A dePEGylated Lipopeptide-Based Pan-Coronavirus Fusion Inhibitor Exhibits Potent and Broad-Spectrum Anti-HIV-1 Activity without Eliciting Anti-PEG Antibodies.

We previously identified a lipopeptide, EK1C4, by linking cholesterol to EK1, a pan-CoV fusion inhibitory peptide via a polyethylene glycol (PEG) linker, which showed potent pan-CoV fusion inhibitory activity. However, PEG can elicit antibodies to PEG in vivo, which will attenuate its antiviral activity. Therefore, we designed and synthesized a dePEGylated lipopeptide, EKL1C, by replacing the PEG linker in EK1C4 with a short peptide. Similar to EK1C4, EKL1C displayed potent inhibitory activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other coronaviruses. In this study, we found that EKL1C also exhibited broad-spectrum fusion inhibitory activity against human immunodeficiency virus type 1 (HIV-1) infection by interacting with the N-terminal heptad repeat 1 (HR1) of viral gp41 to block six-helix bundle (6-HB) formation. These results suggest that HR1 is a common target for the development of broad-spectrum viral fusion inhibitors and EKL1C has potential clinical application as a candidate therapeutic or preventive agent against infection by coronavirus, HIV-1, and possibly other class I enveloped viruses.

HR121 targeting HR2 domain in S2 subunit of spike protein can serve as a broad-spectrum SARS-CoV-2 inhibitor via intranasal administration.

The continuously emerging SARS-CoV-2 variants pose a great challenge to the efficacy of current drugs, this necessitates the development of broad-spectrum antiviral drugs. In the previous study, we designed a recombinant protein, heptad repeat (HR) 121, as a variant-proof vaccine. Here, we found it can act as a fusion inhibitor and demonstrated broadly neutralizing activities against SARS-CoV-2 and its main variants. Structure analysis suggested that HR121 targets the HR2 domain in SARS-CoV-2 spike (S) 2 subunit to block virus-cell fusion. Functional experiments demonstrated that HR121 can bind HR2 at serological-pH and endosomal-pH, highlighting its inhibition capacity when SARS-CoV-2 enters via either cellular membrane fusion or endosomal route. Importantly, HR121 can effectively inhibit SARS-CoV-2 and Omicron variant pseudoviruses entering the cells, as well as block authentic SARS-CoV-2 and Omicron BA.2 replications in human pulmonary alveolar epithelial cells. After intranasal administration to Syrian golden hamsters, it can protect hamsters from SARS-CoV-2 and Omicron BA.2 infection. Together, our results suggest that HR121 is a potent drug candidate with broadly neutralizing activities against SARS-CoV-2 and its variants.

SARS-CoV-2 Omicron XBB subvariants exhibit enhanced fusogenicity and substantial immune evasion in elderly population, but high sensitivity to pan-coronavirus fusion inhibitors.

Numerous emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants have shown significant immune evasion capacity and caused a large number of infections, as well as vaccine-breakthrough infections, especially in elderly populations. Recently emerged Omicron XBB was derived from the BA.2 lineage, but bears a distinct mutant profile in its spike (S) protein. In this study, we found that Omicron XBB S protein drove more efficient membrane-fusion kinetics on human lung-derived cells (Calu-3). Considering the high susceptibility of the elderly to the current Omicron pandemic, we performed a comprehensive neutralization assessment of elderly convalescent or vaccine sera against XBB infection. We found that the sera from elderly convalescent patients who experienced with BA.2 infection or breakthrough infection potently inhibited BA.2 infection, but showed significantly reduced efficacy against XBB. Moreover, recently emerged XBB.1.5 subvariant also showed more significant resistance to the convalescent sera of BA.2- or BA.5-infected elderly. On the other hand, we found that the pan-CoV fusion inhibitors EK1 and EK1C4 can potently block either XBB-S- or XBB.1.5-S-mediated fusion process and viral entry. Moreover, EK1 fusion inhibitor showed potent synergism when combined with convalescent sera of BA.2- or BA.5-infected patients against XBB and XBB.1.5 infection, further indicating that EK1-based pan-CoV fusion inhibitors are promising candidates for development as clinical antiviral agents to combat the Omicron XBB subvariants.

