PARP14 Contributes to the Development of the Tumor-Associated Macrophage Phenotype.

Cancers reprogram macrophages (MΦs) to a tumor-growth-promoting TAM (tumor-associated MΦ) phenotype that is similar to the anti-inflammatory M2 phenotype. Poly(ADP-ribose) polymerase (PARP) enzymes regulate various aspects of MΦ biology, but their role in the development of TAM phenotype has not yet been investigated. Here, we show that the multispectral PARP inhibitor (PARPi) PJ34 and the PARP14 specific inhibitor MCD113 suppress the expression of M2 marker genes in IL-4-polarized primary murine MΦs, in THP-1 monocytic human MΦs, and in primary human monocyte-derived MΦs. MΦs isolated from PARP14 knockout mice showed a limited ability to differentiate to M2 cells. In a murine model of TAM polarization (4T1 breast carcinoma cell supernatant transfer to primary MΦs) and in a human TAM model (spheroids formed from JIMT-1 breast carcinoma cells and THP-1-MΦs), both PARPis and the PARP14 KO phenotype caused weaker TAM polarization. Increased JIMT-1 cell apoptosis in co-culture spheroids treated with PARPis suggested reduced functional TAM reprogramming. Protein profiling arrays identified lipocalin-2, macrophage migration inhibitory factor, and plasminogen activator inhibitor-1 as potential (ADP-ribosyl)ation-dependent mediators of TAM differentiation. Our data suggest that PARP14 inhibition might be a viable anticancer strategy with a potential to boost anticancer immune responses by reprogramming TAMs.

PARP14 is a writer, reader, and eraser of mono-ADP-ribosylation.

PARP14/BAL2 is a large multidomain enzyme involved in signaling pathways with relevance to cancer, inflammation, and infection. Inhibition of its mono-ADP-ribosylating PARP homology domain and its three ADP-ribosyl binding macro domains has been regarded as a potential means of therapeutic intervention. Macrodomains-2 and -3 are known to stably bind to ADP-ribosylated target proteins, but the function of macrodomain-1 has remained somewhat elusive. Here, we used biochemical assays of ADP-ribosylation levels to characterize PARP14 macrodomain-1 and the homologous macrodomain-1 of PARP9. Our results show that both macrodomains display an ADP-ribosyl glycohydrolase activity that is not directed toward specific protein side chains. PARP14 macrodomain-1 is unable to degrade poly(ADP-ribose), the enzymatic product of PARP1. The F926A mutation of PARP14 and the F244A mutation of PARP9 strongly reduced ADP-ribosyl glycohydrolase activity of the respective macrodomains, suggesting mechanistic homology to the Mac1 domain of the SARS-CoV-2 Nsp3 protein. This study adds two new enzymes to the previously known six human ADP-ribosyl glycohydrolases. Our results have key implications for how PARP14 and PARP9 will be studied and how their functions will be understood.

Combining pharmacophore models derived from DNA-encoded chemical libraries with structure-based exploration to predict Tankyrase 1 inhibitors.

DNA-encoded chemical libraries (DECLs) interrogate the interactions of a target of interest with vast numbers of molecules. DECLs hence provide abundant information about the chemical ligand space for therapeutic targets, and there is considerable interest in methods for exploiting DECL screening data to predict novel ligands. Here we introduce one such approach and demonstrate its feasibility using the cancer-related poly-(ADP-ribose)transferase tankyrase 1 (TNKS1) as a model target. First, DECL affinity selections resulted in structurally diverse TNKS1 inhibitors with high potency including compound 2 with an IC50 value of 0.8 nM. Additionally, TNKS1 hits from four DECLs were translated into pharmacophore models, which were exploited in combination with docking-based screening to identify TNKS1 ligand candidates in databases of commercially available compounds. This computational strategy afforded TNKS1 inhibitors that are outside the chemical space covered by the DECLs and yielded the drug-like lead compound 12 with an IC50 value of 22 nM. The study further provided insights in the reliability of screening data and the effect of library design on hit compounds. In particular, the study revealed that while in general DECL screening data are in good agreement with off-DNA ligand binding, unpredictable interactions of the DNA-attachment linker with the target protein contribute to the noise in the affinity selection data.

PARP10 Multi-Site Auto- and Histone MARylation Visualized by Acid-Urea Gel Electrophoresis.

