title: Insights into PARP Inhibitors' Selectivity Using Fluorescence Polarization and Surface Plasmon Resonance Binding Assays.
abstract: PARP inhibitors are an exciting new class of antineoplastic drugs that have been proven to be efficacious as single agents in cancer settings with inherent DNA repair defects, as well as in combination with DNA-damaging chemotherapeutics. Currently, they are designed to target the catalytic domain of PARP-1, the most studied member of the family, with a key role in the DNA-damage repair process. Because PARP inhibitors are substrate (NAD(+)) competitors, there is a need for a deeper understanding of their cross-reactivity. This is particularly relevant for PARP-2, the PARP-1 closest homologue, for which an embryonic lethal phenotype has been observed in double knockout mice. In this study, we describe the development and validation of binding assays based on fluorescence polarization (FP) and surface plasmon resonance (SPR) techniques. PARP-1, PARP-2, PARP-3, and TNKS-1 FP displacement assays are set up by employing ad hoc synthesized probes. These assays are suitable for high-throughput screening (HTS) and selectivity profiling, thus allowing the identification of NAD(+)binding site selective inhibitors. The PARP-1 and PARP-2 complementary SPR binding assays confirm displacement data and the in-depth inhibitor characterization. Moreover, these formats have the potential to be broadly applicable to other members of the PARP family.
authors.affiliation: Nerviano Medical Sciences S.r.l., Nerviano, Italy.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Papeo
authors.affiliation: Nerviano Medical Sciences S.r.l., Nerviano, Italy.
authors.forename: Nilla
authors.initials: N
authors.lastname: Avanzi
authors.affiliation: Nerviano Medical Sciences S.r.l., Nerviano, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Bettoni
authors.affiliation: Nerviano Medical Sciences S.r.l., Nerviano, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Leone
authors.affiliation: Nerviano Medical Sciences S.r.l., Nerviano, Italy.
authors.forename: Mauro
authors.initials: M
authors.lastname: Paolucci
authors.affiliation: Nerviano Medical Sciences S.r.l., Nerviano, Italy.
authors.forename: Rita
authors.initials: R
authors.lastname: Perego
authors.affiliation: Nerviano Medical Sciences S.r.l., Nerviano, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Quartieri
authors.affiliation: Nerviano Medical Sciences S.r.l., Nerviano, Italy.
authors.forename: Federico
authors.initials: F
authors.lastname: Riccardi-Sirtori
authors.affiliation: Nerviano Medical Sciences S.r.l., Nerviano, Italy Evotec Ltd, Milton Park, Abingdon, Oxfordshire, UK.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Thieffine
authors.affiliation: Nerviano Medical Sciences S.r.l., Nerviano, Italy.
authors.forename: Alessia
authors.initials: A
authors.lastname: Montagnoli
authors.affiliation: Nerviano Medical Sciences S.r.l., Nerviano, Italy rosita.lupi@nervianoms.com.
authors.forename: Rosita
authors.initials: R
authors.lastname: Lupi
mesh_terms: D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D005454:Fluorescence Polarization / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D000071137:Poly (ADP-Ribose) Polymerase-1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000067856:Poly(ADP-ribose) Polymerase Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D020349:Surface Plasmon Resonance / Q000379:methods*; D038501:Tankyrases / Q000235:genetics / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D000071137:Poly (ADP-Ribose) Polymerase-1; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases; D011994:Recombinant Proteins; D038501:Tankyrases; C499320:PARP1 protein, human; C483041:PARP2 protein, human; C480979:PARP3 protein, human; C418109:TNKS protein, human
keywords: HTS; PARP; fluorescence polarization (FP); selectivity; surface plasmon resonance (SPR)