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Drug metabolite synthesis by immobilized human FMO3 and whole cell catalysts.

ABSTRACT: BACKGROUND:Sufficient reference standards of drug metabolites are required in the drug discovery and development process. However, such drug standards are often expensive or not commercially available. Chemical synthesis of drug metabolite is often difficulty due to the highly regio- and stereo-chemically demanding. The present work aims to construct stable and efficient biocatalysts for the generation of drug metabolites in vitro. RESULT:In this work, using benzydamine as a model drug, two easy-to-perform approaches (whole cell catalysis and enzyme immobilization) were investigated for the synthesis of FMO3-generated drug metabolites. The whole cell catalysis was carried out by using cell suspensions of E. coli JM109 harboring FMO3 and E. coli BL21 harboring GDH (glucose dehydrogenase), giving 1.2 g/L benzydamine N-oxide within 9 h under the optimized conditions. While for another approach, two HisTrap HP columns respectively carrying His6-GDH and His6-FMO3 were connected in series used for the biocatalysis. In this case, 0.47 g/L benzydamine N-oxide was generated within 2.5 h under the optimized conditions. In addition, FMO3 immobilization at the C-terminal (membrane anchor region) significantly improved its enzymatic thermostability by more than 10 times. Moreover, the high efficiency of these two biocatalytic approaches was also confirmed by the N-oxidation of tamoxifen. CONCLUSIONS:The results presented in this work provides new possibilities for the drug-metabolizing enzymes-mediated biocatalysis.

SUBMITTER: Gao C

PROVIDER: S-EPMC6691536 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Drug metabolite synthesis by immobilized human FMO3 and whole cell catalysts.

Gao Chongliang C Zheng Tingjie T

Microbial cell factories 20190812 1

<h4>Background</h4>Sufficient reference standards of drug metabolites are required in the drug discovery and development process. However, such drug standards are often expensive or not commercially available. Chemical synthesis of drug metabolite is often difficulty due to the highly regio- and stereo-chemically demanding. The present work aims to construct stable and efficient biocatalysts for the generation of drug metabolites in vitro.<h4>Result</h4>In this work, using benzydamine as a model ...[more]

PMID: 31405378

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Project description:BackgroundCurrently, the numerous and versatile applications in pharmaceutical and chemical industry make the recombinant production of cytochrome P450 enzymes (CYPs) of great biotechnological interest. Accelerating the drug development process by simple, quick and scalable access of human drug metabolites is key for efficient and targeted drug development in response to new and sometimes unexpected medical challenges and needs. However, due its biochemical complexity, scalable human CYP (hCYP) production and their application in preparative biotransformations was still in its infancy.ResultsA scalable bioprocess for fine-tuned co-expression of hCYP2C9 and its essential complementary human cytochrome P450 reductase (hCPR) in the yeast Pichia pastoris (Komagataella phaffii) is presented. High-throughput screening (HTS) of a transformant library employing a set of diverse bidirectional expression systems with different regulation patterns and a fluorimetric assay was used in order to fine-tune hCYP2C9 and hCPR co-expression, and to identify best expressing clonal variants. The bioprocess development for scalable and reliable whole cell biocatalyst production in bioreactors was carried out based on rational optimization criteria. Among the different alternatives studied, a glycerol carbon-limiting strategy at high µ showed highest production rates, while methanol co-addition together with a decrease of µ provided the best results in terms of product to biomass yield and whole cell activity. By implementing the mentioned strategies, up to threefold increases in terms of production rates and/or yield could be achieved in comparison with initial tests. Finally, the performance of the whole cell catalysts was demonstrated successfully in biotransformation using ibuprofen as substrate, demonstrating the expected high selectivity of the human enzyme catalyst for 3'hydroxyibuprofen.ConclusionsFor the first time a scalable bioprocess for the production of hCYP2C9 whole cell catalysts was successfully designed and implemented in bioreactor cultures, and as well, further tested in a preparative-scale biotransformation of interest. The catalyst engineering procedure demonstrated the efficiency of the employment of a set of differently regulated bidirectional promoters to identify transformants with most effective membrane-bound hCYP/hCPR co-expression ratios and implies to become a model case for the generation of other P. pastoris based catalysts relying on co-expressed enzymes such as other P450 catalysts or enzymes relying on co-expressed enzymes for co-factor regeneration.

Project description:BackgroundRecently, the metabolite-likeness of the drug space has emerged and has opened a new possibility for exploring human metabolite-like candidates in drug discovery. However, the applicability of metabolite-likeness in drug discovery has been largely unexplored. Moreover, there are no reports on its applications for the repositioning of drugs to possible enzyme modulators, although enzyme-drug relations could be directly inferred from the similarity relationships between enzyme's metabolites and drugs.MethodsWe constructed a drug-metabolite structural similarity matrix, which contains 1,861 FDA-approved drugs and 1,110 human intermediary metabolites scored with the Tanimoto similarity. To verify the metabolite-likeness measure for drug repositioning, we analyzed 17 known antimetabolite drugs that resemble the innate metabolites of their eleven target enzymes as the gold standard positives. Highly scored drugs were selected as possible modulators of enzymes for their corresponding metabolites. Then, we assessed the performance of metabolite-likeness with a receiver operating characteristic analysis and compared it with other drug-target prediction methods. We set the similarity threshold for drug repositioning candidates of new enzyme modulators based on maximization of the Youden's index. We also carried out literature surveys for supporting the drug repositioning results based on the metabolite-likeness.ResultsIn this paper, we applied metabolite-likeness to repurpose FDA-approved drugs to disease-associated enzyme modulators that resemble human innate metabolites. All antimetabolite drugs were mapped with their known 11 target enzymes with statistically significant similarity values to the corresponding metabolites. The comparison with other drug-target prediction methods showed the higher performance of metabolite-likeness for predicting enzyme modulators. After that, the drugs scored higher than similarity score of 0.654 were selected as possible modulators of enzymes for their corresponding metabolites. In addition, we showed that drug repositioning results of 10 enzymes were concordant with the literature evidence.ConclusionsThis study introduced a method to predict the repositioning of known drugs to possible modulators of disease associated enzymes using human metabolite-likeness. We demonstrated that this approach works correctly with known antimetabolite drugs and showed that the proposed method has better performance compared to other drug target prediction methods in terms of enzyme modulators prediction. This study as a proof-of-concept showed how to apply metabolite-likeness to drug repositioning as well as potential in further expansion as we acquire more disease associated metabolite-target protein relations.

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Drug metabolite synthesis by immobilized human FMO3 and whole cell catalysts.

Publications

Drug metabolite synthesis by immobilized human FMO3 and whole cell catalysts.

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