Poly-ADP-ribose polymerase (PARP)-family ADP-ribosyltransferases function in various signaling pathways, predominantly in the nucleus and cytosol. Although PARP inhibitors are in clinical practice for cancer therapy, the enzymatic activities of individual PARP family members are yet insufficiently understood. We studied PARP10, a mono-ADP-ribosyltransferase and potential drug target. Using acid-urea gel electrophoresis, we found that the isolated catalytic domain of PARP10 auto-ADP-ribosylates (MARylates) at eight or more acceptor residues. We isolated individual species with either singular or several modifications and then analyzed them by mass spectrometry. The results confirmed multi-site MARylation in a random order and identified four acceptor residues. The mutagenesis of singular acceptor residues had a minor impact on the overall auto-MARylation level and no effect on the MARylation of histone H3.1. Together, our results suggest that PARP10 automodification may have functions in the regulation of intramolecular or partner binding events, rather than of its enzymatic catalysis. This contributes to a better understanding of PARP10 functions, and, in the long run, to gauging the consequences of PARP inhibitor actions.

System-wide identification and prioritization of enzyme substrates by thermal analysis.

Despite the immense importance of enzyme-substrate reactions, there is a lack of general and unbiased tools for identifying and prioritizing substrate proteins that are modified by the enzyme on the structural level. Here we describe a high-throughput unbiased proteomics method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that the enzymatic post-translational modification of substrate proteins is likely to change their thermal stability. In our proof-of-concept studies, SIESTA successfully identifies several known and novel substrate candidates for selenoprotein thioredoxin reductase 1, protein kinase B (AKT1) and poly-(ADP-ribose) polymerase-10 systems. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, opening opportunities to investigate the effect of post-translational modifications on signal transduction and facilitate drug discovery.

Engineering Af1521 improves ADP-ribose binding and identification of ADP-ribosylated proteins.

Protein ADP-ribosylation is a reversible post-translational modification that regulates important cellular functions. The identification of modified proteins has proven challenging and has mainly been achieved via enrichment methodologies. Random mutagenesis was used here to develop an engineered Af1521 ADP-ribose binding macro domain protein with 1000-fold increased affinity towards ADP-ribose. The crystal structure reveals that two point mutations K35E and Y145R form a salt bridge within the ADP-ribose binding domain. This forces the proximal ribose to rotate within the binding pocket and, as a consequence, improves engineered Af1521 ADPr-binding affinity. Its use in our proteomic ADP-ribosylome workflow increases the ADP-ribosylated protein identification rates and yields greater ADP-ribosylome coverage. Furthermore, generation of an engineered Af1521 Fc fusion protein confirms the improved detection of cellular ADP-ribosylation by immunoblot and immunofluorescence. Thus, this engineered isoform of Af1521 can also serve as a valuable tool for the analysis of cellular ADP-ribosylation under in vivo conditions.

A DNA-Encoded Library of Chemical Compounds Based on Common Scaffolding Structures Reveals the Impact of Ligand Geometry on Protein Recognition.

A DNA-encoded chemical library (DECL) with 1.2 million compounds was synthesized by combinatorial reaction of seven central scaffolds with two sets of 343×492 building blocks. Library screening by affinity capture revealed that for some target proteins, the chemical nature of building blocks dominated the selection results, whereas for other proteins, the central scaffold also crucially contributed to ligand affinity. Molecules based on a 3,5-bis(aminomethyl)benzoic acid core structure were found to bind human serum albumin with a Kd value of 6 nm, while compounds with the same substituents on an equidistant but flexible l-lysine scaffold showed 140-fold lower affinity. A 18 nm tankyrase-1 binder featured l-lysine as linking moiety, while molecules based on d-Lysine or (2S,4S)-amino-l-proline showed no detectable binding to the target. This work suggests that central scaffolds which predispose the orientation of chemical building blocks toward the protein target may enhance the screening productivity of encoded libraries.

Design, synthesis and evaluation of potent and selective inhibitors of mono-(ADP-ribosyl)transferases PARP10 and PARP14.

A series of diaryl ethers were designed and synthesized to discern the structure activity relationships against the two closely related mono-(ADP-ribosyl)transferases PARP10 and PARP14. Structure activity studies identified 8b as a sub-micromolar inhibitor of PARP10 with ∼15-fold selectivity over PARP14. In addition, 8k and 8m were discovered to have sub-micromolar potency against PARP14 and demonstrated moderate selectivity over PARP10. A crystal structure of the complex of PARP14 and 8b shows binding of the compound in a novel hydrophobic pocket and explains both potency and selectivity over other PARP family members. In addition, 8b, 8k and 8m also demonstrate selectivity over PARP1. Together, this study identified novel, potent and metabolically stable derivatives to use as chemical probes for these biologically interesting therapeutic targets.

Identification of Poly(ADP-Ribose) Polymerase Macrodomain Inhibitors Using an AlphaScreen Protocol.

