Identification of polymerase and processivity inhibitors of vaccinia DNA synthesis using a stepwise screening approach.
Nearly all DNA polymerases require processivity factors to ensure continuous incorporation of nucleotides. Processivity factors are specific for their cognate DNA polymerases. For this reason, the vaccinia DNA polymerase (E9) and the proteins associated with processivity (A20 and D4) are excellent therapeutic targets. In this study, we show the utility of stepwise rapid plate assays that (i) screen for compounds that block vaccinia DNA synthesis, (ii) eliminate trivial inhibitors, e.g. DNA intercalators, and (iii) distinguish whether inhibitors are specific for blocking DNA polymerase activity or processivity. The sequential plate screening of 2222 compounds from the NCI Diversity Set library yielded a DNA polymerase inhibitor (NSC 55636) and a processivity inhibitor (NSC 123526) that were capable of reducing vaccinia viral plaques with minimal cellular cytotoxicity. These compounds are predicted to block cellular infection by the smallpox virus, variola, based on the very high sequence identity between A20, D4 and E9 of vaccinia and the corresponding proteins of variola.
Silverman
Janice Elaine Y
JE
Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA.
Ciustea
Mihai
M
Shudofsky
Abigail M Druck
AM
Bender
Florent
F
Shoemaker
Robert H
RH
Ricciardi
Robert P
RP
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D002522:Chlorocebus aethiops; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D006801:Humans; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology*; D014615:Vaccinia / Q000188:drug therapy* / Q000821:virology; D014616:Vaccinia virus / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D014764:Viral Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects*
D000998:Antiviral Agents; D019384:Nucleic Acid Synthesis Inhibitors; D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase