Huihui Chong

Broad Antiviral Activity and Crystal Structure of HIV-1 Fusion Inhibitor Sifuvirtide

Background: Sifuvirtide (SFT) is an HIV peptide fusion inhibitor under phase II clinical trials. Results: Crystal structure of SFT complexed with gp41 NHR peptide and the potent activity of SFT against diverse HIV-1 variants were determined. Conclusion: Crystal structure fully supports earlier peptide design. Significance: Our data present important information for developing SFT for clinical use and for designing novel HIV fusion inhibitors. Sifuvirtide (SFT) is an electrostatically constrained α-helical peptide fusion inhibitor showing potent anti-HIV activity, good safety, and pharmacokinetic profiles, and it is currently under phase II clinical trials in China. In this study, we demonstrate its potent and broad anti-HIV activity by using diverse HIV-1 subtypes and variants, including subtypes A, B, and C that dominate the AIDS epidemic worldwide, and subtypes B′, CRF07_BC, and CRF01_AE recombinants that are currently circulating in China, and those possessing cross-resistance to the first and second generation fusion inhibitors. To elucidate its mechanism of action, we determined the crystal structure of SFT in complex with its target N-terminal heptad repeat region (NHR) peptide (N36), which fully supports our rational inhibitor design and reveals its key motifs and residues responsible for the stability and anti-HIV activity. As anticipated, SFT adopts fully helical conformation stabilized by the multiple engineered salt bridges. The designing of SFT also provide novel inter-helical salt bridges and hydrogen bonds that improve the affinity of SFT to NHR trimer. The extra serine residue and acetyl group stabilize α-helicity of the N-terminal portion of SFT, whereas Thr-119 serves to stabilize the hydrophobic NHR pocket. In addition, our structure demonstrates that the residues critical for drug resistance, located at positions 37, 38, 41, and 43 of NHR, are irreplaceable for maintaining the stable fusogenic six-helix bundle structure. Our data present important information for developing SFT for clinical use and for designing novel HIV fusion inhibitors.

Short-peptide fusion inhibitors with high potency against wild-type and enfuvirtide-resistant HIV-1

Peptides derived from the C‐terminal heptad repeat (C peptides) of HIV‐1 gp41 are potent inhibitors against virus entry. However, development of a short C peptide possessing high anti‐HIV potency is considered a daunting challenge. We recently discovered that the residues Met626 and Thr627 preceding the pocket‐binding domain of the C peptide adopt a unique M‐T hook structure that is crucial for the design of HIV‐1 fusion inhibitors. In this study, we first presented a proof‐of‐concept prototype that the M‐T hook residues can dramatically improve the antiviral activity and thermostability of a short C peptide. We then generated a 24‐mer peptide termed MT‐SC22EK by incorporating the M‐T hook structure to the N terminus of the poorly active short C peptide SC22EK. Amazingly, MT‐SC22EK inhibited HIV‐1‐mediated cell fusion and infection at a level comparable to C34, T1249, SC29EK, and sifuvirtide, and it was highly active against diverse HIV‐1 subtypes and variants, including those T20 (enfuvirtide) and SC29EK‐resistant viruses. The high‐resolution crystal structure of MT‐SC22EK reveals the N‐terminal M‐T hook conformation folded by incorporated Met626 and Thr627 and identifies the C‐terminal boundary critical for the anti‐HIV activity. Collectively, our studies provide new insights into the mechanisms of HIV‐1 fusion and its inhibition.—Chong, H., Yao, X., Qiu, Z., Sun, J., Zhang, M., Waltersperger, S., Wang, M., Liu, S.‐L., Cui, S., and He, Y. Short‐peptide fusion inhibitors with high potency against wild‐type and enfuvirtide‐resistant HIV‐1. FASEB J. 27, 1203–1213 (2013). www.fasebj.org