Macrodomains recognize intracellular adenosine diphosphate (ADP)-ribosylation resulting in either removal of the modification or a protein interaction event. Research into compounds that modulate macrodomain functions could make important contributions. We investigated the interactions of all seven individual macrodomains of the human poly(ADP-ribose) polymerase (PARP) family members PARP9, PARP14, and PARP15 with five mono-ADP-ribosylated (automodified) ADP-ribosyltransferase domains using an AlphaScreen assay. Several mono-ADP-ribosylation-dependent interactions were identified, and they were found to be in the micromolar affinity range using surface plasmon resonance (SPR). We then focused on the interaction between PARP14 macrodomain-2 and the mono-ADP-ribosylated PARP10 catalytic domain, and probed a ~1500-compound diverse library for inhibitors of this interaction using AlphaScreen. Initial hit compounds were verified by concentration-response experiments using AlphaScreen and SPR, and they were tested against PARP14 macrodomain-2 and -3. Two initial hit compounds and one chemical analog each were further characterized using SPR and microscale thermophoresis. In conclusion, our results reveal novel macrodomain interactions and establish protocols for identification of inhibitors of such interactions.

Structural Basis for Potency and Promiscuity in Poly(ADP-ribose) Polymerase (PARP) and Tankyrase Inhibitors.

Selective inhibitors could help unveil the mechanisms by which inhibition of poly(ADP-ribose) polymerases (PARPs) elicits clinical benefits in cancer therapy. We profiled 10 clinical PARP inhibitors and commonly used research tools for their inhibition of multiple PARP enzymes. We also determined crystal structures of these compounds bound to PARP1 or PARP2. Veliparib and niraparib are selective inhibitors of PARP1 and PARP2; olaparib, rucaparib, and talazoparib are more potent inhibitors of PARP1 but are less selective. PJ34 and UPF1069 are broad PARP inhibitors; PJ34 inserts a flexible moiety into hydrophobic subpockets in various ADP-ribosyltransferases. XAV939 is a promiscuous tankyrase inhibitor and a potent inhibitor of PARP1 in vitro and in cells, whereas IWR1 and AZ-6102 are tankyrase selective. Our biochemical and structural analysis of PARP inhibitor potencies establishes a molecular basis for either selectivity or promiscuity and provides a benchmark for experimental design in assessment of PARP inhibitor effects.

Small Molecule Microarray Based Discovery of PARP14 Inhibitors.

Poly(ADP-ribose) polymerases (PARPs) are key enzymes in a variety of cellular processes. Most small-molecule PARP inhibitors developed to date have been against PARP1, and suffer from poor selectivity. PARP14 has recently emerged as a potential therapeutic target, but its inhibitor development has trailed behind. Herein, we describe a small molecule microarray-based strategy for high-throughput synthesis, screening of >1000 potential bidentate inhibitors of PARPs, and the successful discovery of a potent PARP14 inhibitor H10 with >20-fold selectivity over PARP1. Co-crystallization of the PARP14/H10 complex indicated H10 bound to both the nicotinamide and the adenine subsites. Further structure-activity relationship studies identified important binding elements in the adenine subsite. In tumor cells, H10 was able to chemically knockdown endogenous PARP14 activities.

DNA binding to SMC ATPases-trapped for release.

The SMC/Rad50/RecN proteins are universal DNA­associated ABC­type ATPases with crucial functions in genome maintenance. New insights into Rad50-DNA complex structure and cohesin regulation inspire a speculative look at the entire superfamily. Identification of a continuous DNA binding site across the Rad50 dimer interface (Liu et al, 2016; Seifert et al, 2016) suggests a similar site in cohesin. The localization of this site hints a DNA-activated mechanism for cohesin removal from chromosomes.

Sirtuins are Unaffected by PARP Inhibitors Containing Planar Nicotinamide Bioisosteres.

PARP-family ADP-ribosyltransferases (PARPs) and sirtuin deacetylases all use NAD(+) as cosubstrate for ADP-ribosyl transfer. PARP inhibitors are important research tools and several are being evaluated in cancer treatment. With the exception of a few tankyrase inhibitors, all current PARP inhibitors mimic the nicotinamide moiety in NAD(+) and block the nicotinamide binding pocket. We report here that while the activities of the four human sirtuin isoforms SIRT1, SIRT2, SIRT3 and SIRT6 are blocked by sirtuin inhibitor Ex527 in vitro, they are unaffected by the seven clinical and commonly used PARP inhibitors niraparib, olaparib, rucaparib, talazoparib, veliparib, PJ34, and XAV939. These findings indicate that PARP inhibitors containing planar nicotinamide mimetics do not bind to sirtuin cofactor sites. In conclusion, a simple commercially available assay can be used to rule out interference of novel PARP inhibitors with sirtuin NAD(+) binding.

