All PARP inhibitors are not equal: An in vitro mechanistic comparison of PF-01367338 to iniparib.

Abstract

e13576 Background: PARP is a key enzyme involved in base-excision repair. Pre-clinically it has been shown that BRCA1/2 deficient cells are hyper-sensitive to PARP inhibition. These cells can’t repair replication associated double-strand breaks that result from PARP inhibition due to defects in homologous recombination repair. Iniparib (BSI-201) is an allosteric PARP inhibitor that, in its activated form, reacts with PARP in the DNA binding domain. This interaction inhibits DNA damage induced activation of isolated PARP protein resulting in inhibition in vitro. PF-01367338 is an enzymatic active site directed inhibitor. This study compared iniparib with a potent competitive inhibitor, PF-01367338 (formerly AG014699), in a cellular environment. Methods: PARP inhibition was evaluated using purified enzyme and cell extracts in standard biochemical assays. Inhibition of poly(ADP-ribose)(PAR) formation in live cells (MDA-MB-231/436) was determined using an anti-PAR antibody ELISA assay. PARP activity in tumor cell homogenates was measured by radioactive polymer formation assay. Results: PF-01367338 inhibited PARP enzymatic activity and PAR formation in cells with a potency of 5 nM. PF-01367338 inhibited proliferation of MDA-MB-436 cells with an IC50 of 1.2 uM. In addition, PAR formation in tumor-bearing mice was fully inhibited within 30 min at 10 mg/kg. In contrast, iniparib did not inhibit enzymatic activity or PAR formation in vitro. Incubation of iniparib with cell lysates resulted in inhibition of PARP activity with an IC50 of 200 nM, demonstrating that the active nitroso compound can form in these cells. However, iniparib did not induce antiproliferative activity in treated cells. Conclusions: The lack of PARP inhibition by iniparib in two BRCA deficient cell lines suggests that while this inhibitor can inhibit PARP in vitro, it may not be able to inhibit PARP in a complex cellular environment. Additionally, iniparib inhibits other enzymes including GAPDH and caspase-3, suggesting that results seen for iniparib in the clinic may not be due solely to, or reflect PARP inhibition. In contrast, the primary mechanism-of-action of catalytic PARP inhibitors such as PF-01367338 is likely to be related to PARP inhibition.