Assay for detection of viral fusion inhibitors

Details Assay for detection of viral fusion inhibitors Assay for detection of viral fusion inhibitors

2001-02-09

2003-08-12

Park, Hankyel T. (Department: 1648)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S006120, C435S039000, C435S325000, C435S334000, C435S339100

Reexamination Certificate

active

06605427

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention is directed to methods for identifying compounds that inhibit or prevent infection of cells by enveloped viruses such as HIV-1, and the compounds discovered by such methods. The invention also includes using these methods as diagnostic assays to detect antibodies in virus-infected individuals that inhibit the viral entry processes.
2. Related Art
The HIV-1 envelope glycoprotein is a 160 kDa glycoprotein that is cleaved to form the transmembrane (TM) subunit, gp41, which is non-covalently attached to the surface (SU) subunit, gp120 (Allan J. S., et al.,
Science
228:1091-1094 (1985); Veronese F. D., et al.,
Science
229:1402-1405 (1985)). Recent efforts have led to a clearer understanding of the structural components of the HIV-1 envelope system. Such efforts include crystallographic analysis of significant portions of both gp120 and gp41 (Kwong, P. D., et al.,
Nature
(
London
) 393:648-659 (1998); Chan, D. C., et al.,
Cell
89:263-273 (1997); Weissenhorn, W., et al.,
Nature
387:426-430 (1997)).
The surface subunit has been characterized as part of a multi-component complex consisting of the SU protein (the gp120 core absent the variable loops) bound to a soluble form of the cellular receptor CD4 (N-terminal domains 1 and 2 containing amino acid residues 1-181) and an antigen binding fragment of a neutralizing antibody (amino acid residues 1-213 of the light chain and 1-229 of the heavy chain of the 17
b
monoclonal antibody) which blocks chemokine receptor binding (Kwong, P. D., et al.,
Nature
(
London
) 393:648-659 (1998)). Several envelope components believed to exist only in the fusion-active form of gp120 were revealed by the crystallographic analysis including a conserved binding site for the chemokine receptor, a CD4-induced epitope and a cavity-laden CD4-gp120 interface. This supports earlier observations of CD4-induced changes in gp120 conformation.
The gp120/gp41 complex is present as a trimer on the virion surface where it mediates virus attachment and fusion. HIV-1 replication is initiated by the high affinity binding of gp 120 to the cellular receptor CD4 and the expression of this receptor is a primary determinant of HIV-1 cellular tropism in vivo (Dalgleish, A. G., et al.,
Nature
312:763-767 (1984); Lifson, J. D., et al.,
Nature
323:725-728 (1986); Lifson, J. D., et al.,
Science
232:1123-1127 (1986); McDougal, J. S., et al.,
Science
231:382-385 (1986)). The gp120-binding site on CD4 has been localized to the CDR2 region of the N-terminal V1 domain of this four-domain protein (Arthos, J., et al.,
Cell
5:469-481 (1989)). The CD4-binding site on gp120 maps to discontinuous regions of gp120 including the C2, C3 and C4 domains (Olshevsky, U., et al.,
Virol
64:5701-5707 (1990); Kwong, P. D., et al.,
Nature
(
London
) 393:648-659 (1998)). Following attachment to CD4, the virus must interact with a “second” receptor such as a chemokine receptor in order to initiate the fusion process. Recently, researchers have identified the critical role of members of the chemokine receptor family in HIV entry (McDougal J. S., et al.,
Science
231:382-385 (1986); Feng Y., et al.,
Science
272:872-877 (1996); Alkhatib G., et al.,
Science
272:1955-1958 (1996); Doranz B. J., et al.,
Cell
85:1149-1158 (1996); Deng H., et al.,
Nature
381:661-666 (1996); Dragic T., et al.,
Nature
381:667-673 (1996); Choe H., et al.,
Cell
85:1135-1148 (1996); Dimitrov D. S.,
Nat. Med.
2:640-641 (1996); Broder, C. C. and Dimitrov, D. S.,
Pathobiology
64:171-179 (1996)). CCR5 is the chemokine receptor used by macrophage-tropic and many T-cell tropic primary HIV-1 isolates. Most T-cell line-adapted strains use CXCR4, while many T-cell tropic isolates are dual tropic, capable of using both CCR5 and CXCR4.
Binding of gp120 to CD4 and a chemokine receptor initiates a series of conformational changes within the HIV envelope system (Eiden, L. E. and Lifson, J. D.,
Immunol. Today
13:201-206 (1992); Sattentau, Q. J. and Moore J. P.,
