Novel Engineered SARS-CoV-2 HR1 Trimer Exhibits Improved Potency and Broad-Spectrum Activity against SARS-CoV-2 and Its Variants

Abstract

Peptides derived from the SARS-CoV-2 HR1 region are generally poor inhibitors. Here, we constructed a trimeric peptide HR1MFd by fusing the trimerization motif foldon to the C terminus of the HR1 peptide.

Exploring Highly Conserved Regions of SARS-CoV-2 Spike S2 Subunit as Targets for Fusion Inhibition Using Chimeric Proteins

“…Protein or peptide mimics of the gp41 HR1 region have been shown to constitute potent inhibitors of HIV-1, owing to their capacity to tightly bind the HR2 region and interfere with virus–cell fusion [ 24 , 25 , 26 , 31 ]. Our results, together with other reports in the literature [ 22 , 23 ], clearly indicate that HR1 mimetic constructs can also inhibit SARS-CoV-2 by binding to HR2 and presumably blocking the membrane fusion process at some of its stages. According to the classical spring-loaded model of spike-mediated fusion ( Figure 1 a), HR1 forms an exposed trimeric coiled-coil bundle in the extended fusion intermediate of S2, which also transiently exposes HR2 to the action of inhibitors.…”

“…The fusion process can be interfered with by peptides derived from HR2 [ 16 , 17 , 18 ], as observed for other coronaviruses [ 19 ] and for gp41-mediated HIV-1 fusion [ 20 ]. HR1-derived peptides are less potent inhibitors targeting HR2 [ 16 , 19 ] but show strongly increased inhibitory activity if they have a stabilized trimeric coiled-coil structure exposing a stable HR1 groove [ 21 , 22 , 23 ], similar to HIV-1 fusion inhibitors based on the HR1 region of gp41, the HIV protein equivalent to S2 [ 24 , 25 , 26 ].…”

Novel chimeric proteins mimicking SARS-CoV-2 spike epitopes with broad inhibitory activity

“…Our mimetic proteins specifically based on the SARS-CoV-2 S2 HR1 sequence constitute a novel approach to design this type of HR1-based fusion inhibitors targeting the highly conserved HR2 region. This approach is further supported by recent reports of a trimer of S2 HR1 polypeptides stabilized by conjugation to a foldon sequence [25] , and a S2 postfusion-based 5-helix construct [24] , both showing potent and broad inhibitory activities. Our strategy consisted however in engineering a single-chain polypeptide that can fold autonomously to mimic a highly stable HR1 surface, without any chemical modification or addition of external trimerization motifs.…”

“…Moreover, a 5-helix construct based on the S2 6-HB structure but lacking one HR2 region has been reported very recently to inhibit several SARS-CoV-2 variants [24] . Also, trimers of S2 HR1 polypeptides stabilized by conjugation to a foldon sequence (HR1MFd) show broad coronavirus inhibitory activity [25] . All this evidence indicates that both HR1 and HR2 are exposed during coronavirus fusion and susceptible to inhibition, as it has been reported for HIV-1.…”

Potent inhibition of diverse Omicron sublineages by SARS-CoV-2 fusion-inhibitory lipopeptides

“…Several recent works also demonstrated the effectiveness of the lipopeptide-based fusion inhibitors against SARS-CoV-2 and its VOCs, despite only B.1.1.529 was tested in the studies ( Schmitz et al, 2022 ; Xia et al, 2022 ; Lan et al, 2022 ). Additionally, two 5-helix-based and one HR2 trimer-based miniprotein fusion inhibitors also inhibited SARS-CoV-2 and its VOCs similarly but they exhibited much less potent activity overall ( Xing et al, 2022 ; Lin et al, 2022 ; Bi et al, 2022 ). Collectively, it comes out with one more critical thinking: if the Omicron variant prefers endosomal entry over cell surface entry, why does not the shift affect the antiviral activity of fusion-inhibitory lipopeptides?…”