**2. Peptides as a model to build the HIV-1 gp41 fusion core**

HIV-1 uses an envelope protein (ENV) mediated virus-cell membrane fusion to enter host cells for infection (Eckert & Kim 2001). HIV-1 ENV is composed of noncovalently associated gp120/gp41 trimers that form spikes and decorate the viral surface, in which the metastable transmembrane subunit gp41 is sequestered by the cell surface subunit gp120. During HIV-1 infection, gp120 first interacts with the T-cell receptor CD4, ensuring the viruses approach the target cells; then, the coreceptor binding sites in gp120 are sequentially exposed and gp120-coreceptor (CCR5 or CXR4) binding follows (Fig. 1). The resulting dissociation of the gp120-gp41 complex and the release of the unstable gp41 subunit trigger virus-cell membrane fusion. First, gp41 inserts into the target cell membrane using its fusion peptide, resulting in a pre-hairpin intermediate (PHI) in which its C-terminus anchors to the viral membrane and its N-terminus inserts into the host cell membrane, bridging the viral and cellular membranes (Fig. 1). The gp41 PHI automatically undergoes structure rearrangement with its NHR and CHR folding towards each other to form the fusogenic 6-HB. The energetic 6-HB formation drives the juxtaposition of the viral and cellular membrane, and finally results in virus-cell membrane fusion (Fig. 1). Agents that target the presumed gp41 PHI to prevent fusogenic 6-HB formation can terminate the virus-cell membrane fusion processes and be used as fusion inhibitors for antiretroviral therapy (Cai & Jiang 2010).

with their counterparts in gp41 to prevent fusogenic 6-HB formation and inhibit HIV-1-cell membrane fusion, thus preventing HIV-1 infection and replication. T20 (Fuzeon, enfuvirtide), a 36-mer peptide from HIV-1 gp41 CHR, was approved by the USA FDA in 2003 as the first fusion inhibitor for salvage therapy in HIV/AIDS patients unresponsive to common antiretroviral therapy. Its application has been limited by i) the high cost of peptide synthesis, ii) rapid *in vivo* proteolysis, and iii) poor efficacy against emerging T20-resistant strains. These drawbacks have called for a new generation of fusion inhibitors with

In this chapter, we will focus on the development of HIV-1 fusion inhibitors, concentrating on C-peptide fusion inhibitors and their peptidomimetics, which have been used as probes and tools to elucidate gp41 NHR-CHR interactions for future fusion inhibitor design and improve, and in the long run, the development of small molecule inhibitors that can disrupt

HIV-1 uses an envelope protein (ENV) mediated virus-cell membrane fusion to enter host cells for infection (Eckert & Kim 2001). HIV-1 ENV is composed of noncovalently associated gp120/gp41 trimers that form spikes and decorate the viral surface, in which the metastable transmembrane subunit gp41 is sequestered by the cell surface subunit gp120. During HIV-1 infection, gp120 first interacts with the T-cell receptor CD4, ensuring the viruses approach the target cells; then, the coreceptor binding sites in gp120 are sequentially exposed and gp120-coreceptor (CCR5 or CXR4) binding follows (Fig. 1). The resulting dissociation of the gp120-gp41 complex and the release of the unstable gp41 subunit trigger virus-cell membrane fusion. First, gp41 inserts into the target cell membrane using its fusion peptide, resulting in a pre-hairpin intermediate (PHI) in which its C-terminus anchors to the viral membrane and its N-terminus inserts into the host cell membrane, bridging the viral and cellular membranes (Fig. 1). The gp41 PHI automatically undergoes structure rearrangement with its NHR and CHR folding towards each other to form the fusogenic 6-HB. The energetic 6-HB formation drives the juxtaposition of the viral and cellular membrane, and finally results in virus-cell membrane fusion (Fig. 1). Agents that target the presumed gp41 PHI to prevent fusogenic 6-HB formation can terminate the virus-cell membrane fusion processes and be used as

improved antiviral and pharmacokinetic profiles.

Fig. 1. HIV-1 gp41 mediated virus-cell membrane fusion.

**2. Peptides as a model to build the HIV-1 gp41 fusion core** 

fusion inhibitors for antiretroviral therapy (Cai & Jiang 2010).

this important protein-protein interaction.

