A Salt Bridge between an N-terminal Coiled Coil of gp41 and an Antiviral Agent Targeted to the gp41 Core Is Important for Anti-HIV-1 Activity Shibo Jiang 1 and Asim K. Debnath Lindsley F. Kimball Research Institute, New York Blood Center, 310 East 67th Street, New York, New York 10021 Received February 25, 2000 HIV-1 envelope glycoprotein transmembrane subunit gp41 play a critical role in the fusion of viral and target cell membranes. The gp41 C-terminal heptad repeat re- gion interacts with the N-terminal coiled-coil region to form a six-stranded core structure. Peptides derived from gp41 C-terminal heptad repeat region (C-peptides) are potent HIV-1 entry inhibitors by binding to gp41 N-terminal coiled-coil region. Most recently, we have identified two small organic compounds that inhibit HIV-1-mediated membrane fusion by blocking the for- mation of gp41 core. These two active compounds con- tain both hydrophobic and acidic groups while the inac- tive compounds only have hydrophobic groups. Analysis by computer modeling indicate that the acidic groups in the active compounds can form salt bridge with Lys 574 in the N-terminal coiled-coil region of gp41. Asp 632 in a C-peptide can also form a salt bridge with Lys 574. Re- placement of Asp 632 with positively charged residues or hydrophobic residues resulted in significant decrease of HIV-1 inhibitory activity. These results suggest that a salt bridge between an N-terminal coiled coil of the gp41 and an antiviral agent targeted to the gp41 core is im- portant for anti-HIV-1 activity. © 2000 Academic Press Key Words: HIV-1; gp41; salt bridge; coiled coil; anti- viral agent; anti-HIV-1 activity. Infection by the human immunodeficiency virus type 1 (HIV-1) involves fusion of the viral membrane with the target cell membrane, followed by the subsequent transfer of viral genetic material into the cell. HIV-1 envelope glycoproteins gp120 and gp41 play critical roles in these initial events of viral infection, i.e. gp120 recognizes the target cell by binding to both CD4 and a co-receptor (CCR5 or CXCR4) and gp41 then promotes the fusion of viral and cellular membranes (1). The gp41 ectodomain contains three major functional re- gions, the fusion peptide region located at the N-terminus of gp41, followed by two 4 –3 heptad repeat (HR) regions predicted to form coiled coils (2). Peptides derived from the N- and C-terminal HR regions, des- ignated N- and C-peptides, can interact with each other to form a six-stranded -helical bundle, in which three N-helices associate to form the central trimeric coiled coil and three C-helices pack obliquely in an antiparallel manner into the highly conserved hydro- phobic grooves on the surface of the coiled coil (3– 6). The six-stranded -helical bundle was proposed to be the fusion-active gp41 core (3, 4). This was confirmed by using a monoclonal antibody (mAb), NC-1, which specifically recognizes conformational epitopes on the complexes formed between N- and C-peptides and binds to gp41-expressing cells only in the presence of CD4 molecules (7). C-peptides are potent HIV-1 inhibitors (8, 9). They inhibit the membrane fusion step of HIV-1 infection, in a dominant-negative manner, by binding to the N-terminal HR region of gp41 and blocking the formation of the gp41 core (3, 4, 10). Clinical trials demonstrated that C-pep- tides can be potentially used for chemotherapy of HIV-1 infection (11). However, their future clinical application may be constrained because of proteolytic degradation and lack of oral bioavailability. Therefore, it is essential to develop low molecular weight HIV-1 inhibitors having a similar mechanism of action as C-peptides. X-ray crystallographic analysis of the gp41 core structure demonstrated that there is a hydrophobic deep pocket in each groove on the surface of the inner core of gp41 and it was suggested to be an important target for development of novel HIV-1 inhibitors (12). Eckert et al. (13) recently constructed several short cyclic peptides with D-amino acid residues. These pep- Abbreviations used: BCECF, 2',7'-bis-(2-carboxyethyl)-5-and-6- carboxyfluorescein acetoxyethyl ester; CD, circular dichroism; CHR, C-terminal heptad repeat; CPE, cytopathic effect; ELISA, sandwich enzyme-linked immunosorbent assay; HIV-1, the human immunode- ficiency virus type 1; HPLC, high-performance liquid chromatogra- phy; HR, heptad repeat; NHR, N-terminal heptad repeat; IC 50 , 50% effective concentration; MAb, monoclonal antibody. 1 To whom correspondence should be addressed. Fax: 212-570- 3299. E-mail: sjiang@nybc.org. Biochemical and Biophysical Research Communications 270, 153–157 (2000) doi:10.1006/bbrc.2000.2411, available online at http://www.idealibrary.com on 153 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.