima is a shrub plant that belongs to the Asteraceae family in Arabia and Egypt. It is used as folk medicine and is a good source of phenolic acids, flavonoids, and some active compounds. To investigate the anti-cancer effect of A.fragrantissima on breast cancer MCF-7 cells and find the critical mechanism involved in apoptosis. The toxicity and pharmacokinetic studies of ethanolic extract of A.fragrantissima was examined for anti-breast cancer properties. In turn, cytotoxicity and cell viability were achieved by the MTT method. Furthermore, the trypan blue exclusion and microscopy examination proved the presence of apoptotic cells. Again, fluorescent staining such as AO/EtBr, DCFH-DA, Rho-123, and Hoechst-33342 reveals the cellular cytoplasmic disciplines upon A. fragrantissima effect. Moreover, cellular functioning tests like wound healing, colony formation, and Transwell invasion assay were demonstrated. In addition, the qRT-PCR technique authenticates the A. fragrantissima -induced apoptotic network genes (Caspase-3, Caspase-8, Caspase-9, Cytochrome c, BCL-2, BID, BAX, PARP, PTEN, PI3K, and Akt) expression were evaluated. Mainly, the Immunoblot technique proved the expressed level of apoptotic proteins such as cleaved PARP, CYCS, and FADD. This study confirmed that the A. fragrantissima exerts cytotoxicity at 20 µg/mL for 24 hrs in MCF-7 cells. Also, decreases cellular viability, producing apoptotic cells and damaged cellular surfaces with dead matter. Consequently, it creates ROS species accumulation, loss of mitochondrial membrane potential, and fragmentation of DNA in MCF-7 cells. Furthermore, it arrests cell migration, induces colony-forming ability loss, and suppresses cell invasion. In addition, A. fragrantissima significantly upregulates genes such as caspase-3, 9, cytochrome c, BID, BAX, and PTEN while downregulating the Pi3K/ Akt signaling. Nonetheless, A.fragrantissima induced cleaved PARP, CYCS, and FADD proteins in MCF-7 cells to avail apoptosis.

Specific and shared biological functions of PARP2 - is PARP2 really a lil' brother of PARP1?

PARP2, that belongs to the family of ADP-ribosyl transferase enzymes (ART), is a discovery of the millennium, as it was identified in 1999. Although PARP2 was described initially as a DNA repair factor, it is now evident that PARP2 partakes in the regulation or execution of multiple biological processes as inflammation, carcinogenesis and cancer progression, metabolism or oxidative stress-related diseases. Hereby, we review the involvement of PARP2 in these processes with the aim of understanding which processes are specific for PARP2, but not for other members of the ART family. A better understanding of the specific functions of PARP2 in all of these biological processes is crucial for the development of new PARP-centred selective therapies.

Discovery of highly potent PARP7 inhibitors for cancer immunotherapy.

PARP7 has been proven to play an important role in immunity. Substantial upregulation of PARP7 is observed in numerous cancerous cell types, consequently resulting in the inhibition of type Ⅰ interferon signaling pathways. Therefore, inhibiting the activity of PARP7 can enhance type Ⅰ interferon signaling to exert an anti-tumor immune response. In this study, we reported the identification of a newly found PARP7 inhibitor (XLY-1) with higher inhibitory activity (IC50 = 0.6 nM) than that of RBN-2397 (IC50 = 6.0 nM). Additionally, XYL-1 displayed weak inhibitory activity on PARP1 (IC50 > 1.0 µM). Mechanism studies showed that XYL-1 could enhance the type Ⅰ interferon signaling in vitro. Pharmacodynamic experiments showed that 50 mg/kg XYL-1 could significantly inhibit tumor growth (TGI: 76.5 %) and related experiments showed that XYL-1 could restore type Ⅰ interferon signaling and promote T cell infiltration in tumor tissues. Taken together, XYL-1 shows promise as a potential candidate for developing cancer immunotherapy agents.

Deltex family E3 ligases specifically ubiquitinate the terminal ADP-ribose of poly(ADP-ribosyl)ation.