The discovery of potent anti-HIV peptides from HIV-1 gp41 NHR and CHR sequences suggests that gp41 is a target for fusion inhibitors (Wild et al. 1992; Jiang et al. 1993; Wild et al. 1994); these exogenous HIV-1 gp41 peptides interact with their counterparts in the gp41 6-HB, forming an unproductive complex that prevents gp41 fusion core formation. During the membrane fusion process, HIV-1 gp41 progressively undergoes a conformational change, and the gp41 PHI target exists for only a couple of minutes and then rapidly folds into a 6-HB; therefore, gp41 and its ectodomain are not suitable targets for a fusion inhibitor. Efforts to obtain a whole structure of the gp41 ectodomain also have been unsuccessful. So, the identification of a stable target in the PHI or gp41 fusion core is necessary for understanding the mechanism of gp41 mediated virus-cell membrane fusion for fusion inhibitor design and development.

The HIV-1 gp41 fusogenic 6-HB core has been constructed using synthesized peptides from the related gp41 wild-type sequences. Typical resolved crystal structures of the 6-HB fusogenic core include the N36/C34 complex (Chan et al. 1997), the IQNgp41/C43 complex (Weissenhorn et al. 1997), and the N34(L6)C28 trimer (Tan et al. 1997). These crystal structures provide atomic resolution of the interactions between NHR and CHR, verifying that NHR and CHR can be both a target and ligand from which a pharmacophore model can be deduced for fusion inhibitor design and optimization.

The crystal structures show that a parallel coiled-coil trimerized NHR forms the interior core, which is antiparallel packed with three CHR helices, to form a 6-HB (Fig. 2a,2b) (Chan et al. 1997). In the NHR interior core, the N-peptide uses its amino acid residues at the *a* and *d* positions of the heptads for self trimerization to stabilize the core; while the *e* and *g* residues of two adjacent helices form three hydrophobic grooves along the whole NHR trimer, which serve as targets that interact with the *a* and *d* residues of the C-peptides. Each groove contains a particularly deep cavity: Val-570, Lys-574, and Gln-577 from the left N36 (gp41546-581) helix form the left side; Leu-568, Trp- 571, and Gly-572 from the right N36 helix form the right side; and Thr-569, Ile-573, and Leu-576 form the floor, resulting in a pocket of ~16 Å long, 7 Å wide, and 5–6 Å deep (Fig. 2d). With the exception of Ile-573, all of the residues forming the cavity are identical between HIV-1 and SIV. The NHR deep pocket accommodates three hydrophobic residues from the abutting C34 (gp41628-661) helix: Ile-635, Trp-631, and Trp-628 constitute a WWI motif (Fig. 2c). The interaction between the NHR pocket and the WWI motif is predominately hydrophobic. A salt bridge between Lys-574 of NHR and Asp-632 of CHR immediately to the left of the cavity is also important for the NHR-CHR interaction (Chan et al. 1997). In addition to be the main binding sites for the Cpeptide, the deep NHR pocket is also an attractive target for small molecule fusion inhibitors. Besides the deep pocket, the rest of the groove along the NHR helices also makes extensive contact with CHR, providing additional energy to stabilize the 6-HB. The N36/C34 complex shows striking structural similarity to the low-pH-induced conformation of the influenza HA2 subunit (TBHA2) and the TM subunit of Mo-MLV, both of which have been proposed to be in a fusogenic conformation, suggesting a common mechanism of virus-cell membrane fusion among enveloped viruses (Chan et al. 1997).

During 6-HB formation, NHR and CHR are mutual target and ligand, so either can be the target for fusion inhibitor design. In a 6-HB, NHRs form a trimerized interior core that contains three grooves, and each with a deep pocket, which is more like a target, especially for small molecule fusion inhibitors. An electrostatic potential map of the N36 coiled-coil trimer shows that its surface is largely uncharged; and the grooves that are the sites for C34 interaction are aligned with predominantly hydrophobic residues that would be expected to lead to aggregation upon exposure to solvent. In contrast, the N36/C34 complex shows a much more highly charged surface due to acidic residues on the outside of the C34 helices, resulting in greater solubility of the heterodimeric complex (Chan et al. 1997). As a result, Npeptides are prone to aggregate in the absence of C-peptides under physiological conditions. This also accounts for a much weaker inhibitory potency for N-peptides compared to Cpeptides, since they must form a stable discrete trimerized inner core to efficiently interact with the CHR. Thus, construction of a stable and soluble discrete trimerized gp41 NHR core as a target is important for fusion inhibitor design and development.