Targeting the Fusion Process of SARS-CoV-2 Infection by Small Molecule Inhibitors.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a serious threat to global public health, underscoring the urgency of developing effective therapies. Therapeutics and, more specifically, direct-acting antiviral development are still very much in their infancy. Here, we report that two hepatitis C virus (HCV) fusion inhibitors identified in our previous study, dichlorcyclizine and fluoxazolevir, broadly block human coronavirus entry into various cell types. Both compounds were effective against various human-pathogenic CoVs in multiple assays based on vesicular stomatitis virus (VSV) pseudotyped with the spike protein and spike-mediated syncytium formation. The antiviral effects were confirmed in SARS-CoV-2 infection systems. These compounds were equally effective against recently emerged variants, including the delta variant. Cross-linking experiments and structural modeling suggest that the compounds bind to a hydrophobic pocket near the fusion peptide of S protein, consistent with their potential mechanism of action as fusion inhibitors. In summary, these fusion inhibitors have broad-spectrum antiviral activities and may be promising leads for treatment of SARS-CoV-2, its variants, and other pathogenic CoVs. IMPORTANCE SARS-CoV-2 is an enveloped virus that requires membrane fusion for entry into host cells. Since the fusion process is relatively conserved among enveloped viruses, we tested our HCV fusion inhibitors, dichlorcyclizine and fluoxazolevir, against SARS-CoV-2. We performed in vitro assays and demonstrated their effective antiviral activity against SARS-CoV-2 and its variants. Cross-linking experiments and structural modeling suggest that the compounds bind to a hydrophobic pocket in spike protein to exert their inhibitory effect on the fusion step. These data suggest that both dichlorcyclizine and fluoxazolevir are promising candidates for further development as treatment for SARS-CoV-2.

SARS-CoV-2 Omicron subvariants exhibit distinct fusogenicity, but similar sensitivity, to pan-CoV fusion inhibitors.

Continuous emergence of the Omicron variant, along with its subvariants, has caused an increasing number of infections, reinfections, and vaccine-breakthrough infections, seriously threatening human health. Recently, several new Omicron subvariants, such as BA.5, BA.2.75, BA.4.6, and BF.7, bearing distinct mutation profiles in their spike (S) proteins, have significantly increased their capacity to evade vaccine-induced immunity and have shown enhanced infectivity and transmissibility, quickly becoming dominant sublineages. In this study, we found the S proteins of these Omicron subvariants to have 2- to 4-fold more efficient membrane fusion kinetics than that of the original Omicron variant (BA.1), indicating that these novel Omicron subvariants might possess increased pathogenicity. We also identified that peptide-based pan-CoV fusion inhibitors, EK1 and EK1C4, showed equal efficacy against membrane fusion mediated by S proteins of the noted Omicron subvariants and infection by their pseudoviruses. Additionally, either immune sera induced by wild-type (WT) SARS-CoV-2 RBD-based vaccine or BA.2 convalescent sera showed potent synergism with EK1 against both WT SARS-CoV-2 and various Omicron subvariants, further suggesting that EK1-based fusion inhibitors are promising candidates for development as clinical antiviral agents against the currently circulating Omicron subvariants.

Pan-coronavirus fusion inhibitors to combat COVID-19 and other emerging coronavirus infectious diseases.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the currently ongoing coronavirus disease 2019 (COVID-19) pandemic, has posed a serious threat to global public health. Recently, several SARS-CoV-2 variants of concern (VOCs) have emerged and caused numerous cases of reinfection in convalescent COVID-19 patients, as well as breakthrough infections in vaccinated individuals. This calls for the development of broad-spectrum antiviral drugs to combat SARS-CoV-2 and its VOCs. Pan-coronavirus fusion inhibitors, targeting the conserved heptad repeat 1 (HR1) in spike protein S2 subunit, can broadly and potently inhibit infection of SARS-CoV-2 and its variants, as well as other human coronaviruses. In this review, we summarized the most recent development of pan-coronavirus fusion inhibitors, such as EK1, EK1C4, and EKL1C, and highlighted their potential application in combating current COVID-19 infection and reinfection, as well as future emerging coronavirus infectious diseases.

Targeting an evolutionarily conserved "E-L-L" motif in spike protein to identify a small molecule fusion inhibitor against SARS-CoV-2.