Poly(ADP-ribose) polymerases (PARPs) are critical to regulating cellular activities, such as the response to DNA damage and cell death. PARPs catalyze a reversible post-translational modification (PTM) in the form of mono- or poly(ADP-ribosyl)ation. This type of modification is known to form a ubiquitin-ADP-ribose (Ub-ADPR) conjugate that depends on the actions of Deltex family of E3 ubiquitin ligases (DTXs). In particular, DTXs add ubiquitin to the 3'-OH of adenosine ribose' in ADP-ribose, which effectively sequesters ubiquitin and impedes ubiquitin-dependent signaling. Previous work demonstrates DTX function for ubiquitination of protein-free ADPR, mono-ADP-ribosylated peptides, and ADP-ribosylated nucleic acids. However, the dynamics of DTX-mediated ubiquitination of poly(ADP-ribosyl)ation remains to be defined. Here we show that the ADPR ubiquitination function is not found in other PAR-binding E3 ligases and is conserved across DTX family members. Importantly, DTXs specifically target poly(ADP-ribose) chains for ubiquitination that can be cleaved by PARG, the primary eraser of poly(ADP-ribose), leaving the adenosine-terminal ADPR unit conjugated to ubiquitin. Our collective results demonstrate the DTXs' specific ubiquitination of the adenosine terminus of poly(ADP-ribosyl)ation and suggest the unique Ub-ADPR conjugation process as a basis for PARP-DTX control of cellular activities.

PARP1 expression predicts PARP inhibitor sensitivity and correlates with metastatic potential and overall survival in melanoma.

Metastatic melanoma is still a difficult-to-treat cancer type owing to its frequent resistance mechanisms to targeted and immunotherapy. Therefore, we aimed to unravel novel therapeutic strategies for melanoma patients. Preclinical and clinical studies show that melanoma patients may benefit from a treatment with poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi). In this study, we focus on PARP1 as a potential biomarker to predict the response of melanoma cells to PARPi therapy. We found that melanoma cells with high basal PARP1 expression exhibit significantly increased cell death after PARPi treatment owing to higher PARP1 trapping compared with melanoma cells with low PARP1 expression. In addition, we could demonstrate that PARP1 expression levels are low in nonmalignant skin cells, and metastatic melanomas show considerably higher PARP1 levels compared with primary melanomas. Most strikingly, we found that high PARP1 levels correlate with worse overall survival of late stage metastasized melanoma patients. In conclusion, we show that PARP1 might act as a biomarker to predict the response to PARPi therapy, and that in particular the late stage metastasized melanoma patients are especially sensitive to PARPi therapy owing to elevated PARP1 expression. Our data suggest that the PARPi cytotoxicity primarily will affect the high PARP1 expressing melanoma cells, rather than the low PARP1 expressing nonmalignant skin cells resulting in only low side effects.

PARP inhibitors suppress tumours via centrosome error-induced senescence independent of DNA damage response.

BACKGROUND: Poly(ADP-ribose) polymerase (PARP) inhibitors have emerged as promising chemotherapeutic drugs primarily against BRCA1/2-associated tumours, known as synthetic lethality. However, recent clinical trials reported patients' survival benefits from PARP inhibitor treatments, irrelevant to homologous recombination deficiency. Therefore, revealing the therapeutic mechanism of PARP inhibitors beyond DNA damage repair is urgently needed, which can facilitate precision medicine. METHODS: A CRISPR-based knock-in technology was used to establish stable BRCA1 mutant cancer cells. The effects of PARP inhibitors on BRCA1 mutant cancer cells were evaluated by biochemical and cell biological experiments. Finally, we validated its in vivo effects in xenograft and patient-derived xenograft (PDX) tumour mice. FINDINGS: In this study, we uncovered that the majority of clinical BRCA1 mutations in breast cancers were in and near the middle of the gene, rather than in essential regions for DNA damage repair. Representative mutations such as R1085I and E1222Q caused transient extra spindle poles during mitosis in cancer cells. PAR, which is synthesized by PARP2 but not PARP1 at mitotic centrosomes, clustered these transient extra poles, independent of DNA damage response. Common PARP inhibitors could effectively suppress PARP2-synthesized PAR and induce cell senescence by abrogating the correction of mitotic extra-pole error. INTERPRETATION: Our findings uncover an alternative mechanism by which PARP inhibitors efficiently suppress tumours, thereby pointing to a potential new therapeutic strategy for centrosome error-related tumours. FUNDING: Funded by National Natural Science Foundation of China (NSFC) (T2225006, 82272948, 82103106), Beijing Municipal Natural Science Foundation (Key program Z220011), and the National Clinical Key Specialty Construction Program, P. R. China (2023).

