**4.2 Foldamers**

52 Biochemistry

stacking interactions with the pocket (IQN17-Trp571), the substitution of Leu for Ala in the C-terminal flank sequence causes it to be buried an additional 50 Å into the hydrophobic surface area of the pocket, and the new interactions with the flanking sequence do not perturb the pocket-binding structure of the core PIE7 residues. These differences may account for the improved activity of PIE12 over PIE7. CD thermal denaturation showed that the PIE12-trimer forms the more stable complex with IZN17 with a *Tm* of 81 °C in 2 M Guanidine chloride (Gua.HCl), 8 °C higher than that of the PIE7-trimer complex. The anti-HIV-1 breadths of the PIE7-trimer, PIE12-trimer, and PIE12 were tested by a pseudovirion assay against a panel of 23 pseudotyped viruses representing clades A to D, several CRFs, and enfuvirtide-resistant strains. Both PIE7 and PIE12-trimers potently inhibited all strains

Fig. 7. Peptidomimetics used to disrupt the HIV-1 gp41 NHR-CHR interaction. (A) Dpeptides; (B) β-foldamers; (C) α/β-foldamers; (D)/(E) linked peptides; (F) stapled peptides.

Viral passage studies were conducted to select for resistant strains. A strain bearing E560K/V570I mutations, which conferred a 400-fold resistance to PIE7-dimer, was selected with 20 weeks of propagation. These mutations dramatically weaken the binding of Dpeptides to the gp41 pocket but not the C-peptide inhibitor C37. Despite this loss of affinity, the escape mutations had a minimal effect on the potencies of PIE12-dimer and PIE12 trimer. PIE12-dimer and PIE12-trimer resistant virus were identified after 40 and 65 weeks of propagation, respectively, using a much slower escalation strategy; only a Q577R single

tested, though PIE12-trimer was generally a superior inhibitor.

A foldamer is a discrete chain molecule or oligomer that adopts a secondary structure stabilized by noncovalent interactions. Foldamers use unnatural building blocks instead of natural amino acids or nucleotides; as a result, they are more resistant to enzymatic degradation and show enhanced *in vivo* stability. They can mimic the ability of proteins, nucleic acids, and polysaccharides to fold into well-defined conformations, such as helices and β-sheets.

Short β3-foldamers have been designed that mimic the WWI motif of C-peptide fusion inhibitors (Stephens et al. 2005). A β-amino acid contains an additional methylene unit between the amine and carboxylic acid (Fig. 7c), and the amide bonds in β-peptides can resist *in vivo* proteolysis. A set of *β*3-decapeptides, *β-*WWI-1-4 (**26-29**), in which the WWI motif is presented on one face of a short 1,4-helix (Fig. 7b), were designed. Each *β*-peptide was fluorescently labeled at the N-terminus and was used in direct fluorescence polarization experiments to determine its binding affinity to IZN17. *β-*WWI-1-4-Flu bound IZN17 well, with equilibrium affinities of 0.75 ± 0.1, 1.0 ± 0.3, 2.4 ± 0.7, and 1.5 ± 0.4 μM, respectively. A WWI-1 analog *β*-WAI-1-Flu (**30**), containing Ala in place of the central Trp of the WWI motif, bound IZN17 with lower affinity (*K*d > 20 μM), suggesting the WWI motif is critical for pocket binding. The binding affinities are consistent with the cell-cell fusion assay results; *β*-WWI-1-4 inhibited cell-cell fusion with EC50 values of 27 ± 2.5, 15 ± 1.6, 13 ± 1.9, and 5.3 ± 0.5 μM, respectively, whereas *β*-WAI-1 was inactive under the same conditions.

In a follow-up study (Bautista et al. 2009), the second Trp in *β*-W**W**I-4 (**29**) was replaced with unnatural residues to probe steric and electronic effects on the NHR deep pocket binding. Most of the new *β*-peptides (EC50 8.2–19 μM) are more potent than *β*WWI-1 (**26**) (EC50 = 56 μM) at promoting the survival of HIV-infected cells. However, high cytotoxicities, with a selective index (CC50/EC50) <10, render these short β-peptides unsuitable as drug leads.

