Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-03-14
2003-07-22
Housel, James (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C424S160100, C435S004000, C435S005000, C435S007720, C435S007920, C435S007930, C435S007940, C436S501000, C530S388100, C530S388300, C530S389100, C530S389400
Reexamination Certificate
active
06596497
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is directed to a screening assay for antiviral compounds targeted to the HIV-1 gp41 core structure utilizing a conformation-specific monoclonal antibody, which is reactive with fusion active gp41 from human immunodeficiency virus type 1 (“HIV-1”) envelope glycoprotein. The present invention further relates to antiviral compounds targeted to the gp41 core structure of HIV-1.
2. Background Information
The infection of human immunodeficiency virus type I (HIV-1) is initiated by binding of the envelope glycoprotein (Env) surface subunit gp120 to both CD4 and particular chemokine receptors (i.e., CXCR4 and CCR5, etc.) on target cells. The Env transmembrane subunit gp41 concurrently dissociates from gp120 and then mediates the fusion of the viral and cellular membranes (Moore, J. P., B. A. Jameson, R. A. Weiss, and Q. J. Sattentau. 1993, “The HIV-cell fusion reaction”, In
Viral Fusion Mechanisms
, J. Bentz, editors. CRC Press, Boca Raton. pp. 233-289; Berger, E. A. 1997, “HIV-1 entry and tropism: the chemokine receptor connection”,
AIDS,
11 (Supp. A): S3-16; Hunter, E., 1997, “gp41, a multifunctional protein involved in HIV entry and pathogenesis”, In
Human Retroviruses and AIDS,
1997; Korber, B., Hahn, B., Foley, B., Mellors, J. W., Leitner, T., Myers, G., McCutchan, F., Kuiken, C. editors, Los Alamos National Laboratory, Los Alamos, N.M. p. 111-55-111-73; and Chan, D. C. and P. S. Kim, 1998, “HIV entry and its inhibition”,
Cell,
93, 681-684).
HIV-1 gp41 is composed of three domains, an extracellular domain (ectodomain), a transmembrane domain and an intracellular domain (endodomain). The gp41 ectodomain contains three major functional regions, i.e., the fusion peptide located at the N-terminus of gp41, followed by two 4-3 heptad repeats adjacent to the N- and C-terminal portions of the gp41 ectodomain, designated NHR (N-terminal heptad repeat) and CHR (C-terminal heptad repeat), respectively. The N- and C-terminal repeats were also named as “HR1” and “HR2”, respectively, by Rimsky, L. T., D. C. Shugars and T. J. Matthews,
J. Virol.,
72, 986-993.
Both NHR and CHR regions consist of hydrophobic amino acid sequences predicted to form &agr;-helices, denoted N and C helix (Caffrey, M., M. Cal, J. Kaufman, S. J. Stahl, P. T. Wingfield, D. G. Covell, A. M. Gronenborn, and G. M. Clore, 1998, “Three-dimensional solution structure of the 44 kDa ectodomain of SIV gp41
”, EMBO J,
17, 4572-4584), which may function as essential structures required for oligomerization of gp41 and for conformational changes during the process of membrane fusion between HIV-1 and target cells (Gallaher, W. R., J. M. Ball, R. F. Garry, M. C. Griffin, and R. C. Montelaro, 1989, “A general model for the transmembrane proteins of HIV and other retroviruses”,
AIDS Res. Hum. Retroviruses,
5, 431-440; Delwart, E. L., G. Mosialos, and T. Gilmore, 1990, “Retroviral envelope glycoprotein contain a leucine zipper-like repeat”,
AIDS Res. Hum. Retroviruses,
6, 703-706; Wild, C., T. Oas, C. McDanal, D. Bolognesi, and Matthews, T., 1992, “A synthetic peptide inhibitor of human immunodeficiency virus replication: correlation between solution structure and viral inhibition”,
Proc. Natl. Acad. Sci. USA,
89, 10537-10541; Bernstein, H. B., S. P. Tucker, S. R. Kar, S. A. McPherson, D. T. McPherson, J. W. Dubay, J. Lebowitz, R. W. Compans, and E. Hunter, 1995, “Oligomerization of the hydrophobic heptad repeat of gp41
”, J. Virol.,
69, 2745-2750).
