Site directed mutagenesis as a tool to understand the catalytic mechanism of human cytidine deaminase.

Cytidine deaminase (CDA), is one of the enzymes involved in the pyrimidine salvage pathways, which catalyzes the formation of uridine and deoxyuridine by the hydrolytic deamination of cytidine and deoxycytidine, respectively. Human CDA is a tetrameric enzyme of identical 15 kDa subunits, each containing an essential zinc atom in the active site. The substrate binds to each active site independently and the cooperativity between subunits has not been reported. CDA is able to recognize as substrates some antitumor and antiviral cytidine analogs rendering them pharmacologically inactive. In light of the role played by this enzyme, a deep knowledge of CDA active site and mechanism of catalysis is required. Site-directed mutagenesis, associated with molecular modeling studies, may be an important tool to discover the active site structure of an enzyme and consequently its mechanism of action. In this review are summarized the site-directed mutagenesis experiments performed on human CDA: through these studies it was possible to understand the role exerted by specific amino acid residues in CDA active site and in the contacts between subunits. The obtained results may open a way for designing new cytidine based drugs or more potent CDA inhibitors

A multi-element psychosocial intervention for early psychosis (GET UP PIANO TRIAL) conducted in a catchment area of 10 million inhabitants: study protocol for a pragmatic cluster randomized controlled trial

Multi-element interventions for first-episode psychosis (FEP) are promising, but have mostly been conducted in non-epidemiologically representative samples, thereby raising the risk of underestimating the complexities involved in treating FEP in 'real-world' services

Human Cytidine Deaminase (CDA): uncovering the physiological role through its functional genetic polymorphism K27Q

In light of the potential crucial role of CDA polymorphisms in the development of individual therapies designed to maximize response and minimize non-therapeutic side effects, we investigated first of all the CDA polymorphic distributions in healthy individuals from population of central Italy. At this purpose, we developed three polymerase chain reaction (PCR)-based methods for CDA 79A>C (K27Q) and CDA 208G>A (A70T) genotyping. Until now, these SNPs in the CDA gene have been genotyped by allelic discrimination using a TaqMan assay, or by direct sequencing or by High-resolution melting (HRM) analysis of polymerase chain reaction amplicon [...]. A RFLP (restriction fragment length polymorphism) method using RsrII as restriction enzyme was described only for A70T [...].\ud Our PCR–based assay [Allele Specific (AS-PCR) and Restriction Fragment Length Polymorphism (RFLP-PCR)] does not require any special equipment, and it provides rapid, reproducible, and cost effective detection of common CDA mutations. It can be carried out efficiently in a standard molecular genetics laboratory and suitable as a preliminary screen for all patients. Furthermore, we performed a new discontinuous direct HPLC assay to assess the cytidine deaminase activity from whole blood of individuals enrolled in this study, correlating it with CDA K27Q genotypes.\ud Since genetic factors are thought to be responsible for up to 50-60% of the rheumatoid arthritis liability [...] in collaboration with Division of Rheumatology, Catholic University of the Sacred Heart (Rome, Italy), we have tested the genetic association between functional polymorphism of CDA (K27Q, rs2072671) and RA by case-control study.\ud Finally, using mutated recombinant proteins, we performed a kinetic and molecular study on the two functional variants Q27 and K27, assessing the biochemical properties of CDA Q* and K* alleles

PCR-based methods for CDA K27Q and A70T genotyping: genotypes and alleles distribution in a Central Italy population

Cytidine deaminase (CDA) is a pyrimidine salvage pathway enzyme that catalyzes the hydrolytic deamination of cytidine and deoxycytidine to their corresponding uracil nucleosides. CDA also catalyzes the inactivation of some chemotherapeutic nucleoside analogues such as cytosine arabinoside and gemcitabine. CDA 79A>C (K27Q, rs2072671) and 208G>A (A70T, rs60369023) were found to be associated either with clinical outcomes as well as with pharmacokinetics and toxicity of drugs administered to different subsets of patients. In this paper we reported two PCR-based methods for CDA 79A>C (K27Q) and 208G>A (A70T) genotyping and tested their feasibility using DNA extracted from whole blood as well as from buccal swabs. The aim of this study was also to assess the distribution of genotypic variants in a central Italy population. The allele frequencies were 56.3% (K*) and 43.7% (Q*) for K27Q and 100% (A*) and 0% (T*) for A70T. The genotype frequencies were 32.8% (K*/K*), 46.9% (K*/Q*) and 20.3% (Q*/Q*) for K27Q. The genotype frequencies did not deviate from Hardy–Weinberg equilibrium. The results were compared with those of other reported populations. They showed marked ethnic group differences

Rapid allele-specific PCR method for CDA 79A>C (K27Q) genotyping: a useful pharmacogenetic tool and world-wide polymorphism distribution.