As newer variants of SARS-CoV-2 continue to pose major threats to global human health and economy, identifying novel druggable antiviral targets is the key toward sustenance. Here, we identify an evolutionarily conserved "Ex3Lx6L" ("E-L-L") motif present within the HR2 domain of all human and nonhuman coronavirus spike (S) proteins that play a crucial role in stabilizing its postfusion six-helix bundle (6-HB) structure and thus, fusion-mediated viral entry. Mutations within this motif reduce the fusogenicity of the S protein without affecting its stability or membrane localization. We found that posaconazole, an FDA-approved drug, binds to this "E-L-L" motif and impedes the formation of 6-HB, thus effectively inhibiting SARS-CoV-2 infection in cells. While posaconazole exhibits high efficacy in blocking S protein-mediated viral entry, mutations within the "E-L-L" motif rendered the protein completely resistant to the drug, establishing its specificity toward this motif. Our data demonstrate that posaconazole restricts early stages of infection through specific inhibition of membrane fusion and viral genome release into the host cell and is equally effective toward all major variants of concerns of SARS-CoV-2, including Beta, Kappa, Delta, and Omicron. Together, we show that this conserved essential "E-L-L" motif is an ideal target for the development of prophylactic and therapeutic interventions against SARS-CoV-2.

Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation.

Virus fusion process is evolutionarily conserved and provides a promising pan-viral target. Cell-cell fusion leads to syncytial formation and has implications in pathogenesis, virus spread and immune evasion. Drugs that target these processes can be developed into anti-virals. Here, we have developed sensitive, rapid, adaptable fusion reporter gene assays as models for plasma membrane and alternative fusion pathways as well as syncytial fusion in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and have confirmed their specificity using neutralizing antibodies and specific protease inhibitors. The fusion report gene assays are more sensitive and unbiased than morphological fusion assay. The fusion assays can differentiate between transmembrane serine protease 2 (TMPRSS2)-dependency in TMPRSS2(+) cells and trypsin-dependency in angiotensin-converting enzyme 2 (ACE2)(+)TMPRSS2(-) cells. Moreover, we have identified putative novel fusion processes that are triggered by an acidic pH with and without trypsin. Coupled with morphological fusion criteria, we have found that syncytia formation is enhanced by TMPRSS2 or trypsin. By testing against our top drug hits previously shown to inhibit SARS-CoV-2 pseudovirus infection, we have identified several fusion inhibitors including structurally related lopsided kite-shaped molecules. Our results have important implications in the development of universal blockers and synergistic therapeutics and the small molecule inhibitors can provide important tools in elucidating the fusion process.

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

The emergence and rapid spreading of SARS-CoV-2 variants of concern (VOCs) have posed a great challenge to the efficacy of vaccines and therapeutic antibodies, calling for antivirals that can overcome viral evasion. We recently reported that SARS-CoV-2 fusion-inhibitory lipopeptides, IPB02V3 and IPB24, possessed the potent activities against divergent VOCs, including Alpha, Beta, Gamma, Delta, and the initial Omicron strain (B.1.1.529); however, multiple Omicron sublineages have emerged and BA.4/5 is now becoming predominant globally. In this study, we focused on characterizing the functionality of the spike (S) proteins derived from Omicron sublineages and their susceptibility to the inhibition of IPB02V3 and IPB24. We first found that the S proteins of BA.2, BA.2.12.1, BA.3, and BA.4/5 exhibited significantly increased cell fusion capacities compared to BA.1, whereas the pseudoviruses of BA.2.12.1, BA.3, and BA.4/5 had significantly increased infectivity relative to BA.1 or BA.2. Next, we verified that IPB02V3 and IPB24 also maintained their very high potent activities in inhibiting diverse Omicron sublineages, even with enhanced potencies relative to the inhibition on ancestral virus. Moreover, we demonstrated that evolved Omicron mutations in the inhibitor-binding heptad repeat 1 (HR1) site could impair the S protein-driven cell fusogenicity and infectivity, but none of single or combined mutations affected the antiviral activity of IPB02V3 and IPB24. Therefore, we believe that viral fusion inhibitors possess high potential to be developed as effective drugs for fighting SARS-CoV-2 variants including diverse Omicron sublineages.