Peptides derived from the NHR and CHR regions of gp41, designated N- and C-peptides (Chan, D. C, and P. S. Kim, 1998, “HIV entry and its inhibition”,
Cell,
93, 681-684), have potent antiviral activity against HIV-1 infection (Jiang, S., K. Lin, N. Strick, and A. R. Neurath, 1993, “HIV-1 inhibition by a peptide”,
Nature,
365, 113; Wild, C. T., D. C. Shugars, T. K. Greenwell, C. B. McDanal, and T. J. Matthews, 1994, “Peptides corresponding to a predictive alpha-helical domain of human immunodeficiency virus type I gp41 are potent inhibitors of virus infection”,
Proc. Natl. Acad. Sci. USA,
91, 9770-9774; and Lu, M., S. C. Blacklow, and P. S. Kim, 1995, “A trimeric structural domain of the HIV-1 transmembrane glycoprotein”,
Nat. Struct. Biol.,
2, 1075-1082). Previous studies suggest that these peptides inhibit the membrane fusion step of HIV-1 infection, in a dominant-negative manner, by binding to viral gp41 (Chen, C. R, T. J. Matthews, C. B. McDanal, D. P. Bolognesi, and M. L. Greenberg, 1995, “A molecular clasp in the human immunodeficiency virus (HIV) type 1 TM protein determines the anti-HIV activity of gp41 derivatives: implication for viral fusion”,
J. Virol.,
69, 3771-3777; and Furuta, R., C. T. Wild, Y. Weng, and C. D. Weiss, 1998, “Capture of an early fusion-active conformation of HIV-1 gp41
”, Nat. Struct. Biol.,
5:276-279).
Limited proteolysis of a recombinant fragment of the gp41 ectodomain generated an N-peptide and a C-peptide, designated N-51 (spanning residues 540-590) and C-43 (residues 624-666). These two peptides overlap mostly the NHR and CHR regions. Several other N- and C-peptides (i.e., N-36, N-34, C-34 and C-28) were also produced (Lu, M. and P. S. Kim, 1997, “A trimeric structural subdomain of the HIV-1 transmembrane glycoprotein”,
J. Biochem. Struct. Dynamic,
15:465-471). N- and C-peptides mixed at equimolar concentrations form stable &agr;-helical trimers of antiparallel heterodimers, representing the fusion-active (fusogenic) core domain of gp41. Crystallographic studies showed that this core domain is a six-stranded helical bundle. Three N helices associate to form the internal coiled-coil trimer via interaction of the residues at “a” positions in the wheel of one N helix with those at “d” positions (see
FIGS. 2 and 3
) in that of another N helix. Three C helices pack obliquely against the outside grooves of the N helix trimer by the interaction of residues at “a” and “d” positions in C helices with those at “e” and “g” positions (see
FIGS. 2 and 3
) in N helices, respectively (Chan, D. C., D. Fass, J. M. Berger, and P. S. Kim, 1997, “Core structure of gp41 from the HIV envelope glycoprotein”,
Cell,
89, 263-273; Weissenhorn, W., A. Dessen, S. C, Harrison, I. I. Skehel, and D. C. Wiley, 1997, “Atomic Structure of the Ectodomain from HIV-1 gp41
”, Nature,
387, 426-428; and Tan, K., I. Liu, I. Wang, S. Shen, and M. Liu, 1997, “Atomic structure of a thermostable subdomain of HIV-1 gp41
”, Proc. Natl. Acad. Sci. USA,
94, 12303-12308).
The residues at these interaction sites are highly conserved and mutations of these residues may disrupt the six-stranded core structure and abolish HIV-1 infectivity (Cao, J., L. Bergeron, E. Helseth, M. Thali, H. Repke, and I. Sodroski, 1993, “Effects of amino acid changes in the extracellular domain of the human immunodeficiency virus type 1 gp41 envelope glycoprotein”,
J. Virol.,
67, 2747-2755; Chen, S. S., C. N. Lee, W. R. Lee, K. Mcintosh, and T. H. Lee, 1993, “Mutational analysis of the leucine zipper-like motif of the human immunodeficiency virus type 1 envelope transmembrane glycoprotein”,
J. Virol.,
67, 3615-3619; Wild, C., I. W. Dubay, T. Greenwell, T. Baird, Jr., I. G. Oas, C. McDanal, F. Hunter, and T. Matthews, 1994, “Propensity for a leucine zipper-like domain of human immunodeficiency virus type 1 gp41 to form oligomers correlates with a role in virus-induced fusion rather than assembly of the glycoprotein complex”,
Proc. Natl. Acad. Sci. USA,
91, 12676-12680; Poumbourios, P., K. A. Wilson, R. I. Center, R. El Ahmar, and B. E. Kemp, 1997, “Human immunodeficiency virus type 1 envelope glycoprotein oligomerization requires the gp41 amphipathic alpha-helical/leucine zipper-like sequence”,
J. Virol.,
71, 2041-2049).
Each of the grooves on the surface of the N helices has a deep cavity that accommodates three conserved hydrophobic residues (W628, W631 and I635) in C helices. These highly conserved deep hydrophobic cavities have been suggested as attractive targets for development of antiviral lead compounds that
Debnath Asim K.
Jiang Shibo
Frishauf Holtz Goodman & Chick P.C.
Housel James
New York Blood Center Inc.
Winkler Ulrike
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