A solid-phase assay for the binding of peptidic subunit association inhibitors to the herpes simplex virus ribonucleotide reductase large subunit.
The herpes simplex virus (HSV) ribonucleotide reductase is comprised of two nonidentical homodimeric subunits whose association is essential for enzymatic activity. In order to evaluate the affinity of a series of peptidic inhibitors with the enzyme's large subunit (R1), we have developed a sensitive solid-phase binding assay. The assay entails the use of a nonneutralizing antibody directed against the R1 subunit of the enzyme to immobilize either the native holoenzyme from HSV-1-infected cells or a recombinantly expressed HSV-2 R1 subunit. In either case, the radioiodinated peptidic inhibitor 125I-desamino-Tyr-(N-methyl)-Val-Ile-Asp-(gamma-N,N-diethyl)-Asp-Leu demonstrated specific, saturable binding to the HSV R1 that could be competed by the nonapeptide Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu corresponding to the C-terminal sequence of the HSV ribonucleotide reductase small subunit (R2) or by recombinant HSV R2, but not by C-terminally truncated HSV R2 or murine R2. Our results provide direct evidence that inhibitors based on the carboxy-terminal amino acid sequence of HSV R2 compete with intact HSV R2 for a common binding site on HSV R1. The utility and sensitivity of this binding assay were further demonstrated by the ability to detect and discriminate ribonucleotide reductase inhibitors in the low nanomolar range.
Krogsrud
R L
RL
Biochemistry Department, Bio-Mega/Boehringer Ingelheim Research Inc., Laval, Quebec, Canada.
Welchner
E
E
Scouten
E
E
Liuzzi
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000302:isolation & purification; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology*; D004800:Enzymes, Immobilized / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D006825:Hybridomas / Q000378:metabolism; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism* / Q000494:pharmacology*; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D018139:Simplexvirus / Q000201:enzymology*; D014764:Viral Proteins / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D014764:Viral Proteins; D012264:Ribonucleotide Reductases