Structure-guided design and characterization of a clickable, covalent PARP16 inhibitor.

PARP16-the sole ER-resident PARP family member-is gaining attention as a potential therapeutic target for cancer treatment. Nevertheless, the precise function of the catalytic activity of PARP16 is poorly understood. This is primarily due to the lack of inhibitors that are selective for PARP16 over other PARP family members. Herein, we describe a structure-guided strategy for generating a selective PARP16 inhibitor by incorporating two selectivity determinants into a phthalazinone pan-PARP inhibitor scaffold: (i) an acrylamide-based inhibitor (DB008) designed to covalently react with a non-conserved cysteine (Cys169, human numbering) in the NAD+ binding pocket of PARP16 and (ii) a dual-purpose ethynyl group designed to bind in a unique hydrophobic cavity adjacent to the NAD+ binding pocket as well as serve as a click handle. DB008 exhibits good selectivity for PARP16 versus other PARP family members. Copper-catalyzed azide-alkyne cycloaddition (CuAAC) confirmed that covalent labeling of PARP16 by DB008 in cells is dependent on Cys169. DB008 exhibits excellent proteome-wide selectivity at concentrations required to achieve saturable labeling of endogenous PARP16. In-cell competition labeling experiments using DB008 provided a facile strategy for evaluating putative PARP16 inhibitors. Lastly, we found that PARP16 is sequestered into a detergent-insoluble fraction under prolonged amino acid starvation, and surprisingly, treatment with PARP16 inhibitors prevented this effect. These results suggest that the catalytic activity of PARP16 regulates its solubility in response to nutrient stress.

In vivo identification of small molecules mediating Gpr126/Adgrg6 signaling during Schwann cell development.

Gpr126/Adgrg6, an adhesion family G protein-coupled receptor (aGPCR), is required for the development of myelinating Schwann cells in the peripheral nervous system. Myelin supports and insulates vertebrate axons to permit rapid signal propagation throughout the nervous system. In mammals and zebrafish, mutations in Gpr126 arrest Schwann cells at early developmental stages. We exploited the optical and pharmacological tractability of larval zebrafish to uncover drugs that mediate myelination by activating Gpr126 or functioning in parallel. Using a fluorescent marker of mature myelinating glia (Tg[mbp:EGFP-CAAX]), we screened hypomorphic gpr126 mutant larvae for restoration of myelin basic protein (mbp) expression along peripheral nerves following small molecule treatment. Our screens identified five compounds sufficient to promote mbp expression in gpr126 hypomorphs. Using an allelic series of gpr126 mutants, we parsed the ability of small molecules to restore mbp, suggesting differences in drug efficacy dependent on Schwann cell developmental state. Finally, we identify apomorphine hydrochloride as a direct small molecule activator of Gpr126 using combined in vivo/in vitro assays and show that aporphine class compounds promote Schwann cell development in vivo. Our results demonstrate the utility of in vivo screening for aGPCR modulators and identify small molecules that interact with the gpr126-mediated myelination program.

The expanding functional roles and signaling mechanisms of adhesion G protein-coupled receptors.

The adhesion class of G protein-coupled receptors (GPCRs) is the second largest family of GPCRs (33 members in humans). Adhesion GPCRs (aGPCRs) are defined by a large extracellular N-terminal region that is linked to a C-terminal seven transmembrane (7TM) domain via a GPCR-autoproteolysis inducing (GAIN) domain containing a GPCR proteolytic site (GPS). Most aGPCRs undergo autoproteolysis at the GPS motif, but the cleaved fragments stay closely associated, with the N-terminal fragment (NTF) bound to the 7TM of the C-terminal fragment (CTF). The NTFs of most aGPCRs contain domains known to be involved in cell-cell adhesion, while the CTFs are involved in classical G protein signaling, as well as other intracellular signaling. In this workshop report, we review the most recent findings on the biology, signaling mechanisms, and physiological functions of aGPCRs.

Rational Design of Cell-Active Inhibitors of PARP10.

Poly-ADP-ribose polymerases (PARPs 1-16) have emerged as major regulators of diverse cellular processes. PARPs can be subclassified based on their ability to catalyze poly-ADP-ribosylation (PARylation) or mono-ADP-ribosylation (MARylation). While much is known about the cellular roles of PARPs that catalyze PARylation (e.g., PARP1), the function of PARPs that catalyze MARylation (e.g., PARP10) is substantially less understood. This is due in large part to the lack of small-molecule inhibitors that are selective for individual PARP family members that catalyze MARylation. Herein, we describe the rational design and synthesis of selective inhibitors of PARP10. Using structure-based design, we targeted a hydrophobic subpocket within the nicotinamide-binding site of PARP10. We synthesized a series of small molecules based on a 3,4-dihydroisoquinolin-1(2H)-one (dq, 1) scaffold that contain various substituents at the C-5 and C-6 positions designed to exploit this hydrophobic subpocket. We found a dq analogue (22) that contains a methyl group at the C-5 position and a substituted pyridine at the C-6 position that exhibits >10-fold selectivity for PARP10 over a large subset of other PARP family members. The results of this study will serve as a platform for future small-molecule probe development for PARP10 and other PARP family members that catalyze MARylation.

