Synthesis and Evaluation of Ornithine Decarboxylase Inhibitors with Oxime Moiety and MCF-7 Breast Cancer Cells

Abstract

BCPC, an open access journal addition, the biological evaluation of AHPA with MCF-7 (Michigan Cancer Foundation-7) breast cancer cells was shown more potent when compared with DFMO in vitro. Our initial studies investigated the impact of AHPA on proliferation of human breast cancer cells, and the results indicate that inhibition of ODC greatly impairs the ability of these cells to replicate. Therefore, AHPA may have a considerable potential as a cancer chemopreventive and therapeutic agent. Results and Discussion Chemistry We designed and synthesized a 2-amino-5-(hydroxyimino) pentanoic acid 6 (AHPA) inhibitor based on the modified substrate of ornithine decarboxylase with oxime functionality. The Compound 6 (APHA) was synthesized using the procedure described in scheme 2. 2-Amino-5-(hydroxyimino)pentanoic acid (AHPA), 6, was synthesized using L-glutamic acid derivative (S)-5-(tert-butoxy)-4-[(tert-butoxycarbonyl)amino]-5-oxopentanoic acid 1, as the starting material. Esterification of the compound 1 using methyl chloroformate by treatment with triethylamine and a catalytic amount of 4-dimethylaminopyridine (DMAP) in dry methylene chloride provided the ester derivative 2. Synthesis of di-tert-butyl dicarbonate (di-BOC) 3 was carried out by treatment with di-tert-butyl dicarbonate in the presence of DMAP in methylene chloride. The subsequent reduction of ester derivative 3 was performed with diisobutylaluminium hydride (DIBAL-H) in ether to provide the aldehyde 4 [26]. Oxime derivative 5 was prepared for hydroxylamine hydrochloride in methanol under reflux condition. Complete deprotection of the compound, 5 with trifluoroacetic acid (TFA) in methlyene chloride yielded AHPA 6. We have investigated the stabilities of geometric isomers in molecular modeling-docking studies to computationally evaluate the fit between the human and Leishmania donovani ODC (PDB code 2ON3) and the E-and Z-isomers of AHPA [27,28]. Calculated binding data are recorded in table 1. When E-AHPA is bound in an extended conformation, the oxime moiety can make hydrogen bonds with conserved enzyme residues, Lys 69, Arg154, and Glu274. When Z-AHPA is bound, the oxime moiety can make a hydrogen bond with only one conserved residue, Asp364. Thus, the greater hydrogen bonding potential of the E-isomer of the oxime moiety suggests a stability preference for this isomer. Force-field based methods can predict the binding free energy of a protein-ligand complex by adding up individual contributions from different types of interactions. Programs for energetic analysis of receptor-ligand interaction based on force-field scoring functions and terms including van der Waals, electrostatics and hydrogen bonds can be available. Interestingly, Autodock molecular modeling of the structure of human and Leishmania donovani ornithine decarboxylases14 (PDB code 2ON3) with geometric isomers (E/Z) of oxime ligands has shown that a high degree of affinity with E isomer rather than Z isomer (Table 1, Figure 1 and Figure 2). To evaluate the impact of ODC inhibitors on cell proliferation, MCF-7 cancer cells were used. The cells were cultured in the presence or absence of varying doses of AHPA. As shown in figure 3, AHPA treatment significantly reduced cell proliferation. A seven point dilution series was performed, and even at the lowest concentration (0.391 mg/ml), AHPA was found to affect cell viability. At 24 hours post-treatment, the cells were viable and the level of ATP was roughly equivalent for cells under each experimental condition. By 48 hours post-treatment, there was no proliferation detected and viability of all cells that had been exposed to AHPA was decreasing. In contrast, the control cells exhibited healthy and robust proliferation, as evidenced by the increasing amounts of ATP detected. By 96 hours, cells that had been exposed to AHPA at any dose were dead. It thus appears that AHPA was a much more potent inhibitor of cell proliferation than comparable doses of DFMO (difluoromethylornithine), a well-known inhibitor of ornithine decarboxylase. Although there is a possibility that the reduction of cell proliferation by AHPA is due to the compound toxicity, the significant effect of AHPA on cell proliferation and the in silico analysis support the conclusion that the AHPA inhibit the cell proliferation by binding to ODC. `MCF-7 breast cancer cells were cultured in the presence of absence of AHPA at the indicated time points. Cell viability and proliferation was measured via the Cell Titer Glo Assay, and results are expressed as relative light units. As shown in figure 4, MCF-7 cell proliferation was not significantly affected by doses of DFMO lower than 3 mg/ml. At doses higher than 3 mg/ml DFMO, modest reduction of cell proliferation was observed. Proliferation of cells treated with AHPA was dramatically reduced by even the lowest doses of the compound. These results demonstrate that AHPA is a potent inhibitor of cell proliferation. In conclusion, while exploring the design of new therapeutic inhibitors in arginine biosynthetic pathways, AHPA appears to be a potential cancer chemotherapeutic agent based on the observations of in silico docking and cell proliferation experiments. The docking studies have shown that geometric isomers of the ornithine-based Arginase arginine spermidine synthase spermine synthase putrescine spermine spermidine ornithine O OH ODC