Current Topics in Medicinal Chemistry - Volume 11, Issue 24, 2011

The fusion-inducing conformational switch in the extracellular domain of HIV-1 transmembrane protein gp41 began to be elucidated in 1993, through the discovery of highly potent antiviral activity of C-peptides derived from the extracellular domain. In the subsequently determined three dimensional structure, an N-terminal coiled coil core (NHR) anchors three antiparallel C-terminal heptad repeat (CHR) helices in a six-helix bundle (6HB) post-fusion conformation. In 1999, a hydrophobic pocket was identified as a potential hotspot for inhibiting the protein-protein interaction. Since then, numerous studies have focused on peptide and small molecule intervention of HIV-1 fusion. To date, 34 to 36-residue C-peptides with high potency have been developed, and multiple studies have shown that peptide activity could be enhanced by modifications which increased amphiphilic properties, including helical content and salt-bridges on the outer surface. However, there has been very little progress in the development of small molecule alternatives. Currently, only μM activity of small molecules has been achieved. More recently, details of the mechanism of action of 36-residue peptide T20, the sole FDA approved fusion inhibitor, have revealed a critical role for the hydrophobic C-terminal segment in associating with the membrane (and helping to form the fusion pore.) Other studies have shown that the N-terminal coiled coil is itself subject to lipophilic interactions and both C- and N-peptides show enhanced activity if conjugated to lipophilic groups. These studies and others have resulted in novel insights into the mechanism by which gp41 facilitates viral-cell and cell-cell fusion. This issue of Current Topics of Medicinal Chemistry is devoted to reviewing the interaction of gp41 with lipids and model membranes and their role in the transition from the pre- to post-fusion conformation. Structural studies reveal the variability of fusion peptide (FP), membrane-proximal external region (MPER) and NHR structures in different membrane-mimetic environments that most likely play a critical role in the conformational changes associated with gp41 function. The first review from Dr. Robert Blumenthal's group lays the biological framework of the fusion process, and defines the role of gp41 in the processes of not only viral entry, but also bystander apoptosis and HIV pathogenesis. Garg et al have deciphered the important role of gp41 in cellular membrane fusion and its relationship to viral fusion and the spread of infection. The review explores the specific roles of gp41 amphiphilic and lipophilic domains in the process of hemifusion, whereby viral induced apoptosis occurs. They further relate the specific effects of mutations arising from T20 treatment in pathogenicity. Dr. Lifeng Cai's team provides a thorough review of the biochemical and biophysical methods used to study gp41 - lipid interactions, and summarizes the results that have been obtained. The review includes a discussion of gp41 domain and membrane mimetic systems. Each spectroscopic technique is described in terms of the information that can be gleaned from the results. Examples include fluorescence for detection of peptide binding, lipid mixing and fusion, CD and IR for gleaning secondary structure information, NMR and EPR for site specific peptide structure and orientation information, NMR and IR for lipid order, among others. The review goes on to describe the application of these techniques to FP, NHR/CHR, MPER and loop domains of gp41, and paints an intriguing picture of the properties of the various functional domains, while also revealing the complexities and variability in the experimental systems. Dr. Jose Nieva and colleagues consider the role of membrane-transferring peptides from the fusion peptide (FP) domain and membrane-proximal external region (MPER) in contributing to the stability of the six-helix bundle and the fusion pore. They stress the FP as a therapeutic target, reviewing inhibition by oligopeptides optimized for their FP-binding and anti-hemolytic properties, and discussing studies that probe the mechanism by which fusion proceeds past the initial bundle formation to pore formation. The review extends into consideration of peptides derived from the MPER as fusion inhibitors, by virtue of their homo-oligomerization properties, and as immunogens, with consideration of their structure and orientation in the membrane....

HIV gp41 is a metastable protein whose native conformation is maintained in the form of a heterodimer with gp120. The non-covalently associated gp41/gp120 complex forms a trimer on the virus surface. As gp120 engages with HIV's receptor, CD4, and coreceptor, CXCR4 or CCR5, gp41 undergoes several conformational changes resulting in fusion between the viral and cellular membranes. Several lipophilic and amphiphilic domains have been shown to be critical in that process. While the obvious function of gp41 in viral entry is well-established its role in cellular membrane fusion and the link with pathogenesis are only now beginning to appear. Recent targeting of gp41 via fusion inhibitors has revealed an important role of this protein not only in viral entry but also in bystander apoptosis and HIV pathogenesis. Studies by our group and others have shown that the phenomenon of gp41-mediated hemifusion initiates apoptosis in bystander cells and correlates with virus pathogenesis. More interestingly, recent clinical evidence suggests that gp41 mutants arising after Enfuvirtide therapy are associated with CD4 cell increase and immunological benefits. This has in turn been correlated to a decrease in bystander apoptosis in our in vitro as well as in vivo assays. Although a great deal of work has been done to unravel HIV-1 gp41-mediated fusion mechanisms, the factors that regulate gp41-mediated fusion versus hemifusion and the mechanism by which hemifusion initiates bystander apoptosis are not fully understood. Further insight into these issues will open new avenues for drug development making gp41 a critical anti-HIV target both for neutralization and virus attenuation.

Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes ∼2 millions death every year and still defies an effective vaccine. HIV-1 infects host cells through envelope protein - mediated virus-cell fusion. The transmembrane subunit of envelope protein, gp41, is the molecular machinery which facilitates fusion. Its ectodomain contains several distinguishing functional domains, fusion peptide (FP), Nterminal heptad repeat (NHR), C-terminal heptad repeat (CHR) and membrane proximal extracellular region (MPER). During the fusion process, FP inserts into the host cell membrane, and an extended gp41 prehairpin conformation bridges the viral and cell membranes through MPER and FP respectively. Subsequent conformational change of the unstable prehairpin results in a coiled-coil 6-helix bundle (6HB) structure formed between NHR and CHR. The energetics of 6HB formation drives membrane apposition and fusion. Drugs targeting gp41 functional domains to prevent 6HB formation inhibit HIV-1 infection. T20 (enfuvirtide, Fuzeon) was approved by the US FDA in 2003 as the first fusion inhibitor. It is a 36-residue peptide from the gp41 CHR, and it inhibits 6HB formation by targeting NHR and lipids. Development of new fusion inhibitors, especially small molecule drugs, is encouraged to overcome the shortcomings of T20 as a peptide drug. Hydrophobic characteristics and membrane association are critical for gp41 function and mechanism of action. Research in gp41-membrane interactions, using peptides corresponding to specific functional domains, or constructs including several interactive domains, are reviewed here to get a better understanding of gp41 mediated virus-cell fusion that can inform or guide the design of new HIV-1 fusion inhibitors.

The fusogenic function of HIV-1 gp41 transmembrane Env subunit relies on two different kinds of structural elements: i) a collapsible ectodomain structure (the hairpin or six-helix bundle) that opens and closes, and ii) two membrane- transferring regions (MTRs), the fusion peptide (FP) and the membrane-proximal external region (MPER), which ensure coupling of hairpin closure to apposition and fusion of cell and viral membranes. The isolation of naturally produced short peptides and neutralizing IgG-s, that interact with FP and MPER, respectively, and block viral infection, suggests that these conserved regions might represent useful targets for clinical intervention. Furthermore, MTR-derived peptides have been shown to be membrane-active. Here, it is discussed the potential use of these molecules and how the analysis of their membrane activity in vitro could contribute to the development of HIV fusion inhibitors and effective immunogens.

HIV-1 envelope glycoprotein (Env) spikes are supported at the viral membrane interface by a highly conserved and hydrophobic region of gp41, designated the membrane-proximal external region (MPER). The MPER is mandatory for infection of host cells by HIV-1, and is the target of some of the most broadly neutralizing antibodies described to date. As such, the MPER is also of considerable interest for HIV vaccine design. However, structural models indicate that the MPER assumes distinct conformations prior to and leading up to Env-mediated fusion. Thus, the more of these distinct conformations that antibodies and inhibitors can recognize will likely be the better for antiviral potency. In addition to its flexibility, the MPER is lipophilic and its accessibility to bulky macromolecules is limited by steric and kinetic blocks that present particular challenges for eliciting HIV-1 neutralizing antibodies. Moreover, the ability of the MPER and viral membrane to combine as a complex has critical mechanistic implications for molecules that target lipid-bound and/or unbound states. Interestingly, membrane affinity frequently appears to enhance the potency of both fusion inhibitors and antibodies to different sites on gp41. We therefore highlight mechanisms to be harnessed in targeting membraneproximal sites on HIV gp41 for both vaccine and fusion inhibitor design. Such design efforts will likely need to draw upon knowledge of MPER structure and function, and may in turn inform analogous approaches to MPERs of other enveloped viruses and systems.

Small molecule inhibition of HIV fusion has been an elusive goal, despite years of effort by both pharmaceutical and academic laboratories. In this review, we will discuss the amphipathic properties of both peptide and small molecule inhibitors of gp41-mediated fusion. Many of the peptides and small molecules that have been developed target a large hydrophobic pocket situated within the grooves of the coiled coil, a potential hotspot for inhibiting the trimer of hairpin formation that accompanies fusion. Peptide studies reveal molecular properties required for effective inhibition, including elongated structure and lipophilic or amphiphilic nature. The characteristics of peptides that bind in this pocket provide features that should be considered in small molecule development. Additionally, a novel site for small molecule inhibition of fusion has recently been suggested, involving residues of the loop and fusion peptide. We will review the small molecule structures that have been developed, evidence pointing to their mechanism of action and strategies towards improving their affinity. The data points to the need for a strongly amphiphilic character of the inhibitors, possibly as a means to mediate the membrane - protein interaction that occurs in gp41 in addition to the protein - protein interaction that accompanies the fusion-activating conformational transition.