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An assay was developed for the detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) based on real-time quantitative polymerase chain reaction (PCR). A pair of primers and a TaqMan probe were designed that are specific for the recognition of a conservative region in the IHHNV genome. The IHHNV real-time PCR assay had a detection limit of 9 DNA copies, with a dynamic range of detection between 9 x 106 and 9 DNA copies. The primer pairs and probe were specific to IHHNV and did not cross-react with shrimp genomic DNA or other shrimp viruses such as White Spot Syndrome Virus (WSSV), Monodon Baculovirus (MBV), and hepatopancreatic parvovirus (HPV). This assay has a broad application for basic and clinical investigations. For clinical samples, the real-time PCR assay detected all the positive samples screened by conventional PCR, which indicated the sensitivity of the real-time assay. The IHHNV real-time PCR assay with high sensitivity, specificity, wide range of detection ability, and simplicity is particularly useful for screening large numbers of specimens and measuring viral loads to monitor the broodstock.

Human nuclear uracil DNA glycosylase (UNG2) and deoxyuridine triphosphate nucleotidohydrolase (dUTPase) are the primary enzymes that prevent the incorporation and accumulation of deoxyuridine in genomic DNA. These enzymes are desirable targets for small molecule inhibitors given their roles in a wide range of biological processes ranging from chromosomal rearrangements that lead to cancer, viral DNA replication, and the formation of toxic DNA strand breaks during anticancer drug therapy. To accelerate the discovery of such inhibitors, we have developed a high-throughput approach for directed library synthesis and screening. In this efficient technology, a uracil-aldehyde ligand is covalently tethered to one position of a trivalent alkyloxyamine linker via an oxime linkage, and then the vacant linker positions are derivatized with a library of aldehydes. The resulting triskelion oximes were directly screened for inhibitory activity and the most potent of these showed micromolar binding affinities to UNG2 and dUTPase.

Uracil DNA glycosylase (UNG) is an important DNA repair enzyme that recognizes and excises uracil bases in DNA using an extrahelical recognition mechanism. It is emerging as a desirable target for small-molecule inhibitors given its key role in a wide range of biological processes including the generation of antibody diversity, DNA replication in a number of viruses, and the formation of DNA strand breaks during anticancer drug therapy. To accelerate the discovery of inhibitors of UNG we have developed a uracil-directed ligand tethering strategy. In this efficient approach, a uracil aldehyde ligand is tethered via alkyloxyamine linker chemistry to a diverse array of aldehyde binding elements. Thus, the mechanism of extrahelical recognition of the uracil ligand is exploited to target the UNG active site, and alkyloxyamine linker tethering is used to randomly explore peripheral binding pockets. Since no compound purification is required, this approach rapidly identified the first small-molecule inhibitors of human UNG with micromolar to submicromolar binding affinities. In a surprising result, these uracil-based ligands are found not only to bind to the active site but also to bind to a second uncompetitive site. The weaker uncompetitive site suggests the existence of a transient binding site for uracil during the multistep extrahelical recognition mechanism. This very general inhibitor design strategy can be easily adapted to target other enzymes that recognize nucleobases, including other DNA repair enzymes that recognize other types of extrahelical DNA bases.

2-deoxyribonolactone (L) and 2-deoxyribose (AP) are abasic sites that are produced by ionizing radiation, reactive oxygen species and a variety of DNA damaging agents. The biological processing of the AP site has been examined in the yeast Saccharomyces cerevisiae. However, nothing is known about how L is processed in this organism. We determined the bypass and mutagenic specificity of DNA containing an abasic site (AP and L) or the AP analog tetrahydrofuran (F) using an oligonucleotide transformation assay. The tetrahydrofuran analog and L were bypassed at 10-fold higher frequencies than the AP lesions. Bypass frequencies of lesions were greatly reduced in the absence of Rev1 or Polzeta (rev3 mutant), but were only marginally reduced in the absence of Poleta (rad30 mutant). Deoxycytidine was the preferred nucleotide inserted opposite an AP site whereas dA and dC were inserted at equal frequencies opposite F and L sites. In the rev1 and rev3 strains, dA was the predominant nucleotide inserted opposite these lesions. Overall, we conclude that both Rev1 and Polzeta are required for the efficient bypass of abasic sites in yeast.