Αn α/β foldamer with partial β-amino acid replacement was used to modify a highly potent C-peptide fusion inhibitor to increase its *in vivo* stability (Horne et al. 2009). A two-stage design strategy was employed to modify T2635 (**31**), a highly potent third generation peptide fusion inhibitor (Dwyer et al. 2007). A fluorescent polarization binding assay using 5-helix as the target, a cell-cell fusion assay, and a protease K assay were used to assess the peptide and designed foldamer. In the first stage, one amino acid residue in each α-helix turn at the same position was replaced by a β-amino acid. The optimized α/β foldamer (**32**), containing systematic β-amino acid substitutions at positions *c* and *f*, showed weak binding affinity (Ki = 3800 nM) and cell-cell fusion inhibitory activity (IC50 = 390 nM), compared with those of T2635 (Ki < 0.2 nM and IC50 = 9 nM). However, **32** showed a 20-fold increase in half-life (14 min) in the protease K assay, compared with that for T2635. The β-amino acid, with one additional methylene unit, may make the backbone of the α/β foldamer too flexible to adapt a suitable conformation and results in the loss of activity. Accordingly, in the second stage of design, β-amino acids at certain positions were replaced with cyclic βamino acids to restore the rigidity of the backbone. The resulting α/β foldamer (**33**) had an IC50 value of 5 nM in the cell-cell fusion assay, similar to that of T2635; its binding affinity to 5-helix was 9 nM, similar to its cell-cell fusion inhibitory potency, despite its significantly weaker binding affinity to 5-helix than T2635. The α/β foldamer (**33**) showed a half-life of 200 min in the protease K assay, a 280-fold increase from T2635. The anti-HIV-1 infection activity of the α/β foldamer was also similar to T2635, as measured by HIV-1 infection of TZM-bl (JC53BL) cells using both R5 and X4 HIV-1 strains.

X-ray crystallography was used to characterize the structures of N36/T2635 and N36/**33** complexes. The N36/T2635 6-HB structure is almost identical to that of the wild-type N36/C34 6-HB, with a rmsd of 0.73 Å for the Cα atoms. However, the N36/**33** complex showed large structure distortion in the N-terminus (4.2 Å Cα rmsd for residues 2–15); the side chains of Trp3 and Trp5 were not resolved in electron density, suggesting a high degree of disorder, indicating that the N-terminal segment of **33** does not engage the NHR binding pocket in the complex. However, removal of the first ten residues of **33**, where the WWI motif is located, causes the loss of binding to 5-helix (*K*i >10 μM), indicating that the N-terminal segment of **33** is essential for high-affinity 5-helix binding. The N36/**34**  complex, maintaining the intact WWI motif in the foldamer sequence (**8**), was crystallized and resolved to 2.8 Å resolution. Relative to **33**, **34** tracks much more closely with T2635, with a 1.4 Å Cα rmsd for residues 2–33 between the two structures. The side chains of the WWI motif in the N-terminal segment of **34** show the expected packing into the binding pocket on the NHR core trimer. The above results suggest that the lack of direct contact between the N-terminal portion of **33** and the NHR trimer in the N36/**33** complex may be an artifact of crystal packing.

#### **4.3 Covalent-linked constrained peptides**

Helical structure is critical for C-peptide fusion inhibitors to make proper contacts with the NHR binding sites to elicit potent inhibition. Constraining methods that add structural constraints into the peptide sequence by covalently cross-linking amino acid residues at suitable positions can promote the formation of the α-helical conformation, even in short peptides. The covalent linker can be a longer linker between the *i* and *i* + 7 residues, or a short linker called a stapler between the *i* and *i* + 4 residues.