The M-T Hook Structure Is Critical for Design of HIV-1 Fusion Inhibitors

Background: We recently found that N-terminal residues Met-626 and Thr-627 of HIV-1 fusion inhibitor CP621-652 adopt a unique hook-like structure, termed the M-T hook. Results: The structure and function of the M-T hook have been characterized. Conclusion: The M-T hook is critical for the stability and antiviral activity of HIV-1 fusion inhibitors. Significance: Our data provide important information for designing novel HIV-1 fusion inhibitors. CP621-652 is a potent HIV-1 fusion inhibitor peptide derived from the C-terminal heptad repeat of gp41. We recently identified that its N-terminal residues Met-626 and Thr-627 adopt a unique hook-like structure (termed M-T hook) thus stabilizing the interaction of the inhibitor with the deep pocket on the N-terminal heptad repeat. In this study, we further demonstrated that the M-T hook structure is a key determinant of CP621-652 in terms of its thermostability and anti-HIV activity. To directly define the structure and function of the M-T hook, we generated the peptide MT-C34 by incorporating Met-626 and Thr-627 into the N terminus of the C-terminal heptad repeat-derived peptide C34. The high resolution crystal structure (1.9 Å) of MT-C34 complexed by an N-terminal heptad repeat-derived peptide reveals that the M-T hook conformation is well preserved at the N-terminal extreme of the inhibitor. Strikingly, addition of two hook residues could dramatically enhance the binding affinity and thermostability of 6-helix bundle core. Compared with C34, MT-C34 exhibited significantly increased activity to inhibit HIV-1 envelope-mediated cell fusion (6.6-fold), virus entry (4.5-fold), and replication (6-fold). Mechanistically, MT-C34 had a 10.5-fold higher increase than C34 in blocking 6-helix bundle formation. We further showed that MT-C34 possessed higher potency against T20 (Enfuvirtide, Fuzeon)-resistant HIV-1 variants. Therefore, this study provides convincing data for our proposed concept that the M-T hook structure is critical for designing HIV-1 fusion inhibitors.

Biophysical Property and Broad Anti-HIV Activity of Albuvirtide, a 3-Maleimimidopropionic Acid-Modified Peptide Fusion Inhibitor

Albuvirtide (ABT) is a 3-maleimimidopropionic acid (MPA)-modified peptide HIV fusion inhibitor that can irreversibly conjugate to serum albumin. Previous studies demonstrated its in vivo long half-life and potent anti-HIV activity. Here, we focused to characterize its biophysical properties and evaluate its antiviral spectrum. In contrast to T20 (Enfuvirtide, Fuzeon), ABT was able to form a stable α-helical conformation with the target sequence and block the fusion-active six-helix bundle (6-HB) formation in a dominant-negative manner. It efficiently inhibited HIV-1 Env-mediated cell membrane fusion and virus entry. A large panel of 42 HIV-1 pseudoviruses with different genotypes were constructed and used for the antiviral evaluation. The results showed that ABT had potent inhibitory activity against the subtypes A, B and C that predominate the worldwide AIDS epidemics, and subtype B′, CRF07_BC and CRF01_AE recombinants that are currently circulating in China. Furthermore, ABT was also highly effective against HIV-1 variants resistant to T20. Taken together, our data indicate that the chemically modified peptide ABT can serve as an ideal HIV-1 fusion inhibitor.

Discovery of critical residues for viral entry and inhibition through structural Insight of HIV-1 fusion inhibitor CP621-652.

Background: CP621–652 is a HIV-1 fusion inhibitor peptide containing the gp41 621QIWNNMT627 motif. Results: The crystal structure of CP621–652 in complex with T21 was determined and mutational analyses were performed. Conclusion: The residues Met626 and Thr627 in the gp41 621QIWNNMT627 motif are critical for HIV-1 entry and inhibition. Significance: Our data provide important information for designing novel HIV fusion inhibitors. The core structure of HIV-1 gp41 is a stable six-helix bundle (6-HB) folded by its trimeric N- and C-terminal heptad repeats (NHR and CHR). We previously identified that the 621QIWNNMT627 motif located at the upstream region of gp41 CHR plays critical roles for the stabilization of the 6-HB core and peptide CP621–652 containing this motif is a potent HIV-1 fusion inhibitor, however, the molecular determinants underlying the stability and anti-HIV activity remained elusive. In this study, we determined the high-resolution crystal structure of CP621–652 complexed by T21. We find that the 621QIWNNMT627 motif does not maintain the α-helical conformation. Instead, residues Met626 and Thr627 form a unique hook-like structure (denoted as M-T hook), in which Thr627 redirects the peptide chain to position Met626 above the left side of the hydrophobic pocket on the NHR trimer. The side chain of Met626 caps the hydrophobic pocket, stabilizing the interaction between the pocket and the pocket-binding domain. Our mutagenesis studies demonstrate that mutations of the M-T hook residues could completely abolish HIV-1 Env-mediated cell fusion and virus entry, and significantly destabilize the interaction of NHR and CHR peptides and reduce the anti-HIV activity of CP621–652. Our results identify an unusual structural feature that stabilizes the six-helix bundle, providing novel insights into the mechanisms of HIV-1 fusion and inhibition.