Leveraging shape screening and molecular dynamics simulations to optimize PARP1-Specific chemo/radio-potentiators for antitumor drug design.

PARP1 plays a pivotal role in DNA repair within the base excision pathway, making it a promising therapeutic target for cancers involving BRCA mutations. Current study is focused on the discovery of PARP inhibitors with enhanced selectivity for PARP1. Concurrent inhibition of PARP1 with PARP2 and PARP3 affects cellular functions, potentially causing DNA damage accumulation and disrupting immune responses. In step 1, a virtual library of 593 million compounds has been screened using a shape-based screening approach to narrow down the promising scaffolds. In step 2, hierarchical docking approach embedded in Schrödinger suite was employed to select compounds with good dock score, drug-likeness and MMGBSA score. Analysis supplemented with decomposition energy, molecular dynamics (MD) simulations and hydrogen bond frequency analysis, pinpointed that active site residues; H862, G863, R878, M890, Y896 and F897 are crucial for specific binding of ZINC001258189808 and ZINC000092332196 with PARP1 as compared to PARP2 and PARP3. The binding of ZINC000656130962, ZINC000762230673, ZINC001332491123, and ZINC000579446675 also revealed interaction involving two additional active site residues of PARP1, namely N767 and E988. Weaker or no interaction was observed for these residues with PARP2 and PARP3. This approach advances our understanding of PARP-1 specific inhibitors and their mechanisms of action, facilitating the development of targeted therapeutics.

Targeting PARP14 with lomitapide suppresses drug resistance through the activation of DRP1-induced mitophagy in multiple myeloma.

Multiple myeloma (MM) is a hematological malignancy that remains incurable, primarily due to the high likelihood of relapse or development of resistance to current treatments. To explore and discover new medications capable of overcoming drug resistance in MM, we conducted cell viability inhibition screens of 1504 FDA-approved drugs. Lomitapide, a cholesterol-lowering agent, was found to exhibit effective inhibition on bortezomib-resistant MM cells in vitro and in vivo. Our data also indicated that lomitapide decreases the permeability of the mitochondrial outer membrane and induces mitochondrial dysfunction in MM cells. Next, lomitapide treatment upregulated DRP1 and PINK1 expression levels, coupled with the mitochondrial translocation of Parkin, leading to MM cell mitophagy. Excessive mitophagy caused mitochondrial damage and dysfunction induced by lomitapide. Meanwhile, PARP14 was identified as a direct target of lomitapide by SPR-HPLC-MS, and we showed that DRP1-induced mitophagy was crucial in the anti-MM activity mediated by PARP14. Furthermore, PARP14 is overexpressed in MM patients, implying that it is a novel therapeutic target in MM. Collectively, our results demonstrate that DRP1-mediated mitophagy induced by PARP14 may be the cause for mitochondrial dysfunction and damage in response to lomitapide treatment.

Cooperative nucleic acid binding by Poly ADP-ribose polymerase 1.

Poly (ADP)-ribose polymerase 1 (PARP1) is an abundant nuclear protein well-known for its role in DNA repair yet also participates in DNA replication, transcription, and co-transcriptional splicing, where DNA is undamaged. Thus, binding to undamaged regions in DNA and RNA is likely a part of PARP1's normal repertoire. Here we describe analyses of PARP1 binding to two short single-stranded DNAs, a single-stranded RNA, and a double stranded DNA. The investigations involved comparing the wild-type (WT) full-length enzyme with mutants lacking the catalytic domain (∆CAT) or zinc fingers 1 and 2 (∆Zn1∆Zn2). All three protein types exhibited monomeric characteristics in solution and formed saturated 2:1 complexes with single-stranded T20 and U20 oligonucleotides. These complexes formed without accumulation of 1:1 intermediates, a pattern suggestive of positive binding cooperativity. The retention of binding activities by ∆CAT and ∆Zn1∆Zn2 enzymes suggests that neither the catalytic domain nor zinc fingers 1 and 2 are indispensable for cooperative binding. In contrast, when a double stranded 19mer DNA was tested, WT PARP1 formed a 4:1 complex while the ∆Zn1Zn2 mutant binding saturated at 1:1 stoichiometry. These deviations from the 2:1 pattern observed with T20 and U20 oligonucleotides show that PARP's binding mechanism can be influenced by the secondary structure of the nucleic acid. Our studies show that PARP1:nucleic acid interactions are strongly dependent on the nucleic acid type and properties, perhaps reflecting PARP1's ability to respond differently to different nucleic acid ligands in cells. These findings lay a platform for understanding how the functionally versatile PARP1 recognizes diverse oligonucleotides within the realms of chromatin and RNA biology.

