Conversions of Tricyclic Antidepressants and Antipsychotics with Selected P450s fromSorangium cellulosumSo ce56

Abstract

Human cytochromes P450 (P450s) play a major role in the biotransformation of drugs. The generated metabolites are important for pharmaceutical, medical, and biotechnological applications and can be used for derivatization or toxicological studies. The availability of human drug metabolites is restricted and alternative ways of production are requested. For this, microbial P450s turned out to be a useful tool for the conversion of drugs and related derivatives. Here, we used 10 P450s from the myxobacterium Sorangium cellulosum So ce56, which have been cloned, expressed, and purified. The P450s were investigated concerning the conversion of the antidepressant drugs amitriptyline, clomipramine, imipramine, and promethazine; the antipsychotic drugs carbamazepine, chlorpromazine, and thioridazine, as well as their precursors, iminodibenzyl and phenothiazine.Amitriptyline, chlorpromazine, clomipramine, imipramine, and thioridazine are efficiently converted during the in vitro reaction and were chosen to upscale the production by an Escherichia coli-based whole-cell bioconversion system. Two different approaches, a whole-cell system using M9CA medium and a system using resting cells in buffer, were used for the production of sufficient amounts of metabolites for NMR analysis. Amitriptyline, clomipramine, and imipramine are converted to the corresponding 10-hydroxylated products, whereas the conversion of chlorpromazine and thioridazine leads to a sulfoxidation in position 5. It is shown for the first time that myxobacterial P450s are efficient to produce known human drug metabolites in a milligram scale, revealing their ability to synthesize pharmaceutically important compounds.

CYP267A1 and CYP267B1 from Sorangium cellulosum So ce56 are Highly Versatile Drug Metabolizers

“…The genes of CYP267A1 and CYP267B1 were cloned into a pET22b plasmid (Novagen, Darmstadt, Germany) as described elsewhere (Litzenburger et al, 2015). The pKKHC plasmids for the heterologous expression of the autologous redox partners ferredoxin 8 (Fdx8) and ferredoxin-NADP + reductase B (FdR_B) originate from previous work done in our laboratory .…”

“…To obtain sufficient amounts of products for structure elucidation via NMR analysis, the previously described whole-cell conversions were up-scaled to a total volume of 2.5 l. After 48 hours, the reaction was stopped with the same volume of the appropriate solvent (see Supplemental Table 0). Before extraction of dmd.aspetjournals.org the product of substrate 13, the culture was set to a pH of 11 using 2 M KOH and subsequently purified as described previously (Litzenburger et al, 2015). The purification of product 13a was done in the absence of trifluoroacetic acid.…”

“…For the expression of the redox partners in the whole-cell system, the pCDF_dFA plasmid from Litzenburger et al (2015) was used and changed as follows. The gene encoding ferredoxin NADP + reductase (Fpr) was excised using the restriction enzymes NdeI and KpnI and substituted with the FdR_B gene obtained from pKKHC_FdR_B using the same restriction enzymes.…”

“…These bacterial P450 enzymes are considered to be active drug metabolizers for several drugs including amitriptyline, chlorpromazine (2), and diclofenac (5) (Prior et al, 2010;Kulig et al, 2015). In addition, we have previously shown that CYP264A1 was able to convert tricyclic drug molecules (Litzenburger et al, 2015), and CYP265A1 and CYP266A1 were able to hydroxylate the antitumor drug epothilone D . As a result, our homology study with the drug metabolizing bacterial P450 enzymes suggested that the two members of the CYP267 family (in addition to CYP265A1 and CYP266A1) are potential candidates for metabolizing certain drugs.…”

“…Among others, the two members of the CYP267 family, CYP267A1 and CYP267B1, were found to be potential candidates. Although purified CYP267A1 and CYP267B1 have been previously shown to convert certain drugs Litzenburger et al, 2015) and CYP267A1 was found to catalyze the hydroxylation of fatty acids , a broader analysis of their substrate spectrum has never been tested. Therefore, in this study, we have tested the in vitro and whole-cell conversion of 22 widely used drugs ( Fig.…”