Fig. 2. Crystal structures of the HIV-1 gp41 fusion core. (A) 6-HB structure of the gp41 N36/C34 fusion core; (B) the top to bottom view of the N36/C34 6-HB structure (Chan et al. 1997); (C) the deep pocket in the NHR groove interacts with the WWI motif of CHR (Chan et al. 1998); (D) the NHR deep pocket.

The key for constructing an efficient NHR target is to promote trimerization of N-peptides without changing their native binding sites and conformation. Addition of physicochemical restraints in N-peptides has been shown to be an efficient way to construct a stable and discrete NHR trimer. Typical NHR constructs include: IQN17 (**3**) and IZN17 (**4**) (Eckert & Kim 2001), 5-helix (Root et al. 2001; Frey et al. 2006), and Env2.0 (**5**) and Env5.0 (**6**) (Cai & Gochin 2007; Cai et al. 2009). These stable NHR-trimers can be efficient targets for fusion inhibitor discovery and development. Through forming discrete and stable trimers, they are also highly potent HIV-1 fusion inhibitors by themselves. The sequences of the N-peptide targets are shown in Fig. 3.

IQN17/IZN17 (Fig. 4): A trimeric coiled-coil GCN4 isoleucine zipper was used to construct the first HIV-1 gp41 fusion core for an x-ray crystallographic study (Weissenhorn et al. 1997). IQN17 was constructed by fusing a modified GCN4-pIQI peptide sequence to the 17 mer N-peptide gp41565-581 (N17) that comprises the gp41 hydrophobic pocket (Eckert et al. 1999). The resulting peptide, IQN17, is a fully helical discrete trimer in solution, as determined by circular dichroism (CD) and sedimentation equilibrium experiments. The crystal structure of the IQN17/D10-p1 complex, a cyclic D-peptide fusion inhibitor, showed that the overall architecture of the HIV-1 gp41 hydrophobic pocket in the complex is almost

trimer shows that its surface is largely uncharged; and the grooves that are the sites for C34 interaction are aligned with predominantly hydrophobic residues that would be expected to lead to aggregation upon exposure to solvent. In contrast, the N36/C34 complex shows a much more highly charged surface due to acidic residues on the outside of the C34 helices, resulting in greater solubility of the heterodimeric complex (Chan et al. 1997). As a result, Npeptides are prone to aggregate in the absence of C-peptides under physiological conditions. This also accounts for a much weaker inhibitory potency for N-peptides compared to Cpeptides, since they must form a stable discrete trimerized inner core to efficiently interact with the CHR. Thus, construction of a stable and soluble discrete trimerized gp41 NHR core

Fig. 2. Crystal structures of the HIV-1 gp41 fusion core. (A) 6-HB structure of the gp41 N36/C34 fusion core; (B) the top to bottom view of the N36/C34 6-HB structure (Chan et al. 1997); (C) the deep pocket in the NHR groove interacts with the WWI motif of CHR (Chan et

The key for constructing an efficient NHR target is to promote trimerization of N-peptides without changing their native binding sites and conformation. Addition of physicochemical restraints in N-peptides has been shown to be an efficient way to construct a stable and discrete NHR trimer. Typical NHR constructs include: IQN17 (**3**) and IZN17 (**4**) (Eckert & Kim 2001), 5-helix (Root et al. 2001; Frey et al. 2006), and Env2.0 (**5**) and Env5.0 (**6**) (Cai & Gochin 2007; Cai et al. 2009). These stable NHR-trimers can be efficient targets for fusion inhibitor discovery and development. Through forming discrete and stable trimers, they are also highly potent HIV-1 fusion inhibitors by themselves. The sequences of the N-peptide

IQN17/IZN17 (Fig. 4): A trimeric coiled-coil GCN4 isoleucine zipper was used to construct the first HIV-1 gp41 fusion core for an x-ray crystallographic study (Weissenhorn et al. 1997). IQN17 was constructed by fusing a modified GCN4-pIQI peptide sequence to the 17 mer N-peptide gp41565-581 (N17) that comprises the gp41 hydrophobic pocket (Eckert et al. 1999). The resulting peptide, IQN17, is a fully helical discrete trimer in solution, as determined by circular dichroism (CD) and sedimentation equilibrium experiments. The crystal structure of the IQN17/D10-p1 complex, a cyclic D-peptide fusion inhibitor, showed that the overall architecture of the HIV-1 gp41 hydrophobic pocket in the complex is almost

al. 1998); (D) the NHR deep pocket.

targets are shown in Fig. 3.

