Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Virus or component thereof
Reexamination Certificate
2000-02-29
2003-09-23
Scheiner, Laurie (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Virus or component thereof
C530S324000
Reexamination Certificate
active
06623741
ABSTRACT:
1. FIELD OF THE INVENTION
The present invention relates to peptides derived from protein regions or domains referred to herein as heptad repeat regions. In particular, the invention relates to a peptide referred to herein as DP178 or, alternatively, as T20. DP178 corresponds to amino acid residues 638 to 673 of the HIV-1
LAI
transmembrane protein (TM) gp41 which correspond to a heptad repeat region referred to as HR2. The invention also relates to portions or analogs of DP178 which exhibit anti-membrane fusion capability, antiviral activity (for example the ability to inhibit HIV transmission to uninfected CD4
+
cells) and/or an ability to modulate intracellular processes involving coiled-coil peptide structures.
The present invention also releates to a peptide referred to herein as DP107 or, alternatively, as T21. DP107 corresponds to amino acid residues 558 to 595 of the HIV-1
LAI
transmembrane protein gp41 which correspond to a heptad region referred to as HR1. The invention also relates to portions or analogs of DP107 which exhibit anti-membrane fusion capability, antiviral activity (for example the ability to inhibit HIV transmission to uninfected CD4
+
cells) and/or an ability to modulate intracellular process involving coiled-coil peptide structures.
The invention further relates to other DP178-like and DP107-like peptides derived, e.g., from other species of virus, and which correspond to HR1 and HR2 regions analogous to the HR1 and HR2 regions of the HIV-1 transmembrane protein gp41. Such peptides include, e.g., peptides derived from amino acid sequences of HR1 and HR2 regions of the respiratory syncytial virus (RSV) F1 fusion protein (F1-protein) which are described herein.
As described herein, the HR1 and HR2 regions of proteins such as HIV gp41 and the RSV F1-protein interact (non-covalently) with each other and/or with peptides derived therefrom (such as T20 and T21). This interaction is required for normal infectivity of viruses such as RSV and HIV.
The present invention therefore additionally relates to methods for identifying compounds, including small molecule compounds, that disrupt the interaction between DP178 and DP107 and/or between DP107-like and DP178-like peptides. In one embodiment, such methods relate to identification and utilization of modified DP178, DP178-like, DP107 and DP107-like peptides and peptide pairs that interact with each other at a lower affinity than the affinity exhibited by corresponding “parent” or “native” peptides. Further, the invention relates to the use of DP178, DP178 portions, DP107, DP107 portions and/or analogs and other modulators, including small molecule modulators, of DP178/DP107, DP178-like/DP107-like or HR1/HR2 interactions as antifusogenic or antiviral compounds or as inhibitors of intracellular events involving coiled-coil peptide structures.
The invention is demonstrated, first, by way of an Example, wherein DP178 and a peptide whose sequence is homologous to DP178 are each shown to be potent, non-cytotoxic inhibitors of HIV-1 transfer to uninfected CD4
+
cells. The invention is further demonstrated by Examples wherein peptides having structural and/or amino acid motif similarity to DP107 and DP178 are identified in a variety of viral and nonviral organisms, including RSV, and in Examples wherein a number of such identified peptides derived from several different viral systems are demonstrated to exhibit antiviral activity. The invention is still further demonstrated by way of other Examples wherein other DP178-like and DP107-like peptides are identified that interact with their corresponding HR1 and HR2 domains with a lower affinity than the affinity exhibited by the native DP178 or DP107 peptide from which they are derived. The invention is still further demonstrated by way of an additional Example wherein DP178-like and DP107-like peptides from the RSV F1-protein are identified and structural studies are carried out demonstrating that these peptides associate non-covalently to form the coiled-coil structure typical of an HR1/HR2 interaction.
2. BACKGROUND OF THE INVENTION
2.1. Membrane Fusion Events
Membrane fusion is a ubiquitous cell biological process (for a review, see White, J. M., 1992, Science 258:917-924). Fusion events which mediate cellular housekeeping functions, such as endocytosis, constitutive secretion, and recycling of membrane components, occur continuously in all eukaryotic cells.
