**5.2 Small molecules**

56 Biochemistry

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

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

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

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

orally bioavailable and also provide clues for small molecule fusion inhibitor design.

therapeutic potential of natural bioactive polypeptides.

**5. Small molecule helix mimetics** 

fusion inhibitor design and development.

**5.1 Small molecule-peptide conjugates** 

A small molecule α-helical mimetic based on a substituted terphenyl scaffold was designed to inhibit the assembly of the 6-HB core (Ernst et al. 2002). Tris-functionalized 3,2',2'' terphenyl derivatives can serve as effective mimics of the surface functionality projected along one face of an α-helix. Compound 1a (**48**) was designed to mimic the side chains of an *i*, *i*+4, *i*+7 hydrophobic surface in an α-helix, using the branched alkyl substituents isobutyl and isopropyl (to avoid complications from chirality in a sec-butyl group) to mimic the side chains of the most prevalent Leu and Ile in the *a* and *d* positions of a 3-4 heptad repeat. Terminal carboxylate groups were also added to mimic the anionic character of the Cpeptide and to improve the aqueous solubility. The ability of 1a (**48**) to disrupt the gp41 core was studied by CD spectroscopy, using a N36/C34 6-HB model (*Tm* = 66 °C). Titration of 1a into a 10 μM solution of N36/C34 resulted in a decrease of the CD signal at 222 and 208 nm, which corresponds to a reduction in the helicity of the 6-HB. A plot of θ222 versus inhibitor concentration shows saturation at approximately three equivalents of 1a. The CD spectrum with excess 1a was similar to the theoretical addition of the individual N36 and C34 spectra at the same concentration; and the thermal denaturation curve of the gp41 core in the presence of 50 μM 1a shows a significant 18 °C drop in the *Tm* value and closely resembles the melting transition of N36 alone at the same concentration. These data suggest that the 6- HB structure is completely disrupted by helix mimetic 1a. Both the hydrophobic and electrostatic features of 1a are important for its ability to disrupt the bundle. Analogs lacking the key alkyl side chains or carboxylic groups have little effect on the CD spectrum of the protein, even at high concentrations. Mimetic 1a effectively disrupts N36/C34 complexation with an IC50 value of 13.18 ± 2.54 µg.mL-1, as measured by an ELISA assay using NC-1 (Jiang et al. 1998). Compound 1a inhibits HIV-1 mediated cell-to-cell fusion with an IC50 value of 15.70 ± 1.30 μg.mL-1, using a dye-transfer cell fusion assay. In comparison, analogs lacking hydrophobic side chains or carboxylic groups had no inhibitory activity and proved to be cytotoxic at similar concentrations. Compound 1b (**49**), with larger hydrophobic groups than 1a, showed marginally enhanced activity than 1a.

Cai and Gochin identified a set of small molecule fusion inhibitors from a peptidomimetic library using a fluorescent biochemistry assay using Env2.0 as the target (Cai & Gochin 2007). Compounds **54** [3,5] and **55** [6,11] showed Ki values of 1.51 ± 0.16 and 1.34 ± 0.19 μM, respectively, in a competitive binding assay using Env2.0 as the target; and an IC50 value of ~8 μM in an HIV-1 gp41 mediated cell-cell fusion assay using a CCR5/CXR4 dual dependent target cell line (JI et al. 2006). These compounds contain two units that are covalently linked by an amide bond, and each unit contains two aromatic rings that may bind into the gp41 NHR hydrophobic pocket. A carboxyl group provides electrostatic interaction with K574 in the binding pocket and is critical for the activity of these small molecules; methylation of the carboxyl group resulted in loss of activities of the compounds in both the biochemical assay and cell-cell fusion assay. Three-unit compounds are prone to form aggregates under the assay conditions used and showed no activity, while single-unit compounds, such as M1 (**56**), display submillimolar inhibitory activity (Cai et al. 2009). Compound 1 (**57**), based on M1, was developed, which displayed an IC50 value of 4.5 ± 0.5 and 3.2 ± 0.5 μM in a fluorescence biochemical assay and a cell-cell fusion assay, respectively (Zhou et al. 2010). Compound 1 (**57**) showed very low cytotoxcity (IC50 > 500 μM); with a relatively small size, it is a promising lead for fusion inhibitor design.

Others have reported well-characterized small molecule fusion inhibitors targeting gp41, including SDS-J1 (**50**) (Debnath et al. 1999), NB64 (**51**), NB2 (**52**) (Jiang et al. 2004), and 4M041 (**53**) (Frey et al. 2006). These fusion inhibitors were selected from an active compound library by visual screening, then identified by high-throughput screening, and finally verified by a cell-cell fusion assay or HIV-1 infection assay. They usually showed low micromolar IC50 values for fusion inhibition; however, the following work to optimize the structures to obtain more potent fusion inhibitors were less fruitful, resulting in the identification of more small molecules with similar activity (Jiang et al. 2011). Also, their exact binding model with the gp41 NHR deep pocket still needs to be verified.

Fig. 8. Small molecule fusion inhibitors.

#### **6. Conclusion**

Peptides and peptidomimetics are efficient tools to study the HIV-1 gp41 NHR-CHR interaction, a key protein-protein interaction for HIV-1 gp41 mediated virus-cell membrane fusion, which enables HIV-1 enters and ultimately infects host cells. Peptides derived from wild-type HIV-1 gp41 sequences have been used to model the HIV-1 gp41 fusogenic core, a 6-HB formed by the NHR trimer as the inner core, and anti-parallel bind with three CHRs. Crystallographic structure analysis of the 6-HB has uncovered structure details for the gp41 NHR-CHR interaction. A deep pocket in the surface of NHR is a hot spot for the NHR-CHR interaction and a potential target for small molecule fusion inhibitors. N-peptides can be efficient targets for screening fusion inhibitors targeting the gp41 deep pocket by adding structural modulators to promote the trimeration of N-peptide.

Natural C-peptides can efficiently inhibit the gp41 NHR-CHR interaction by interacting with their counterpart in the gp41 6-HB; therefore, they can be used as fusion inhibitors against HIV-1 ENV mediated virus-cell fusion. They use residues at the *a* and *d* positions in heptad registration to bind the NHR hydrophobic grooves. The WWI motif of C-peptide provides a critical interaction with the NHR deep pocket, and an additional interaction between the C-peptide and NHR groove is required for a highly potent peptide fusion inhibitor of 30–40 residues. The *b*, *c*, *f*, and *g* residues in the C-peptide that form the predominantly hydrophilic surface in the 6-HB can be modified for increasing the secondary structure and solubility of the C-peptide, in order to increase its anti-HIV potency. The knowledge gained has been tested by artificial design of highly potent peptide fusion inhibitors with few similarities from known peptide sequences.

Peptidomimetics using unnatural building blocks have been successfully employed to mimic the molecular structures involved in the gp41 NHR-CHR interaction, resulting in highly potent HIV-1 fusion inhibitors with extraordinary *in vivo* stability to overcome the weakness of peptide drugs with potential oral administration possibilities. The achievements of the high potency peptidomimetic fusion inhibitors can also be used to guide small molecule fusion inhibitor design to disrupt this important protein-protein interaction.

In summary, HIV-1 fusion inhibitor development provides a model for using peptides as tools to probe protein-protein interactions for small molecule PPII design and development. The methods and results described in this chapter not only provide clues for future HIV-1 fusion inhibitor design, but also can be used for other viruses using a familiar virus-cell membrane fusion mechanism, as well as to guide other PPII design and development.