The DNA repair enzyme uracil DNA glycosylase (UDG) utilizes base flipping to recognize and remove unwanted uracil bases from the genome but does not react with its structural congener, thymine, which differs by a single methyl group. Two factors that determine whether an enzyme flips a base from the duplex are its shape and hydrogen bonding properties. To probe the role of these factors in uracil recognition by UDG, we have synthesized a DNA duplex that contains a single difluorophenyl (F) nucleotide analogue that is an excellent isostere of uracil but possesses no hydrogen bond donor or acceptor groups. By using binding affinity measurements, solution 1 9 F NMR, and solid state 3 1 P{ 1 9 F} rotational-echo double-resonance (REDOR) NMR measurements, we establish that UDG partially unstacks F from the duplex. However, due to the lack of hydrogen bonding groups that are required to support an open-to-closed conformational transition in UDG, F cannot stably dock in the UDG active site. We propose that F attains a metastable unstacked state that mimics a previously detected intermediate on the uracil-flipping pathway and suggest structural models of the metastable state that are consistent with the REDOR NMR measurements.

Uracil DNA glycosylase (UDG) removes uracil from U·A or U·G base pairs in genomic DNA by extruding the aberrant uracil from the DNA base stack. A question in enzymatic DNA repair is whether UDG and related glycosylases also use an extrahelical recognition mechanism to inspect the integrity of undamaged base pairs. Using NMR imino proton exchange measurements we find that UDG substantially increases the equilibrium constant for opening of T-A base pairs by almost two orders of magnitude relative to free B-DNA. This increase is brought about by enzymatic stabilization of an open state of the base pair without increasing the rate constant for spontaneous base pair opening. These findings indicate a passive search mechanism in which UDG uses the spontaneous opening dynamics of DNA to inspect normal base pairs in a rapid genome-wide search for uracil in DNA.

The DNA repair enzyme uracil DNA glycosylase has been crystallized with a cationic 1-aza-2'-deoxyribose-containing DNA that mimics the ultimate transition state of the reaction in which the water nucleophile attacks the anomeric center of the oxacarbenium ion-uracil anion reaction intermediate. Comparison with substrate and product structures, and the previous structure of the intermediate determined by kinetic isotope effects, reveals an exquisite example of geometric strain, least atomic motion, and electrophile migration in biological catalysis. This structure provides a rare opportunity to reconstruct the detailed structural transformations that occur along an enzymatic reaction coordinate.

Trifluoroacetic acid and benzenesulfonic acid in dichloromethane at ambient temperature have been found to be efficient co-catalysts for the epimerization of C -nucleosides with electron-withdrawing substituents. This approach provides a convenient route to the β anomer of the nucleosides with high yields in the range 54–78%. Synthesis of the 3′-phosphoramidite of 2,4-difluorophenyl C -nucleoside suitable for solid-phase DNA synthesis is described.

We have investigated the inhibition of the DNA repair enzyme uracil DNA glycosylase (UDG) by an 11-mer oligonucleotide (AIA) containing a cationic 1-aza-deoxyribose (I) residue designed to be a stable mimic of the high-energy oxacarbenium ion reaction intermediate [Werner, R. M., and Stivers, J. T. (2000) Biochemistry 39, 14054-14064]. Inhibition kinetics and direct binding studies indicate that AIA binds weakly to the free enzyme (K(D) = 2 microM) but binds 4000-fold more tightly to the enzyme-uracil anion (EU) product complex (K(D) = 500 pM). The importance of the positive charge on the 1-nitrogen in binding is established by the observation that AIA binds30 000-fold more tightly to the EU complex than the corresponding neutral tetrahydrofuran (F) abasic site product analogue (AFA). The unusual inhibition mechanism for AIA results in a time dependence that resembles slow-onset inhibition even though the apparent on-rate of the inhibitor for the EU(-) binary product complex is moderate (1 microM(-1) x s(-1)). Accordingly, the low K(D) of AIA for the EU complex is largely due its very slow off-rate (5 x 10(-4) x s(-1)). These results support previous kinetic isotope effect measurements that indicate UDG stabilizes a discrete oxacarbenium ion-uracil anion intermediate. This oxacarbenium ion mimic represents the tightest binding inhibitor of UDG yet identified.

