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.

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.

Background: CP621-652 is a HIV-1 fusion inhibitor peptide containing the gp41 621 QIWNNMT 627 motif. Results: The crystal structure of CP621-652 in complex with T21 was determined and mutational analyses were performed. Conclusion: The residues Met 626 and Thr 627 in the gp41 621 QIWNNMT 627 motif are critical for HIV-1 entry and inhibition. Significance: Our data provide important information for designing novel HIV fusion inhibitors.

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.

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.