Engineering of Saccharomyces pastorianus Old Yellow Enzyme 1 for the Synthesis of Pharmacologically Active (S)-Profen Derivatives

<a>2-Arylpropionic acid </a><a>derivatives</a>, such as ibuprofen, constitute an important group of non-steroidal anti-inflammatory drugs (NSAIDs). Biocatalytic asymmetric reduction of<a> 2-arylacrylic acid</a> derivatives by ene reductases (EREDs) is a valuable approach for synthesis of these derivatives. However, previous bioreduction of <a>2-arylacrylic acid derivatives</a> by either ERED wild-types or variants resulted solely in nonpharmacological (<i>R</i>)-enantiomers as the products. <a></a><a>Here, </a>we present the engineering of <i>Saccharomyces pastorianus</i> old yellow enzyme 1 (OYE1) into (<i>S</i>)-stereoselective enzymes, which afford pharmacologically active (<i>S</i>)-profen derivatives. By structural comparison of substrate recognition in related EREDs and analysis of non-covalent contacts in the pro-<i>S</i> model of OYE1, the key residues of OYE1 that switch its stereoselectivity to an (<i>S</i>)-stereopreference were identified. Systematic site-directed mutagenesis screening at these positions successfully provided the (<i>S</i>)-stereoselective OYE1 variants, which catalyzed stereoselective bioreduction of various profen precursors to afford pharmacologically active (<i>S</i>)-derivatives including (<i>S</i>)-ibuprofen and (<i>S</i>)-naproxen methyl esters with up to >99% <i>ee</i> values. <a>Moreover, the key residues and mutation strategy obtained from OYE1 </a>could be further transferred to OYE 2.6 (from <i>Pichia stipitis</i>) and KnOYE1 (from <i>Kazachstania naganishii</i>) to create the (<i>S</i>)-stereoselective EREDs. Our results may provide a generalizable strategy for stereocontrol of OYEs and set the basis for biocatalytic production of (<i>S</i>)-profens.

Engineering of Saccharomyces pastorianus Old Yellow Enzyme 1 for the Synthesis of Pharmacologically Active (S)-Profen Derivatives

<a>2-Arylpropionic acid </a><a>derivatives</a>, such as ibuprofen, constitute an important group of non-steroidal anti-inflammatory drugs (NSAIDs). Biocatalytic asymmetric reduction of<a> 2-arylacrylic acid</a> derivatives by ene reductases (EREDs) is a valuable approach for synthesis of these derivatives. However, previous bioreduction of <a>2-arylacrylic acid derivatives</a> by either ERED wild-types or variants resulted solely in nonpharmacological (<i>R</i>)-enantiomers as the products. <a></a><a>Here, </a>we present the engineering of <i>Saccharomyces pastorianus</i> old yellow enzyme 1 (OYE1) into (<i>S</i>)-stereoselective enzymes, which afford pharmacologically active (<i>S</i>)-profen derivatives. By structural comparison of substrate recognition in related EREDs and analysis of non-covalent contacts in the pro-<i>S</i> model of OYE1, the key residues of OYE1 that switch its stereoselectivity to an (<i>S</i>)-stereopreference were identified. Systematic site-directed mutagenesis screening at these positions successfully provided the (<i>S</i>)-stereoselective OYE1 variants, which catalyzed stereoselective bioreduction of various profen precursors to afford pharmacologically active (<i>S</i>)-derivatives including (<i>S</i>)-ibuprofen and (<i>S</i>)-naproxen methyl esters with up to >99% <i>ee</i> values. <a>Moreover, the key residues and mutation strategy obtained from OYE1 </a>could be further transferred to OYE 2.6 (from <i>Pichia stipitis</i>) and KnOYE1 (from <i>Kazachstania naganishii</i>) to create the (<i>S</i>)-stereoselective EREDs. Our results may provide a generalizable strategy for stereocontrol of OYEs and set the basis for biocatalytic production of (<i>S</i>)-profens.

In Silico Evaluation of Ibuprofen and Two Benzoylpropionic Acid Derivatives with Potential Anti-Inflammatory Activity

Inflammation is a complex reaction involving cellular and molecular components and an unspecific response to a specific aggression. The use of scientific and technological innovations as a research tool combining multidisciplinary knowledge in informatics, biotechnology, chemistry and biology are essential for optimizing time and reducing costs in the drug design. Thus, the integration of these in silico techniques makes it possible to search for new anti-inflammatory drugs with better pharmacokinetic and toxicological profiles compared to commercially used drugs. This in silico study evaluated the anti-inflammatory potential of two benzoylpropionic acid derivatives (MBPA and DHBPA) using molecular docking and their thermodynamic profiles by molecular dynamics, in addition to predicting oral bioavailability, bioactivity and toxicity. In accordance to our predictions the derivatives proposed here had the potential capacity for COX-2 inhibition in the human and mice enzyme, due to containing similar interactions with the control compound (ibuprofen). Ibuprofen showed toxic predictions of hepatotoxicity (in human, mouse and rat; toxicophoric group 2-arylacetic or 3-arylpropionic acid) and irritation of the gastrointestinal tract (in human, mouse and rat; toxicophoric group alpha-substituted propionic acid or ester) confirming the literature data, as well as the efficiency of the DEREK 10.0.2 program. Moreover, the proposed compounds are predicted to have a good oral bioavailability profile and low toxicity (LD50 < 700 mg/kg) and safety when compared to the commercial compound. Therefore, future studies are necessary to confirm the anti-inflammatory potential of these compounds.

Biodegradation of micropollutant naproxen with a selected fungal strain and identification of metabolites

Pharmaceuticals are widely used for treating human and animal diseases. Naproxen [(S) 6-methoxy-α-methyl-2-naphthalene acetic acid] and its sodium salt are members of the α-arylpropionic acid group of nonsteroidal anti-inflammatory drugs. Due to excessive usage of naproxen, this drug has been determined even in drinking water. In this study, four fungal strains Phanerochaete chrysosporium, Funalia trogii, Aspergillus niger, and Yarrowia lipolytica were investigated in terms of naproxen removal abilities. According to LC/MS data, A. niger was found the most efficient strain with 98% removal rate. Two main by-products of fungal transformation, O-desmethylnaproxen and 7-hydroxynaproxen, were identified by using LC/MS, 1HNMR, and 13CNMR. Our results showed that O-demethylation and hydroxylation of naproxen is catalyzed by cytochrome P450 enzyme system.