J. Exp. Med.
174:407-415 (1991); Allan J. S., et al.,
AIDS Res Hum Retroviruses
8:2011-2020 (1992); Clapham, P. R., et al.,
J. Virol.
66:3531-3537 (1992)). These changes occur in both the surface and transmembrane subunits and result in the formation of envelope structures which are necessary for virus entry. The functions of gp41 and gp120 appear to involve positioning the virus and cell membranes in close proximity thereby facilitating membrane fusion (Bosch M. L., et al.,
Science
244:694-697 (1989); Slepushkin, V. A. et al.,
AIDS Res Hum Retroviruses
8:9-(1992); Freed E. O. et al.,
Proc. Natl. Acad. Sci. USA
87:4650-4654 (1990)).
A good deal of structural information is available with respect to the HIV-1 transmembrane glycoprotein (gp41). This protein contains a number of well-characterized functional regions. See FIG.
3
. For example, the N-terminal region consists of a glycine-rich sequence referred to as the fusion peptide which is believed to function by insertion into and disruption of the target cell membrane (Bosch, M. L., et al.,
Science
244:694-697 (1989); Slepushkin, V. A., et al.,
AIDS Res. Hum. Retrovirus
8:9-18 (1992); Freed, E. O., et al.,
Proc. Natl. Acad. Sci. USA
87:4650-4654 (1990); Moore, J. P., et al., “The HIV-cell Fusion Reaction,” in
Viral Fusion Mechanism,
Bentz, J., ed., CRC Press, Inc., Boca Raton, Fla.). Another region, characterized by the presence of disulfide linked cysteine residues, has been shown to be immunodominant and is suggested as a contact site for the surface (gp120) and transmembrane glycoproteins (Gnann, J. W., Jr., et al.,
J. Virol.
61:2639-2641 (1987); Norrby, E., et al.,
Nature
329:248-250 (1987); Xu, J. Y., et al.,
J. Virol.
65:4832-4838 (1991)). Other regions in the gp41 ectodomain have been associated with escape from neutralization (Klasse, P. J., et al.,
Virology
196:332-337 (1993); Thali, M., et al.,
J. Virol.
68:674-680 (1994); Stem, T. L., et al.,
J. Virol.
69:1860-1867 (1995)), immunosuppression (Cianciolo, G. J., et al.,
Immunol. Lett.
19:7-13 (1988); Ruegg, C. L., et al.,
J. Virol.
63:3257-3260 (1989)), and target cell binding (Qureshi, N. M., et al.,
AIDS
4:553-558 (1990); Ebenbichler, C. F., et al.,
AIDS
7:489-495 (1993); Henderson, L. A. and Qureshi, M. N.,
J. Biol. Chem.
268:15291-15297 (1993)).
Recent work has increased knowledge of the structural components of the HIV-1 transmembrane glycoprotein, however, the immunogenic nature of gp41 remains poorly understood. It is known that one of two immunodominant regions present in the HIV-1 envelope complex is located in gp41 (Xu, J. Y., et al.,
J. Virol.
65:4832-4838 (1991)). This region (TM residues 597-613) is associated with a strong, albeit non-neutralizing, humoral response in a large number of HIV+ individuals.
Two regions of the ectodomain of gp41 have been shown to be critical to virus entry. Primary sequence analysis predicted that these regions (termed the N-helix (residues 558-595 of the HIV-1
LAI
sequence) and C-helix (residues 643-678 of the HIV-1
LAI
sequence)) model the &agr;-helical secondary structure. Experimental efforts stemming from previous structural studies of synthetic peptide mimics established that the sequence analysis predictions were generally correct (Wild, C., et al.,
Proc. Natl. Acad. Sci. USA
89:10537-10541 (1992); Wild, C. T., et al.,
Proc. Natl. Acad. Sci. USA
91:9770-9774 (1994); Gallaher, W. R., et al.,
AIDS Res. Hum. Retroviruses
5:431-440 (1989); Delwart, E. L., et al.,
AIDS Res. Hum. Retroviruses
6:703-704 (1990)). Subsequent structural analysis determined that these regions of the transmembrane protein interact in a specific fashion to form a higher order structure characterized as a trimeric six-helix bundle (Chan, D. C., et al.,
Cell
89:263-273 (1997); Weissenhorn, W., et al.,
Nature
387:426-430 (1997)). This trimeric structure consists of an interior parallel coiled-coil trimeric core (region one, N-helix) which associates with three identical &agr;-helices (region

Affiliated with

Also associated with

Rating

Assay for detection of viral fusion inhibitors does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Assay for detection of viral fusion inhibitors, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Assay for detection of viral fusion inhibitors will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3082258