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Abstract Mono-ADP-ribosyltransferases of the PARP/ARTD enzyme family are enzymes catalyzing the transfer of a single ADP-ribose unit to target proteins. The enzymes have various roles in vital cellular processes such as DNA repair and transcription, and many of the enzymes are linked to cancer-relevant functions. Thus inhibition of the enzymes is a potential way to discover and develop new drugs against cancer. Here we describe an activity-based screening assay for mono-ADP-ribosyltransferases. The assay utilizes the natural substrate of the enzymes, NAD+, and it is based on chemically converting the leftover substrate to a fluorophore and measuring its relative concentration after the enzymatic reaction. The assay is homogenous, robust, and cost-effective and, most importantly, applicable to mono-ADP-ribosyltransferases as well as poly-ADP-ribosyltransferases for screening of small-molecule inhibitors against the enzymes.
Abstract The scaffold of TIQ-A, a previously known inhibitor of human poly-ADP-ribosyltransferase PARP1, was utilized to develop inhibitors against human mono-ADP-ribosyltransferases through structure-guided design and activity profiling. By supplementing the TIQ-A scaffold with small structural changes, based on a PARP10 inhibitor OUL35, selectivity changed from poly-ADP-ribosyltransferases towards mono-ADP-ribosyltransferases. Binding modes of analogs were experimentally verified by determining complex crystal structures with mono-ADP-ribosyltransferase PARP15 and with poly-ADP-ribosyltransferase TNKS2. The best analogs of the study achieved 10–20-fold selectivity towards mono-ADP-ribosyltransferases PARP10 and PARP15 while maintaining micromolar potencies. The work demonstrates a route to differentiate compound selectivity between mono- and poly-ribosyltransferases of the human ARTD family.
Abstract Human Diphtheria toxin-like ADP-ribosyltranferases (ARTD) 10 is an enzyme carrying out mono-ADP-ribosylation of a range of cellular proteins and affecting their activities. It shuttles between cytoplasm and nucleus and influences signaling events in both compartments, such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling and S phase DNA repair. Furthermore, overexpression of ARTD10 induces cell death. We recently reported on the discovery of a hit compound, OUL35 (compound 1), with 330 nM potency and remarkable selectivity towards ARTD10 over other enzymes in the human protein family. Here we aimed at establishing a structure-activity relationship of the OUL35 scaffold, by evaluating an array of 4-phenoxybenzamide derivatives. By exploring modifications on the linker between the aromatic rings, we identified also a 4-(benzyloxy)benzamide derivative, compound 32, which is potent (IC50 = 230 nM) and selective, and like OUL35 was able to rescue HeLa cells from ARTD10-induced cell death. Evaluation of an enlarged series of derivatives produced detailed knowledge on the structural requirements for ARTD10 inhibition and allowed the discovery of further tool compounds with submicromolar cellular potency that will help in understanding the roles of ARTD10 in biological systems.
Abstract Tankyrases 1 and 2 are central biotargets in the WNT/β-catenin signaling and Hippo signaling pathways. We have previously developed tankyrase inhibitors bearing a 1,2,4-triazole moiety and binding predominantly to the adenosine binding site of the tankyrase catalytic domain. Here we describe a systematic structure-guided lead optimization approach of these tankyrase inhibitors. The central 1,2,4-triazole template and trans-cyclobutyl linker of the lead compound 1 were left unchanged, while side-group East, West, and South moieties were altered by introducing different building blocks defined as point mutations. The systematic study provided a novel series of compounds reaching picomolar IC30 inhibition in WNT/β-catenin signaling cellular reporter assay. The novel optimized lead 13 resolves previous atropisomerism, solubility, and Caco-2 efflux liabilities. 13 shows a favorable ADME profile, including improved Caco-2 permeability and oral bioavailability in mice, and exhibits antiproliferative efficacy in the colon cancer cell line COLO 320DM in vitro.
Abstract Compounds 13 and 14 were evaluated against eleven PARP isoforms to reveal that both 13 and 14 were more potent and isoform-selective towards inhibiting tankyrases (TNKSs) than the “standard” inhibitor 1 (XAV939)5, i.e. IC50 = 100 pM vs. TNKS2 and IC50 = 6.5 μM vs. PARP1 for 14. In cellular assays, 13 and 14 inhibited Wnt-signaling, enhanced insulin-stimulated glucose uptake and inhibited the proliferation of DLD-1 colorectal adenocarcinoma cells to a greater extent than 1.
