Duplexed CeTEAM drug biosensors reveal determinants of PARP inhibitor selectivity in cells

Maria J Pires; Seher Alam; Alen Lovric; Emanuele Fabbrizi; Dante Rotili; Mikael Altun; Nicholas C K Valerie

doi:10.1016/j.jbc.2025.108361

Duplexed CeTEAM drug biosensors reveal determinants of PARP inhibitor selectivity in cells

J Biol Chem. 2025 Feb 26;301(4):108361. doi: 10.1016/j.jbc.2025.108361. Online ahead of print.

Authors

Maria J Pires¹, Seher Alam¹, Alen Lovric¹, Emanuele Fabbrizi², Dante Rotili³, Mikael Altun⁴, Nicholas C K Valerie⁵

Affiliations

¹ Division of Clinical Physiology, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Sweden.
² Department of Chemistry and Technology of Drugs, Sapienza University of Rome Roma RM, Italy.
³ Department of Science, "Roma Tre" University, Rome, Italy; INBB - Biostructures and Biosystems National Institute, Rome, Italy.
⁴ Science for Life Laboratory, Division of Clinical Physiology, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Sweden. Electronic address: mikael.altun@ki.se.
⁵ Science for Life Laboratory, Division of Clinical Physiology, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Sweden. Electronic address: nicholas.valerie@ki.se.

Abstract

Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) targeting PARP1 and PARP2 have revolutionized cancer therapy by selectively killing cancer cells with defective DNA repair. However, achieving PARP1 or PARP2-selective inhibitors is difficult due to structural homology. Selectivity profiling is typically done with purified proteins, but these lack the complexity of intracellular environments and could therefore be inaccurate. Here, we duplex PARP1 L713F-GFP and PARP2 L269A-mCherry cellular target engagement by accumulation of mutant (CeTEAM) drug biosensors to systematically characterize binding and cell cycle alterations of 27 PARPi. Our results reveal that most PARPi are equipotent for both PARPs, including the next-generation drug, senaparib. However, benzimidazole carboxamide (niraparib) derivatives demonstrated PARP1-selective tendencies, while phthalazinones (olaparib) favored PARP2. AZD5305, a reported PARP1-selective inhibitor with characteristics of both series, was the exception and appears ∼1600-fold more potent toward PARP1. In agreement with current understanding, we see that trapping-associated S/G2-phase transitions positively correlate with PARP1/2 binding potency, while some potent binders, such as veliparib, did not - likely reflecting their allosteric influence on DNA retention. We also assessed the effect of the PARP1/2 active site component, histone PARylation factor 1, on intracellular PARPi binding and see that its depletion elicits slight deviations in apparent binding potency, while contributing additively to trapping-like phenotypes. The PARP1/2 CeTEAM platform thus provides a structural roadmap for the development of selective PARPi and should facilitate the discovery of targeted therapies. Furthermore, our results highlight that multiplexing CeTEAM biosensors and layered genetic perturbations can systematically profile determinants of intracellular drug selectivity.

Keywords: CeTEAM; HPF1; PARP inhibitors; biosensor; cellular target engagement; drug discovery; drug selectivity; fluorescence; pharmacology; protein engineering.