Real experiences of uHTS: a prototypic 1536-well fluorescence anisotropy-based uHTS screen and application of well-level quality control procedures.

This paper describes, for the first time, a true ultra-high throughput screen (uHTS) based upon fluorescence anisotropy and performed entirely in 1536-well assay plates. The assay is based upon binding and displacement of a BODIPY-FL-labeled antibiotic to a specific binding site on 70S ribosomes from Escherichia coli (Kd approximately 15 nM). The screen was performed at uHTS rates (i.e., >100,000 assay wells/24 h) using entirely commercially available equipment. In order to examine the reproducibility of detection of test compound effects, assays were performed in duplicate. Both overall assay statistics and reproducibility for individual compound results were excellent, at least equivalent to conventional HTS assays. Interference artifacts occurred mainly as a result of autofluorescence from test compounds. Well-level quality control procedures were developed to detect, eliminate, or even correct for such effects. Moreover, development of a brighter, longer wavelength probe (based upon Cy3B) markedly reduced such interferences. Overall, the data demonstrate that fluorescence anisotropy-based uHTS is now a practical reality.

FlashPlate scintillation proximity assays for characterization and screening of DNA polymerase, primase, and helicase activities.

DNA replication proteins represent a class of extremely well-established anti-infective drug targets for which improvements in assay technology are required in order to support enzyme characterization, HTS, and structure-activity relationship studies. Replication proteins are conventionally assayed using precipitation/filtration or gel-based techniques, and are not yet all suitable for conversion into homogeneous fluorescence-based formats. We have therefore developed radiometric assays for these enzymes based upon FlashPlate technology that can be applied to a wide range of targets using a common set of reagents. This approach has allowed the rapid characterization of DNA polymerase, DNA primase, and DNA helicase activities. The resultant 96-/384-well microplate assays are suitable for primary HTS, hit selectivity determination, and/or elucidating the mechanism of action of inhibitors. In all cases, biotinylated DNA oligonucleotide substrates were tethered to streptavidin-coated scintillant-embedded FlashPlate wells. Various adaptations were employed for each enzyme activity. For DNA polymerase, a short complementary oligonucleotide primer was annealed to the longer tethered oligonucleotide, and polymerization was measured by incorporation of [(3)H]-dNTPs onto the growing primer 3' end. For DNA primase, direct synthesis of short oligoribonucleotides complementary to the tethered DNA strand was measured by incorporation of [(3)H]-rNTPs or by subsequent polymerase extension with [(3)H]-dNTPs from unlabeled primers. For DNA helicase, unwinding of a [(33)P]-labeled oligonucleotide complementary to the tethered oligonucleotide was measured. This robust and flexible system has a number of substantial advantages over conventional assay techniques for this difficult class of enzymes.

CD23 (FcepsilonRII) release from cell membranes is mediated by a membrane-bound metalloprotease.

CD23 (low-affinity IgE receptor, FcepsilonRII) is expressed as a Type II extracellular protein on a variety of cells such as B-cells, monocytes and macrophages and is cleaved from the cell surface to generate several distinct fragments. The expression of CD23 on the cell surface as well as the generation of soluble fragments of CD23 has been shown to be involved in the regulation of IgE synthesis. Here we report that the release of CD23 from the cell surface is mediated by a metalloprotease. An assay utilizing purified CD23 and an neo-epitope antibody specific for one of the known cleavage products is described and used to demonstrate unambiguously the cleavage of CD23 by a distinct protease. Characterization of the mechanism of CD23 processing shows that the protease exists as an integral membrane protein with a functional molecular mass of approx. 63 kDa as determined by gel-filtration chromatography. The CD23-cleaving activity found in enriched plasma membranes from RPMI 8866 cells is inhibited by the metalloprotease inhibitors 1, 10-phenanthroline and imidazole and by the matrix metalloprotease inhibitor batimastat, but not by inhibitors of cysteine proteases, serine proteases or acid proteases. The same or a similar activity that cleaves CD23 to the known 33 kDa fragment and is inhibited by batimastat is present in diverse cell types such as unstimulated fibroblasts and monocytic cell lines not expressing CD23, as well as in the Epstein-Barr virus-transformed B-cell line, RPMI 8866, which constitutively expresses CD23.

Characterisation of the nucleotide and DNA coeffector binding sites of the herpes simplex virus type 1 (HSV-1) encoded helicase-primase complex and UL9 origin binding protein.

Nucleotide and DNA coeffector substrate binding site characterizations were performed on two HSV-1 DNA helicases fulfilling different roles in DNA replication. Single ATP-binding sites were identified for helicase-primase and UL9 protein (Km(ATP) 0.62 mM and 0.54 mM, respectively). Analysis of structural requirements for DNA-dependent NTP hydrolysis revealed comparatively stringent requirements for helicase-primase in accommodating base-modified NTP analogs whereas the UL9 protein was much more permissive in this respect; neither enzyme was dependent on the ribose 2' or 3' hydroxyls for NTP hydrolysis. Both helicase-primase and UL9 protein ATPase activities were inhibited by ADP or GDP; this effect was competitive rather than allosteric. The enhancement of ATPase activity on a single stranded (ss) DNA substrate as opposed to double stranded (ds) DNA was much more marked for helicase-primase than for the UL9 protein (Km(dsDNA)/Km(ssDNA) 60 and 9, respectively). The triphosphates of the antiviral agents acyclovir and penciclovir were not effective substrates for either helicase-primase or UL9 protein.