Network integration of thermal proteome profiling with multi-omics data decodes PARP inhibition.

Complex disease phenotypes often span multiple molecular processes. Functional characterization of these processes can shed light on disease mechanisms and drug effects. Thermal Proteome Profiling (TPP) is a mass-spectrometry (MS) based technique assessing changes in thermal protein stability that can serve as proxies of functional protein changes. These unique insights of TPP can complement those obtained by other omics technologies. Here, we show how TPP can be integrated with phosphoproteomics and transcriptomics in a network-based approach using COSMOS, a multi-omics integration framework, to provide an integrated view of transcription factors, kinases and proteins with altered thermal stability. This allowed us to recover consequences of Poly (ADP-ribose) polymerase (PARP) inhibition in ovarian cancer cells on cell cycle and DNA damage response as well as interferon and hippo signaling. We found that TPP offers a complementary perspective to other omics data modalities, and that its integration allowed us to obtain a more complete molecular overview of PARP inhibition. We anticipate that this strategy can be used to integrate functional proteomics with other omics to study molecular processes.

Cannabinoids induce cell death in leukaemic cells through Parthanatos and PARP-related metabolic disruptions.

BACKGROUND: Several studies have described a potential anti-tumour effect of cannabinoids (CNB). CNB receptor 2 (CB2) is mostly present in hematopoietic stem cells (HSC). The present study evaluates the anti-leukaemic effect of CNB. METHODS: Cell lines and primary cells from acute myeloid leukaemia (AML) patients were used and the effect of the CNB derivative WIN-55 was evaluated in vitro, ex vivo and in vivo. RESULTS: We demonstrate a potent antileukemic effect of WIN-55 which is abolished with CB antagonists. WIN-treated mice, xenografted with AML cells, had better survival as compared to vehicle or cytarabine. DNA damage-related genes were affected upon exposure to WIN. Co-incubation with the PARP inhibitor Olaparib prevented WIN-induced cell death, suggesting PARP-mediated apoptosis which was further confirmed with the translocation of AIF to the nucleus observed in WIN-treated cells. Nicotinamide prevented WIN-related apoptosis, indicating NAD+ depletion. Finally, WIN altered glycolytic enzymes levels as well as the activity of G6PDH. These effects are reversed through PARP1 inhibition. CONCLUSIONS: WIN-55 exerts an antileukemic effect through Parthanatos, leading to translocation of AIF to the nucleus and depletion of NAD+, which are reversed through PARP1 inhibition. It also induces metabolic disruptions. These effects are not observed in normal HSC.

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.

CRABP2 reduces the sensitivity of Olaparib in ovarian cancer by downregulating Caspase-8 and decreasing the production of reactive oxygen species.

Poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors, such as Olaparib, have been pivotal in treating BRCA-deficient ovarian cancer. However, their efficacy is limited in over 40% of BRCA-deficient patients, with acquired resistance posing new clinical challenges. To address this, we employed bioinformatics methods to identify key genes impacting Olaparib sensitivity in ovarian cancer. Through comprehensive analysis of public databases including GEO, CPTAC, Kaplan Meier Plotter, and CCLE, we identified CRABP2 as significantly upregulated at both mRNA and protein levels in ovarian cancer, correlating with poor prognosis and decreased Olaparib sensitivity. Using colony formation and CCK-8 assays, we confirmed that CRABP2 knockdown in OVCAR3 and TOV112D cells enhanced sensitivity to Olaparib. Additionally, 4D label-free quantitative proteomics analysis, GSEA, and GO/KEGG analysis revealed CRABP2's involvement in regulating oxidation signals. Flow cytometry, colony formation assays, and western blotting demonstrated that CRABP2 knockdown promoted ROS production by activating Caspase-8, thereby augmenting Olaparib sensitivity and inhibiting ovarian cancer cell proliferation. Moreover, in xenograft models, CRABP2 knockdown significantly suppressed tumorigenesis and enhanced Olaparib sensitivity, with the effect being reversed upon Caspase-8 knockdown. These findings suggest that CRABP2 may modulate Olaparib sensitivity in ovarian cancer through the Caspase-8/ROS axis, highlighting its potential as a target for Olaparib sensitization.

