Abstract: Although human blood plasma contains molecules that inhibit the activity of HIV-1, their identity is largely unknown. Münch et al., 2007Münch J. Ständker L. Adermann K. Schulz A. Schindler M. Chinnadurai R. Pöhlmann S. Chaipan C. Biet T. Peters T. et al.Cell. 2007; (this issue)Google Scholar now identify a peptide corresponding to a portion of α1-antitrypsin that potently inhibits entry of HIV-1 into host cells by binding to a hydrophobic segment of the viral envelope glycoprotein gp41. Although human blood plasma contains molecules that inhibit the activity of HIV-1, their identity is largely unknown. Münch et al., 2007Münch J. Ständker L. Adermann K. Schulz A. Schindler M. Chinnadurai R. Pöhlmann S. Chaipan C. Biet T. Peters T. et al.Cell. 2007; (this issue)Google Scholar now identify a peptide corresponding to a portion of α1-antitrypsin that potently inhibits entry of HIV-1 into host cells by binding to a hydrophobic segment of the viral envelope glycoprotein gp41. A number of molecules have been discovered that can inhibit the entry of HIV-1 and other viruses into cells of their host (reviewed in Dimitrov, 2004Dimitrov D.S. Nat. Rev. Microbiol. 2004; 2: 109-122Crossref PubMed Scopus (312) Google Scholar, Liu et al., 2007Liu S. Wu S. Jiang S. Curr. Pharm. Des. 2007; 13: 143-162Crossref PubMed Scopus (120) Google Scholar). Perhaps the most well-known are the anti-HIV peptides SJ-2176 (Jiang et al., 1993Jiang S. Lin K. Strick N. Neurath A.R. Nature. 1993; 365: 113Crossref PubMed Scopus (461) Google Scholar) and T20 (Wild et al., 1994Wild C.T. Shugars D.C. Greenwell T.K. McDanal C.B. Matthews T.J. Proc. Natl. Acad. Sci. USA. 1994; 91: 9770-9774Crossref PubMed Scopus (864) Google Scholar) derived from the C-terminal heptad repeat region of the viral envelope glycoprotein gp41. T20 remains the only HIV-1 entry inhibitor approved for clinical use by the US Food and Drug Administration. The search for peptides with similar or greater potency and crossreactivity and improved pharmacokinetics has yet to yield additional peptide-based therapeutics of clinical utility. Following the example of T20, research has remained largely focused on peptides derived from viruses. However, human blood plasma contains naturally occurring low-molecular-weight protein inhibitors of HIV-1 infection. Although the possibility of finding inhibitory peptides in human blood plasma has long been recognized, until recently the task of identifying them from a mixture of more than one million different peptides and small proteins has been daunting. Now, using a peptide library extracted from 10,000 liters of human hemofiltrate, Münch et al., 2007Münch J. Ständker L. Adermann K. Schulz A. Schindler M. Chinnadurai R. Pöhlmann S. Chaipan C. Biet T. Peters T. et al.Cell. 2007; (this issue)Google Scholar have identified a peptide—VIRIP for VIRUS-INHIBITORY PEPTIDE—that potently inhibits HIV-1 entry into cells. Münch et al. used a straightforward, systematic, and labor-intensive approach to identify VIRIP hidden among an enormous number of other peptide molecules. They fractionated peptides from hemofiltrate by cation-exchange and reverse-phase chromatography into 322 fractions and identified one that inhibited HIV-1 activity without cellular toxicity. Further analysis of this fraction revealed VIRIP, a 20-residue peptide corresponding to the C-proximal region of the most abundant circulating serine protease inhibitor α1-antitrypsin. VIRIP inhibited primary isolates of HIV-1 from different clades and inhibited HIV isolates that use different coreceptors for entry. VIRIP also inhibited variants of HIV resistant to protease and reverse transcriptase inhibitors as well variants resistant to the fusion inhibitor T20. The inhibitory activity of VIRIP was further improved by a systematic analysis of the relationship between structure and activity. Residues critical for its activity were initially identified, and then a series of more than 600 chemically synthesized VIRIP analogs were analyzed. Several of these VIRIP derivatives containing intramolecular disulfide bonds and other modifications exhibited neutralizing activity toward HIV in vitro up to two orders of magnitude higher than that of VIRIP. Although the neutralizing activity of these VIRIP derivatives is comparable to that of T20, T20 inhibits cell fusion at significantly lower concentrations than these peptides. What causes these differences is not clear and may depend on the assay used and the isolate tested. Cell-to-cell transmission of HIV-1 is much more efficient than transmission by cell-free virus (Dimitrov et al., 1993Dimitrov D.S. Willey R.L. Sato H. Chang L.J. Blumenthal R. Martin M.A. J. Virol. 