STUDIO DI ENZIMI D’INTERESSE MEDICO ED INDUSTRIALE (2010)

Three main projects were carried out in the time span of my doctorate. Two of them consist in the identification of enzymes necessary to perform mid- or final steps in drugs preparation; the third is related to recombinant human tyrosinase expression, partial purification and characterization. In collaboration with Fabbrica Italiana Sintetici (FIS), a critical step in the synthesis of a Moxifloxacin building block has been identified. An alternative enzymatic route is proposed that allows the resolution of a dicarboxylic esters racemic mixture. To this aim an enzyme that selectively hydrolyse one of the two enantiomers was searched. More precisely we aimed to use this enzyme to produce an enantiopure acid form, easy to separate from the unreacted enantiomer by organic phase extraction and used the desired enantiomer to prepare the Moxifloxacin building block (4as,7as)-ottaidro-1H-pirrolo[3,4-b]piridina. After trying several commercially available enzymes that have as substrates racemic mixtures, using different analytical approaches (NMR and HPLC-MS techniques), we succeeded in finding Candida antarctica lipase B as a highly selective enzymes that partially hydrolysed the mixture of dimethyl 1-acetylpiperidine-2,3-dicarboxylate in aqueous buffer. Enantiomer selectivity was evaluated by chiral HPLC analysis on a preparative scale, confirming that hydrolysis produces a high enantiomeric excess of the 2R, 3S form. Furthermore, MS analysis confirmed that only one of the two methyl esters was hydrolyzed by the lipase, conferring at the enzyme regioselectivity too. The reaction product was further characterized by NMR techniques (COSY, HMQC, HMBC), which indicate that hydrolysis take place on methyl group in position 3. Kinetics analysis by NMR was used to estimate the values of Km and Vmax of the hydrolysis reaction. The enzyme was used by FIS for grams-scale Moxifloxacin precursor synthesis. This process has been submitted for patent. A second project, again in collaboration with FIS, was focus on overcoming a critical step in the organic synthesis of the hormone testosterone (TS) by a regioand stereo-specific conversion from 4-androstene-3,17-dione (AD). According to literature, no commercially available enzymes have been found that properly catalyse this transformation. Since both the substrate and the product are natural compounds, we focused the research on identification of natural enzymes that catalyze analogous reactions. The enzymatic category that catalysed this transformation is generally classified as 17b-hydroxysteroid dehydrogenases (E.C.:1.1.1.51). However these enzymes, in different organisms, differ in terms of region-selectivity and rate on catalysis. A combined bioinformatics and literature screening resulted in two candidates that were selected for recombinant expression: the 17β-hydrosteroid dehydrogenase type 5 from mouse and the YMR226c gene product of Saccaromiyces cereviseae. The coding sequence of enzymes were cloned in suitable vectors for expression, in fusion with an histidine-tag, in E.coli. Small scale preparations of the recombinant enzyme were initially set up and activity tests on the desired substrate were performed. Only the mouse enzyme showed activity towards AD and it was subjected of further studies. A small scale purifications allowed verification of the product obtained in terms of regio- and stereo-selectivity and yield, evaluation of thermal stability of the enzyme, kinetic analysis, cofactor preference (NADPH or NADH), and choice of an adequate partial organic mixture to work with, for increase substrate solubility in water. A protocol for purification of the enzyme from medium scale cultures was then designed and performed. The amount of enzyme obtained from these preparations, in combination with the commercially available D-glucose dehydrogenase required for the recycling of the cofactor NADPH, allowed the conversion of milligrams of androstendione. Also this process was submitted for patent. The third project is on the membrane associate human tyrosinase. This enzyme is implicated in different pathologies and it is a potentially targets for drugs aimed to treat melanin synthesis disorder, melanoma and ROS associated diseases. The protein was expressed in two recombinant forms using insect cell sf9 and baculovirus system (Bac to Bac ®, Invitrogen), a system capable of glycosilation. Both forms were efficiently expressed by the cells: the wild type and a form lacking the signal sequence and the transmembrane domain. The two proteins are observed initially by western blot. The effort to define a purification protocol was focused on wild type form. It appears as a membrane associated protein and a screening with several detergents indicates that only Triton 100X, ndodecyl-β-D-maltoside and SDS were able to solubilize the protein. Following solubilisation, tests were performed in order to check its activity towards natural substrate L-DOPA and L-tyrosine, by spectrophotometeric analysis. This activity absent in not infected cells or expressing human tyrosinase in soluble form. This latter control confirmed the importance of glycosilation in correct folding of the protein. The low efficiency in the purification of protein expressed and its instability forced us to try to improve the purification protocol, cell expression and activity test. However, the low expression level of tyrosinase remained the critical point of the procedure. For this reason we decided to directly purify the protein using SDS-PAGE gel techniques. An activity test was developed using 96 well plate to determine kinetic constants of natural or artificial substrates, as well as known inhibitors. The screening was run in parallel using soluble extract of human tyrosinase and the purified tyrosinase from S. Antibioticus, an enzyme with a high level of homology with the crystallized tyrosinase from S. castaneoglobusporus. This will allow to directly compare kinetics data obtain for the two tyrosinases.