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

Torun Ekblad; Patricia Verheugd; Anders E Lindgren; Tomas Nyman; Mikael Elofsson; Herwig Schüler

doi:10.1177/2472555217750870

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

SLAS Discov. 2018 Apr;23(4):353-362. doi: 10.1177/2472555217750870. Epub 2018 Jan 9.

Authors

Torun Ekblad^{1

2}, Patricia Verheugd^{1

2}, Anders E Lindgren³, Tomas Nyman⁴, Mikael Elofsson³, Herwig Schüler¹

Affiliations

¹ 1 Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
² Present addresses: For TE, Mabtech AB, 131 52 Nacka Strand, Sweden; for PV, Institute of Biochemistry and Molecular Biology, Medical School, RWTH Aachen University, 52057 Aachen, Germany.
³ 2 Department of Chemistry, Umeå University, Umeå, Sweden.
⁴ 3 Protein Science Facility, Karolinska Institutet, Stockholm, Sweden.

PMID: 29316839
DOI: 10.1177/2472555217750870

Abstract

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.

Keywords: ADP-ribosylation; AlphaScreen; PARP; macrodomain inhibitor; protein–protein interaction.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

ADP Ribose Transferases / metabolism
ADP-Ribosylation / drug effects
Adenosine Diphosphate Ribose / metabolism
Biological Assay / methods*
Humans
Pentosyltransferases
Poly(ADP-ribose) Polymerase Inhibitors / pharmacology*
Poly(ADP-ribose) Polymerases / metabolism*

Substances

Poly(ADP-ribose) Polymerase Inhibitors
Adenosine Diphosphate Ribose
ADP Ribose Transferases
Pentosyltransferases
Poly(ADP-ribose) Polymerases