Design and Activity of Novel Oxadiazole Based Compounds That Target Poly(ADP-ribose) Polymerase

Abstract

Novel PARP inhibitors with selective mode-of-action have been approved for clinical use. Herein, oxadiazole based ligands that are predicted to target PARP-1 have been synthesized and screened for the loss of cell viability in mammary carcinoma cells, wherein seven compounds were observed to possess significant IC₅₀ values in the range of 1.4 to 25 µM. Furthermore, compound 5u, inhibited the viability of MCF-7 cells with an IC₅₀ value of 1.4µM, when compared to Olaparib (IC₅₀ = 3.2 µM). Compound 5s also decreased cell viability in MCF-7 and MDA-MB-231 cells with IC₅₀ values of 15.3 and 19.2 µM, respectively. Treatment of MCF-7 cells with compounds 5u and 5s produced PARP cleavage, H2AX phosphorylation and CASPASE-3 activation comparable to that observed with Olaparib. Compounds 5u and 5s also decreased foci-formation and 3D Matrigel growth of MCF-7 cells equivalent to or greater than that observed with Olaparib. Finally, in silico analysis demonstrated binding of compound 5s towardsthe catalytic site of PARP-1, indicating that these novel oxadiazoles synthesized herein may serve as exemplars for the development of new therapeutics in cancer.

Keywords: human breast cancer; nicotinamide; oxadiazole; poly(ADP-ribose) polymerase.

Figure 1

Design of a new generation of oxadiazoles, presumed to bind to the adenosine binding pocket of PARP1. The blue and red colored structures were used as effective PARP1 binding motifs.

Scheme 1

Synthesis of novel substituted oxadiazoles from 5a–x. Ar/Het–Ar groups and structures of 4a–c are mentioned in Table 1.

Figure 2

Lead compounds 5u, 5s, and Olaparib produce loss of viability of ER+ breast cancer (MCF-7) and triple-negative breast cancer (MDA-MB-231) cells. MCF-7 and MDA-MB-231 were treated with 5u, 5s and Olaparib (0, 0.01, 0.1, 1, 10, 100 µM) for 72 h, respectively. The viability of MCF-7 (A) and MDA-MB-231 cells (B) were analyzed using a microplate reader to measure the fluorescence of Alamar Blue reagent (excitation wavelength is between 530–560 nm and emission wavelength is 590 nm). The results are means of triplicate determinations.

Figure 3

Inhibition of PARP1 catalytic activity by oxadiazoles and 3-aminobenzamide. Graphical representation of the colorimetric readout of the inhibition of catalytic activity of PARP−1 by oxadiazoles and 3-aminobenzamide.

Figure 4

The effect of compounds 5u, 5s, and Olaparib on PARP1 cleavage (upper panel of A,B) phosphorylation of H2A-X (Ser139) (lower panel of A,B), and CASPASE-3 activity (C,D) in MCF-7 and MDA-MB-231 cells. Cancer cells were treated with or without compounds 5u, 5s,and Olaparib (1 or 10 µM). The protein levels after treatment were observed by western blot analysis. GAPDH was used as input control. The data are expressed as mean ± SEM. ** p ≤ 0.01; *** p ≤ 0.001.

Figure 5

Compounds 5u, 5s, and Olaparib inhibited the foci formation of MCF-7 (left panel of A) and MDA-MB-231 (left panel of B) cells. Visualization of foci formed was achieved by use of Crystal Violet. Inhibition of foci formation was also quantified (right panels of both A,B) The data are expressed as mean ± SEM. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Figure 6

Compounds 5u, 5s, and Olaparib inhibited the growth of MCF-7 (A) and MDA-MB-231 (B) breast cancer cells in 3-D Matrigel. Cells were grown in a medium containing 4% Matrigel. Cells were treated with vehicles or compounds 5s, 5u, or Olaprib (1 or 10 µM), after colonies had formed. Compounds were added into the culture medium every 3 days for a total of 2 cycles. Alamar Blue assay was used for quantification of live cells. The data are expressed as mean ± SEM. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Figure 7

Structure of compound 33 and in silico molecular interaction studies of co-crystal ligand ((A), compound 33) and compound 5s (B) with the PARP catalytic domain. The surface view of merged ligands is also presented for clarity (C). The protein bound ligands were presented in stick (compound 5s) and ball and stick (compound 33) mode for better visualization.