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

SARS-CoV-2 spike (S) protein mediates virus attachment to the cells and fusion between viral and cell membranes. Membrane fusion is driven by mutual interaction between the highly conserved heptad-repeat regions 1 and 2 (HR1 and HR2) of the S2 subunit of the spike. For this reason, these S2 regions are interesting therapeutic targets for COVID-19. Although HR1 and HR2 have been described as transiently exposed during the fusion process, no significant antibody responses against these S2 regions have been reported. Here we designed chimeric proteins that imitate highly stable HR1 helical trimers and strongly bind to HR2. The proteins have broad inhibitory activity against WT B.1 and BA.1 viruses. Sera from COVID-19 convalescent donors showed significant levels of reactive antibodies (IgG and IgA) against the HR1 mimetic proteins, whereas these antibody responses were absent in sera from uninfected donors. Moreover, both inhibitory activity and antigenicity of the proteins correlate positively with their structural stability but not with the number of amino acid changes in their HR1 sequences, indicating a conformational and conserved nature of the involved epitopes. Our results reveal previously undetected spike epitopes that may guide the design of new robust COVID-19 vaccines and therapies.

Carbohydrate-binding protein from stinging nettle as fusion inhibitor for SARS-CoV-2 variants of concern.

Urtica dioica agglutinin (UDA) is a carbohydrate-binding small monomeric protein isolated from stinging nettle rhizomes. It inhibits replication of a broad range of viruses, including coronaviruses, in multiple cell types, with appealing selectivity. In this work, we investigated the potential of UDA as a broad-spectrum antiviral agent against SARS-CoV-2. UDA potently blocks transduction of pseudotyped SARS-CoV-2 in A549.ACE2+-TMPRSS2 cells, with IC50 values ranging from 0.32 to 1.22 µM. Furthermore, UDA prevents viral replication of the early Wuhan-Hu-1 strain in Vero E6 cells (IC50 = 225 nM), but also the replication of SARS-CoV-2 variants of concern, including Alpha, Beta and Gamma (IC50 ranging from 115 to 171 nM). In addition, UDA exerts antiviral activity against the latest circulating Delta and Omicron variant in U87.ACE2+ cells (IC50 values are 1.6 and 0.9 µM, respectively). Importantly, when tested in Air-Liquid Interface (ALI) primary lung epithelial cell cultures, UDA preserves antiviral activity against SARS-CoV-2 (20A.EU2 variant) in the nanomolar range. Surface plasmon resonance (SPR) studies demonstrated a concentration-dependent binding of UDA to the viral spike protein of SARS-CoV-2, suggesting interference of UDA with cell attachment or subsequent virus entry. Moreover, in additional mechanistic studies with cell-cell fusion assays, UDA inhibited SARS-CoV-2 spike protein-mediated membrane fusion. Finally, pseudotyped SARS-CoV-2 mutants with N-glycosylation deletions in the S2 subunit of the spike protein remained sensitive to the antiviral activity of UDA. In conclusion, our data establish UDA as a potent fusion inhibitor for the current variants of SARS-CoV-2.

SARS-CoV-2 fusion-inhibitory lipopeptides maintain high potency against divergent variants of concern including Omicron.

The emergence of SARS-CoV-2 Omicron and other variants of concern (VOCs) has brought huge challenges to control the COVID-19 pandemic, calling for urgent development of effective vaccines and therapeutic drugs. In this study, we focused on characterizing the impacts of divergent VOCs on the antiviral activity of lipopeptide-based fusion inhibitors that we previously developed. First, we found that pseudoviruses bearing the S proteins of five VOCs (Alpha, Beta, Gamma, Delta, and Omicron) and one variant of interest (Lambda) exhibited greatly decreased infectivity relative to the wild-type (WT) strain or single D614G mutant, especially the Omicron pseudovirus. Differently, the most of variants exhibited an S protein with significantly enhanced cell fusion activity, whereas the S protein of Omicron still mediated decreased cell-cell fusion. Next, we verified that two lipopeptide-based fusion inhibitors, IPB02V3 and IPB24, maintained the highly potent activities in inhibiting various S proteins-driven cell fusion and pseudovirus infection. Surprisingly, both IPB02V3 and IPB24 lipopeptides displayed greatly increased potencies against the infection of authentic Omicron strain relative to the WT virus. The results suggest that Omicron variant evolves with a reduced cell fusion capacity and is more sensitive to the inhibition of fusion-inhibitory lipopeptides; thus, IPB02V3 and IPB24 can be further developed as potent, broad-spectrum antivirals for combating Omicron and the potential future outbreak of other emerging variants.

