Discovery of 2-Amide-3-methylester Thiophenes that Target SARS-CoV-2 Mac1 and Repress Coronavirus Replication, Validating Mac1 as an Antiviral Target.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has made it clear that further development of antiviral therapies will be needed. Here, we describe small-molecule inhibitors for SARS-CoV-2 Mac1, which counters ADP-ribosylation-mediated innate immune responses. Three high-throughput screening hits had the same 2-amide-3-methylester thiophene scaffold. We studied the compound binding mode using X-ray crystallography, allowing us to design analogues. Compound 27 (MDOLL-0229) had an IC50 of 2.1 µM and was selective for CoV Mac1 proteins after profiling for activity against a panel of viral and human proteins. The improved potency allowed testing of its effect on virus replication, and indeed, 27 inhibited replication of both murine hepatitis virus (MHV) prototypes CoV and SARS-CoV-2. Sequencing of a drug-resistant MHV identified mutations in Mac1, further demonstrating the specificity of 27. Compound 27 is the first Mac1-targeted small molecule demonstrated to inhibit coronavirus replication in a cell model.

High-throughput screening assay for PARP-HPF1 interaction inhibitors to affect DNA damage repair.

ADP-ribosyltransferases PARP1 and PARP2 play a major role in DNA repair mechanism by detecting the DNA damage and inducing poly-ADP-ribosylation dependent chromatin relaxation and recruitment of repair proteins. Catalytic PARP inhibitors are used as anticancer drugs especially in the case of tumors arising from sensitizing mutations. Recently, a study showed that Histone PARylation Factor (HPF1) forms a joint active site with PARP1/2. The interaction of HPF1 with PARP1/2 alters the modification site from Aspartate/Glutamate to Serine, which has been shown to be a key ADP-ribosylation event in the context of DNA damage. Therefore, disruption of PARP1/2-HPF1 interaction could be an alternative strategy for drug development to block the PARP1/2 activity. In this study, we describe a FRET based high-throughput screening assay to screen inhibitor libraries against PARP-HPF1 interaction. We optimized the conditions for FRET signal and verified the interaction by competing the FRET pair in multiple ways. The assay is robust and easy to automate. Validatory screening showed the robust performance of the assay, and we discovered two compounds Dimethylacrylshikonin and Alkannin, with µM inhibition potency against PARP1/2-HPF1 interaction. The assay will facilitate the discovery of inhibitors against HPF1-PARP1/2 complex and to develop potentially new effective anticancer agents.

Synthetically accessible de novo design using reaction vectors: Application to PARP1 inhibitors.

De novo design has been a hotly pursued topic for many years. Most recent developments have involved the use of deep learning methods for generative molecular design. Despite increasing levels of algorithmic sophistication, the design of molecules that are synthetically accessible remains a major challenge. Reaction-based de novo design takes a conceptually simpler approach and aims to address synthesisability directly by mimicking synthetic chemistry and driving structural transformations by known reactions that are applied in a stepwise manner. However, the use of a small number of hand-coded transformations restricts the chemical space that can be accessed and there are few examples in the literature where molecules and their synthetic routes have been designed and executed successfully. Here we describe the application of reaction-based de novo design to the design of synthetically accessible and biologically active compounds as proof-of-concept of our reaction vector-based software. Reaction vectors are derived automatically from known reactions and allow access to a wide region of synthetically accessible chemical space. The design was aimed at producing molecules that are active against PARP1 and which have improved brain penetration properties compared to existing PARP1 inhibitors. We synthesised a selection of the designed molecules according to the provided synthetic routes and tested them experimentally. The results demonstrate that reaction vectors can be applied to the design of novel molecules of biological relevance that are also synthetically accessible.

[1,2,4]Triazolo[3,4-b]benzothiazole Scaffold as Versatile Nicotinamide Mimic Allowing Nanomolar Inhibition of Different PARP Enzymes.

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.

