Identification of polymerase and processivity inhibitors of vaccinia DNA synthesis using a stepwise screening approach

• Published in: Antiviral research 2008-11, Vol.80 (2), p.114-123
• Main Authors: Silverman, Janice Elaine Y., Ciustea, Mihai, Shudofsky, Abigail M. Druck, Bender, Florent, Shoemaker, Robert H., Ricciardi, Robert P.
• Format: Article
• Language: English
• Subjects: Animals; Antibiotics. Antiinfectious agents. Antiparasitic agents; Antiviral agents; Antiviral Agents - pharmacology; Antiviral inhibitors; Biological and medical sciences; Cell Line; Cercopithecus aethiops; DNA polymerase; DNA-Directed DNA Polymerase - genetics; DNA-Directed DNA Polymerase - metabolism; Drug Evaluation, Preclinical; High-throughput screening; Human viral diseases; Humans; Infectious diseases; Medical sciences; Nucleic Acid Synthesis Inhibitors - pharmacology; Pharmacology. Drug treatments; Poxvirus; Processivity factor; Rapid plate assay; Smallpox; Smallpox, monkey pox, tanapox; Tropical viral diseases; Vaccinia - drug therapy; Vaccinia - virology; Vaccinia virus; Vaccinia virus - drug effects; Vaccinia virus - genetics; Vaccinia virus - physiology; Variola; Viral diseases; Viral diseases with cutaneous or mucosal lesions and viral diseases of the eye; Viral Proteins - antagonists & inhibitors; Viral Proteins - genetics; Viral Proteins - metabolism; Virus Replication - drug effects
• ISSN: 0166-3542; 1872-9096
• DOI: 10.1016/j.antiviral.2008.05.010

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.