Structurally distinct PARP7 inhibitors provide new insights into the function of PARP7 in regulating nucleic acid-sensing and IFN-ß signaling.

The mono-ADP-ribosyltransferase PARP7 has emerged as a key negative regulator of cytosolic NA-sensors of the innate immune system. We apply a rational design strategy for converting a pan-PARP inhibitor into a potent selective PARP7 inhibitor (KMR-206). Consistent with studies using the structurally distinct PARP7 inhibitor RBN-2397, co-treatment of mouse embryonic fibroblasts with KMR-206 and NA-sensor ligands synergistically induced the expression of the type I interferon, IFN-ß. In mouse colon carcinoma (CT-26) cells, KMR-206 alone induced IFN-ß. Both KMR-206 and RBN-2397 increased PARP7 protein levels in CT-26 cells, demonstrating that PARP7's catalytic activity regulates its own protein levels. Curiously, treatment with saturating doses of KMR-206 and RBN-2397 achieved different levels of PARP7 protein, which correlated with the magnitude of type I interferon gene expression. These latter results have important implications for the mechanism of action of PARP7 inhibitors and highlights the usefulness of having structurally distinct chemical probes for the same target.

Quantification of PARP7 Protein Levels and PARP7 Inhibitor Target Engagement in Cells Using a Split Nanoluciferase System.

PARP7 is an enzyme that catalyzes mono-ADP-ribosylation (MARylation), is a critical regulator of type I interferon signaling, and has emerged as an immune-oncology drug candidate. PARP7 is a labile protein that is regulated in a proteasome-dependent manner. Indeed, endogenous PARP7 levels are undetectable by western blot in most cells. Intriguingly, treatment of cells with orthosteric small molecule inhibitors of PARP7 can increase endogenous PARP7 protein to detectable levels. This characteristic of PARP7 inhibitors could potentially be exploited to assess target engagement-and thus cellular efficacy-of PARP7 inhibitors; however, no method exists to quantitatively monitor endogenous PARP7 levels in a high-throughput manner. In this protocol, we describe an assay using a split Nanoluciferase (NanoLuc) system for quantifying endogenous PARP7 protein levels and PARP7 inhibitor target engagement in cells in a 96-well plate format. We show that this assay can be used to quantify PARP7 protein levels under various cellular treatments and can assess cellular PARP7 inhibitor target engagement. We envision this split NanoLuc PARP7 assay can be used not only for evaluating the cellular efficacy of PARP7 inhibitors in a high-throughput manner but also for uncovering the mechanisms regulating PARP7 protein levels in cells.

Allosteric regulation of DNA binding and target residence time drive the cytotoxicity of phthalazinone-based PARP-1 inhibitors.

Allosteric coupling between the DNA binding site to the NAD+-binding pocket drives PARP-1 activation. This allosteric communication occurs in the reverse direction such that NAD+ mimetics can enhance PARP-1's affinity for DNA, referred to as type I inhibition. The cellular effects of type I inhibition are unknown, largely because of the lack of potent, membrane-permeable type I inhibitors. Here we identify the phthalazinone inhibitor AZ0108 as a type I inhibitor. Unlike the structurally related inhibitor olaparib, AZ0108 induces replication stress in tumorigenic cells. Synthesis of analogs of AZ0108 revealed features of AZ0108 that are required for type I inhibition. One analog, Pip6, showed similar type I inhibition of PARP-1 but was ∼90-fold more cytotoxic than AZ0108. Washout experiments suggest that the enhanced cytotoxicity of Pip6 compared with AZ0108 is due to prolonged target residence time on PARP-1. Pip6 represents a new class of PARP-1 inhibitors that may have unique anticancer properties.

PASTA: PARP activity screening and inhibitor testing assay.

Small-molecule inhibitors have been instrumental in uncovering the biological importance of poly-ADP-ribose polymerases (PARPs), a family of enzymes involved in wide-ranging aspects of cell biology. However, few PARP inhibitors are tested against the entire family of PARPs. This makes it impossible to confidently assess the role of a single PARP in cellular processes using small molecules. Here, we detail a PARP activity screening and inhibitor testing assay (PASTA) for determining relative selectivity of PARP inhibitors. For complete details on the use and execution of this protocol, please refer to Kirby et al. (2018).

Coronavirus infection and PARP expression dysregulate the NAD metabolome: An actionable component of innate immunity.