Background: The CDA 79ANC (K27Q, rs2072671) functional SNP has recently shown a crucial role in the pharmacogenetics of cytidine-based anticancer drugs widely administered to different subsets of patients. Current gold standard in screening for the CDA rs2072671 is the sequence-based genotyping method. Here we developed a novel, rapid Allele-Specific PCR method for CDA rs2072671 genotyping. Methods: DNA was extracted from 324 healthy individuals from two different populations (Italian and Han Chinese). CDA rs2072671 genotyping was performed by Allele-Specific PCR. Sequencing was performed to validate the test results. Results obtained from population screening were compared to that already available in HapMap and in the literature. Results: Samples analyzed were successfully genotyped and the resultswere confirmed by sequencing.Genotype distribution does not differ significantly from that previously reported for each relative ethnic group. Also, the world-wide distribution of the CDA rs2072671 SNP is reported. A striking difference is present among the main ethnicities (p=1.715×10−77), with CDA*27Q allele showing the lowest frequency in African group (9.7%) and the highest in Caucasians (35.9%). Conclusion: This Allele-Specific PCR method is a useful tool in pharmacogenetics research and a valid and reliable alternative for CDA rs2072671 screening where sequencing or Real-Time PCR is not available

Delineation of the molecular mechanisms of nucleoside recognition by cytidine deaminase through virtual screening.

Cytidine deaminase (EC 3.5.4.5, CDA), an enzyme of the pyrimidine salvage pathways, is responsible for the degradation and inactivation of several cytidine-based antitumor drugs such as cytarabine, gemcitabine, decitabine, and azacytidine. Thus, CDA inhibitors are highly sought after as compounds to be coadministered with said drugs to improve their effectiveness. Alternatively, the design of antitumor drugs not susceptible to the action of CDA is also regarded as an attractive solution. Herein we describe a virtual screen for CDA ligands based on chemical similarity and molecular docking. The campaign led to the identification of three novel inhibitors and one novel substrate, with a 19% hit rate, and allowed a significant extension of the structure–activity relationships, also in light of the compounds that resulted inactive. The most active compound identified through the screen is the inhibitor pseudoisocytidine, which has the potential to serve as a lead for highly stable compounds. The study also delineated the detrimental effect of 5-aza and 6-aza substitutions, the incompatibility of the presence of an amino group at the 3’-position, as well as the presence of very strict steric requirements around the 2’- arabino position and, even more, the N4-position. Importantly, these features can be exploited for the design of novel antineoplastic agents resistant to the action of CDA

Role of NAD+ metabolism in protection from protein misfolding induced aging in a yeast model.

Introduction Cells have developed complex interconnecting biosynthetic pathways to regulate intracellular NAD+ levels. Aberrant NAD+ metabolism has been linked to cancer, metabolic disorders and neurodegenerative diseases. The role played by NAD+ metabolism in protection from protein misfolding induced aging has been studied in wild type yeast cells (YWT) in comparison with in yeast cells expressing polyQ huntingtin (YPQ), designed to recapitulate the crucial events that are manifested during the course of human aging, including protein misfolding and aggregation. Methods In the yeast model the protein responsible of cellular toxicity consists of an amino-terminal FLAG-tagged construct containing the first 17 amino acids of Htt followed by the polyQ tract and green fluorescent protein (GFP). The lifespan of yeast cultures were followed both by monitoring the absorbance of liquid cultures at 600 nm, and by counting the number of colonies grown in agar plates. Quantification of NAD related metabolites has been performed by RP-HPLC in yeast cells extracts, after extraction with boiling buffered ethanol. Nicotinamide mononucleotide adenylyltransferase (NMNAT) enzymatic activity has been determined through a spectrophotometric assay. Results The growth curves of both yeast cells expressing polyQ huntingtin (YPQ) and wild type yeast cells (YWT) have shown that the mutant protein is not lethal in the proliferative life span, while can affect negatively the stationary phase. The evaluation of the NAD metabolites in yeast cells has shown a significant decrease of nicotinamide and NAD in YPQ cells in comparison with the control yeast cells, while NMNAT specific activity in YPQ cells was higher than in YWT cells. Conclusions The presence of polyQ huntingtin can affect the chronological life span of S. cerevisiae yeast cells. The observed response of NMNAT enzymatic activity in YPQ cells could be related to a possible role of the enzyme as molecular chaperone

Human cytidine deaminase: A biochemical characterization of its naturally occurring variants.

Human cytidine deaminase is an enzyme of the pyrimidine salvage pathways that metabolizes severalcytosine nucleoside analogs used as prodrugs in chemotherapy. We carried out a characterization ofthe cytidine deaminase 79A>C and 208G>A Single Nucleotide Polymorphisms, in order to highlight theirfunctional role and provide data that could help fine-tune the chemotherapic use of cytosine nucleosidesin patients carrying the above mentioned SNPs. The 79A>C SNP results in a K27Q change in a proteinregion not involved in the catalytic event. The 208G>A SNP produces an alanine to threonine substitution(A70T) within the conserved catalytic domain. Q27 variant is endowed with a greater catalytic efficiencytoward the natural substrates and the antileukemic agent cytarabine (Ara-C), when compared to K27variant. Molecular modeling, protein stability experiments and site-directed mutagenesis suggest thatK27 variant may have an increased stability with respect to Q27 due to an ionic interaction between alysine residue at position 27 and a glutamate residue at position 24. The T70 variant has a lower catalyticefficiency toward the analyzed substrates when compared to the A70 variant, suggesting that patientscarrying the 208G>A SNP may have a greater exposure to cytosine based pro drugs, with possible toxicityconsequences