as a target is important for fusion inhibitor design and development.

identical to that in the wild-type HIV-1 gp41 N36/C34 structure, with a Cα root mean square deviation (rmsd) of 0.65 Å. In follow-up studies, a new version, IZN17, was designed using the same strategy. IZN17 is more thermally stable than IQN17, with a *Tm* > 100 °C, compared with ~100 °C for IQN17; the enhancement of thermal stability was further confirmed by measuring the *Tm* in 2 M guanidine chloride, with a *Tm* of 66 °C and 74 °C for IQN17 and QZN17, respectively. IZN17 is also more soluble than IQN17 under physiological conditions (Eckert & Kim 2001). Both IQN17 and IZN17 were used as targets in a mirror-image phage display experiment to identify D-peptide fusion inhibitors (Eckert et al. 1999; Welch et al. 2007; Welch et al. 2010).


Fig. 3. NHR target sequences. The sequences and groups responsible for physicochemical constraint are shown in grey.

5-Helix (Fig. 4): 5-Helix was designed using the 6-HB as a motif (Root et al. 2001). In 5-helix, five of the six helices that make up the 6-HB core structure are connected by short peptide linkers. The 5-helix protein lacks a third C-peptide helix, and this vacancy is expected to create a high-affinity binding site for the gp41 CHR. Under physiological conditions, 5-helix is soluble and a well folded protein that adopts >95% helical content, as expected from the design, and is extremely stable. In addition, denaturation was not observed, even at 96 °C or in 8 M guanidine chloride. 5-Helix interacts strongly and specifically with C-peptides, inducing a helical conformation in the bound C-peptide as judged by CD. 5-Helix was successfully used as the target in a fluorescence polarization assay to identify small molecule fusion inhibitors (Frey et al. 2006).

Env2.0/Env5.0 (Fig. 4): A trivalent coordination metal complex was used to fortify the gp41 NHR trimer (Gochin et al. 2003). 5-Carboxy-2,2'-bipyridine (BPY) was attached to an Npeptide that contains a deep pocket. Addition of a metal ion such as Fe2+ or Ni2+ resulted in the formation of a tris-BPY metal complex, which stabilizes the coiled-coil structure. The resulting magenta Fe2+(BPY)3 complex solution was due to a Fe2+–BPY charge transfer band at 545 nm and confirmed Fe2+-BPY binding. The apo-Env2.0 displayed 40% α-helical structure that increased to 89% upon the addition of Fe2+ ions, as measured by CD. The integrity of the binding grooves in Fe2+(Env2.0)3 was confirmed by its efficient binding with a matched C-peptide, as shown by CD and NMR (Gochin et al. 2006). The 545 nm absorbance agrees well with the emission maxima of the fluorophores fluorescein and Lucifer yellow. Fluorescence quenching by fluorescence resonance energy transfer (FRET) should occur if the fluorophore is brought close to the Fe2+–BPY center. This enables direct determination of binding by using a fluorophore labeled C-peptide as the probe. Compounds which are able to bind to the NHR target and displace the probe can be measured with a competitive inhibition assay by following the recovery of probe fluorescence intensity (Cai & Gochin 2007). The BPY-metal complex FRET strategy is generally applicable to different interacting peptide pairs, as long as the two peptide sequences are matched. This has been confirmed by the development of a longer gp41 Npeptide/C-peptide pair, Env5.0 and CP5; the peptide pair showed nanomolar binding affinity and can be used to screen more potent fusion inhibitors. Env5.0 contains the whole groove, and it has been used to identify ligands that interact with the range of the groove outside of the deep pocket by designing suitable probes (Cai et al. 2009). In addition, Env2.0 has been successfully used as a target for a screening assay to identify small molecule fusion inhibitors (Cai & Gochin 2007; Zhou et al. 2010).

Fig. 4. Designed soluble and discrete NHR target.

In summary, peptides have been used to construct the HIV-1 gp41 fusion core, which is a 6- HB. Crystal structure analysis showed that the gp41 NHR trimer forms an interior core, which contains three hydrophobic grooves as the binding site for C-peptide. A deep pocket in the groove is a hot spot for the NHR-CHR interaction, and can be a target for small molecule fusion inhibitors. The NHR target can be constructed by adding physicochemical constraints in the N-peptides to promote the formation of a soluble and discrete NHR trimer, which can be used for screening HIV-1 fusion inhibitors targeting gp41 NHR or the deep pocket.