Discovery of 2-amide-3-methylester thiophenes that target SARS-CoV-2 Mac1 and repress coronavirus replication, validating Mac1 as an anti-viral target.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has made it clear that further development of antiviral therapies will be needed to combat additional SARS-CoV-2 variants or novel CoVs. Here, we describe small molecule inhibitors for SARS-CoV-2 Mac1, which counters ADP-ribosylation mediated innate immune responses. The compounds inhibiting Mac1 were discovered through high-throughput screening (HTS) using a protein FRET-based competition assay and the best hit compound had an IC50 of 14 µM. Three validated HTS hits have the same 2-amide-3-methylester thiophene scaffold and the scaffold was selected for structure-activity relationship (SAR) studies through commercial and synthesized analogs. We studied the compound binding mode in detail using X-ray crystallography and this allowed us to focus on specific features of the compound and design analogs. Compound 27 (MDOLL-0229) had an IC50 of 2.1 µM and was generally selective for CoV Mac1 proteins after profiling for activity against a panel of viral and human ADP-ribose binding proteins. The improved potency allowed testing of its effect on virus replication and indeed, 27 inhibited replication of both MHVa prototype CoV, and SARS-CoV-2. Furthermore, sequencing of a drug-resistant MHV identified mutations in Mac1, further demonstrating the specificity of 27. Compound 27 is the first Mac1 targeted small molecule demonstrated to inhibit coronavirus replication in a cell model. This, together with its well-defined binding mode, makes 27 a good candidate for further hit/lead-optimization efforts.

An Evolutionary Perspective on the Origin, Conservation and Binding Partner Acquisition of Tankyrases.

Tankyrases are poly-ADP-ribosyltransferases that regulate many crucial and diverse cellular processes in humans such as Wnt signaling, telomere homeostasis, mitotic spindle formation and glucose metabolism. While tankyrases are present in most animals, functional differences across species may exist. In this work, we confirm the widespread distribution of tankyrases throughout the branches of multicellular animal life and identify the single-celled choanoflagellates as earliest origin of tankyrases. We further show that the sequences and structural aspects of TNKSs are well-conserved even between distantly related species. We also experimentally characterized an anciently diverged tankyrase homolog from the sponge Amphimedon queenslandica and show that the basic functional aspects, such as poly-ADP-ribosylation activity and interaction with the canonical tankyrase binding peptide motif, are conserved. Conversely, the presence of tankyrase binding motifs in orthologs of confirmed interaction partners varies greatly between species, indicating that tankyrases may have different sets of interaction partners depending on the animal lineage. Overall, our analysis suggests a remarkable degree of conservation for tankyrases, and that their regulatory functions in cells have likely changed considerably throughout evolution.

Protein engineering approach to enhance activity assays of mono-ADP-ribosyltransferases through proximity.

Human mono-ADP-ribosylating PARP enzymes have been linked to several clinically relevant processes and many of these PARPs have been suggested as potential drug targets. Despite recent advances in the field, efforts to discover inhibitors have been hindered by the lack of tools to rapidly screen for high potency compounds and profile them against the different enzymes. We engineered mono-ART catalytic fragments to be incorporated into a cellulosome-based octavalent scaffold. Compared to the free enzymes, the scaffold-based system results in an improved activity for the tested PARPs due to improved solubility, stability and the proximity of the catalytic domains, altogether boosting their activity beyond 10-fold in the case of PARP12. This allows us to measure their activity using a homogeneous NAD+ conversion assay, facilitating its automation to lower the assay volume and costs. The approach will enable the discovery of more potent compounds due to increased assay sensitivity.

Potent 2,3-dihydrophthalazine-1,4-dione derivatives as dual inhibitors for mono-ADP-ribosyltransferases PARP10 and PARP15.

