Modification of the Rapid Mechanistic Plate Assay for HTS

1. The original rapid mechanistic plate assay for KSHV Pol-8/PF-8 DNA synthesis assay (9) described above (Subheadings 3.1.-3.9.) can be modified for HTS. The assay is modified so that known herpesvirus polymerase inhibitors cause at least a 50% reduction in DNA synthesis at micromolar concentrations. Lowering the compound test dose to the micromolar range is one of our critical goals for optimization, to avoid selecting promiscuous inhibitors of KSHV Pol-8/PF-8, as it has previously been cautioned that higher doses of test compounds commonly result in many nonspecific screening hits from molecular target-based in vitro HTS assays (13).

2. Cidofovir diphosphate, a phosphorylated form of cidofovir, has been selected as a reference inhibitory compound, since cidofovir has been shown to suppress KSHV DNA production in cell-based assays (14-17) (cidofovir was a kind gift from Dr. Michael Hitchcock of Gilead Sciences, Foster City, CA; its diphosphate form was synthesized by TriLink BioTechnologies, San Diego, CA).

3. The KSHV Pol-8/PF-8 HTS assay thus employs a reaction buffer containing 50 mM (NH4)2SO4, 20 mM Tris-HCl, pH 7.5, 3 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, 2% glycerol, 40 |g/mL BSA, in addition to 0.625 |M dNTPs, 0.125 |M DIG-dUTP, and KSHV Pol-8 and PF-8. Z-factors of the modified KSHV Pol-8/Pf-8 assay generally ranged from 0.5 to 0.8.

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