Nitro-Reduction of Nitro-Containing Pharmaceuticals Using Bacterial Enzymes

Abstract

Many Nitroaromatic or nitro containing drug groups have a wide application in the health care and biotechnology sectors. Many of these compounds have to undergo different bio-transformations via enzymatic reactions which makes them act as a pharmacophore or toxicophore. In this research, we investigated the nitro reduction biotransformation of nisoldipine, nimodipine, and entacapone using NfsA, NfsB, Ntr1, and Ntr2. This was done by conducting whole cell-based and in vitro assays. For the in vivo assay, an E. coli BL21 Gold (DE3) strain containing the plasmid with the genes of interest was grown with 0.1 mM IPTG to induce protein expression. In the case of in vitro assays, protein purification was done to extract the proteins of interest. For both cases, HPLC and LC-MS analysis was done to confirm the bio-transformation of these drugs. The whole cell-based control assay of E. coli BL21 (DE3) gold with nimodipine showed a very low conversion activity, this made the strain a suitable platform for conducting the experiments because activity of the enzymes could more easily be identified with the evidence of higher peak intensities. The whole cell-based assay experiments showed that at least three enzymes (i.e., NfsA, NfsB, Ntr2) could reduce nimodipine and entacapone. The anaerobic in vitro assay experiments also showed that at least three enzymes (i.e., NfsA, NfsB, Ntr2) could reduce nimodipine only and not the other drugs. The difference in enzyme activity in both cases may be due to the of amino residues which the enzymes have that are essential for its activity.