While human poly-ADP-ribose chain generating poly-ARTs, PARP1 and 2 and TNKS1 and 2, have been widely characterized, less is known on the pathophysiological roles of the mono-ADP-ribosylating mono-ARTs, partly due to the lack of selective inhibitors. In this context, we have focused on the development of inhibitors for the mono-ART PARP10, whose overexpression is known to induce cell death. Starting from OUL35 (1) and its 4-(benzyloxy)benzamidic derivative (2) we herein report the design and synthesis of new analogues from which the cyclobutyl derivative 3c rescued cells most efficiently from PARP10 induced apoptosis. Most importantly, we also identified 2,3-dihydrophthalazine-1,4-dione as a new suitable nicotinamide mimicking PARP10 inhibitor scaffold. When it was functionalized with cycloalkyl (8a-c), o-fluorophenyl (8h), and thiophene (8l) rings, IC50 values in the 130-160 nM range were obtained, making them the most potent PARP10 inhibitors reported to date. These compounds also inhibited PARP15 with low micromolar IC50s, but none of the other tested poly- and mono-ARTs, thus emerging as dual mono-ART inhibitors. Compounds 8a, 8h and 8l were also able to enter cells and rescue cells from apoptosis. Our work sheds more light on inhibitor development against mono-ARTs and identifies chemical probes to study the cellular roles of PARP10 and PARP15.

Preparation of screening assays for ADP-ribosyl readers and erasers using the GAP-tag as a binding probe.

Here, we describe a protocol to set up a screening assay for ADP-ribosyl binding proteins including proteins that possess O-glycosidase or N-glycosidase activities. The FRET-based assay measures the interaction of any ADP-ribosyl binding protein fused to CFP with a cysteine-ADP-ribosylated GAP-tag fused to YFP. Recombinant PtxS1 and PARP2 are used to mono-ADP-ribosylate and poly-ADP-ribosylate the GAP-tag. The protocol does not require specialized compounds or substrates, making it accessible and easy to adapt in any laboratory or for other proteins of interest. For complete details on the use and execution of this profile, please refer to Sowa et al. (2021).

The Tankyrase Inhibitor OM-153 Demonstrates Antitumor Efficacy and a Therapeutic Window in Mouse Models.

The catalytic enzymes tankyrase 1 and 2 (TNKS1/2) alter protein turnover by poly-ADP-ribosylating target proteins, which earmark them for degradation by the ubiquitin-proteasomal system. Prominent targets of the catalytic activity of TNKS1/2 include AXIN proteins, resulting in TNKS1/2 being attractive biotargets for addressing of oncogenic WNT/ß-catenin signaling. Although several potent small molecules have been developed to inhibit TNKS1/2, there are currently no TNKS1/2 inhibitors available in clinical practice. The development of tankyrase inhibitors has mainly been disadvantaged by concerns over biotarget-dependent intestinal toxicity and a deficient therapeutic window. Here we show that the novel, potent, and selective 1,2,4-triazole-based TNKS1/2 inhibitor OM-153 reduces WNT/ß-catenin signaling and tumor progression in COLO 320DM colon carcinoma xenografts upon oral administration of 0.33-10 mg/kg twice daily. In addition, OM-153 potentiates anti-programmed cell death protein 1 (anti-PD-1) immune checkpoint inhibition and antitumor effect in a B16-F10 mouse melanoma model. A 28-day repeated dose mouse toxicity study documents body weight loss, intestinal damage, and tubular damage in the kidney after oral-twice daily administration of 100 mg/kg. In contrast, mice treated oral-twice daily with 10 mg/kg show an intact intestinal architecture and no atypical histopathologic changes in other organs. In addition, clinical biochemistry and hematologic analyses do not identify changes indicating substantial toxicity. The results demonstrate OM-153-mediated antitumor effects and a therapeutic window in a colon carcinoma mouse model ranging from 0.33 to at least 10 mg/kg, and provide a framework for using OM-153 for further preclinical evaluations. Significance: This study uncovers the effectiveness and therapeutic window for a novel tankyrase inhibitor in mouse tumor models.

Development of a 1,2,4-Triazole-Based Lead Tankyrase Inhibitor: Part II.

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/ß-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 IC50 inhibition in a cellular (HEK293) WNT/ß-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.

Analogs of TIQ-A as inhibitors of human mono-ADP-ribosylating PARPs.

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.

A molecular toolbox for ADP-ribosyl binding proteins.