The first selected gp41 C-peptide was truncated T20 that lacks the LBD sequence, called HIV35 (gp41638-665, **35**) (Judice et al. 1997). A covalent cross-linker between the *i* and *i* + 7 residues of the polypeptide chain locks the intervening residues into a α-helical conformation. Residues at adjacent *f* positions on the opposite face of the helix were selected for cross-linking to enforce the residues at positions *a* and *d* to adopt a suitable conformation for target binding. Analogs of HIV35 were prepared containing either one, HIV24 (**36**), or two, HIV31 (**37**), tethers to impart increasing helicity. A control peptide, HIV30 (**38**), was prepared in which a tether was introduced between successive *d* residues to stabilize the helicity while blocking potential binding interactions across the *a-d* face. HIV24 and HIV30 were partially α-helical as measured by CD. By contrast, the doubly constrained analog HIV31 was mostly α-helical. HIV35 showed very weak inhibitory activity against HIV-1 in primary infectivity assays by using peripheral blood mononuclear cells with the virus JRCSF, a nonsyncytium-inducing strain, and BZ167, a syncytium-inducing HIV-1 strain. Single restrained HIV24 is more potent than HIV35, partially restoring the inhibitory

amino acids to restore the rigidity of the backbone. The resulting α/β foldamer (**33**) had an IC50 value of 5 nM in the cell-cell fusion assay, similar to that of T2635; its binding affinity to 5-helix was 9 nM, similar to its cell-cell fusion inhibitory potency, despite its significantly weaker binding affinity to 5-helix than T2635. The α/β foldamer (**33**) showed a half-life of 200 min in the protease K assay, a 280-fold increase from T2635. The anti-HIV-1 infection activity of the α/β foldamer was also similar to T2635, as measured by HIV-1 infection of

X-ray crystallography was used to characterize the structures of N36/T2635 and N36/**33** complexes. The N36/T2635 6-HB structure is almost identical to that of the wild-type N36/C34 6-HB, with a rmsd of 0.73 Å for the Cα atoms. However, the N36/**33** complex showed large structure distortion in the N-terminus (4.2 Å Cα rmsd for residues 2–15); the side chains of Trp3 and Trp5 were not resolved in electron density, suggesting a high degree of disorder, indicating that the N-terminal segment of **33** does not engage the NHR binding pocket in the complex. However, removal of the first ten residues of **33**, where the WWI motif is located, causes the loss of binding to 5-helix (*K*i >10 μM), indicating that the N-terminal segment of **33** is essential for high-affinity 5-helix binding. The N36/**34**  complex, maintaining the intact WWI motif in the foldamer sequence (**8**), was crystallized and resolved to 2.8 Å resolution. Relative to **33**, **34** tracks much more closely with T2635, with a 1.4 Å Cα rmsd for residues 2–33 between the two structures. The side chains of the WWI motif in the N-terminal segment of **34** show the expected packing into the binding pocket on the NHR core trimer. The above results suggest that the lack of direct contact between the N-terminal portion of **33** and the NHR trimer in the N36/**33** complex may be

Helical structure is critical for C-peptide fusion inhibitors to make proper contacts with the NHR binding sites to elicit potent inhibition. Constraining methods that add structural constraints into the peptide sequence by covalently cross-linking amino acid residues at suitable positions can promote the formation of the α-helical conformation, even in short peptides. The covalent linker can be a longer linker between the *i* and *i* + 7 residues, or a

The first selected gp41 C-peptide was truncated T20 that lacks the LBD sequence, called HIV35 (gp41638-665, **35**) (Judice et al. 1997). A covalent cross-linker between the *i* and *i* + 7 residues of the polypeptide chain locks the intervening residues into a α-helical conformation. Residues at adjacent *f* positions on the opposite face of the helix were selected for cross-linking to enforce the residues at positions *a* and *d* to adopt a suitable conformation for target binding. Analogs of HIV35 were prepared containing either one, HIV24 (**36**), or two, HIV31 (**37**), tethers to impart increasing helicity. A control peptide, HIV30 (**38**), was prepared in which a tether was introduced between successive *d* residues to stabilize the helicity while blocking potential binding interactions across the *a-d* face. HIV24 and HIV30 were partially α-helical as measured by CD. By contrast, the doubly constrained analog HIV31 was mostly α-helical. HIV35 showed very weak inhibitory activity against HIV-1 in primary infectivity assays by using peripheral blood mononuclear cells with the virus JRCSF, a nonsyncytium-inducing strain, and BZ167, a syncytium-inducing HIV-1 strain. Single restrained HIV24 is more potent than HIV35, partially restoring the inhibitory

TZM-bl (JC53BL) cells using both R5 and X4 HIV-1 strains.

an artifact of crystal packing.