Additional fusion events occur in specialized cells. Intracellularly, for example, fusion events are involved in such processes as occur in regulated exocytosis of hormones, enzymes and neurotransmitters. Intercellularly, such fusion events feature prominently in, for example, sperm-egg fusion and myoblast fusion.
Fusion events are also associated with disease states. For example, fusion events are involved in the formation of giant cells during inflammatory reactions, the entry of all enveloped viruses into cells, and, in the case of human immunodeficiency virus (HIV), for example, are responsible for the virally induced cell-cell fusion which leads to cell death.
2.2. The Human Immunodeficiency Virus
The human immunodeficiency virus (HIV) has been implicated as the primary cause of the slowly degenerative immune system disease termed acquired immune deficiency syndrome (AIDS) (Barre-Sinoussi, F. et al., 1983, Science 220:868-870; Gallo, R. et al., 1984, Science 224:500-503). There are at least two distinct types of HIV: HIV-1 (Barre-Sinoussi, F. et al., 1983, Science 220:868-870; Gallo R. et al., 1984, Science 224:500-503) and HIV-2 (Clavel, F. et al., 1986, Science 233:343-346; Guyader, M. et al., 1987, Nature 326:662-669). Further, a large amount of genetic heterogeneity exists within populations of each of these types. Infection of human CD-4
+
T-lymphocytes with an HIV virus leads to depletion of the cell type and eventually to opportunistic infections, neurological dysfunctions, neoplastic growth, and ultimately death.
HIV is a member of the lentivirus family of retroviruses (Teich, N. et al., 1984, RNA Tumor Viruses, Weiss, R. et al., eds., CSH-Press, pp. 949-956). Retroviruses are small enveloped viruses that contain a diploid, single-stranded RNA genome, and replicate via a DNA intermediate produced by a virally-encoded reverse transcriptase, an RNA-dependent DNA polymerase (Varmus, H., 1988, Science 240:1427-1439). Other retroviruses include, for example, oncogenic viruses such as human T-cell leukemia viruses (HTLV-I,-II,-III), and feline leukemia virus.
The HIV viral particle consists of a viral core, composed of capsid proteins, that contains the viral RNA genome and those enzymes required for early replicative events. Myristylated Gag protein forms an outer viral shell around the viral core, which is, in turn, surrounded by a lipid membrane enveloped derived from the infected cell membrane. The HIV enveloped surface glycoproteins are synthesized as a single 160 Kd precursor protein which is cleaved by a cellular protease during viral budding into two glycoproteins, gp41 and gp120. gp41 is a transmembrane protein and gp120 is an extracellular protein which remains non-covalently associated with gp41, possibly in a trimeric or multimeric form (Hammarskjold, M. and Rekosh, D., 1989, Biochem. Biophys. Acta 989:269-280).
HIV is targeted to CD-4
+
cells because the CD-4 cell surface protein acts as the cellular receptor for the HIV-1 virus (Dalgleish, A. et al., 1984, Nature 312:763-767; Klatzmann et al., 1984, Nature 312:767-768; Maddon et al., 1986, Cell 47:333-348). Viral entry into cells is dependent upon gp120 binding the cellular CD-4
+
receptor molecules (McDougal, J. S. et al., 1986, Science 231:382-385; Maddon, P. J. et al., 1986, Cell 47:333-348) and thus explains HIV's tropism for CD-4
+
cells, while gp41 anchors the enveloped glycoprotein complex in the viral membrane.
2.3. HIV Treatment
HIV infection is pandemic and HIV associated diseases represent a major world health problem. Although considerable effort is being put into the successful design
Antczak James B.
Delmedico Mary K.
Erickson Joel B.
Lambert Dennis M.
Sista Prakash
Parkin Jeffrey S.
Pennie & Edmonds LLP
Scheiner Laurie
Trimeris, Inc.
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