Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-03-19
2001-09-25
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007900, C435S007920, C435S007940, C435S974000, C436S531000, C530S350000, C530S826000
Reexamination Certificate
active
06294341
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for detecting substances having an activity to inhibit HIV infection. More particularly, the invention relates to an improved method for detecting substances having an activity to inhibit action of gp41, which is responsible for the infection of HIV, in order to develop a therapeutic agent for Acquired Immune Deficiency Syndrome (AIDS). This method is quicker, more economical and safer than the prior art.
2. Description of the Prior Art
HIV Infection is mediated by two types of protein, gp120 and gp41, which present on the envelope membrane of the virus. gp120 recognizes a cell to be infected and induces initial binding to the cell, while gp41 causes the fusion between the envelope membrane of the virus and the cytoplasmic membrane of the cell.
The tertiary structure of these proteins is unknown, but studies of their primary structure revealed that gp41 comprises a fusion peptide (FP) capable of interacting with a target cell membrane, an amino-terminal helical structure (N-&agr;H), carboxy-terminal helical structure (C-&agr;H), a transmembrane segment (TM) and a cytoplasmic domain as shown in FIG.
1
. In
FIG. 1
, the fusion peptide (FP) is represented by a black square, both helical structure N-&agr;H and C-&agr;H are represented by gray squares, and transmembrane segment (TM) is represented by a black square.
The peptides derived from two helical region of the ectodomain of gp41 outside the cell strongly bind to each other to form a stable six-helical bundle complex composed of a trimer of two interacting peptides (See, Chan et al., Cell 89, 263-273, 1997). It is presumed that the interaction of two helical domains plays a key role in the structural stability or function of the gp41 protein. Therefore, a substance that can inhibit the interaction between two helical structures of the gp41 proteins may inhibit HIV infection by inhibiting the action of gp41. Thus, the substance can be utilized as a therapeutic agent for AIDS.
The substances having such inhibitory activity include, for example, peptides derived from two helical domains of gp41 (See, Wild et al., Pro. Natl. Acad. Sci. USA 91, 9770-9774, 1994). These peptides bind to one of the two helical domains to inhibit binding or interaction between the two helical structures of gp41. Consequently, they inhibit the function of gp41 and, as a result, inhibit HIV infection. Therefore, if a method that easily detects the interaction between two helical domains of gp41 is developed, such method will be able to be used to detect substances that inhibit the function of gp41 or HIV infection.
Currently used methods for detecting substances to inhibit the function of gp41 use a system comprising culturing an animal cell in which receptors of gp120 are expressed, infecting the cell with HIV or vaccinia virus harboring env gene encoding gp 120 and gp41, and inducing cell fusion between the infected cells (See, Nussbaum et al., J. Virol. 68, 5411-5422, 1994). Anti-HIV activity of a substance is measured by investigating the inhibitory effect on the fusion between cells infected with HIV or a vaccinia virus. However, this method has the following problems: First, it is complex, difficult to carry out and requires the animal cell culture, which is expensive. Second, since it uses a living HIV or vaccinia virus, there is a possibility that an experimenter could be exposed to or infected with the harmful virus. Moreover, special expensive equipment is required for preventing such infection. Third, in addition to the considerable amount of time that is required to culture the cell, since numerous processing steps are required as well, it is difficult to screen a number of compounds that can inhibit gp41. That is, the known method for detecting a substance to inhibit the activity of gp41 is expensive, time consuming and requires special equipment. Therefore, there exists the need for a safe, inexpensive, easily manageable and time efficient method for detecting an inhibitor of gp41 activity from the known compounds.
Considering the problems associated with conventional methods, the present inventors investigated a method for rapidly, economically and safely detecting a substance having an inhibitory activity of HIV infection. As the result, the present inventors found that a substance capable of inhibiting gp41 activity can be detected by using the interaction between one variant protein, Trx-N, and another variant protein, GST-C, wherein Trx-N is prepared by connecting the N-terminal helical domain of gp41 to Trx (thioredoxin), and GST-C is prepared by connecting the C-terminal helical domain of gp41 to GST (Glutathione S-transferase).
Therefore, the method of the invention comprises preparing the variant proteins, identifying the presence of interaction between the two helical domains of gp41 by determining the interaction between two variant proteins, developing immunoassay using two variant proteins having such interaction and detecting a substance having an inhibitory activity of HIV infection by the immunoassay.
REFERENCES:
patent: 92/13955 (1992-08-01), None
Pharmacia catalog, “Molecular and Cell Biology Catalog 1993”.*
David C. Chan, e t al., “Core Structure of gp41 From the HIV Envelope Glycoprotein”, Cell, vol. 89, Apr. 18, 1997, pp. 263-273.
C. T. Wild, et al., “Peptides Correspoding to a Predictive ∝-Helical Domain of Human Immunodeficiency Virus Type 1 gp41 Are Potent Inhibitors of Virus Infection”, Proc. Natl. Acad. Sci. USA, vol. 91, Oct. 1994, pp. 9770-9974.
W. Weissenhorn, et al., “Atomic Structure of the Ectodomain From HIV-1 gp41”, letters to nature, Nature, vol. 387, No. 22, May 1997, pp. 426-430.
D.M. Lambert, et al., “Peptides From Conserved Regions of Paramyxovirus Fusion (F) Proteins Are Potent Inhibitors of Viral Fusion”, Proc. Natl. Acad. Sci. USA, vol. 93, Mar. 1996, pp. 2186-2191.
O. Nussbaum, et al., “Fusogenic Mechanisms of Enveloped-Virus Glycoproteins Analyzed by a Novel Recombinant Vaccinia Virus-Based Assay Quantitating Cell Fusion-Dependent Reporter Gene Activation”, Journal of Virology, Sep. 1994, pp. 5411-5422.
Kim Sung-Hou
Ryu Jae-Ryeon
Yu Yeon Gyu
Korea Institute of Science and Technology
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Stucker Jeffrey
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