# US 5,708,137 A · Reagent and method for determining activity of herpes protease

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- Number: US 5,708,137 A
- Document ID: US5708137A
- Status: Granted
- Kind: A
- Application number: 08/544,519
- Filed: 1995-10-18
- Granted: 1998-01-13
- Earliest claimed: 1994-04-29
- Assignees: G.D. Searle & Co.
- Inventors: Mihaly V. Toth; Arthur J. Wittwer; Barry C. Holwerda
- Web page: https://patentgrep.com/patent/US5708137A

## CPC

- C12Q 1/37: involving peptidase or proteinase
- G01N 2333/03: Herpetoviridae, e.g. pseudorabies virus
- G01N 2333/045: Cytomegalovirus
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

A class of compounds is described for use as fluorogenic substrates for herpesvirus proteases and for use in an assay to identify inhibitors of herpesvirus proteases. ##STR1## wherein W is selected from glycine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminocaproic acid and 7-aminoheptanoic acid; wherein Y is selected from EDANS, Abz, DANSYL, nicotinic acid, 4-guanidino-benzoic acid, N-methyl-Abz, 4-chloro-Abz, 5-chloro-Abz, 6-chloro-Abz, 3,5-dibromo-Abz, 6-amino-nicotinic acid, 2-amino-nicotinic acid, 2-chloronicotinic acid, niflumic acid and fluorogenic derivatives thereof; and wherein Z is selected from tryptophan, tyrosine, phenylalanine, p-nitrophenylalanine, m-nitrophenylalanine, DABSYL, DABCYL and halogenated derivatives thereof.

## Claims

### Claim 1

A compound of Formula I
Z--(W).sub.m --X--(V).sub.n --Y (I)
wherein X is an amino acid sequence sufficient for substrate recognition by a herpesvirus protease;
wherein V and W are independently selected from aminoalkylcarboxylic acids;
wherein m and n are numbers independently selected from 0 and 1; and
wherein one of Y and Z is a fluorogenic donor radical and the other is an acceptor radical.

### Claim 2 (depends on 1)

Compound of claim 1 wherein X is a peptide containing six to sixteen amino acids, inclusive;
wherein V and W are independently selected from lower aminoalkylcarboxylic acids;
wherein the fluorogenic donor radical is a fluorescence emitting radical; and
wherein the acceptor radical is an aromatic radical which absorbs the fluorescence energy of the fluorogenic donor radical when the acceptor radical and the fluorogenic donor radical are covalently held in close proximity.

### Claim 3 (depends on 1)

Compound of claim 1 wherein X is selected from -Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-›SEQ ID NO:1!, -Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-›SEQ ID NO:2!, -Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-›SEQ ID NO:3!, -Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-›SEQ ID NO:4!, -Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-›SEQ ID NO:5!, -Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-Thr-›SEQ ID NO:6!, -Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-›SEQ ID NO:7!, -Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-›SEQ ID NO:8!, -Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-Thr-›SEQ ID NO:9!, -Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-Thr-Ala-›SEQ ID NO:10!, -Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-›SEQ ID NO:11!, -Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-›SEQ ID NO:12!, -Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-Thr-›SEQ ID NO:13!, -Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-Thr-Ala-›SEQ ID NO:14!, -Arg-Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-›SEQ ID NO:15!, -Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala›SEQ ID NO:16!, -Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-Thr-›SEQ ID NO:17!, -Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-Thr-Ala-›SEQ ID NO:18!, -Glu-Arg-Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-›SEQ ID NO:19!, -Arg-Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-›SEQ ID NO:20!, -Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala -Thr-›SEQ ID NO:21!, -Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-Thr-Ala-›SEQ ID NO:22!, -Ala-Glu-Arg-Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-›SEQ ID NO:23!, -Glu-Arg-Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-›SEQ ID NO:24!, -Arg-Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-Thr-›SEQ ID NO:25!, -Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-Thr-Ala-›SEQ ID NO:26!, -Val-Ala-Glu-Arg-Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-›SEQ ID NO:27!, -Ala-Glu-Arg-Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-›SEQ ID NO:28!, -Glu-Arg-Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-Thr-›SEQ ID NO:29! and -Arg-Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-Thr-Ala-›SEQ ID NO:30! and homologs thereof;
wherein V and W are independently selected from glycine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminocaproic acid and 7-aminoheptanoic acid;
wherein the fluorogenic radical is selected from EDANS, Abz, DANSYL, nicotinic acid, 4-guanidino-benzoic acid, N-methyl-Abz, 4-chloro-Abz, 5-chloro-Abz, 6-chloro-Abz, 3,5-dibromo-Abz, 6-amino-nicotinic acid, 2-amino-nicotinic acid, 2-chloronicotinic acid, niflumic acid and fluorogenic derivatives thereof; and
wherein the acceptor radical is selected from tryptophan, tyrosine, phenylalanine, p-nitrophenylalanine, m-nitrophenylalanine, DABSYL, DABCYL and halogenated derivatives thereof.