Design of a highly potent HIV-1 fusion inhibitor targeting the gp41 pocket.

Objective:T20 (Enfuvirtide), which is a 36-residue peptide derived from the C-terminal heptad repeat (CHR) of gp41, is the only clinically available HIV-1 fusion inhibitor, but it easily induces drug resistance, which calls for next-generation drugs. Design:We recently demonstrated that the M-T hook structure can be used to design a short CHR peptide that specifically targets the conserved gp41 pocket rather than the T20-resistant sites. We attempted to develop more potent HIV-1 fusion inhibitors based on the structure–activity relationship of MT-SC22EK. Methods:Multiple biophysical and functional approaches were performed to determine the structural features, binding affinities and anti-HIV activities of the inhibitors. Results:The 23-residue peptide HP23, which mainly contains the M-T hook structure and pocket-binding sequence, showed a helical and trimeric state in solution. HP23 had dramatically improved binding stability and antiviral activity, and it was the most potent inhibitor of the M-T hook-modified and unmodified control peptides. More promisingly, HP23 was highly active in the inhibition of diverse HIV-1 subtypes, including T20 and MT-SC22EK resistant HIV-1 mutants, and it exhibited a high genetic barrier to the development of resistance. Conclusion:Our studies delivered an ideal HIV-1 fusion inhibitor that specifically targeted the highly conserved gp41 pocket and possessed potent binding and antiviral activity. Furthermore, HP23 can serve as a critical tool to explore the mechanisms of HIV-1 fusion and inhibition.

The M-T hook structure increases the potency of HIV-1 fusion inhibitor sifuvirtide and overcomes drug resistance

OBJECTIVES Peptides derived from the C-terminal heptad repeat (CHR) of HIV-1 gp41 are potent fusion inhibitors. We have recently demonstrated that the unique M-T hook structure preceding the pocket-binding motif of CHR peptide-based inhibitors can greatly improve their antiviral activity. In this study, we applied the M-T hook structure to optimize sifuvirtide (SFT), a potent CHR-derived inhibitor currently under Phase III clinical trials in China. METHODS The peptide MT-SFT was generated by incorporating two M-T hook residues (Met-Thr) into the N-terminus of sifuvirtide. Multiple structural and functional approaches were used to determine the biophysical properties and antiviral activity of MT-SFT. RESULTS The high-resolution crystal structure of MT-SFT reveals a highly conserved M-T hook conformation. Compared with sifuvirtide, MT-SFT exhibited a significant improvement in the ability to bind to the N-terminal heptad repeat, to block the formation of the six helix bundle and to inhibit HIV-1 Env-mediated cell fusion, viral entry and infection. Importantly, MT-SFT was fully active against sifuvirtide- and enfuvirtide (T20)-resistant HIV-1 variants and displayed a high genetic barrier to developing drug resistance. CONCLUSIONS Our studies have verified that the M-T hook structure offers a general strategy for designing novel HIV-1 fusion inhibitors and provide new insights into viral entry and inhibition.

