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We recently reported a β3-decapeptide, βWWI-1, that binds a validated gp41 model in vitro and inhibits gp41-mediated fusion in cell culture. Here we report six analogs of βWWI-1 containing a variety of non-natural side chains in place of the central tryptophan of the WWI-epitope. These analogs were compared on the basis of both gp41 affinity in vitro and fusion inibition in live, HIV-infected cells. One new β3-peptide, βWXI-a, offers a significantly improved CC50/EC50 ratio in the live cell assay.
Linear peptides derived from the C-terminus of HIV-1 gp41 (C-peptides) are potent HIV fusion inhibitors1. These molecules bind to the gp41 N-peptide region and inhibit an intramolecular protein-protein interaction that drives fusion of viral and host cell membranes2–4. Previous work has shown that the protein-protein interface consists of a highly conserved pocket on the N-peptide surface that is occupied by three C-peptide side chains: Trp-628, Trp-631 and Ile-6353–5. These three residues comprise the WWI epitope3–5. Simple6–9 and constrained10–13 α-peptides, aromatic foldamers14, peptide-small molecule conjugates15, and small molecules16, 17 that bind this N-peptide surface pocket inhibit gp41-mediated cell fusion with IC50 values ranging from 250 pM for α-peptides to 5 μM for small molecules. We previously reported a set of β3-decapeptides that present a WWI epitope on one face of a salt bridge18–21 and macrodipole-stabilized22 14-helix23, 24. One of these molecules, βWWI-1, binds a validated gp41 model in vitro and inhibits gp41-mediated fusion in cell culture25. Past work by Chan and co-workers6 demonstrated the importance of the three epitope residues, particularly the central tryptophan, in both gp41 affinity and viral infectivity. Here we report six analogs of βWWI-1 containing a variety of nonnatural side chains in place of the central tryptophan of the WWI-epitope. These analogs were compared on the basis of both gp41 affinity in vitro and fusion inibition in live, HIV-infected cells. One new β3-peptide, βWXI-a, offers a significantly improved CC50/EC50 ratio in the live cell assay.
We synthesized a small collection of β3-decapeptides (βWXI-a—f) containing a variety of nonnatural side chains in place of the central tryptophan of the WWI-epitope (Figure 1). These nonnatural residues included those with both entended or alternative π-systems (βWXI-b,d) and halogen-substituted aromatic rings (βWXI-a,c,e,f) to probe the steric and electronic requirements of the N-peptide surface pocket in the context of a β3-peptide. βWWI-1, a previously described β-peptide HIV fusion inhibitor25, was synthesized as a positive control.
Each β-peptide was labeled at the N-terminus with 6-(fluorescein-5(6)-carboxamido) hexanoic acid N-hydroxy-succinimidyl ester (Flu) and employed in a direct fluorescence polarization (FP) assay to determine its affinity for IQN17, a fusion protein containing 17-residues from the gp41 N-terminus joined to a 29 residue isoleucine zipper10. IQN17 exists as a stable trimer in solution10 and effectively recreates the N-peptide surface pocket for C-peptide-like ligands. β-peptides βWXI-a-fFlu bound IQN17 with equilibrium dissociation constants between 12.1 μM (βWXI-d) and 105.4 mM (βWXI-b) (Table 1, Figure 1A). With the exception of pyridyl-containing βWXI-b, all new β-peptides bound IQN17 about as well as βWWI-1 (KD = 16.5 ± 0.6 μM). These results are significant if not surprising, given the loss off affinity that typically results from altering the central tryptophan residue6, 25.
All seven β-peptides were evaluated for the ability to promote cell survival in an MTT colorimetric assay26, 27. In this method, MT-2 human T-cells are plated with varying concentrations of β-peptide inhibitor and cultured with wild-type HIV-1 IIIB28–30. After 5 days incubation, the number of live cells that remain is determined by addition of (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). MTT is reduced in the mitochondria of live cells to formazan (λmax = 595 nm) and quantified by UV. The EC50 values reported represent the β-peptide concentration required to achieve 50% survival of infected cells (Figure 2; Table 1).
The EC50 values of β-peptides βWXI-a through f vary between 8.2 μM (βWXI-d) and > 250 μM (βWXI-b). With the exception of βWXI-b, which is inactive (EC50> 250 μM), all of the new β-peptides (8.2 μM ≤ EC50 ≤ 19 μM) are more potent than βWWI-1 (EC50 = 56 μM) at promoting the survival of HIV-infected cells. Interestingly, two of the most potent new β-peptides (βWXI-c and f) share little structural similarity, with halogen substituents at para- and ortho- positions, respectively. βWXI-a and e, with EC50 = 18–19 μM, share a fluorine-containing substituent at the meta position of the phenyl side chain.
We also compared the new β-peptides in terms of cytotoxicity, determined as the viability of uninfected cells in the presence of inhibitor alone (Figure S1, Table 1). The CC50 values reported represent the β-peptide concentration required to inhibit MT-2 cell growth by 50%. CC50 values range from 31 μM (βWXI-f) to > 250 μM (βWXI-b), with a value of 100 μM for βWWI-1. Interestingly, although βWXI-d and f are characterized by the lowest EC50 values, each was cytotoxic at concentrations close to this value, with CC50/EC50 ratios less than 4. Importantly, one new β-peptide, βWXI-a, exhibits an CC50/EC50 ratio of 8, representing a significant improvement relative to βWXI-1 as well as βWXI-c-f.
The ability of βWXI-a to bind IQN17 and inhibit fusion in the MTT assay may be partially rationalized by a simple model in which the indole side chain of the central tryptophan is replaced by the central aromatic side chains of our β-peptides (Figure 3). A crystal structure of the gp41 fusion peptide solved by Sia et. al.11 depicts the epitope-containing β-peptide C14linkmid bound to IQN17 and clearly shows association between the indole side chain and the N-peptide surface pocket. Substitution of the Trp indole ring of C14linkmid with the m-trifluoromethylphenyl side chain in βWXI-a suggests that the trifluoro-methylbenzene side chain is a reasonable structural mimic of the indole ring, whereas the 3-pyridyl side chain is not. Although βWXI-a is not as potent as Fuzeon in the MTT assay (EC50 = 37.5 nM), it has a significantly lower mass (1457 Da vs. 4492 for Fuzeon), and higher metabolic and proteolytic stablity31–35. Furthermore, due to the ability of the 14-helical scaffold to tolerate changes to the epitope face, it may be possible to identify β3-peptides with further improved activity and decreased toxicity through combinatorial optimization36, 37.
This work was partially supported by NIH grant GM49551 (KSA) and a research award from Bristol-Myers Squibb (ADB). ADB thanks EJP and JSA for helpful discussion.
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