Substrates for human cytomegalovirus protease

Details Substrates for human cytomegalovirus protease Substrates for human cytomegalovirus protease

1998-10-20

2001-02-27

Salimi, Ali R. (Department: 1648)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S023000, C435S024000, C530S326000, C530S327000, C530S328000, C530S329000, C530S330000

Reexamination Certificate

active

06194143

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to substrates for herpesvirus proteases, to methods of determining the activity of herpesvirus proteases and to methods of identifying inhibitors of herpesvirus proteases.
BACKGROUND OF THE INVENTION
Herpesviruses inflict a wide range of diseases against humans and animals. For instance, herpes simplex viruses, types 1 and 2 (HSV-1 and HSV-2), are responsible for cold sores and genital lesions, respectively; varicella zoster virus (VZV) causes chicken pox and shingles; Epstein-Barr virus (EBV) causes infectious mononucleosis; and the human cytomegalovirus (HCMV, a &bgr;-herpesvirus) is a serious pathogen in immunocompromised individuals, including AIDS patients, neonates and organ transplant recipients {Mocarski, E. S. (1996) in Virology (Fields, B. N., Knipe, D. M., Howley, P. M., Eds.), pp. 2447-2492, Lippincott-Raven Publishers, Philadelphia; and Britt, W. J., Alford, C. A. (1996) in Virology (Fields, B. N., Knipe, D. M., Howley, P. M., Eds.), pp. 2493-2523, Lippincott-Raven Publishers, Philadelphia}. The most widely used antiherpes agents to date are acyclovir and ganciclovir (purine and pyrimidine nucleoside analogs) and foscarnet. These agents have found only limited success in treating herpesvirus infections, and safer and more effective treatment agents are required.
As a member of the Herpesvirus family, HCMV encodes a unique serine protease which is involved in capsid assembly and essential for the production of infectious virions (Preston, V. G., Coates, J. A. V., Rixon, F. J. (1983)
J. Virol.
45, 1056-1064; Gao, M., Matusick-Kumar, L., Hurlburt, W., DiTusa, S. F., Newcomb, W. W., Brown, J. C., McCann, P. J., III, Deckman, I., Colonno, R. J. (1994)
J. Virol.
68, 3702-3712; Matusick-Kumar, L., McCann, P. J., III, Robertson, B. J., Newcomb, W. W., Brown, J. C., Gao, M. 1995
J. Virol.
69, 7113-7121; Gibson, W., Welch, A. R., Hall, M. R. T. (1995)
Perspect. Drug Discovery Des.
2, 413-426; and Liu, F., Roizman, B. (1991)
J. Virol.,
65, 5149-5156). This enzyme is responsible for the processing of the assembly protein whose function is analogous to that of the “scaffolding” protein of bacteriophages {Casjens, S., King, J. (1975)
Annu. Rev. Biochem.
44, 555-611). Failure to process the assembly protein results in accumulation of only aberrant, non-infectious capsids (Preston, V. G. et al., vide infra). This protease therefore represents an attractive target for the development of new antiviral agents.
In order to identify inhibitors of herpesvirus proteases, assays that allow the measurement of protease activity are required. Initial assays were based on the electrophoretic separation of the cleavage products (Gibson, W. et al., vide infra and Liu, F. et al., vide infra). However, these assays were very slow and cumbersome. More rapid and high-throughput assays using fluorogenic substrates for the HCMV protease were subsequently developed (Holskin, B. P., Bukhtiyarova, M., Dunn, B. M., Baur, P., de Chastonay, J., Pennington, M. W. (1995)
Anal. Biochem.
227, 148-155; Pinko, C., Margosiak, S. A., Vanderpool, D., Gutowski, J. C., Condon, B., Kan, C.-C. (1995)
J. Biol. Chem.
270, 23634-23640; Handa, B. K., Keech, E., Conway, E. A., Broadhurst, A., Ritchie, A. (1995)
Antivir. Chem. Chemother.
6, 255-261; and Toth, M. V., Wittner, A. J., Holwerda, B. C., U.S. Pat. No. 5,506,115 issued Apr. 9, 1996}. These fluorogenic substrates are based on the maturation cleavage site of the enzyme (the “M-site”) and exploit the spectral overlap properties of fluorescent donor/acceptor pairs such as EDANS/DABCYL and 2-aminobenzoic acid/3-nitrotyrosine {Matayoshi, E. D., Wang, G. T., Krafft, G., Erickson, J. (1990)
Science
247, 954-958; and Meldal, M., Breddam, K. (1991)
Anal. Biochem.
