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.

Design and characterization of novel SARS-CoV-2 fusion inhibitors with N-terminally extended HR2 peptides.

Development of potent and broad-spectrum antivirals against SARS-CoV-2 remains one of top priorities, especially in the case of that current vaccines cannot effectively prevent viral transmission. We previously generated a group of fusion-inhibitory lipopeptides, with one formulation being evaluated under clinical trials. In this study, we dedicated to characterize the extended N-terminal motif (residues 1161-1168) of the so-called spike (S) heptad repeat 2 (HR2) region. Alanine scanning analysis of this motif verified its critical roles in S protein-mediated cell-cell fusion. Using a panel of HR2 peptides with the N-terminal extensions, we identified a peptide termed P40, which contained four extended N-terminal residues (VDLG) and exhibited improved binding and antiviral activities, whereas the peptides with further extensions had no such effects. Then, we developed a new lipopeptide P40-LP by modifying P40 with cholesterol, which exhibited dramatically increased activities in inhibiting SARS-CoV-2 variants including divergent Omicron sublineages. Moreover, P40-LP displayed a synergistic effect with IPB24 lipopeptide that was designed containing the C-terminally extended residues, and it could effectively inhibit other human coronaviruses, including SARS-CoV, MERS-CoV, HCoV-229E, and HCoV-NL63. Taken together, our results have provided valuable insights for understanding the structure-function relationship of SARS-CoV-2 fusion protein and offered novel antiviral strategies to fight against the COVID-19 pandemic.

Cyclic lipopeptides as membrane fusion inhibitors against SARS-CoV-2: New tricks for old dogs.

With the resurgence of the coronavirus pandemic, the repositioning of FDA-approved drugs against coronovirus and finding alternative strategies for antiviral therapy are both important. We previously identified the viral lipid envelope as a potential target for the prevention and treatment of SARS-CoV-2 infection with plant alkaloids (Shekunov et al., 2021). Here, we investigated the effects of eleven cyclic lipopeptides (CLPs), including well-known antifungal and antibacterial compounds, on the liposome fusion triggered by calcium, polyethylene glycol 8000, and a fragment of SARS-CoV-2 fusion peptide (816-827) by calcein release assays. Differential scanning microcalorimetry of the gel-to-liquid-crystalline and lamellar-to-inverted hexagonal phase transitions and confocal fluorescence microscopy demonstrated the relation of the fusion inhibitory effects of CLPs to alterations in lipid packing, membrane curvature stress and domain organization. The antiviral effects of CLPs were evaluated in an in vitro Vero-based cell model, and aculeacin A, anidulafugin, iturin A, and mycosubtilin attenuated the cytopathogenicity of SARS-CoV-2 without specific toxicity.

Development of highly effective LCB1-based lipopeptides targeting the spike receptor-binding motif of SARS-CoV-2.

LCB1 is a computationally designed 56-mer miniprotein targeting the spike (S) receptor-binding motif of SARS-CoV- 2 with high potent activity (Science, 2020; Cell host microbe, 2021); however, recent studies have demonstrated that emerging SARS-CoV-2 variants are highly resistant to LCB1's inhibition. In this study, we first identified a truncated peptide termed LCB1v8, which maintained the high antiviral potency. Then, a group of lipopeptides were generated by modifying LCB1v8 with diverse lipids, and of two lipopeptides, the C-terminally stearicacid-conjugtaed LCB1v17 and cholesterol-conjugated LCB1v18, were highly effective in inhibiting both S protein-pseudovirus and authentic SARS-CoV-2 infections. We further showed that LCB1-based inhibitors had similar α-helicity and thermostability in structure and bound to the target-mimic RBD protein with high affinity, and the lipopeptides exhibited greatly enhanced binding with the viral and cellular membranes, improved inhibitory activities against emerging SARS-CoV-2 variants. Moreover, LCB1v18 was validated with high preventive and therapeutic efficacies in K18-hACE2 transgenic mice against lethal SARS-CoV-2 challenge. In conclusion, our studies have provided important information for understanding the structure and activity relationship (SAR) of LCB1 inhibitor and would guide the future development of novel antivirals.

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.

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.

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.

Novel Synthetic Lipopeptides as Potential Mucosal Adjuvants Enhanced SARS-CoV-2 rRBD-Induced Immune Response.

