A Potent and Selective PARP11 Inhibitor Suggests Coupling between Cellular Localization and Catalytic Activity

Kirby, Ilsa T; Kojic, Ana; Arnold, Moriah R.; Thorsell, A.G.; Karlberg, T.; Vermehren-Schmaedick, Anke; Sreenivasan, Raashi; Schultz, Carsten; Schüler, H.; Cohen, Michael S.

doi:10.1016/j.chembiol.2018.09.011

Cited by 57 publications

(62 citation statements)

References 33 publications

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“…The high complexity of ADP-ribosyl associated pathways makes the involved proteins notoriously hard to study (Bonfiglio et al, 2020;Lüscher et al, 2018). While potent and specific inhibitors against many of the ADP-ribosyl transferases fundamentally helped to broaden our understanding of these enzymes (Durkacz et al, 1980;Huang et al, 2009;Kirby et al, 2018;Venkannagari et al, 2016;Wang et al, 2018), such inhibitors against ADP-ribosyl readers and erasers are still scarcely available or in early stages of development (Harrision et al, 2020;James et al, 2016;Liu et al, 2020;Palazzo and Ahel, 2018;Schuller et al, 2017). It was suggested that a possible bottleneck for the discovery of inhibitors is due to the lack of accessible high-throughput technologies (Schuller et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

A molecular toolbox for ADP-ribosyl binding proteins

Sowa

Galera‐Prat

Wazir

et al. 2021

Preprint

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Proteins interacting with ADP-ribosyl groups are often involved in disease-related pathways or in viral infections, which makes them attractive targets for the development of inhibitors. Our goal was to develop a robust and accessible assay technology that is suitable for high-throughput screening and applicable to a wide range of proteins acting as either hydrolysing or non-hydrolysing binders of mono- and poly-ADP-ribosyl groups. As a foundation of our work, we developed a C-terminal protein fusion 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 as well as chemical ADP-ribosyl analogs. By fusion of the GAP-tag and ADP-ribosyl binders to fluorescent proteins, we were able to generate robust FRET signals and the interaction with 22 previously described ADP-ribosyl-binders was confirmed. To demonstrate the applicability of this binding assay for high-throughput screening, we utilized it to screen for inhibitors of the SARS-CoV-2 nsp3 macrodomain and identified the drug suramin as a moderate yet unspecific inhibitor of this protein. To complement the binding technology, we prepared high-affinity ADP-ribosyl binders fused to a nanoluciferase, which enabled simple blot-based detection of mono- and poly-ADP-ribosylated proteins. These tools can be expressed recombinantly in E. coli using commonly available agents and will help to investigate ADP-ribosylation systems and aid in drug discovery.

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Section: Discussionmentioning

confidence: 99%

A molecular toolbox for ADP-ribosyl binding proteins

Sowa

Galera‐Prat

Wazir

et al. 2021

Preprint

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“…The clinical success of PARP inhibitors in BRCA1/2-mutated breast and ovarian cancers has ignited a push for more widespread use of these compounds in cancers with a molecular signature of defective HR, irrespec-ADP-ribose mechanisms and disease 7 tive of which HR gene is mutated and in which tissue the tumour originated (Pilie et al, 2019). Similarly, novel inhibitors that selectively target different PARPs, including PARP3, PARP5a/5b, PARP7, PARP10, PARP11 and PARP14 are under investigation for the targeted treatment of cancers with alterations in particular pathways (Ishida et al, 2006;Lindgren et al, 2013;Iwata et al, 2016;Wang, Y. Q. et al, 2016b;Ferri et al, 2017;Yoneyama-Hirozane et al, 2017;Kirby et al, 2018;Moustakim et al, 2018;Murthy et al, 2018).…”

Section: Parp1 and Cell Deathmentioning

confidence: 99%

ADP-ribosylation: from molecular mechanisms to human disease

Hoch

Polo

2020

Genet. Mol. Biol.

