Analysis of bacterial isocytosine deaminases

The two main objects of this master’s thesis are enzymes Vcz and KANOS – deaminases from the metagenomic libraries, discovered at the Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Vilnius University. It is a well-known fact that the chemotherapeutic drug 5-fluorouracil can be a product of a deaminase-catalyzed reaction. Hence, it was decided to investigate the applicability of Vcz and KANOS deaminases for targeted cancer therapy. In vitro enzyme evolution methods were used to improve the kinetic parameters of Vcz deaminase using isocytosine as its substrate. The effectiveness of the methods applied has been demonstrated, but due to the lack of a proper mutant enzyme selection system, the improved variant of Vcz deaminase could not be obtained. The ability of KANOS deaminase to catalyze the deamination of both cytosine and isocytosine was compared with that of a typical cytosine deaminase CodA from E. coli. The kinetic parameters of KANOS for isocytosine deamination were ~1.5 times higher than those of CodA, suggesting that this enzyme is suitable for further application in targeted cancer therapy. Also, a hybrid system that uses a two-enzyme (Vcz deaminase and D8 amidohydrolase) hybrid and a single prodrug (5-fluoro-N2-acetylisocytosine) is proposed. It was shown in vivo that uracil can be obtained from N2-acetylisocytosine in a two-step reaction, using both Vcz deaminase and D8 amidohydrolase in a hybrid enzyme. Using complex prodrugs that need several enzymatic steps to be converted into the active anti-cancer drug (5-fluorouracil) should reduce the severity of the side effects that accompany enzyme-prodrug cancer therapies such as cytosine deaminase / 5-fluorocytosine in their clinical application

Importance of the putative furin recognition site 742RNRR745 for antiangiogenic Sema3C activity in vitro

Angiogenesis is one of the key processes in the growth and development of tumors. Class-3 semaphorins (Sema3) are characterized as axon guidance factors involved in tumor angiogenesis by interacting with the vascular endothelial growth factor signaling pathway. Sema3 proteins convey their regulatory signals by binding to neuropilins and plexins receptors, which are located on the effector cell. These processes are regulated by furin endoproteinases that cleave RXRR motifs within the Sema, plexin-semaphorins-integrin, and C-terminal basic domains of Sema3 protein. Several studies have shown that the furin-mediated processing of the basic domain of Sema3F and Sema3A is critical for association with receptors. It is unclear, however, if this mechanism can also be applied to other Sema3 proteins, including the main subject of this study, Sema3C. To address this question, we generated a variant of the full-length human Sema3C carrying point mutation R745A at the basic domain at the hypothetical furin recognition site 742RNRR745, which would disable the processing of Sema3C at this specific location. The effects produced by this mutation were tested in an in vitro angiogenesis assay together with the wild-type Sema3C, Sema3A, and Sema3F proteins. Our results showed that the inhibitory effect of Sema3C on microcapillary formation by human umbilical vein endothelial cells could be abrogated upon mutation at the Sema3C basic domain within putative furin cleavage site 742RNRR745, indicating that this site was essential for the Sema3 biological activity

Bacterial amidohydrolases and modified 5-fluorocytidine compounds: Novel enzyme-prodrug pairs.

Gene-directed enzyme prodrug therapy is an emerging strategy for cancer treatment based on the delivery of a gene that encodes an enzyme that is able to convert a prodrug into a potent cytotoxin exclusively in target cancer cells. However, it is limited by the lack of suitable enzyme variants and a scarce choice of chemical bonds that could be activated. Therefore, this study is aimed to determine the capability of bacterial amidohydrolases YqfB and D8_RL to activate novel prodrugs and the effect such system has on the viability of eukaryotic cancer cells. We have established cancer cell lines that stably express the bacterial amidohydrolase genes and selected several N4-acylated cytidine derivatives as potential prodrugs. A significant decrease in the viability of HCT116 human colon cancer cell lines expressing either the YqfB or the D8_RL was observed after exposure to the novel prodrugs. The data we acquired suggests that bacterial YqfB and D8_RL amidohydrolases, together with the modified cytidine-based prodrugs, may serve as a promising enzyme-prodrug system for gene-directed enzyme prodrug therapy

Biocatalytic synthesis of asymmetric water-soluble indirubin derivatives /

A method for the synthesis of asymmetric carboxy-substituted indirubins is presented. It employs indole-5-carboxylic acid or indole-6-carboxylic acid and 2-indolinone derivatives as substrates for bacterial monooxygenase-driven enzymatic bioconversion in different bacterial hosts. This bioconversion system achieved the highest titer of monocarboxyindirubin production of up to 327 mg L−1 for 5-bromoindirubin-6′-carboxylic acid during the 16-h incubation period. The purified monocarboxyindirubins exhibited high solubility in water, up to three orders of magnitude higher than that of indirubin. In addition, several monocarboxyindirubins, namely 1-methylindirubin-5′-carboxylic acid, possess potent antiproliferative activity against different cancer cell lines. Therefore, the synthesis method for monocarboxyindirubins described herein is an efficient and environmentally friendly bioconversion system and the synthesized monocarboxyindirubins show great promise due to their high water solubility and potential antiproliferative activity