The site-specific DNA cleavage and religation activities of the vaccinia virus type IB topoisomerase at (C/T)CCTT(+1)X(-1) sites in duplex DNA have allowed detailed investigations of the chemical and conformational steps on the reaction pathway of this enzyme (see accompanying article (Kwon, K., and Stivers, J. T. (2002) J. Biol. Chem. 277, 345-352)). To extend these studies to the DNA substrate, we have performed 19F NMR experiments using substrates in which the +1 T has been replaced with the NMR-sensitive thymidine base analogue 5-fluoro-2'-deoxyuridine (5-F-dUrd). Substitution of 5-F-dUrd has little effect on the binding affinity of topoisomerase I for DNA, results in small changes in the cleavage and religation rate constants, and produces a net 3-fold decrease in the cleavage equilibrium constant as compared with the CCCTT consensus DNA. One-dimensional 19F NMR experiments show that the +1 5-F-dUrd is in a dynamic equilibrium between a stacked and unstacked state in both the noncovalent complex and the covalent phosphotyrosine complex. These NMR observations are supported by the selective sensitivity of the +1 T and +1 5-F-dUrd to KMnO4 oxidation. A role for localized DNA distortion in the topoisomerase I mechanism is suggested.

The DNA repair enzyme uracil DNA glycosylase (UDG) is a powerful N-glycohydrolase that cleaves the glycosidic bond of deoxyuridine in DNA. We have investigated the role of substrate binding energy in catalysis by systematically dismantling the optimal substrate Ap(+1)UpA(-1)pA(-2) by replacing the nucleotides at the +1, -1, or -2 position with a tetrahydrofuran abasic site nucleotide (D), a 3-hydroxypropyl phosphodiester spacer (S), a phosphate monoester (p), or a hydroxyl group (h). Contrary to previous reports, the minimal substrate for UDG is 2'-deoxyuridine (hUh). UDG has a significant catalytic efficiency (CE) for hUh of 4 x 10(7) M(-1) [CE = (k(cat)/K(m))(1/k(non)), where k(non) is the rate of the spontaneous hydrolysis reaction of hUh at 25 degrees C]. Addition of +1 and -1 phosphate monoanions to form pUp increases k(cat)/K(m) by 45-fold compared to that of hUh. The k(cat)/K(m) for pUp, but not pU or Up, is found to decrease by 20-fold over the pH range of 6-9 with a pK(a) of 7.1, which is identical to the pK(a) values for deprotonation of the +1 and -1 phosphate groups determined by the pH dependence of the (31)P NMR chemical shifts. This pH dependence indicates that binding of the pUp tetraanion is disfavored, possibly due to unfavorable desolvation or electrostatic properties of the highly charged +1 and -1 phosphate groups. Addition of flexible hydroxypropyl groups to the +1 and -1 positions to make SpUpS increases k(cat)/K(m) by more than 10(5)-fold compared to that of hUh, which is a 20-fold greater effect than observed with rigid D substituents in these positions (i.e., DpUpD). The -2 phosphoester or nucleotide is found to increase the reactivity of trimer substrates with rigid furanose rings or nucleotides in the +1 and -1 positions by 1300-270000-fold (i.e., DpUpD --DpUpDpA or ApUpA --ApUpApA). In contrast, the -2 nucleotide provides only an 8-fold rate enhancement when appended to the substrate containing the more flexible +1 and -1 S substituents (SpUpS --SpUpSpA). These context-dependent effects of a -2 nucleotide are interpreted in terms of a mechanism in which the binding energy of this "handle" is used drive the rigid +1 and -1 A or D substituents into their binding pockets, resulting in a net catalytic benefit of -4.3 to -7.5 kcal/mol. Taken together, these results systematically track how UDG uses distant site binding interactions to produce an overall four billion-fold increase in CE compared to that of the minimal substrate hUh.