Poly(ADP-ribose) polymerase (PARP) superfamily members covalently link either a single ADP-ribose (ADPR) or a chain of ADPR units to proteins using NAD as the source of ADPR. Although the well-known poly(ADP-ribosylating) (PARylating) PARPs primarily function in the DNA damage response, many noncanonical mono(ADP-ribosylating) (MARylating) PARPs are associated with cellular antiviral responses. We recently demonstrated robust up-regulation of several PARPs following infection with murine hepatitis virus (MHV), a model coronavirus. Here we show that SARS-CoV-2 infection strikingly up-regulates MARylating PARPs and induces the expression of genes encoding enzymes for salvage NAD synthesis from nicotinamide (NAM) and nicotinamide riboside (NR), while down-regulating other NAD biosynthetic pathways. We show that overexpression of PARP10 is sufficient to depress cellular NAD and that the activities of the transcriptionally induced enzymes PARP7, PARP10, PARP12 and PARP14 are limited by cellular NAD and can be enhanced by pharmacological activation of NAD synthesis. We further demonstrate that infection with MHV induces a severe attack on host cell NAD+ and NADP+ Finally, we show that NAMPT activation, NAM, and NR dramatically decrease the replication of an MHV that is sensitive to PARP activity. These data suggest that the antiviral activities of noncanonical PARP isozyme activities are limited by the availability of NAD and that nutritional and pharmacological interventions to enhance NAD levels may boost innate immunity to coronaviruses.

Mechanisms governing PARP expression, localization, and activity in cells.

Poly-(ADP)-ribose polymerases (PARPs) are a family of 17 enzymes in humans that have diverse roles in cell physiology including DNA damage repair, transcription, innate immunity, and regulation of signaling pathways. The modular domain architecture of PARPs gives rise to this functional diversity. PARPs catalyze the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD+) to targets-proteins and poly-nucleic acids. This enigmatic post-translational modification comes in two varieties: the transfer of a single unit of ADP-ribose, known as mono-ADP-ribosylation (MARylation) or the transfer of multiple units of ADP-ribose, known as poly-ADP-ribosylation (PARylation). Emerging data shows that PARPs are regulated at multiple levels to control when and where PARP-mediated M/PARylation occurs in cells. In this review, we will discuss the latest knowledge regarding the regulation of PARPs in cells: from transcription and protein stability to subcellular localization and modulation of catalytic activity.

Coronavirus infection and PARP expression dysregulate the NAD Metabolome: an actionable component of innate immunity.

Poly-ADP-ribose polymerase (PARP) superfamily members covalently link either a single ADP-ribose (ADPR) or a chain of ADPR units to proteins using nicotinamide adenine dinucleotide (NAD) as the source of ADPR. While the well-known poly-ADP-ribosylating (PARylating) PARPs primarily function in the DNA damage response, many non-canonical mono-ADP-ribosylating (MARylating) PARPs are associated with cellular antiviral responses. We recently demonstrated robust upregulation of several PARPs following infection with Murine Hepatitis Virus (MHV), a model coronavirus. Here we show that SARS-CoV-2 infection strikingly upregulates MARylating PARPs and induces the expression of genes encoding enzymes for salvage NAD synthesis from nicotinamide (NAM) and nicotinamide riboside (NR), while downregulating other NAD biosynthetic pathways. We show that overexpression of PARP10 is sufficient to depress cellular NAD and that the activities of the transcriptionally induced enzymes PARP7, PARP10, PARP12 and PARP14 are limited by cellular NAD and can be enhanced by pharmacological activation of NAD synthesis. We further demonstrate that infection with MHV induces a severe attack on host cell NAD+ and NADP+. Finally, we show that NAMPT activation, NAM and NR dramatically decrease the replication of an MHV virus that is sensitive to PARP activity. These data suggest that the antiviral activities of noncanonical PARP isozyme activities are limited by the availability of NAD, and that nutritional and pharmacological interventions to enhance NAD levels may boost innate immunity to coronaviruses.

Identification and characterization of diverse OTU deubiquitinases in bacteria.

Manipulation of host ubiquitin signaling is becoming an increasingly apparent evolutionary strategy among bacterial and viral pathogens. By removing host ubiquitin signals, for example, invading pathogens can inactivate immune response pathways and evade detection. The ovarian tumor (OTU) family of deubiquitinases regulates diverse ubiquitin signals in humans. Viral pathogens have also extensively co-opted the OTU fold to subvert host signaling, but the extent to which bacteria utilize the OTU fold was unknown. We have predicted and validated a set of OTU deubiquitinases encoded by several classes of pathogenic bacteria. Biochemical assays highlight the ubiquitin and polyubiquitin linkage specificities of these bacterial deubiquitinases. By determining the ubiquitin-bound structures of two examples, we demonstrate the novel strategies that have evolved to both thread an OTU fold and recognize a ubiquitin substrate. With these new examples, we perform the first cross-kingdom structural analysis of the OTU fold that highlights commonalities among distantly related OTU deubiquitinases.