Abstract Tankyrases (TNKSs) are enzymes specialized in catalyzing poly-ADP-ribosylation of target proteins. Several studies have validated TNKSs as anti-cancer drug targets due to their regulatory role in Wnt/β-catenin pathway. Recently a lot of effort has been put into developing more potent and selective TNKS inhibitors and optimizing them towards anti-cancer agents. We noticed that some 2-phenylquinazolinones (2-PQs) reported as CDK9 inhibitors were similar to previously published TNKS inhibitors. In this study, we profiled this series of 2-PQs against TNKS and selected kinases that are involved in the Wnt/β-catenin pathway. We found that they were much more potent TNKS inhibitors than they were CDK9/kinase inhibitors. We evaluated the compound selectivity to tankyrases over the ARTD enzyme family and solved co-crystal structures of the compounds with TNKS2. Comparative structure-based studies of the catalytic domain of TNKS2 with selected CDK9 inhibitors and docking studies of the inhibitors with two kinases (CDK9 and Akt) revealed important structural features, which could explain the selectivity of the compounds towards either tankyrases or kinases. We also discovered a compound, which was able to inhibit tankyrases, CDK9 and Akt kinases with equal μM potency.
Abstract Intracellular ADP-ribosyltransferases catalyze mono- and poly-ADP-ribosylation and affect a broad range of biological processes. The mono-ADP-ribosyltransferase PARP10 is involved in signaling and DNA repair. Previous studies identified OUL35 as a selective, cell permeable inhibitor of PARP10. We have further explored the chemical space of OUL35 by synthesizing and investigating structurally related analogs. Key synthetic steps were metal-catalyzed cross-couplings and functional group modifications. We identified 4-(4-cyanophenoxy)benzamide and 3-(4-carbamoylphenoxy)benzamide as PARP10 inhibitors with distinct selectivities. Both compounds were cell permeable and interfered with PARP10 toxicity. Moreover, both revealed some inhibition of PARP2 but not PARP1, unlike clinically used PARP inhibitors, which typically inhibit both enzymes. Using crystallography and molecular modeling the binding of the compounds to different ADP-ribosyltransferases was explored regarding selectivity. Together, these studies define additional compounds that interfere with PARP10 function and thus expand our repertoire of inhibitors to further optimize selectivity and potency.
Abstract Tankyrase 1 and 2 (TNKS1/2) catalyze post-translational modification by poly-ADP-ribosylation of a plethora of target proteins. In this function, TNKS1/2 also impact the WNT/β-catenin and Hippo signaling pathways that are involved in numerous human disease conditions including cancer. Targeting TNKS1/2 with small-molecule inhibitors shows promising potential to modulate the involved pathways, thereby potentiating disease intervention. Based on our 1,2,4-triazole-based lead compound 1 (OM-1700), further structure–activity relationship analyses of East-, South- and West-single-point alterations and hybrids identified compound 24 (OM-153). Compound 24 showed picomolar IC59 inhibition in a cellular (HEK293) WNT/β-catenin signaling reporter assay, no off-target liabilities, overall favorable absorption, distribution, metabolism, and excretion (ADME) properties, and an improved pharmacokinetic profile in mice. Moreover, treatment with compound 24 induced dose-dependent biomarker engagement and reduced cell growth in the colon cancer cell line COLO 320DM.
Abstract A structure-guided hybridization approach using two privileged substructures gave instant access to a new series of tankyrase inhibitors. The identified inhibitor <strong>16</strong> displays high target affinity on tankyrase 1 and 2 with biochemical and cellular IC50 values of 29 nM, 6.3 nM and 19 nM, respectively, and high selectivity toward other poly (ADP-ribose) polymerase enzymes. The identified inhibitor shows a favorable in vitro ADME profile as well as good oral bioavailability in mice, rats, and dogs. Critical for the approach was the utilization of an appropriate linker between 1,2,4-triazole and benzimidazolone moieties, whereby a cyclobutyl linker displayed superior affinity compared to a cyclohexane and phenyl linker.
Abstract We report [1,2,4]triazolo[3,4-b]benzothiazole (TBT) as a new inhibitor scaffold, which competes with nicotinamide in the binding pocket of human poly- and mono-ADP-ribosylating enzymes. The binding mode was studied through analogues and cocrystal structures with TNKS2, PARP2, PARP14, and PARP15. Based on the substitution pattern, we were able to identify 3-amino derivatives 21 (OUL243) and 27 (OUL232) as inhibitors of mono-ARTs PARP7, PARP10, PARP11, PARP12, PARP14, and PARP15 at nM potencies, with 27 being the most potent PARP10 inhibitor described to date (IC50 of 7.8 nM) and the first PARP12 inhibitor ever reported. On the contrary, hydroxy derivative 16 (OUL245) inhibits poly-ARTs with a selectivity toward PARP2. The scaffold does not possess inherent cell toxicity, and the inhibitors can enter cells and engage with the target protein. This, together with favorable ADME properties, demonstrates the potential of TBT scaffold for future drug development efforts toward selective inhibitors against specific enzymes.