Kang Lin

A screening assay for antiviral compounds targeted to the HIV-1 gp41 core structure using a conformation-specific monoclonal antibody.

The human immunodeficiency virus type 1 (HIV-1) gp41 plays an important role in membrane fusion between viruses and target cells. The gp41 ectodomain contains two heptad repeat regions adjacent to the N and C-termini. Peptides derived from these two regions, designated N and C-peptides, are potent inhibitors of HIV-1 infection and can interact with each other to form a six-stranded coiled-coil, representing the fusogenic core structure of gp41. A monoclonal antibody was generated, designated NC-1, which specifically binds to the complex formed by the N and C-peptides, but not to the individual peptides. An enzyme linked immunosorbent assay (ELISA) was developed using NC-1 for detecting complex formed by N and C-peptides and for screening of organic compounds for antiviral agents that may interfere with complex formation and inhibit HIV-1 infection. Single point mutations in the C-peptides abolish the complex formation also eliminate their anti-HIV-1 activity. A phenylazo-naphthalene sulfonic acid derivative, designated ADS-J1, was found to inhibit both formation of NC-1 detectable complex and HIV-1-mediated membrane fusion, suggesting that the described ELISA is applicable to rapid screening of libraries of organic compounds for HIV-1 inhibitors targeted to the HIV-1 gp41 core structure.

3-Hydroxyphthaloyl-β-Lactoglobulin. I. Optimization of Production and Comparison with other Compounds Considered for Chemoprophylaxis of Mucosally Transmitted Human Immunodeficiency Virus Type 1

Modification of the major bovine whey protein, β-lactoglobulin (β-LG) by 3-hydroxyphthalic anhydride (3HP) leads to the generation of a potent inhibitor of infection by human immunodeficiency virus (HIV) types 1 and 2, designated 3HP-β-LG. 3HP-β-LG also has antiviral activity against herpesviruses, albeit at concentrations exceeding those required for inhibition of HIV-1 infection. The topical application of 3HP-β-LG to decrease the rate of sexual transmission of HIV and other sexually transmitted viruses worldwide is being considered. Results presented here: (i) define the conditions for chemical modification of β-LG by 3HP, resulting in 3HP-β-LG with optimum anti-HIV-1 activity; (ii) show that β-LG, prior to chemical modification, or 3HP-β-LG can be exposed to the elevated temperatures used to pasteurize milk without adversely affecting anti-HIV-1 activity; (iii) provide evidence that 3HP-β-LG is a more potent anti-HIV-1 compound than sulphated polysaccharides, other candidate compounds considered as prophylactic agents to prevent sexual transmission of HIV-1; and (iv) confirm that the primary target for 3HP-β-LG is CD4, although binding to the HIV-1 envelope protein gp120 was also observed and contributed to the antiviral activity of 3HP-β-LG.

3-Hydroxyphthaloyl-β-Lactoglobulin. II. Anti-Human Immunodeficiency Virus Type 1 Activity in in Vitro Environments Relevant to Prevention of Sexual Transmission of the Virus

It is anticipated that the rate of sexual transmission of viruses could be substantially decreased by the use of topical chemical barrier methods. Chemical modification of bovine (β-lactoglobulin (β-LG), the major protein of whey, led to the generation of a potent inhibitor (designated 3HP-β-LG) of human immunodeficiency virus type 1 (HIV-1) infection which was also active against herpesviruses. Compounds intended for topical application to prevent sexual transmission of viruses need to maintain their antiviral activity at pH <<7, corresponding to an acidic vaginal environment, and in the presence of seminal fluid. Results presented here show that the binding of 3HP-β-LG to the CD4 receptor for HIV, involved in the anti-HIV-1 activity of this compound, decreases with decreasing pH. The presence of seminal fluid also decreased the binding of 3HP-β-LG to CD4 and diminished the inhibitory effect of the compound on CD4-gp120 binding. 3HP-β-LG was shown to bind Zn++, and the inhibitory effect of seminal fluid could be substantially diminished by chelating Zn++ with ethylenediaminetetraacetate. Saliva had no effect on 3HP-β-LG binding to CD4 or on its interference with gp120-CD4 binding. The decreased 3HP-β-LG-CD4 binding and the concomitant reduction of gp120-CD4 binding inhibition by 3HP-β-LG at low pH and in the presence of seminal fluid could be compensated for by an increase of the 3HP-β-LG concentration and by adding Zn++ chelators to 3HP-β-LG. These results provide a background for the design of 3HP-β-LG formulations for topical use.

Tin Protoporphyrin IX Used in Control of Heme Metabolism in Humans Effectively Inhibits HIV-1 Infection

Recent observations indicated that several porphyrins bound to the V3 loop of the envelope glycoprotein gp120 of the human immunodeficiency virus type 1 (HIV-1) and inhibited infection of cells by HIV-1. The tin derivative of protoporphyrin IX (Sn-PTP-IX) has already been used clinically in humans to suppress hyperbilirubinemia. It was therefore of interest to determine whether Sn-PTP-IX has anti-HIV-1 activity. It is demonstrated here that Sn-PTP-IX effectively inhibited infection by several HIV-1 isolates (HIB, MN, RF, SF-2 and two isolates resistant to azidothymidine). This was surprising, since earlier studies indicated that incorporation of other metals into porphyrins markedly decreased their antiviral activity. Sn-PTP-IX blocked the binding to gp120 of anti-V3-loop-specific antibodies and of monoclonal antibodies specific for the CD4 binding site on gp120. The latter effect appeared to be allosteric and was not observed with a deletion mutant of gp 120 lacking the V3 loop sequence. This suggests that Sn-PTP-IX binds to the V3 loop and distorts the native conformation of the HIV-1 envelope, thereby preventing infection. These results merit the consideration of Sn-PTP-IX as a prophylactic and chemotherapeutic agent against HIV-1.