Proteins interacting with ADP-ribosyl groups are often involved in disease-related pathways or viral infections, making them attractive drug targets. We present a robust and accessible assay applicable to both hydrolyzing or non-hydrolyzing binders of mono- and poly-ADP-ribosyl groups. This technology relies on a C-terminal tag based on a Gi protein alpha subunit peptide (GAP), which allows for site-specific introduction of cysteine-linked mono- and poly-ADP-ribosyl groups or analogs. By fusing the GAP-tag and ADP-ribosyl binders to fluorescent proteins, we generate robust FRET partners and confirm the interaction with 22 known ADP-ribosyl binders. The applicability for high-throughput screening of inhibitors is demonstrated with the SARS-CoV-2 nsp3 macrodomain, for which we identify suramin as a moderate-affinity yet non-specific inhibitor. High-affinity ADP-ribosyl binders fused to nanoluciferase complement this technology, enabling simple blot-based detection of ADP-ribosylated proteins. All these tools can be produced in Escherichia coli and will help in ADP-ribosylation research and drug discovery.

Evaluation of 3- and 4-Phenoxybenzamides as Selective Inhibitors of the Mono-ADP-Ribosyltransferase PARP10.

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.

Activation of PARP2/ARTD2 by DNA damage induces conformational changes relieving enzyme autoinhibition.

Human PARP2/ARTD2 is an ADP-ribosyltransferase which, when activated by 5'-phosphorylated DNA ends, catalyses poly-ADP-ribosylation of itself, other proteins and DNA. In this study, a crystal structure of PARP2 in complex with an activating 5'-phosphorylated DNA shows that the WGR domain bridges the dsDNA gap and joins the DNA ends. This DNA binding results in major conformational changes, including reorganization of helical fragments, in the PARP2 regulatory domain. A comparison of PARP1 and PARP2 crystal structures reveals how binding to a DNA damage site leads to formation of a catalytically competent conformation. In this conformation, PARP2 is capable of binding substrate NAD+ and histone PARylation factor 1 that changes PARP2 residue specificity from glutamate to serine when initiating DNA repair processes. The structure also reveals how the conformational changes in the autoinhibitory regulatory domain would promote the flexibility needed by the enzyme to reach the target macromolecule for ADP-ribosylation.

Divergent CPEB prion-like domains reveal different assembly mechanisms for a generic amyloid-like fold.

BACKGROUND: Amyloids are ordered, insoluble protein aggregates, characterized by a cross-ß sheet quaternary structure in which molecules in a ß-strand conformation are stacked along the filament axis via intermolecular interactions. While amyloids are typically associated with pathological conditions, functional amyloids have also been identified and are present in a wide variety of organisms ranging from bacteria to humans. The cytoplasmic polyadenylation element-binding (CPEB) prion-like protein is an mRNA-binding translation regulator, whose neuronal isoforms undergo activity-dependent aggregation, a process that has emerged as a plausible biochemical substrate for memory maintenance. CPEB aggregation is driven by prion-like domains (PLD) that are divergent in sequence across species, and it remains unknown whether such divergent PLDs follow a similar aggregating assembly pathway. Here, we describe the amyloid-like features of the neuronal Aplysia CPEB (ApCPEB) PLD and compare them to those of the Drosophila ortholog, Orb2 PLD. RESULTS: Using in vitro single-molecule and bulk biophysical methods, we find transient oligomers and mature amyloid-like filaments that suggest similarities in the late stages of the assembly pathway for both ApCPEB and Orb2 PLDs. However, while prior to aggregation the Orb2 PLD monomer remains mainly as a random coil in solution, ApCPEB PLD adopts a diversity of conformations comprising α-helical structures that evolve to coiled-coil species, indicating structural differences at the beginning of their amyloid assembly pathways. CONCLUSION: Our results indicate that divergent PLDs of CPEB proteins from different species retain the ability to form a generic amyloid-like fold through different assembly mechanisms.

Cohesin-dockerin code in cellulosomal dual binding modes and its allosteric regulation by proline isomerization.