195, 141-147}. The separation between the pair in these HCMV protease substrates varies from 10 to 11 amino acids and the specificity constant, k
cat
/K
m
, ranges from 800 to 3000 M
−1
s
−1
(Table I).
TABLE I
PRIOR REPORTS OF HCMV PROTEASE
FLUOROGENIC SUBSTRATES
k
cat
k
cat
/K
M
Substrate
(s
−1
)
k
M
(&mgr;M)
(M
−1
s
−1
)
DABCYL-RGVVNA-SSRLA-EDANS
a
nd
nd
 796
DABCYL-RRVVNA-S-Aba-
nd
  3.0
nd
RLD(EDANS)-NH
2
b
ABz-GVVNA-SSRLAY(3-NO
2
)G
c
0.37
134 
2750
Y(3-NO
2
)GVVNA-SSRLA-ABz-K
c
nd
nd
3167
a
Holskin, B. P., Bukhtiyarova, M., Dunn, B. M., Baur, P., de Chastonay, J., Pennington, M. W. (1995) Anal. Biochem. 227, 148-155.
b
Handa, B. K., Keech, E., Conway, E. A., Broadhurst, A., Ritchie, A. (1995) Antivir. Chem. Chemother. 6, 255-261
c
Pinko, C., Margosiak, S. A., Vanderpool, D., Gutowski, J. C., Condon, B., Kan, C. -C. (1995) J. Biol. Chem. 270, 23634-23640.
These numbers reflect both the extensive requirement of herpesvirus proteases for a long peptide chain and their relatively low activity compared to other viral proteases (Gibson, W. et al., vide infra).
As disclosed herein, a new class of substrates, modeled on the maturation site of the enzyme, have been developed. The kinetic properties of the new substrates of this class are suitable for the elaboration of, for example, a fluorometric, chromogenic or radiometric assay for screening and mechanistic studies of HCMV protease inhibitors. If one is to achieve rapid progress in the rational design of peptide-based inhibitors, a more active substrate than those found in prior reports is required. The benefits accruing to a more active substrate are a significant reduction of enzyme concentration in the assay and the allowance of an unequivocal determination of inhibitor potency. The requirement of improved activity has now been met by the new substrates disclosed herein.
These new substrates are specifically useful for high performance liquid chromatography (HPLC), radiometric, chromogenic and fluorometric-based assays. The latter type of assay usually offers significant advantages over the other approaches, such as greater throughput, higher sensitivity and continuous monitoring of the hydrolytic process. Most recently, the growing interest in the design of new fluorogenic substrates is exemplified by their numerous application in a variety of enzymatic protocols {Matayoshi, E. D. et al.,vide infra; Wang, G. T., Chung, C. C., Holzman, T. F., Krafft, G. A. (1993)
Anal. Biochem.
210, 351-359; Pennington, N. W., Zaydenberg, I., Byrnes, M. E., de Chastonay, J., Malcolm, B. A., Swietnicki, W., Farmerie, W. G., Scarborough, P. E., Dunn, B. M. (1993) in Peptides 1992: Proceedings of the 22nd European Peptide Symposium (Schneider, C. H., Eberle, A. N., Eds), pp. 936-937, Escom, Leiden, Netherlands; Pennington, M. W., Thornberry, N. A. (1994)
Peptide Res.
7, 72-76; Knight, G. C. (1995)
Meth. Enzymol.
248, 18-34 (and references therein); and Jean, F., Basak, A., DiMaio, J., Seidah, N. G., Lazure, C. (1995),
Biochem. J.
307, 689-695}.
SUMMARY OF THE INVENTION
One aspect of this invention involves a substrate, suitable for cleavage by a herpesvirus protease, represented by formula I:
X-Tbg-Tbg-Y-Ala-Z (I) [SEQ ID NO:1]
wherein X is a fluorescent donor radical, a fluorescent acceptor radical, an affinity tag, a detectable label or an N-terminal capping group; or X is an amino acid sequence, or derivative thereof, sufficient for substrate recognition and to which a fluorescent donor radical, a fluorescent acceptor radical, an affinity tag, a detectable label or an N-terminal capping group, is optionally attached;
Y is a divalent radical NHCH{CH
2
C(O)N(R
1
)(R
2
)}C(O) wherein R
1
and R
2
are independently selected from hydrogen, lower alkyl, lower cycloalkyl or (lower cycloalkyl)-(lower alkyl), or R
1
and R
2
together with the nitrogen atom to which they are attached form a pyrrolidino, piperidino or morpholino; and Z is an amino acid sequence or derivative thereof sufficient for substrate recognition and to which a fluorescent donor radical, a fluorescent acceptor radical, an affinity tag or a detectable label is optionally attached; or Z is an aromatic amino or phenoxy compound

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