Combining Chemical Genetics with Proximity-Dependent Labeling Reveals Cellular Targets of Poly(ADP-ribose) Polymerase 14 (PARP14).

Poly(ADP-ribose) polymerase 14 (PARP14) is a member of the PARP family of enzymes that transfer ADP-ribose from NAD+ to nucleophilic amino acids on target proteins, a process known as mono-ADP-ribosylation (MARylation). PARP14 is involved in normal immune function through the IL-4 signaling pathway and is a prosurvival factor in multiple myeloma and hepatocellular carcinoma. A mechanistic understanding of the physiological and pathophysiological roles of PARP14 has been limited by the dearth of PARP14-specific MARylation targets. Herein we engineered a PARP14 variant that uses an NAD+ analog that is orthogonal to wild-type PARPs for identifying PARP14-specific MARylation targets. Combining this chemical genetics approach with a BioID approach for proximity-dependent labeling of PARP14 interactors, we identified 114 PARP14-specific protein substrates, several of which are RNA regulatory proteins. One of these targets is PARP13, a protein known to play a role in regulating RNA stability. PARP14 MARylates PARP13 on several acidic amino acids. This study not only reveals crosstalk among PARP family members but also highlights the advantage of using disparate approaches for identifying the direct targets of individual PARP family members.

A Simple, Sensitive, and Generalizable Plate Assay for Screening PARP Inhibitors.

Poly-ADP-ribose polymerases (also known as ADP-ribosyltransferases or ARTDs) are a family of 17 enzymes in humans that catalyze the reversible posttranslational modification known as ADP-ribosylation. PARPs are implicated in diverse cellular processes, from DNA repair to the unfolded protein response. Small-molecule inhibitors of PARPs have improved our understanding of PARP-mediated biology and, in some cases, have emerged as promising treatments for cancers and other human diseases. However these advancements are hindered, in part, by a poor understanding of inhibitor selectivity across the PARP family. Here, we describe a simple, sensitive, and generalizable plate assay to test the potency and selectivity of small molecules against several PARP enzymes in vitro. In principle, this assay can be extended to all active PARPs, providing a convenient and direct comparison of inhibitors across the entire PARP enzyme family.

Detecting Protein ADP-Ribosylation Using a Clickable Aminooxy Probe.

ADP-ribosylation, a posttranslational modification catalyzed by a family of enzymes known as poly(ADP-ribose) polymerases (PARPs, 17 in humans), regulates diverse cellular processes. To aid in understanding the functions of ADP-ribosylation in cells, we developed a clickable aminooxy probe, AO-alkyne, which detects ADP-ribosylation of acidic amino acids. AO-alkyne can be used to detect auto-ADP-ribosylation of PARP10 in cells following Cu-catalyzed click conjugation to an azide reporter. This method can be extended to other PARP family members that catalyze ADP-ribosylation on acidic amino acids, providing a convenient and direct readout of PARP activity in cells.

Biosensor reveals multiple sources for mitochondrial NAD⁺.

Nicotinamide adenine dinucleotide (NAD(+)) is an essential substrate for sirtuins and poly(adenosine diphosphate-ribose) polymerases (PARPs), which are NAD(+)-consuming enzymes localized in the nucleus, cytosol, and mitochondria. Fluctuations in NAD(+) concentrations within these subcellular compartments are thought to regulate the activity of NAD(+)-consuming enzymes; however, the challenge in measuring compartmentalized NAD(+) in cells has precluded direct evidence for this type of regulation. We describe the development of a genetically encoded fluorescent biosensor for directly monitoring free NAD(+) concentrations in subcellular compartments. We found that the concentrations of free NAD(+) in the nucleus, cytoplasm, and mitochondria approximate the Michaelis constants for sirtuins and PARPs in their respective compartments. Systematic depletion of enzymes that catalyze the final step of NAD(+) biosynthesis revealed cell-specific mechanisms for maintaining mitochondrial NAD(+) concentrations.

Identifying Family-Member-Specific Targets of Mono-ARTDs by Using a Chemical Genetics Approach.

