| Área: Genética e Biologia Molecular ( Divisão N ) BIOCHEMICAL AND GENETIC STUDIES OF HUMAN URIDINE PHOSPHORYLASE 1 (EC 2.4.2.3) AS A TARGET FOR THE DEVELOPMENT OF NEW INHIBITORS FOR CANCER CHEMOTHERAPY Daiana Renck (INCT-TB); Rodrigo Gay Ducati (INCT-TB); Diógenes Santiago Santos (INCT-TB); Luiz Augusto Basso (INCT-TB)
ResumoPyrimidines can be synthesized either through the de novo or salvage pathways. Uridine phosphorylase (UP; EC 2.4.2.3) is a pyrimidine nucleoside phosphorylase that belongs to the nucleoside phosphorylase (NP) super-family of proteins in the NP-1 subset. UP is an enzyme that is present in the pyrimidine salvage pathway and catalyzes the phosphorolysis of uridine to uracil. Although UP is present in most normal tissues and in tumors, its activity as well as its expression is elevated at the second. This enzyme regulates strictly the concentration of uridine in plasma and tissues and affects activation and catabolism of several nucleoside analogues used in chemotherapy of cancer, such as 5-fluorouracil (5-FU). Clinical studies have demonstrated that uridine can be used to reduce 5-FU toxicity, leading to an increased therapeutic index, and to selectively protect normal tissues from this host toxicity; high doses of exogenous uridine are not well tolerated in humans. In humans there are two UP isoforms, hUP1 and hUP2. Therefore, there is a significant interest in the development of compounds that inhibit the hUP1, since it is more widely distributed and the elevated levels of activity in tumor cells may contribute to selectivity. In this study, we describe the amplification and cloning of the gene, and expression and purification of recombinant and kinetic studies of hUP1.
The open reading frame of the UPP1 gene was PCR-amplified with, the PCR-product was cloned into pCR-Blunt cloning vector, subcloned into pET-23a(+) expression vector, and sequenced to ensure gene identity. The recombinant protein was expressed in Escherichia coli Rosetta (DE3) electrocompetent cells, grown in TB medium, at 30°C, up to 36h without induction. The homogeneous protein was obtained with through a single-step purification protocol, using a cation exchange column, and mass spectrometry analysis and N-terminal amino acid sequencing revealed evidence for the identity of the recombinant hUP1. Steady-state initial velocity studies were carried out at varying concentration of one substrate and several fixed-varied concentrations of the co-substrate, and the product inhibition patterns were determined at varying concentrations of one substrate, fixed concentrations of the co-substrate (near the KM) and fixed-varying concentrations of products (either ribose-1-phosphate or uracil). These experiments will determine the kinetic constants and the probable kinetic mechanism for hUP1 catalyzed reaction, which may be useful to the rational design of new inhibitors for this enzyme, and, thereby, contribute for chemotherapy of cancer. The perspectives of this work are to determine the hUP1 kinetic mechanism; binding assays will allow the determination of dissociation constants (Kd). We also intend to performe Real-Time PCR for mRNA expression determination of both human enzymes in normal and tumor colorectal samples.
Palavras-chave: PYRIMIDINE SALVAGE PATHWAY, URIDINE PHOSPHORYLASE 1, CHEMOTHERAPY OF CANCER, NEW INHIBITORS |