Cellulose is the most abundant organic molecule on Earth and represents a renewable and practically everlasting feedstock for the production of biofuels and chemicals. Self-assembled owing to the high-affinity cohesin-dockerin interaction, cellulosomes are huge multi-enzyme complexes with unmatched efficiency in the degradation of recalcitrant lignocellulosic substrates. The recruitment of diverse dockerin-borne enzymes into a multicohesin protein scaffold dictates the three-dimensional layout of the complex, and interestingly two alternative binding modes have been proposed. Using single-molecule fluorescence resonance energy transfer and molecular simulations on a range of cohesin-dockerin pairs, we directly detect varying distributions between these binding modes that follow a built-in cohesin-dockerin code. Surprisingly, we uncover a prolyl isomerase-modulated allosteric control mechanism, mediated by the isomerization state of a single proline residue, which regulates the distribution and kinetics of binding modes. Overall, our data provide a novel mechanistic understanding of the structural plasticity and dynamics of cellulosomes.

Activity-Based Screening Assay for Mono-ADP-Ribosylhydrolases.

ADP-ribosylation is a post-translational modification involved in the regulation of many vital cellular processes. This posttranslational modification is carried out by ADP-ribosyltransferases converting ß-NAD+ into nicotinamide and a protein-linked ADP-ribosyl group or a chain of PAR. The reverse reaction, release of ADP-ribose from the acceptor molecule, is catalyzed by ADP-ribosylhydrolases. Several hydrolases contain a macrodomain fold, and activities of human macrodomain protein modules vary from reading or erasing mono- and poly-ADP-ribosylation. Macrodomains have been linked to diseases such as cancer, making them potential drug targets. Discovery of inhibitors requires robust biochemical tools mostly lacking for hydrolases, and here we describe an inhibitor screening assay against mono-ADP-ribosylhydrolyzing enzymes. The activity-based assay uses an α-NAD+, anomer of ß-NAD+, which is accepted as a substrate by MacroD1, MacroD2, and ARH3 due to its resemblance to the protein-linked ADP-ribose. The amount of α-NAD+ present after hydrolysis is measured by chemically converting it on a microtiter plate to a fluorescent compound. We optimized the assay for MacroD2 and performed a proof-of-concept compound screening. Three compounds were identified as screening hits with micromolar potency. However, further characterization of the compounds identified them as protein destabilizers, excluding further follow-up studies. Validation and screening demonstrated the usability of the in vitro assay for MacroD2, and we also demonstrate the applicability of the assay as a tool for other human ADP-ribosylhydrolases.

A FRET-based high-throughput screening platform for the discovery of chemical probes targeting the scaffolding functions of human tankyrases.

Tankyrases catalyse poly-ADP-ribosylation of their binding partners and the modification serves as a signal for the subsequent proteasomal degradation of these proteins. Tankyrases thereby regulate the turnover of many proteins involved in multiple and diverse cellular processes, such as mitotic spindle formation, telomere homeostasis and Wnt/ß-catenin signalling. In recent years, tankyrases have become attractive targets for the development of inhibitors as potential therapeutics against cancer and fibrosis. Further, it has become clear that tankyrases are not only enzymes, but also act as scaffolding proteins forming large cellular signalling complexes. While many potent and selective tankyrase inhibitors of the poly-ADP-ribosylation function exist, the inhibition of tankyrase scaffolding functions remains scarcely explored. In this work we present a robust, simple and cost-effective high-throughput screening platform based on FRET for the discovery of small molecule probes targeting the protein-protein interactions of tankyrases. Validatory screening with the platform led to the identification of two compounds with modest binding affinity to the tankyrase 2 ARC4 domain, demonstrating the applicability of this approach. The platform will facilitate identification of small molecules binding to tankyrase ARC or SAM domains and help to advance a structure-guided development of improved chemical probes targeting tankyrase oligomerization and substrate protein interactions.

Preclinical Lead Optimization of a 1,2,4-Triazole Based Tankyrase Inhibitor.

Tankyrases 1 and 2 are central biotargets in the WNT/ß-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 IC50 inhibition in WNT/ß-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.