**4.3 Covalent-linked constrained peptides** 

short linker called a stapler between the *i* and *i* + 4 residues.

activity of T20. Doubly constrained HIV31 shows dramatically higher potency, and its activity was comparable with T20 in both HIV-1 infection assays.

In another report, a 14-residue C-peptide C14 (gp41626-639, **39**) was selected (Sia et al. 2002). A cell–cell fusion assay was used to evaluate the biological activity of the peptides. Two strategies were employed, substitution with 2-aminoisobutyric acid (Aib) or a diaminoalkane crosslinker, to stabilize the helical conformation of C14. Six peptides were designed and produced, C14linkmid (**41**) was the most potent inhibitor against syncytia formation (IC50 = 35 μM), followed by C14Aib (**40**) (IC50 = 144 μM). C14linkmid and C14Aib bind to IQN17 with a *K*d of 1.2 μM, respectively, as measured by ITC. The efficacy of the cross-linker on the inhibitory activities depends on its position in the peptide sequence, Nterminal cross-linked C14linkN does not inhibit cell–cell fusion, whereas the middle crosslinked C14linkmid inhibits cell–cell fusion at micromolar concentrations. The cell–cell fusion inhibitory activities of the peptides generally correlated with their NHR binding affinities, although the cell–cell fusion activities were consistently ~10-fold less potent than the Kd of NHR binding. Additional factors, other than binding affinity to the target, may be necessary for blocking viral entry. The crystal structure of the C14linkmid/IQN17 complex showed that C14linkmid binds to the gp41 hydrophobic pocket in essentially the same conformation as the pocket-binding region of C34, demonstrating that the crosslink imparts no detectable distortion on the backbone of the C14 peptide in the bound conformation.

Chemical staples have been used to fortify peptides to overcome the proteolytic shortcomings of highly potent peptide HIV fusion inhibitors as therapeutics. As an example, chemical staples were inserted at the N- or C-termini of T649v (**43**) by substituting (*S*)-2- (((9H-fluoren-9-yl)methoxy)carbonylamino)-2-methyl-hept-6-enoic acid at select (*i* and *i* + 4) positions, followed by ruthenium-catalyzed olefin metathesis (Bird et al. 2010). Sites for unnatural amino acid insertion were carefully selected to avoid disruption of the critical hydrophobic interface between NHR and CHR helices as delineated by the crystal structure of N36/C34. Three stapled peptides were designed by inserting single or double staples at selected positions. The activities of related peptides were measured using a luciferase-based HIV-1 infectivity assay, using viruses derived from HXBc2 and the neutralization-resistant primary R5 isolate, YU2. All of the peptides showed low nanomolar IC50 values against HXBc2 strains, suggesting that chemical modification in the stapled peptides does not disrupt its NHR interaction. Moreover, all of the stapled peptides showed higher inhibitory activities against drug resistant HIV-1 isolates, such as YU2 and the HIV-1 HXBc2 virus bearing the T20-resistant V38A/N42T or V38E/N42S double mutations in gp41 NHR, with a rank order of SAH-gp41626–662 (**46**) > **44** > **45** > T649v > enfuvirtide. SAH-gp41626–662 (**46**) displayed medium to low nanomolar IC50 values for all of the viruses tested, including T20 and the T649v-resistant YU2 isolate.