Transcription-replication conflicts underlie sensitivity to PARP inhibitors.

An important advance in cancer therapy has been the development of poly(ADP-ribose) polymerase (PARP) inhibitors for the treatment of homologous recombination (HR)-deficient cancers1-6. PARP inhibitors trap PARPs on DNA. The trapped PARPs are thought to block replisome progression, leading to formation of DNA double-strand breaks that require HR for repair7. Here we show that PARP1 functions together with TIMELESS and TIPIN to protect the replisome in early S phase from transcription-replication conflicts. Furthermore, the synthetic lethality of PARP inhibitors with HR deficiency is due to an inability to repair DNA damage caused by transcription-replication conflicts, rather than by trapped PARPs. Along these lines, inhibiting transcription elongation in early S phase rendered HR-deficient cells resistant to PARP inhibitors and depleting PARP1 by small-interfering RNA was synthetic lethal with HR deficiency. Thus, inhibiting PARP1 enzymatic activity may suffice for treatment efficacy in HR-deficient settings.

Poly(ADP-ribosyl)ation of TIMELESS limits DNA replication stress and promotes stalled fork protection.

Poly(ADP-ribosyl)ation (PARylation), catalyzed mainly by poly(ADP-ribose) polymerase (PARP)1, is a key posttranslational modification involved in DNA replication and repair. Here, we report that TIMELESS (TIM), an essential scaffold of the replisome, is PARylated, which is linked to its proteolysis. TIM PARylation requires recognition of auto-modified PARP1 via two poly(ADP-ribose)-binding motifs, which primes TIM for proteasome-dependent degradation. Cells expressing the PARylation-refractory TIM mutant or under PARP inhibition accumulate TIM at DNA replication forks, causing replication stress and hyper-resection of stalled forks. Mechanistically, aberrant engagement of TIM with the replicative helicase impedes RAD51 loading and protection of reversed forks. Accordingly, defective TIM degradation hypersensitizes BRCA2-deficient cells to replication damage. Our study defines TIM as a substrate of PARP1 and elucidates how the control of replisome remodeling by PARylation is linked to stalled fork protection. Therefore, we propose a mechanism of PARP inhibition that impinges on the DNA replication fork instability caused by defective TIM turnover.

Excavatolide C/cisplatin combination induces antiproliferation and drives apoptosis and DNA damage in bladder cancer cells.

Excavatolide C (EXCC), a marine coral-derived compound, exhibits an antiproliferation effect on bladder cancer cells. The present study evaluated the improvement in the antiproliferation ability of EXCC by co-treatment with cisplatin in bladder cancer cells. EXCC/cisplatin (12.5 and 1 µg/mL) showed higher antiproliferation effects on bladder cancer cells than single treatments (EXCC or cisplatin alone) in the 48 h ATP assay. EXCC/cisplatin also enhanced the increase in subG1, annexin V-mediated apoptosis, and activation of poly (ADP-ribose) polymerase (PARP) and several caspases (caspases 3, 8, and 9) compared to the single treatments. Cellular and mitochondrial oxidative stress was enhanced with EXCC/cisplatin compared to the single treatments according to analyses of reactive oxygen species (ROS), mitochondrial superoxide, and mitochondrial membrane potential; in addition, cellular antioxidants, such as glutathione (GSH), and the mRNA expressions of antioxidant signaling genes (catalase and NFE2-like bZIP transcription factor 2) were downregulated. EXCC/cisplatin treatment produced more DNA damage than the single treatments, as indicated by γH2AX and 8-hydroxy-2'-deoxyguanosine levels. Moreover, several DNA repair genes for homologous recombination (HR) and non-homologous end joining (NHEJ) were downregulated in EXCC/cisplatin compared to others. The addition of the GSH precursor N-acetylcysteine, which has ROS scavenging activity, attenuated all EXCC/cisplatin-induced changes. Notably, EXCC/cisplatin showed lower antiproliferation, apoptosis, ROS induction, GSH depletion, and γH2AX DNA damage in normal cells than in bladder cancer cells. Therefore, the co-treatment of EXCC/cisplatin reduces the proliferation of bladder cancer cells via oxidative stress-mediated mechanisms with normal cell safety.

