Development of therapeutic proteins and compounds against viruses with class-i fusion machines

Resumen

Zoonotic diseases have caused the world’s biggest pandemics in recent history. HIV was first identified in 1981 and, since then, 70 million people have been infected with HIV and more than 37 million have died from AIDS-related illnesses. 40 years after, the SARS-CoV-2 outbreak let the world shocked crushing economies and killing more than 6 million people within the ongoing COVID-19 global pandemic. Preventing those viruses from entering the host’s cells constitutes a major goal in the development of effective anti-viral therapies. As enveloped viruses, HIV and SARS-CoV-2 need to fuse their membranes with those of human cells in order to infect them. Their class-I fusion machines, the HIV Envelope and the SARS-CoV-2 Spike respectively are responsible for membrane fusion. In this process the N-terminal Heptad Repeat (NHR or HR1) regions of gp41 and S2 subunits stand out because of their high conservation and because their interaction with the C-terminal Heptad Repeat (CHR or HR2) region is the driving force that ultimately leads to membrane fusion. Consequently, HR1 and HR2 represent vulnerable sites for the development of broad and effective therapies. In previous works, we designed and studied chimeric miniproteins (named covNHR) that mimic the exposed NHR of HIV gp41 in its pre-hairpin conformation as fusion inhibitors and as molecular models to explore the complexity of the interactions governing their function. In this thesis: We have used the covNHR miniproteins in combination with a small-molecule fluorescent probe to investigate the conformational and dynamic properties of a conserved hydrophobic pocket (HP) located in the NHR-groove, which is a promising target for the development of small-molecule inhibitors. We demonstrated that the HP is conformationally flexible and connected allosterically to other NHR regions, which strongly influence the binding of potential ligands. We have studied the role of buried polar residues in the interior of the NHR coil by mutating them to a hydrophobic amino acid, Isoleucine. The mutants show an extreme thermostability but an enhanced self-association and unaltered HIV-1 inhibitory activity. The results support a role of buried core polar residues in maintaining structural uniqueness and promoting an energetic coupling between conformational stability and NHR–CHR binding. We have further stabilized covNHR proteins with the addition of an intra-chain disulfide-bond. The new disulfide-bonded strongly stabilizes the miniprotein, increases binding affinity for the CHR region and strongly improves HIV broad inhibitory activity, without targeting the preserved HP motif of gp41. The results suggest new strategies to inhibit HIV. Finally, we have translated covNHR’s approach to design and characterize chimeric proteins that imitate HR1 helical trimers of SARS-CoV-2 S2. The proteins bind strongly HR2 and have broad inhibitory activity against WT and Omicron viruses. Moreover, they are recognized by antibodies present in sera from COVID-19 patients, revealing previously undetected Spike epitopes that may guide the design of new vaccine and therapies.