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

The ongoing pandemic of COVID-19, caused by SARS-CoV-2, has substantially increased the risk to global public health. Multiple vaccines and neutralizing antibodies (nAbs) have been authorized for preventing and treating SARS-CoV-2 infection. However, the emergence and spread of the viral variants may limit the effectiveness of these vaccines and antibodies. Fusion inhibitors targeting the HR1 domain of the viral S protein have been shown to broadly inhibit SARS-CoV-2 and its variants. In theory, peptide inhibitors targeting the HR2 domain of the S protein should also be able to inhibit viral infection. However, previously reported HR1-derived peptide inhibitors targeting the HR2 domain exhibit poor inhibitory activities. Here, we engineered a novel HR1 trimer (HR1MFd) by conjugating the trimerization motif foldon to the C terminus of the HR1-derived peptide. HR1MFd showed significantly improved inhibitory activity against SARS-CoV-2, SARS-CoV-2 variants of concern (VOCs), SARS-CoV, and MERS-CoV. Mechanistically, HR1MFd possesses markedly increased α-helicity, thermostability, higher HR2 domain binding affinity, and better inhibition of S protein-mediated cell-cell fusion compared to the HR1 peptide. Therefore, HR1MFd lays the foundation to develop HR1-based fusion inhibitors against SARS-CoV-2. IMPORTANCE 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. HR1MFd was highly effective in blocking transductions by SARS-CoV-2, SARS-CoV-2 variants, SARS-CoV, and MERS-CoV pseudoviruses. In comparison with HR1M, HR1MFd adopted a much higher helical conformation, better thermostability, increased affinity to the viral HR2 domain, and better inhibition of S protein-mediated cell-cell fusion. Overall, HR1MFd provides the information to develop effective HR1-derived peptides as fusion inhibitors against SARS-CoV-2 and its variants.

Potency of Fusion-Inhibitory Lipopeptides against SARS-CoV-2 Variants of Concern.

The ability of SARS-CoV-2 to evolve in response to selective pressures poses a challenge to vaccine and antiviral efficacy. The S1 subunit of the spike (S) protein contains the receptor-binding domain and is therefore under selective pressure to evade neutralizing antibodies elicited by vaccination or infection. In contrast, the S2 subunit of S is only transiently exposed after receptor binding, which makes it a less efficient target for antibodies. As a result, S2 has a lower mutational frequency than S1. We recently described monomeric and dimeric SARS-CoV-2 fusion-inhibitory lipopeptides that block viral infection by interfering with S2 conformational rearrangements during viral entry. Importantly, a dimeric lipopeptide was shown to block SARS-CoV-2 transmission between ferrets in vivo. Because the S2 subunit is relatively conserved in newly emerging SARS-CoV-2 variants of concern (VOCs), we hypothesize that fusion-inhibitory lipopeptides are cross-protective against infection with VOCs. Here, we directly compared the in vitro efficacies of two fusion-inhibitory lipopeptides against VOC, in comparison with a set of seven postvaccination sera (two doses) and a commercial monoclonal antibody preparation. For the beta, delta, and omicron VOCs, it has been reported that convalescent and postvaccination sera are less potent in virus neutralization assays. Both fusion-inhibitory lipopeptides were equally effective against all five VOCs compared to ancestral virus, whereas postvaccination sera and therapeutic monoclonal antibody lost potency to newer VOCs, in particular to omicron BA.1 and BA.2. The neutralizing activity of the lipopeptides is consistent, and they can be expected to neutralize future VOCs based on their mechanism of action. IMPORTANCE SARS-CoV-2, the causative agent of COVID-19, continues to spread globally, with waves resulting from new variants that evade immunity generated by vaccines and previous strains and escape available monoclonal antibody therapy. Fusion-inhibitory peptides may provide an intervention strategy that is not similarly affected by this viral evolution.

