Failure of iniparib to inhibit poly(ADP-Ribose) polymerase in vitro

Abstract

Purpose: Poly(ADP-ribose) polymerase (PARP) inhibitors are undergoing extensive clinical testing for their single-agent activity in homologous recombination (HR)-deficient tumors and ability to enhance the action of certain DNA-damaging agents. Compared with other PARP inhibitors in development, iniparib (4-iodo-3-nitrobenzamide) is notable for its simple structure and the reported ability of its intracellular metabolite 4-iodo-3-nitrosobenzamide to covalently inhibit PARP1 under cell-free conditions. The present preclinical studies were conducted to compare the actions iniparib with the more extensively characterized PARP inhibitors olaparib and veliparib.

Experimental design: The abilities of iniparib, olaparib, and veliparib to (i) selectively induce apoptosis or inhibit colony formation in HR-deficient cell lines, (ii) selectively sensitize HR-proficient cells to topoisomerase I poisons, and (iii) inhibit formation of poly(ADP-ribose) polymer (pADPr) in intact cells were compared.

Results: Consistent with earlier reports, olaparib and veliparib selectively induced apoptosis and inhibited colony formation in cells lacking BRCA2 or ATM. Moreover, like earlier generation PARP inhibitors, olaparib and veliparib sensitized cells to the topoisomerase I poisons camptothecin and topotecan. Finally, olaparib and veliparib inhibited formation of pADPr in intact cells. In contrast, iniparib exhibited little or no ability to selectively kill HR-deficient cells, sensitize cells to topoisomerase I poisons, or inhibit pADPr formation in situ. In further experiments, iniparib also failed to sensitize cells to cisplatin, gemcitabine, or paclitaxel.

Conclusions: While iniparib kills normal and neoplastic cells at high (>40 μmol/L) concentrations, its effects are unlikely to reflect PARP inhibition and should not be used to guide decisions about other PARP inhibitors.

Figure 1. Structures of nicotinamide and the three drugs used in this study

Figure 2. Effects of veliparib, olaparib and iniparib on PEO1 and PEO4 cells

A, PEO1 and PEO4 treated with diluent, 20 µM veliparib (left), 5 µM olaparib (middle) or 80 µM iniparib (right) were stained with propidium iodide (PI) and subjected to flow microfluorimetry. B, summary of results obtained in panel A and additional concentrations. Inset in B, whole cell lysates were subjected to immunoblotting with antibody to BRCA2 and, as a loading control, PARP1. C, effects of various concentrations of veliparib (left), olaparib (middle) and iniparib (right) on colony formation. Error bars in these and subsequent colony forming assays, ± SD of triplicate aliquots. All results are representative of ≥ 3 independent experiments.

Figure 3. Effects of veliparib, olaparib and iniparib on paired GM16666 (ATM-deficient) and GM16667 (ATM restored) human fibroblasts

A, cells treated with diluent, 20 µM veliparib (left), 5 µM olaparib (middle) or 80 µM iniparib (right) were stained with PI and examined by flow microfluorimetry as in Fig. 2A. B, effects of veliparib (left), olaparib (middle) and iniparib (right) on colony formation. Inset in B, whole cell lysates were subjected to immunoblotting for ATM and, as a loading control, heat shock protein 90β (HSP90β). C, Atm^−/− and wildtype MEFs examined by colony forming assays.

Figure 4. Inability of iniparib to sensitize cells to topoisomerase I poisons

MEFs (A, B) or SKOV3 cells (C) were treated continuously with the indicated concentrations of camptothecin (A, B) or topotecan (C) in the presence of 100 nM veliparib, 100 nM olaparib or 10 µM iniparib, then examined for colony formation.

Figure 5. Effects of iniparib in combination with cisplatin, gemcitabine, paclitaxel or etoposide

SKOV3 cells were treated continuously with the indicated concentrations of cisplatin (A), gemcitabine (C), paclitaxel (E) or etoposide (F) in the presence of diluent or 10 µM iniparib and examined for colony formation. In panels B and D, the ATR inhibitor VE-821 (1 µM), which is known to sensitize other cells to cisplatin (34), or the Chk1 inhibitor AZD 7762 (100 nM), which is known to sensitize to gemcitabine (35), was substituted for imiparib as a positive control. Error bars in each panel, ± SD of triplicate samples.

Figure 6. Effects of veliparib, olaparib and iniparib on pADPr synthesis

A, following a 4-h treatment with the indicated agent, SKOV3 cells were treated with MMS in the continued presence of the small molecule inhibitor, fixed, stained for pADPr and examined by confocal microscopy. B, mean fluorescence of pADPr in nuclei after treatment of cells with diluent (open bar) or 1 mM MMS (closed bars) in the presence of diluent or the indicated agent at 1, 10 and 100 µM. Error bars, ± SEM for 30 nuclei.