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Post-translational modification of proteins by ADP-ribosylation, catalysed by poly (ADP-ribose) polymerases (PARPs) using NAD + as a substrate, plays central roles in DNA damage signalling and repair, modulates a range of cellular signalling cascades and initiates programmed cell death by parthanatos. Here, we present mechanistic aspects of ADP-ribose modification, PARP activation and the cellular functions of ADP-ribose signalling, and discuss how this knowledge is uncovering therapeutic avenues for the treatment of increasingly prevalent human diseases such as cancer, ischaemic damage and neurodegeneration.The PARP1 active site is formed between the catalytic domain (ART domain) and the helical domain (HD) Figure 1 -Schematic mechanism of ADP ribosylation reaction and the catalytic domain of DNA-dependant PARPs. A) A simplified overview of the (ADP)-ribosylation reactions catalysed by PARPs. The final products depend on the acceptor residue acting as a nucleophile (Nu, in blue). PARP1 active-site residues interacting with the ribose-nicotinamide moiety of NAD + are illustrated in orange. B) The NAD + (modelled based on the human PARP1 bound to benzamide adenine dinucleotide [PDB: 6BHV], carbon atoms in yellow) in an extended conformation, bound to the catalytic domain of human PARP1 (ART in cartoon, orange, [PDB: 6BHV]). The residues involved in the catalysis are presented as sticks. C) Superposed cartoon view of human PARP-1 ART domain (orange, [PDB: 6BHV]), PARP1 (light blue, [PDB: 5WS1]) and PARP2 (green, [PDB: 3KJD]) showing the structure of the entire catalytic domains (ART and HD). The modelled NAD + (in yellow) denotes the donor site, while a molecule of ADP (modelled by superimposing the structures of chicken PARP1 [PDB: 1A26] to the human PARP1 [PDB: 3KJD]) indicates the acceptor site. Donor loop (D-loop) and acceptor loop are labelled. D) Surface representation of human PARP1 [PDB: 3KJD] with NAD + modelled into the active site. The ribose group to be attacked is exposed to the solvent. ADP-ribose mechanisms and disease 3 ADP-ribose mechanisms and disease 13

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“…1 Ribon Therapeutics Inc., Cambridge, MA, USA 2 Evotec SE, Hamburg, Germany inhibitors, and of those published, potencies and intra-PARP family selectivity are modest. [3][4][5][6][7][8][9][10] Approved drugs that target PARP1 and PARP2 (niraparib and talazoparib) or PARP1, PARP2, and PARP3 (olaparib and rucaparib) are now in use for the treatment of a variety of cancers, and potent and selective inhibitors of PARP5a and PARP5b (tankyrases 1 and 2) have been reported to have antiproliferative activity in cancer cell lines and in vivo models. 11 MonoPARPs have been reported to have important roles in immunology, inflammation, and cancer; [12][13][14] however, these studies have relied almost exclusively on genetic perturbation techniques that often do not distinguish loss of catalytic activity from loss of the entire protein.…”

Section: Research-article2019mentioning

confidence: 99%

Forced Self-Modification Assays as a Strategy to Screen MonoPARP Enzymes

et al. 2020

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Mono(ADP-ribosylation) (MARylation) and poly(ADP-ribosylation) (PARylation) are posttranslational modifications found on multiple amino acids. There are 12 enzymatically active mono(ADP-ribose) polymerase (monoPARP) enzymes and 4 enzymatically active poly(ADP-ribose) polymerase (polyPARP) enzymes that use nicotinamide adenine dinucleotide (NAD+) as the ADP-ribose donating substrate to generate these modifications. While there are approved drugs and clinical trials ongoing for the enzymes that perform PARylation, MARylation is gaining recognition for its role in immune function, inflammation, and cancer. However, there is a lack of chemical probes to study the function of monoPARPs in cells and in vivo. An important first step to generating chemical probes for monoPARPs is to develop biochemical assays to enable hit finding, and determination of the potency and selectivity of inhibitors. Complicating the development of enzymatic assays is that it is poorly understood how monoPARPs engage their substrates. To overcome this, we have developed a family-wide approach to developing robust high-throughput monoPARP assays where the enzymes are immobilized and forced to self-modify using biotinylated-NAD+, which is detected using a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) readout. Herein we describe the development of assays for 12 monoPARPs and 3 polyPARPs and apply them to understand the potency and selectivity of a focused library of inhibitors across this family.

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A Potent and Selective PARP11 Inhibitor Suggests Coupling between Cellular Localization and Catalytic Activity

Abstract: Highlights d Structure-guided design of PARP inhibitors d Identification of a potent and selective PARP11 inhibitor (ITK7) d ITK7 inhibits PARP11 auto-MARylation in cells d ITK7 causes PARP11 to dissociate from the nuclear envelope

Cited by 57 publications

References 33 publications

A molecular toolbox for ADP-ribosyl binding proteins

A molecular toolbox for ADP-ribosyl binding proteins

ADP-ribosylation: from molecular mechanisms to human disease

Forced Self-Modification Assays as a Strategy to Screen MonoPARP Enzymes

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