ADP-ribosyltransferases (ARTD1-16) have emerged as major downstream effectors of NAD(+) signaling in the cell. Most ARTDs (ARTD7 and 8, 10-12, and 14-17) catalyze the transfer of a single unit of ADP-ribose from NAD(+) to target proteins, a process known as mono-ADP-ribosylation (MARylation). Progress in understanding the cellular functions of MARylation has been limited by the inability to identify the direct targets for individual mono-ARTDs. Here, we engineered mono-ARTDs to use an NAD(+) analog that is orthogonal to wild-type ARTDs. We profiled the MARylomes of ARTD10 and ARTD11 in vitro, identifying isoform-specific targets and revealing a potential role for ARTD11 in nuclear pore complex biology. We found that ARTD11 targeting is dependent on both its regulatory and catalytic domains, which has important implications for how ARTDs recognize their targets. We anticipate that our chemical genetic strategy will be generalizable to all mono-ARTD family members based on the similarity of the mono-ARTD catalytic domains.

Selective inhibition of PARP10 using a chemical genetics strategy.

The lack of inhibitors that are selective for individual poly-ADP-ribose polymerase (PARP) family members has limited our understanding of their roles in cells. Here, we describe a chemical genetics approach for generating selective inhibitors of an engineered variant of PARP10. We synthesized a series of C-7 substituted 3,4-dihydroisoquinolin-1(2H)-one (dq) analogues designed to selectively inhibit a mutant of PARP10 (LG-PARP10) that contains a unique pocket in its active site. A dq analogue containing a bromo at the C-7 position demonstrated a 10-fold selectivity for LG-PARP10 compared to its WT counterpart. This study provides a platform for the development of selective inhibitors of individual PARP family members that will be useful for decoding their cellular functions.

A Clickable Aminooxy Probe for Monitoring Cellular ADP-Ribosylation.

ADP-ribosylation is essential for cell function, yet there is a dearth of methods for detecting this post-translational modification in cells. Here, we describe a clickable aminooxy alkyne (AO-alkyne) probe that can detect cellular ADP-ribosylation on acidic amino acids following Cu-catalyzed conjugation to an azide-containing reporter. Using AO-alkyne, we show that PARP10 and PARP11 are auto-ADP-ribosylated in cells. We also demonstrate that AO-alkyne can be used to monitor stimulus-induced ADP-ribosylation in cells. Functional studies using AO-alkyne support a previously unknown mechanism for ADP-ribosylation on acidic amino acids, wherein a glutamate or aspartate at the initial C1'-position of ADP-ribose transfers to the C2' position. This new mechanism for ADP-ribosylation has important implications for how glutamyl/aspartyl-ADP-ribose is recognized by proteins in cells.

Engineering the substrate specificity of ADP-ribosyltransferases for identifying direct protein targets.

Adenosine diphosphate ribosyltransferases (ARTDs; ARTD1-17 in humans) are emerging as critical regulators of cell function in both normal physiology and disease. These enzymes transfer the ADP-ribose moiety from its substrate, nicotinamide adenine dinucleotide (NAD(+)), to amino acids of target proteins. The functional redundancy and overlapping target specificities among the 17 ARTDs in humans make the identification of direct targets of individual ARTD family members in a cellular context a formidable challenge. Here we describe the rational design of orthogonal NAD(+) analogue-engineered ARTD pairs for the identification of direct protein targets of individual ARTDs. Guided by initial inhibitor studies with nicotinamide analogues containing substituents at the C-5 position, we synthesized an orthogonal NAD(+) variant and found that it is used as a substrate for several engineered ARTDs (ARTD1, -2, and -6) but not their wild-type counterparts. Comparing the target profiles of ARTD1 (PARP1) and ARTD2 (PARP2) in nuclear extracts highlighted the semi-complementary, yet distinct, protein targeting. Using affinity purification followed by tandem mass spectrometry, we identified 42 direct ARTD1 targets and 301 direct ARTD2 targets. This represents a powerful new technique for identifying direct protein targets of individual ARTD family members, which will facilitate studies delineating the pathway from ARTD activation to a given cellular response.

Evidence that ubiquinone is a required intermediate for rhodoquinone biosynthesis in Rhodospirillum rubrum.

Rhodoquinone (RQ) is an important cofactor used in the anaerobic energy metabolism of Rhodospirillum rubrum. RQ is structurally similar to ubiquinone (coenzyme Q or Q), a polyprenylated benzoquinone used in the aerobic respiratory chain. RQ is also found in several eukaryotic species that utilize a fumarate reductase pathway for anaerobic respiration, an important example being the parasitic helminths. RQ is not found in humans or other mammals, and therefore inhibition of its biosynthesis may provide a parasite-specific drug target. In this report, we describe several in vivo feeding experiments with R. rubrum used for the identification of RQ biosynthetic intermediates. Cultures of R. rubrum were grown in the presence of synthetic analogs of ubiquinone and the known Q biosynthetic precursors demethylubiquinone, demethoxyubiquinone, and demethyldemethoxyubiquinone, and assays were monitored for the formation of RQ(3). Data from time course experiments and S-adenosyl-l-methionine-dependent O-methyltransferase inhibition studies are discussed. Based on the results presented, we have demonstrated that Q is a required intermediate for the biosynthesis of RQ in R. rubrum.