Towards small molecule inhibitors of mono-ADP-ribosyltransferases.

Protein ADP-ribosylation is a post-translational modification involved in DNA repair, protein degradation, transcription regulation, and epigenetic events. Intracellular ADP-ribosylation is catalyzed predominantly by ADP-ribosyltransferases with diphtheria toxin homology (ARTDs). The most prominent member of the ARTD family, poly(ADP-ribose) polymerase-1 (ARTD1/PARP1) has been a target for cancer drug development for decades. Current PARP inhibitors are generally non-selective, and inhibit the mono-ADP-ribosyltransferases with low potency. Here we describe the synthesis of acylated amino benzamides and screening against the mono-ADP-ribosyltransferases ARTD7/PARP15, ARTD8/PARP14, ARTD10/PARP10, and the poly-ADP-ribosyltransferase ARTD1/PARP1. The most potent compound inhibits ARTD10 with sub-micromolar IC50.

Identification of structure-activity relationships from screening a structurally compact DNA-encoded chemical library.

Methods for the rapid and inexpensive discovery of hit compounds are essential for pharmaceutical research and DNA-encoded chemical libraries represent promising tools for this purpose. We here report on the design and synthesis of DAL-100K, a DNA-encoded chemical library containing 103 200 structurally compact compounds. Affinity screening experiments and DNA-sequencing analysis provided ligands with nanomolar affinities to several proteins, including prostate-specific membrane antigen and tankyrase 1. Correlations of sequence counts with binding affinities and potencies of enzyme inhibition were observed and enabled the identification of structural features critical for activity. These results indicate that libraries of this type represent a useful source of small-molecule binders for target proteins of pharmaceutical interest and information on structural features important for binding.

Design, synthesis, crystallographic studies, and preliminary biological appraisal of new substituted triazolo[4,3-b]pyridazin-8-amine derivatives as tankyrase inhibitors.

Searching for selective tankyrases (TNKSs) inhibitors, a new small series of 6,8-disubstituted triazolo[4,3-b]piridazines has been synthesized and characterized biologically. Structure-based optimization of the starting hit compound NNL (3) prompted us to the discovery of 4-(2-(6-methyl-[1,2,4]triazolo[4,3-b]pyridazin-8-ylamino)ethyl)phenol (12), a low nanomolar selective TNKSs inhibitor working as NAD isostere as ascertained by crystallographic analysis. Preliminary biological data candidate this new class of derivatives as a powerful pharmacological tools in the unraveling of TNKS implications in physiopathological conditions.

Pharmacology of ADP-ribosylation.

ADP-ribosyltransferase ARTD1/PARP1 is a target for cancer and ischemia drug development. Several other ARTD-family enzymes have been characterized in recent years, and it has become clear that their inhibition might also have therapeutic value. This minireview series summarizes current knowledge of pharmacological inhibition of ADP-ribosyltransferases by a compound class called PARP inhibitors and the prospects for drug development.

PARP inhibitor with selectivity toward ADP-ribosyltransferase ARTD3/PARP3.

Inhibiting ADP-ribosyl transferases with PARP-inhibitors is considered a promising strategy for the treatment of many cancers and ischemia, but most of the cellular targets are poorly characterized. Here, we describe an inhibitor of ADP-ribosyltransferase-3/poly(ADP-ribose) polymerase-3 (ARTD3), a regulator of DNA repair and mitotic progression. In vitro profiling against 12 members of the enzyme family suggests selectivity for ARTD3, and crystal structures illustrate the molecular basis for inhibitor selectivity. The compound is active in cells, where it elicits ARTD3-specific effects at submicromolar concentration. Our results show that by targeting the nicotinamide binding site, selective inhibition can be achieved among the closest relatives of the validated clinical target, ADP-ribosyltransferase-1/poly(ADP-ribose) polymerase-1.

PARP inhibitors: polypharmacology versus selective inhibition.

Inhibition of ADP-ribosyltransferases with diphtheria toxin homology (ARTD), widely known as the poly(ADP-ribose) polymerase (PARP) family, is a strategy under development for treatment of various conditions, including cancers and ischemia. Here, we give a brief summary of ARTD enzyme functions and the implications for their potential as therapeutic targets. We present an overview of the PARP inhibitors that have been used in clinical trials. Finally, we summarize recent insights from structural biology, and discuss the molecular aspects of PARP inhibitors in terms of broad-range versus selective inhibition of ARTD family enzymes.