Two M-T hook residues greatly improve the antiviral activity and resistance profile of the HIV-1 fusion inhibitor SC29EK

BackgroundPeptides derived from the C-terminal heptad repeat (CHR) of HIV-1 gp41 such as T20 (Enfuvirtide) and C34 are potent viral fusion inhibitors. We have recently found that two N-terminal residues (Met115 and Thr116) of CHR peptides form a unique M-T hook structure that can greatly enhance the binding and anti-HIV activity of inhibitors. Here, we applied two M-T hook residues to optimize SC29EK, an electrostatically constrained peptide inhibitor with a potent anti-HIV activity.ResultsThe resulting peptide MT-SC29EK showed a dramatically increased binding affinity and could block the six-helical bundle (6-HB) formation more efficiently. As expected, MT-SC29EK potently inhibited HIV-1 entry and infection, especially against those T20- and SC29EK-resistant HIV-1 variants. More importantly, MT-SC29EK and its short form (MT-SC22EK) suffered from the difficulty to induce HIV-1 resistance during the in vitro selection, suggesting their high genetic barriers to the development of resistance.ConclusionsOur studies have verified the M-T hook structure as a vital strategy to design novel HIV-1 fusion inhibitors and offered an ideal candidate for clinical development.

Development of potent and long-acting HIV-1 fusion inhibitors.

Background:T20 (enfuvirtide) is the first approved HIV entry inhibitor and currently the only viral fusion inhibitor, but its low efficacy and genetic barrier to resistance significantly limit its application, calling for a next-generation drug. Design:On the basis of the M-T hook structure, we recently developed a short-peptide named HP23, which mainly targets the deep pocket site of gp41 and possesses highly potent antiviral activity. To improve the pharmaceutical properties of a peptide-based inhibitor, we modified HP23 by different classes of lipids including fatty acid, cholesterol, and sphingolipids. To avoid the potential problem of oxidation, the methionine residue in the M-T hook sequence of HP23 was replaced with leucine. Methods:Peptides were synthesized and their anti-HIV activity and biophysical properties were determined. Results:A group of lipopeptides were generated with greatly improved anti-HIV activity. Promisingly, a fatty acid-conjugated lipopeptide named LP-11 showed potent and broad inhibitory activity against diverse primary HIV-1 isolates and clinically drug-resistant mutants, and it had dramatically increased ex-vivo antiviral activity and extended half-life. Also, LP-11 displayed highly enhanced &agr;-helicity and thermal stability, and it was physically stable under high temperature and humidity. Conclusion:LP-11 has high potentials for clinical development and it can serve as an ideal tool for exploring the mechanisms of HIV-1 fusion and inhibition.

Structural basis of potent and broad HIV-1 fusion inhibitor CP32M

Background: CP32M is a newly designed HIV-1 fusion inhibitor. Results: The crystal structure of CP32M and its potent activity against diverse HIV-1 variants were determined. Conclusion: The crystal structure reveals the mechanistic insights of CP32M. Significance: Our data provide important information for designing potent HIV-1 fusion inhibitors. CP32M is a newly designed peptide fusion inhibitor possessing potent anti-HIV activity, especially against T20-resistant HIV-1 strains. In this study, we show that CP32M can efficiently inhibit a large panel of diverse HIV-1 variants, including subtype B′, CRF07_BC, and CRF01_AE recombinants and naturally occurring or induced T20-resistant viruses. To elucidate its mechanism of action, we determined the crystal structure of CP32M complexed with its target sequence. Differing from its parental peptide, CP621-652, the 621VEWNEMT627 motif of CP32M folds into two α-helix turns at the N terminus of the pocket-binding domain, forming a novel layer in the six-helix bundle structure. Prominently, the residue Asn-624 of the 621VEWNEMT627 motif is engaged in the polar interaction with a hydrophilic ridge that borders the hydrophobic pocket on the N-terminal coiled coil. The original inhibitor design of CP32M provides several intra- and salt bridge/hydrogen bond interactions favoring the stability of the helical conformation of CP32M and its interactions with N-terminal heptad repeat (NHR) targets. We identified a novel salt bridge between Arg-557 on the NHR and Glu-648 of CP32M that is critical for the binding of CP32M and resistance against the inhibitor. Therefore, our data present important information for developing novel HIV-1 fusion inhibitors for clinical use.