The pharmacokinetic properties of **44** were evaluated in a mouse model (Bird et al. 2010). The total body clearance of **44** (1.0 mL/min/kg) was 10-fold more slow than that of the unmodified T649v peptide (9.5 mL/min/kg). A proteolysis assay using both chymotrypsin and pepsin suggested that the striking protease resistance of stapled peptides is conferred by a combination of (1) decreased rate of proteolysis due to induction of α-helical structure and (2) complete blockage of peptidase cleavage at sites localized within or immediately adjacent to the (*i*, *i* + 4)-crosslinked segment. In addition, a pilot study was undertaken to compare the oral absorption of T649v and **44** using a mouse model. Measurable concentrations of the full-length peptide were found in plasma samples from all **44** treated animals after oral dosing, and the concentration was dose dependent; no T649v was detected in plasma under the same conditions. The hydrocarbon double-stapling confers striking protease resistance of the peptide fusion inhibitor, which translates into markedly improved pharmacokinetic properties, including oral absorption, thus unlocking the therapeutic potential of natural bioactive polypeptides.

In summary, highly potent peptidomimetic HIV-1 fusion inhibitors have been discovered based on peptide fusion inhibitors, including: D-peptide fusion inhibitors discovered by mirror-image phage display using a D-amino acid form of the HIV-1 gp41 target; foldamers constructed from highly potent C-peptide fusion inhibitors by proper substitution of selected residues with β-amino acid residues; and structurally constrained peptides by covalently linking two residues at the same positions in a helical turn to promote α-helical structure formation. More like small molecule drugs, these peptidomimetics are potentially orally bioavailable and also provide clues for small molecule fusion inhibitor design.

#### **5. Small molecule helix mimetics**

The ultimate goal for drug development is small molecule drugs; it is also the main challenge in PPII development. The NHR deep pocket is a hot spot for the NHR-CHR interaction; it has an internal volume of roughly 400 Å3, and could be filled by a molecule with a molecular weight of approximately 500 Da, raising the possibility that it could be targeted by small molecule drugs (Chan et al. 1997). Several groups have identified small molecules that show low micromolar inhibitory potency against HIV-1 ENV mediated cellcell fusion and virus infection (Debnath et al. 1999; Frey et al. 2006; Cai & Gochin 2007; Zhou et al. 2010), however no direct evidence supports that these small molecule fusion inhibitors bind to the deep pocket (Gochin & Cai 2009; Cai & Jiang 2010). Therefore, providing direct structural evidence that a small molecule can bind to the NHR deep pocket, so that a small molecule pharmacophore model can be deduced, is highly desired for small molecule HIV fusion inhibitor design and development.

#### **5.1 Small molecule-peptide conjugates**

To identify small molecule ligands that specifically bind to the gp41 NHR deep pocket, Harrison's group has synthesized a biased peptide conjugate library (Ferrer et al. 1999). It contained ~60,000 compounds and used three small molecule building blocks to replace the WWI motif in C-peptide and links to the same peptide sequence. The library was synthesized and screened against 5-helix (Weissenhorn et al. 1997) using an on-bead affinity-based assay. A small molecule moiety was identified, which sequentially contained cyclopentyl propionic acid–ε-glutamic acid–p-(N-carboxyethyl) aminomethyl benzoic acid (Fig. 8) (**47**). The moiety alone had no activity based on an HIV-1 ENV mediated cell-cell fusion assay. However, when conjugated to a 30-mer C-peptide C30 (gp41636-665) without the PBD sequence, the resulting conjugate peptide showed an IC50 value of 0.3 µM, which was 20-fold increase compared with the IC50 value of 7 µM for C30. The conjugated peptide still had a much lower potency than a 38-mer (gp41628-665) Cpeptide containing the PBD that showed an IC50 value of 3 nM. The conjugated peptide could form a stable complex with N-peptide, as shown by size exclusion chromatography and native N-PAGE. This indicated that the small molecule moiety could partially mimic the WWI motif of C-peptide to occupy the deep binding pocket of the NHR, while structure modification is needed to optimize the binding.

Crystallography was used to characterize the interaction between the conjugated peptide and gp41 NHR (Zhou et al. 2000). The full length of the non-peptide moiety is visible in electron density maps, but unexpectedly in two orientations, each with about 50% occupancy. The two binding modes share the same aminobenzoic acid position (F1) but diverge at the two more distal building blocks. Also, the electron density for the amino acid at the connection to the non-peptide moiety is poor, suggesting disorder in the peptide linkage to the non-peptide moiety.