Use of isotopically chiral [4'-13C]penciclovir and 13C NMR to determine the specificity and absolute configuration of penciclovir phosphate esters formed in HSV-1 and HSV-2 infected cells and by HSV-1-encoded thymidine kinase.

Penciclovir is a potent antiherpesvirus agent which is highly selective due to its phosphorylation only in virus infected cells. Phosphorylation of one of the hydroxymethyl groups of penciclovir (PCV) creates a chiral centre leading to the possible formation of (R)- and (S)-enantiomers. The absolute configuration and stereospecificity of the PCV-phosphates produced in cells infected with herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2), as well as by HSV-1-encoded thymidine kinase, were determined using isotopically chiral [4'-13C]PCV precursors and 13C NMR spectroscopy of the isolated metabolites. The absolute configuration of penciclovir-triphosphate (PCV-TP) produced in HSV-1 infected cells was shown to be S with an enantiomeric purity of greater than 95%. However, in contrast fo HSV-1-infected cells in which none of the (R) enantiomer was detected, about 10% of (R)-PCV-TP was produced in HSV-2-infected cells. Phosphorylation of PCV by HSV-1-encoded thymidine kinase was found to give 75% (S)- and 25% (R)-PCV-monophosphate. The proportion of the (S)-isomer appears to be amplified in the subsequent phosphorylations leading to the triphosphate.

Mode of antiviral action of penciclovir in MRC-5 cells infected with herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus.

The metabolism and mode of action of penciclovir [9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine; BRL 39123] were studied and compared with those of acyclovir. In uninfected MRC-5 cells, low concentrations of the triphosphates of penciclovir and acyclovir were occasionally just detectable, the limit of detection being about 1 pmol/10(6) cells. In contrast, in cells infected with either herpes simplex virus type 2 (HSV-2) or varicella-zoster virus (VZV), penciclovir was phosphorylated quickly to give high concentrations of the triphosphate ester. Following the removal of penciclovir from the culture medium, penciclovir-triphosphate remained trapped within the cells for a long time (half-lives, 20 and 7 h in HSV-2- and VZV-infected cells, respectively). In HSV-2-infected cells, acyclovir was phosphorylated to a lesser extent and the half-life of the triphosphate ester was only 1 h. We were unable to detect any phosphates of acyclovir in VZV-infected cells. (S)-Penciclovir-triphosphate inhibited HSV-1 and HSV-2 DNA polymerase competitively with dGTP, the Ki values being 8.5 and 5.8 microM, respectively, whereas for acyclovir-triphosphate, the Ki value was 0.07 microM for the two enzymes. Both compounds had relatively low levels of activity against the cellular DNA polymerase alpha, with Ki values of 175 and 3.8 microM, respectively. (S)-Penciclovir-triphosphate did inhibit DNA synthesis by HSV-2 DNA polymerase with a defined template-primer, although it was not an obligate chain terminator like acyclovir-triphosphate. These results provide a biochemical rationale for the highly selective and effective inhibition of HSV-2 and VZV DNA synthesis by penciclovir and for the greater activity of penciclovir than that of acyclovir when HSV-2-infected cells were treated for a short time.

Demonstration of RNA polymerase multiplicity in Trypanosoma brucei. Characterization and purification of alpha-amanitin-resistant and -sensitive enzymes.

We have isolated, characterized and substantially purified two distinct RNA polymerase activities from the flagellate protozoan parasite Trypanosoma brucei. RNA polymerases from this organism were resolved poorly on DEAE-Sephadex, but could be separated with CM-Sephadex. One form was totally resistant to alpha-amanitin, whereas the second was 50% inhibited by 10-20 micrograms of the drug/ml. The enzymes had different salt optima, but both were of high Mr (greater than 480,000) and demonstrated the template preference: poly[d(A-T)] greater than denatured DNA greater than native DNA, and both were more active with Mn2+ than with Mg2+. The amanitin-resistant enzyme, polymerase R, was partially purified by chromatography on CM-Sephadex, DEAE-Sephadex and heparin-Sepharose. This enzyme was very labile, and activity yields were around 9%; after purification, one or two protein bands could be discerned after electrophoresis under non-denaturing conditions, but about 20 polypeptides were resolved on denaturing gels, including a major component (not thought to be part of the enzyme) of Mr 65,000. Polymerase S, sensitive to low alpha-amanitin concentrations, was more extensively purified, with an 18% recovery, and yielded a single major band with two minor ones after native gel electrophoresis. Analysis under denaturing conditions permitted a possible subunit structure for this enzyme to be ascribed.

Unusual RNA polymerase content of Trypanosoma brucei nuclei.

Nuclei were isolated from bloodstream forms of Trypanosoma brucei by nitrogen cavitation and sedimentation through percoll density gradients. Transcription studies with these nuclei in vitro demonstrated features not seen with other eukaryotes: RNA synthesis was much greater in the presence of Mn2+ than with Mg2+ and was sensitive to high concentrations (10-100 micrograms/ml) of alpha-amanitin at all salt concentrations tested (25-300 mM ammonium sulphate). RNA polymerase extracted from nuclei by sonication at high ionic strength chromatographed as a single peak, sensitive to high alpha-amanitin concentrations, on DEAE-sephadex under conditions which resolved the classic three RNA polymerase forms when rat liver nuclear extracts were used.