1993; 67: 2182-2190Crossref PubMed Google Scholar) and could be dominant in vivo. In contrast to other inhibitors including T20, HIV-1 was not able to generate resistant strains to one of the most extensively characterized VIRIP variants (VIR-576). If replicated in humans this property alone of VIR-576 would set it apart from existing inhibitors that affect other stages of the HIV-1 life cycle. Remarkably, the target for VIRIP turned out to be the fusion peptide region of HIV-1 gp41, a glycoprotein that forms a trimer with gp120. The identification of the hydrophobic fusion peptide as the target is surprising because its role in the fusion cascade leading to virus entry has been elusive (Gallo et al., 2003Gallo S.A. Finnegan C.M. Viard M. Raviv Y. Dimitrov A. Rawat S.S. Puri A. Durell S. Blumenthal R. Biochim. Biophys. Acta. 2003; 1614: 36-50Crossref PubMed Scopus (333) Google Scholar). Moreover, raising antibodies against this region has proven exceedingly difficult (C.C. Broder, personal communication). According to the “sticky finger” model, drawn in many cartoons of the HIV-1 fusion reaction, the fusion peptide inserts into the target cell following the change of conformation of gp120-gp41, called “triggering,” that occurs subsequent to binding of gp120 to host cell CD4 and coreceptors. Yet, if the fusion peptide is a target for VIRIP, which binds to the aqueous form of the fusion peptide, this poses a conundrum. This is due to the fact that gp41 may rapidly dip its sticky finger into the bilayer once the trigger has been pulled. Consequently, the sticky finger should avoid interference by VIRIP because the bilayer-inserted fusion peptide will no longer be available for binding to VIRIP. Indeed, attempts have been made to inhibit the HIV-1 gp41 fusion process after insertion of the fusion peptide into the membrane by designing membrane-anchored fusion peptide analogs (Gerber et al., 2004Gerber D. Pritsker M. Gunther-Ausborn S. Johnson B. Blumenthal R. Shai Y. J. Biol. Chem. 2004; 279: 48224-48230Crossref PubMed Scopus (28) Google Scholar). Exposing the sticky finger prior to triggering would have deleterious consequences for gp120-gp41 (such as aggregation). Because we do not have a high-resolution structure of the gp120-gp41 trimer we can only speculate about the disposition of the fusion peptide in the overall structure. One speculation is that after the gp120 interaction with CD4 and coreceptors the fusion peptide spends some time between the viral and cell membranes disrupting the water structure and the respective repulsive hydration forces, and this leads to spontaneous local fusion (Dimitrov, 1996Dimitrov D.S. Nat. Med. 1996; 2: 640-641Crossref PubMed Scopus (31) Google Scholar). Further studies addressing the timing and location of VIRIP action may not only uncover new features of how it inhibits HIV entry but may also shed light on the elusive role of the fusion peptide itself. Among the many implications of this study perhaps the most important and urgent question, especially for individuals carrying HIV-1 isolates resistant to other drugs, is whether VIRIP variants, such as VIR-576, have therapeutic potential. Although only clinical trials could definitely answer this question, one could speculate that such a possibility exists, with T20 providing an example of success. The unique mechanism of inhibition by VIRIP combined with a hopefully low level of resistant mutants holds promise for the use of this inhibitor in combination with other drugs. VIRIP and its variants are also the first HIV-1 fusion inhibitors identified by the systematic screening of naturally occurring mixtures of very high complexity. Even if the discovery of VIRIP does not lead to a new HIV-1 therapeutic, the methodology will undoubtedly have a profound impact on future research. Although labor intensive, the ability to screen human blood plasma and select peptides with predefined properties promises to yield new compounds of importance for other diseases. Finally, these results suggest yet another possible mechanism of resistance to HIV infection—the induction of large amounts of inhibitory peptides during inflammation. How significant this mechanism might be in delaying the progression of HIV disease is an intriguing question that remains to be answered. We thank Dr. Christopher Broder for helpful comments. The authors are supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research and by the NIH Intramural AIDS Targeted Antiviral Program (IATAP). Discovery and Optimization of a Natural HIV-1 Entry Inhibitor Targeting the gp41 Fusion PeptideMünch et al.CellApril 20, 2007In BriefA variety of molecules in human blood have been implicated in the inhibition of HIV-1. However, it remained elusive which circulating natural compounds are most effective in controlling viral replication in vivo. To identify natural HIV-1 inhibitors we screened a comprehensive peptide library generated from human hemofiltrate. The most potent fraction contained a 20-residue peptide, designated VIRUS-INHIBITORY PEPTIDE (VIRIP), corresponding to the C-proximal region of α1-antitrypsin, the most abundant circulating serine protease inhibitor. Full-Text PDF Open Archive