Peptide-Based Dual HIV and Coronavirus Entry Inhibitors.

The continued HIV/AIDS epidemic worldwide and the battle against emerging infectious diseases caused by coronaviruses underscore the need for the development of an ever-expanding repertoire of antiviral drugs. Entry inhibitors are of particular interest because of their potential to be used as therapeutic or prophylactic treatments for blocking viral invasion. HIV and coronaviruses utilize class I fusion proteins to facilitate their entry and membrane fusion. Discovery of a common hexameric coiled-coil fusion complex resulting from the packing of three C-terminal heptad repeat region from the fusion-mediating subunit of viral fusion proteins against trimeric coiled-coil made up by their N-terminal heptad repeat prompted the search for peptides mimicking the heptad repeat regions that could potentially inhibit viral entry. This has led to the development of effective peptides that are specific to the virus that is developed for. In this review, we focus on peptide-based entry dual inhibitors that block fusion process not only of HIV but also coronaviruses through interrupting their fusogenic six-helical bundle core and which hopefully will help to gain insight into the α-helical secondary structure- and coiled-coil superstructure-based strategies to design entry inhibitors with broad-spectrum antiviral activity against enveloped viruses with class I fusion proteins.

A Modified Fibronectin Type III Domain-Conjugated, Long-Acting Pan-Coronavirus Fusion Inhibitor with Extended Half-Life.

The coronavirus disease 2019 (COVID-19) pandemic caused by infection of SARS-CoV-2 and its variants has posed serious threats to global public health, thus calling for the development of potent and broad-spectrum antivirals. We previously designed and developed a peptide-based pan-coronavirus (CoV) fusion inhibitor, EK1, which is effective against all human CoVs (HCoV) tested by targeting the HCoV S protein HR1 domain. However, its relatively short half-life may limit its clinical use. Therefore, we designed, constructed, and expressed a recombinant protein, FL-EK1, which consists of a modified fibronectin type III domain (FN3) with albumin-binding capacity, a flexible linker, and EK1. As with EK1, we found that FL-EK1 could also effectively inhibit infection of SARS-CoV-2 and its variants, as well as HCoV-OC43. Furthermore, it protected mice from infection by the SARS-CoV-2 Delta variant and HCoV-OC43. Importantly, the half-life of FL-EK1 (30 h) is about 15.7-fold longer than that of EK1 (1.8 h). These results suggest that FL-EK1 is a promising candidate for the development of a pan-CoV fusion inhibitor-based long-acting antiviral drug for preventing and treating infection by current and future SARS-CoV-2 variants, as well as other HCoVs.

A Five-Helix-Based SARS-CoV-2 Fusion Inhibitor Targeting Heptad Repeat 2 Domain against SARS-CoV-2 and Its Variants of Concern.

The prolonged duration of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic has resulted in the continuous emergence of variants of concern (VOC, e.g., Omicron) and variants of interest (VOI, e.g., Lambda). These variants have challenged the protective efficacy of current COVID-19 vaccines, thus calling for the development of novel therapeutics against SARS-CoV-2 and its VOCs. Here, we constructed a novel fusion inhibitor-based recombinant protein, denoted as 5-Helix, consisting of three heptad repeat 1 (HR1) and two heptad repeat 2 (HR2) fragments. The 5-Helix interacted with the HR2 domain of the viral S2 subunit, the most conserved region in spike (S) protein, to block homologous six-helix bundle (6-HB) formation between viral HR1 and HR2 domains and, hence, viral S-mediated cell-cell fusion. The 5-Helix potently inhibited infection by pseudotyped SARS-CoV-2 and its VOCs, including Delta and Omicron variants. The 5-Helix also inhibited infection by authentic SARS-CoV-2 wild-type (nCoV-SH01) strain and its Delta variant. Collectively, our findings suggest that 5-Helix can be further developed as either a therapeutic or prophylactic to treat and prevent infection by SARS-CoV-2 and its variants.

Discovery of Highly Potent Fusion Inhibitors with Potential Pan-Coronavirus Activity That Effectively Inhibit Major COVID-19 Variants of Concern (VOCs) in Pseudovirus-Based Assays.

