Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms23052794/s1.Acknowledgments: M.C.-M. acknowledges a grant from Youth Employment Operative Program of
the Andalusia Government and the European Social Fund (ESF). S.C. acknowledges an exchange
studentship from the ERASMUS+ program of the European Union. The results shown are included
as part of M.C.-M. doctoral thesis.Inhibition of the HIV-1 fusion process constitutes a promising strategy to neutralize the
virus at an early stage before it enters the cell. In this process, the envelope glycoprotein (Env)
plays a central role by promoting membrane fusion. We previously identified a vulnerability at
the flexible C-terminal end of the gp41 C-terminal heptad repeat (CHR) region to inhibition by
a single-chain miniprotein (named covNHR-N) that mimics the first half of the gp41 N-terminal
heptad repeat (NHR). The miniprotein exhibited low stability, moderate binding to its complementary
CHR region, both as an isolated peptide and in native trimeric Envs, and low inhibitory activity
against a panel of pseudoviruses. The addition of a disulfide bond stabilizing the miniprotein
increased its inhibitory activity, without altering the binding affinity. Here, to further study the
effect of conformational stability on binding and inhibitory potency, we additionally stabilized
these miniproteins by engineering a second disulfide bond stapling their N-terminal end, The
new disulfide-bond strongly stabilizes the protein, increases binding affinity for the CHR target
and strongly improves inhibitory activity against several HIV-1 strains. Moreover, high inhibitory
activity could be achieved without targeting the preserved hydrophobic pocket motif of gp41. These
results may have implications in the discovery of new strategies to inhibit HIV targeting the gp41
CHR region.Grants BIO2016-76640-R and PID2019.107515RB.C21 from the
Spain’s State Research AgencySRA/10.13039/501100011033, co-funded by ERDF/ESF, “A way to
make Europe”/“Investing in your future