A synergistic two-drug therapy specifically targets a DNA repair dysregulation that occurs in p53-deficient colorectal and pancreatic cancers.

The tumor-suppressor p53 is commonly inactivated in colorectal cancer and pancreatic ductal adenocarcinoma, but existing treatment options for p53-mutant (p53Mut) cancer are largely ineffective. Here, we report a therapeutic strategy for p53Mut tumors based on abnormalities in the DNA repair response. Investigation of DNA repair upon challenge with thymidine analogs reveals a dysregulation in DNA repair response in p53Mut cells that leads to accumulation of DNA breaks. Thymidine analogs do not interrupt DNA synthesis but induce DNA repair that involves a p53-dependent checkpoint. Inhibitors of poly(ADP-ribose) polymerase (PARPis) markedly enhance DNA double-strand breaks and cell death induced by thymidine analogs in p53Mut cells, whereas p53 wild-type cells respond with p53-dependent inhibition of the cell cycle. Combinations of trifluorothymidine and PARPi agents demonstrate superior anti-neoplastic activity in p53Mut cancer models. These findings support a two-drug combination strategy to improve outcomes for patients with p53Mut cancer.

P. gingivalis-Induced TLR2 Interactome Analysis Reveals Association with PARP9.

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium strongly associated with periodontal disease. Toll-like receptor 2 (TLR2) is indispensable for the host response to P. gingivalis, but P. gingivalis escapes from immune clearance via TLR2-dependent activation of phosphoinositide-3-kinase (PI3K). To probe the TLR2-dependent escape pathway of P. gingivalis, we analyzed the TLR2 interactome induced following P. gingivalis infection or activation by a synthetic lipopeptide TLR2/1 agonist on human macrophages overexpressing TLR2. Interacting proteins were stabilized by cross-linking and then immunoprecipitated and analyzed by mass spectrometry. In total, 792 proteins were recovered and network analysis enabled mapping of the TLR2 interactome at baseline and in response to infection. The P. gingivalis infection-induced TLR2 interactome included the poly (ADP-ribose) polymerase family member mono-ADP-ribosyltransferase protein 9 (PARP9) and additional members of the PARP9 complex (DTX3L and NMI). PARP9 and its complex members are highly upregulated in macrophages exposed to P. gingivalis or to the synthetic TLR2/1 ligand Pam3Cys-Ser-(Lys)4 (PAM). Consistent with its known role in virally induced interferon production, PARP9 knockdown blocked type I interferon (IFN-I) production in response to P. gingivalis and reduced inflammatory cytokine production. We found that P. gingivalis drives signal transducer and activation of transcription (STAT) 1 (S727) phosphorylation through TLR2-PARP9, explaining PARP9's role in the induction of IFN-I downstream of TLR2. Furthermore, PARP9 knockdown reduced PI3K activation by P. gingivalis, leading to improved macrophage bactericidal activity. In summary, PARP9 is a novel TLR2 interacting partner that enables IFN-I induction and P. gingivalis immune escape in macrophages downstream of TLR2 sensing.

Novel modifications of PARP inhibitor veliparib increase PARP1 binding to DNA breaks.

Catalytic poly(ADP-ribose) production by PARP1 is allosterically activated through interaction with DNA breaks, and PARP inhibitor compounds have the potential to influence PARP1 allostery in addition to preventing catalytic activity. Using the benzimidazole-4-carboxamide pharmacophore present in the first generation PARP1 inhibitor veliparib, a series of 11 derivatives was designed, synthesized, and evaluated as allosteric PARP1 inhibitors, with the premise that bulky substituents would engage the regulatory helical domain (HD) and thereby promote PARP1 retention on DNA breaks. We found that core scaffold modifications could indeed increase PARP1 affinity for DNA; however, the bulk of the modification alone was insufficient to trigger PARP1 allosteric retention on DNA breaks. Rather, compounds eliciting PARP1 retention on DNA breaks were found to be rigidly held in a position that interferes with a specific region of the HD domain, a region that is not targeted by current clinical PARP inhibitors. Collectively, these compounds highlight a unique way to trigger PARP1 retention on DNA breaks and open a path to unveil the pharmacological benefits of such inhibitors with novel properties.