A colorimetric assay for high-throughput screening of inhibitors of herpes simplex virus type 1 alkaline nuclease.
Herpes simplex virus type 1 (HSV-1) encodes a deoxyribonuclease that is frequently referred to as alkaline nuclease (AN) because of its elevated pH optimum. Studies with recombinant viruses which contain deletions in the HSV-1 gene encoding AN have indicated that this enzyme is required for efficient virus replication and therefore represents a potential target for novel antiviral therapies. A simple colorimetric assay for deoxyribonuclease activity employing a DNA-methyl green substrate was adapted for use in a high-throughput screen to identify small molecule inhibitors of this enzyme. This screen identified 1,2-benzoisothiazolin-3-one as a specific inhibitor of AN, since it exhibited activity against AN but was completely inactive against bovine pancreatic DNaseI. Subsequent studies revealed that this compound most likely inhibited AN by forming disulfide linkages with one or more exposed cysteine residues on the surface of the enzyme and that AN was sensitive to sulfhydryl-group-modifying reagents in general. These results demonstrated the utility of this DNA-methyl green substrate-based assay in both the rapid identification and the characterization of novel small molecule inhibitors of the AN encoded by HSV-1 and other herpesviruses.
Bronstein
J C
JC
Infectious Diseases Section, Pfizer Global Research and Development, Ann Arbor, Michigan 48105, USA.
Weber
P C
PC
D000818:Animals; D002417:Cattle; D003124:Colorimetry / Q000379:methods*; D004247:DNA / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D005033:Ethylmaleimide / Q000378:metabolism / Q000494:pharmacology; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D008739:Methyl Green / Q000378:metabolism; D015394:Molecular Structure; D012260:Ribonucleases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000378:metabolism / Q000494:pharmacology; D013844:Thiazoles / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology
D004220:Disulfides; D004791:Enzyme Inhibitors; D013439:Sulfhydryl Reagents; D013844:Thiazoles; D008739:Methyl Green; D004247:DNA; D012260:Ribonucleases; C066964:viral alkaline nuclease; D003850:Deoxyribonuclease I; C015699:1,2-benzisothiazoline-3-one; D005033:Ethylmaleimide