We report the discovery of several highly potent small molecules with low-nM potency against severe acute respiratory syndrome coronavirus (SARS-CoV; lowest half-maximal inhibitory concentration (IC50: 13 nM), SARS-CoV-2 (IC50: 23 nM), and Middle East respiratory syndrome coronavirus (MERS-CoV; IC50: 76 nM) in pseudovirus-based assays with excellent selectivity index (SI) values (>5000), demonstrating potential pan-coronavirus inhibitory activities. Some compounds showed 100% inhibition against the cytopathic effects (CPE; IC100) of an authentic SARS-CoV-2 (US_WA-1/2020) variant at 1.25 µM. The most active inhibitors also potently inhibited variants of concern (VOCs), including the UK (B.1.1.7) and South African (B.1.351) variants and the Delta variant (B.1.617.2) originally identified in India in pseudovirus-based assay. Surface plasmon resonance (SPR) analysis with one potent inhibitor confirmed that it binds to the prefusion SARS-CoV-2 spike protein trimer. These small-molecule inhibitors prevented virus-mediated cell-cell fusion. The absorption, distribution, metabolism, and excretion (ADME) data for one of the most active inhibitors, NBCoV1, demonstrated drug-like properties. An in vivo pharmacokinetics (PK) study of NBCoV1 in rats demonstrated an excellent half-life (t1/2) of 11.3 h, a mean resident time (MRT) of 14.2 h, and oral bioavailability. We expect these lead inhibitors to facilitate the further development of preclinical and clinical candidates.

25-Hydroxycholesterol-Conjugated EK1 Peptide with Potent and Broad-Spectrum Inhibitory Activity against SARS-CoV-2, Its Variants of Concern, and Other Human Coronaviruses.

The COVID-19 pandemic caused by SARS-CoV-2 infection poses a serious threat to global public health and the economy. The enzymatic product of cholesterol 25-hydroxylase (CH25H), 25-Hydroxycholesterol (25-HC), was reported to have potent anti-SARS-CoV-2 activity. Here, we found that the combination of 25-HC with EK1 peptide, a pan-coronavirus (CoV) fusion inhibitor, showed a synergistic antiviral activity. We then used the method of 25-HC modification to design and synthesize a series of 25-HC-modified peptides and found that a 25-HC-modified EK1 peptide (EK1P4HC) was highly effective against infections caused by SARS-CoV-2, its variants of concern (VOCs), and other human CoVs, such as HCoV-OC43 and HCoV-229E. EK1P4HC could protect newborn mice from lethal HCoV-OC43 infection, suggesting that conjugation of 25-HC with a peptide-based viral inhibitor was a feasible and universal strategy to improve its antiviral activity.

Plant Alkaloids Inhibit Membrane Fusion Mediated by Calcium and Fragments of MERS-CoV and SARS-CoV/SARS-CoV-2 Fusion Peptides.

To rationalize the antiviral actions of plant alkaloids, the ability of 20 compounds to inhibit calcium-mediated fusion of lipid vesicles composed of phosphatidylglycerol and cholesterol was investigated using the calcein release assay and dynamic light scattering. Piperine, tabersonine, hordenine, lupinine, quinine, and 3-isobutyl-1-methylxanthine demonstrated the most potent effects (inhibition index greater than 50%). The introduction of phosphatidylcholine into the phosphatidylglycerol/cholesterol mixture led to significant changes in quinine, hordenine, and 3-isobutyl-1-methylxanthine efficiency. Comparison of the fusion inhibitory ability of the tested alkaloids, and the results of the measurements of alkaloid-induced alterations in the physical properties of model membranes indicated a potent relationship between a decrease in the cooperativity of the phase transition of lipids and the ability of alkaloids to prevent calcium-mediated vesicle fusion. In order to use this knowledge to combat the novel coronavirus pandemic, the ability of the most effective compounds to suppress membrane fusion induced by fragments of MERS-CoV and SARS-CoV/SARS-CoV-2 fusion peptides was studied using the calcein release assay and confocal fluorescence microscopy. Piperine was shown to inhibit vesicle fusion mediated by both coronavirus peptides. Moreover, piperine was shown to significantly reduce the titer of SARS-CoV2 progeny in vitro in Vero cells when used in non-toxic concentrations.