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

• Published in: SLAS discovery Vol. 23; no. 4; pp. 353 - 362
• Main Authors: Ekblad, Torun, Verheugd, Patricia, Lindgren, Anders E., Nyman, Tomas, Elofsson, Mikael, Schüler, Herwig
• Format: Journal Article
• Language: English
• Published: Los Angeles, CA SAGE Publications 01.04.2018
• Subjects: Adenosine Diphosphate Ribose - metabolism; ADP Ribose Transferases - metabolism; ADP-ribosylation; ADP-Ribosylation - drug effects; AlphaScreen; Biological Assay - methods; Humans; macrodomain inhibitor; Medicin och hälsovetenskap; PARP; Pentosyltransferases; Poly(ADP-ribose) Polymerase Inhibitors - pharmacology; Poly(ADP-ribose) Polymerases - metabolism; protein-protein interaction; protein–protein interaction; PARP; AlphaScreen; macrodomain inhibitor; ADP-ribosylation
• ISSN: 2472-5552; 2472-5560; 2472-5560
• DOI: 10.1177/2472555217750870

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.