The DNA repair enzyme uracil DNA glycosylase (UDG) pinches the phosphodiester backbone of damaged DNA using the hydroxyl side chains of a conserved trio of serine residues, resulting in flipping of the deoxyuridine from the DNA helix into the enzyme active site. We have investigated the energetic role of these serine-phosphodiester interactions using the complementary approaches of crystallography, directed mutagenesis, and stereospecific phosphorothioate substitutions. A new crystal structure of UDG bound to 5'-HO-dUAAp-3' (which lacks the 5' phosphodiester group that interacts with the Ser88 pinching finger) shows that the glycosidic bond of dU has been cleaved, and that the enzyme has undergone the same specific clamping motion that brings key active site groups into position as previously observed in the structures of human UDG bound to large duplex DNA substrates. From this structure, it may be concluded that glycosidic bond cleavage and the induced fit conformational change in UDG can occur without the 5' pinching interaction. The S88A, S189A, and S192G "pinching" mutations exhibit 360-, 80-, and 21-fold damaging effects on k(cat)/K(m), respectively, while the S88A/S189A double mutant exhibits an 8200-fold damaging effect. A free energy analysis of the combined effects of nonbridging phosphorothioate substitution and mutation at these positions reveals the presence of a modest amount of strain energy between the compressed 5' and 3' phosphodiester groups flanking the bound uridine. Overall, these results indicate a role for these serine-phosphodiester interactions in uracil flipping and preorganization of the sugar ring into a reactive conformation. However, in contrast to a recent proposal [Parikh, S. S., et al. (2000) Proc Natl. Acad. Sci. 94, 5083], there is no evidence that conformational strain of the glycosidic bond induced by serine pinching plays a major role in the 10(12)-fold rate enhancement brought about by UDG.

The title compound, C 7 H 10 N 2 O 2 , displays extensive hydrogen-bonding interactions which give rise to connected 10- and 11-membered rings throughout the lattice.

Background Esterases are often overexpressed in cancer cells and can have chiral specificities different from that of the corresponding normal tissues. For this reason, ester prodrugs could be a promising approach in chemotherapy. In this study, we focused on the identification and characterization of differentially expressed esterases between non-tumorigenic and tumorigenic prostate epithelial cells. Methods Cellular lysates from LNCaP, DU 145, and PC3 prostate cancer cell lines, tumorigenic RWPE-2 prostate epithelial cells, and non-tumorigenic RWPE-1 prostate epithelial cells were separated by native polyacrylamide gel electrophoresis (n-PAGE) and the esterase activity bands visualized using α-naphthyl acetate or α-naphthyl-N-acetylalaninate (ANAA) chiral esters and Fast Blue RR salt. The esterases were identified using nanospray LC/MS-MS tandem mass spectrometry and confirmed by Western blotting, native electroblotting, inhibition assays, and activity towards a known specific substrate. The serine protease/esterase oxidized protein hydrolase (OPH) was overexpressed in COS-7 cells to verify our results. Results The major esterase observed with the ANAA substrates within the n-PAGE activity bands was identified as OPH. OPH (EC 3.4.19.1) is a serine protease/esterase and a member of the prolyl oligopeptidase family. We found that LNCaP lysates contained approximately 40% more OPH compared to RWPE-1 lysates. RWPE-2, DU145 and PC3 cell lysates had similar levels of OPH activity. OPH within all of the cell lysates tested had a chiral preference for the S-isomer of ANAA. LNCaP cells were stained more intensely with ANAA substrates than RWPE-1 cells and COS-7 cells overexpressing OPH were found to have a higher activity towards the ANAA and AcApNA than parent COS-7 cells. Conclusions These data suggest that prodrug derivatives of ANAA and AcApNA could have potential as chemotherapeutic agents for the treatment of prostate cancer tumors that overexpress OPH.