Advertisement

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

  • Ilsa T. Kirby
  • Rory K. Morgan
  • Michael S. Cohen
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1813)

Abstract

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.

Key words

ADP-ribosylation PARPs ARTDs Small-molecule inhibitor Screen Click chemistry 6-a-NAD+ 

Notes

Acknowledgments

We thank members of the Cohen lab for many helpful discussions. We thank H. Schuler for the construct for PARP14wwe-cat. We thank J. Pascal (Université de Montréal) for helpful discussions regarding PARP3 enzyme activity. This work was funded by the NIH (NIH 1R01NS088629) and a grant from the Pew Charitable Trust (M.S.C.).

References

  1. 1.
    Mehrotra P, Hollenbeck A, Riley JP et al (2013) Poly (ADP-ribose) polymerase 14 and its enzyme activity regulates TH2 differentiation and allergic airway disease. J Allergy Clin Immunol 131:521–531.e12. https://doi.org/10.1016/j.jaci.2012.06.015 CrossRefPubMedGoogle Scholar
  2. 2.
    Barbarulo A, Iansante V, Chaidos A et al (2013) Poly(ADP-ribose) polymerase family member 14 (PARP14) is a novel effector of the JNK2-dependent pro-survival signal in multiple myeloma. Oncogene 32:4231–4242. https://doi.org/10.1038/onc.2012.448 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Wahlberg E, Karlberg T, Kouznetsova E et al (2012) Family-wide chemical profiling and structural analysis of PARP and tankyrase inhibitors. Nat Biotechnol 30:283–288. https://doi.org/10.1038/nbt.2121 CrossRefPubMedGoogle Scholar
  4. 4.
    Venkannagari H, Verheugd P, Koivunen J et al (2016) Small-molecule chemical probe rescues cells from mono-ADP-ribosyltransferase ARTD10/PARP10-induced apoptosis and sensitizes cancer cells to DNA damage. Cell Chem Biol 23:1251–1260. https://doi.org/10.1016/j.chembiol.2016.08.012 CrossRefPubMedGoogle Scholar
  5. 5.
    Thorsell A-G, Ekblad T, Karlberg T et al (2017) Structural basis for potency and promiscuity in poly(ADP-ribose) polymerase (PARP) and tankyrase inhibitors. J Med Chem 60:1262–1271. https://doi.org/10.1021/acs.jmedchem.6b00990 CrossRefPubMedGoogle Scholar
  6. 6.
    Carter-OConnell I, Jin H, Morgan RK et al (2014) Engineering the substrate specificity of ADP-ribosyltransferases for identifying direct protein targets. J Am Chem Soc 136:5201–5204. https://doi.org/10.1021/ja412897a CrossRefGoogle Scholar
  7. 7.
    Morgan RK, Cohen MS (2015) A clickable aminooxy probe for monitoring cellular ADP-ribosylation. ACS Chem Biol 10:1778–1784. https://doi.org/10.1021/acschembio.5b00213 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Venkannagari H, Fallarero A, Feijs KLH et al (2013) Activity-based assay for human mono-ADP-ribosyltransferases ARTD7/PARP15 and ARTD10/PARP10 aimed at screening and profiling inhibitors. Eur J Pharm Sci 49:148–156. https://doi.org/10.1016/j.ejps.2013.02.012 CrossRefPubMedGoogle Scholar
  9. 9.
    Cambronne XA, Stewart ML, Kim D et al (2016) Biosensor reveals multiple sources for mitochondrial NAD. Science 352:1474–1477. https://doi.org/10.1126/science.aad5168 CrossRefPubMedGoogle Scholar
  10. 10.
    Menear KA, Adcock C, Boulter R et al (2008) 4-[3-(4-cyclopropanecarbonylpiperazine-1-carbonyl)-4-fluorobenzyl]-2H-phthalazin-1-one: a novel bioavailable inhibitor of poly(ADP-ribose) polymerase-1. J Med Chem 51:6581–6591. https://doi.org/10.1021/jm8001263 CrossRefPubMedGoogle Scholar
  11. 11.
    Thomas HD, Calabrese CR, Batey MA et al (2007) Preclinical selection of a novel poly(ADP-ribose) polymerase inhibitor for clinical trial. Mol Cancer Ther 6:945–956. https://doi.org/10.1158/1535-7163.MCT-06-0552 CrossRefPubMedGoogle Scholar
  12. 12.
    Langelier M-F, Riccio AA, Pascal JM (2014) PARP-2 and PARP-3 are selectively activated by 5′ phosphorylated DNA breaks through an allosteric regulatory mechanism shared with PARP-1. Nucleic Acids Res 42:7762–7775. https://doi.org/10.1093/nar/gku474 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Ilsa T. Kirby
    • 1
  • Rory K. Morgan
    • 1
  • Michael S. Cohen
    • 1
  1. 1.Program in Chemical Biology, Department of Physiology and PharmacologyOregon Health and Science UniversityPortlandUSA

Personalised recommendations