As TLR2 agonists, several lipopeptides had been proved to be candidate vaccine adjuvants. In our previous study, lipopeptides mimicking N-terminal structures of the bacterial lipoproteins were also able to promote antigen-specific immune response. However, the structure-activity relationship of lipopeptides as TLR2 agonists is still unclear. Here, 23 synthetic lipopeptides with the same lipid moiety but different peptide sequences were synthesized, and their TLR2 activities in vitro and mucosal adjuvant effects to OVA were evaluated. LP1-14, LP1-30, LP1-34 and LP2-2 exhibited significantly lower cytotoxicity and stronger TLR2 activity compared with Pam2CSK4, the latter being one of the most potent TLR2 agonists. LP1-34 and LP2-2 assisted OVA to induce more profound specific IgG in sera or sIgA in BALF than Pam2CSK4. Furthermore, the possibility of LP1-34, LP2-2 and Pam2CSK4 as the mucosal adjuvant for the SARS-CoV-2 recombinant RBD (rRBD) was investigated. Intranasally immunized with rRBD plus either the novel lipopeptide or Pam2CSK4 significantly increased the levels of specific serum and respiratory mucosal IgG and IgA, while rRBD alone failed to induce specific immune response due to its low immunogenicity. The novel lipopeptides, especially LP2-2, significantly increased levels of rRBD-induced SARS-CoV-2 neutralizing antibody in sera, BALF and nasal wash. Finally, Support vector machine (SVM) results suggested that charged residues in lipopeptides might be beneficial to the agonist activity, while lipophilic residues might adversely affect the agonistic activity. Figuring out the relationship between peptide sequence in the lipopeptide and its TLR2 activity may lay the foundation for the rational design of novel lipopeptide adjuvant for COVID-19 vaccine.

A Palmitic Acid-Conjugated, Peptide-Based pan-CoV Fusion Inhibitor Potently Inhibits Infection of SARS-CoV-2 Omicron and Other Variants of Concern.

Our previous studies have shown that cholesterol-conjugated, peptide-based pan-coronavirus (CoV) fusion inhibitors can potently inhibit human CoV infection. However, only palmitic acid (C16)-based lipopeptide drugs have been tested clinically, suggesting that the development of C16-based lipopeptide drugs is feasible. Here, we designed and synthesized a C16-modified pan-CoV fusion inhibitor, EK1-C16, and found that it potently inhibited infection by SARS-CoV-2 and its variants of concern (VOCs), including Omicron, and other human CoVs and bat SARS-related CoVs (SARSr-CoVs). These results suggest that EK1-C16 could be further developed for clinical use to prevent and treat infection by the currently circulating MERS-CoV, SARS-CoV-2 and its VOCs, as well as any future emerging or re-emerging coronaviruses.

Lipopeptides against COVID-19 RNA-dependent RNA polymerase using molecular docking.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is caused by a novel virus that is responsible for the largest pandemic in recent times. Although numerous studies have explored methods to cope with COVID-19 and targeted drugs and vaccines have been developed, the spread of disease remains rapid due to the high infectivity and mutation capability of SARS-CoV-2, the causative virus of COVID-19. Therefore, there is an urgent necessity to seek more efficient treatments and approaches to combat the disease. METHODS: In this study, molecular docking was used to predict the binding of different lipopeptides, which exhibit significant biological functions, to the RNA-dependent RNA polymerase (also known as nsp12) of SARS-CoV-2, the central component of coronaviral replication and transcription machinery. RESULTS: The results showed that seven lipopeptides bound to nsp12 at the same location as the FDA-approved drug remdesivir, with higher affinities. Notably, iron-chelating ferrocin A (ferrocin A-iron complex [FAC]) bound to nsp12 most tightly, releasing up to 9.1 kcal mol-1 of free energy. Protein-ligand interaction analysis revealed that FAC formed four hydrogen bonds, two hydrophobic interactions, and three salt bridges with nsp12. These active amino acids are mainly distributed in the fingers and thumb subdomains of nsp12 and are highly conserved. CONCLUSIONS: Our findings suggest that the abovementioned lipopeptides can tightly bind to nsp12, and thus represent promising drug candidates for anti-coronaviral treatments with the potential to fight SARS-CoV-2.

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.

Intranasal fusion inhibitory lipopeptide prevents direct-contact SARS-CoV-2 transmission in ferrets.

Containment of the COVID-19 pandemic requires reducing viral transmission. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is initiated by membrane fusion between the viral and host cell membranes, which is mediated by the viral spike protein. We have designed lipopeptide fusion inhibitors that block this critical first step of infection and, on the basis of in vitro efficacy and in vivo biodistribution, selected a dimeric form for evaluation in an animal model. Daily intranasal administration to ferrets completely prevented SARS-CoV-2 direct-contact transmission during 24-hour cohousing with infected animals, under stringent conditions that resulted in infection of 100% of untreated animals. These lipopeptides are highly stable and thus may readily translate into safe and effective intranasal prophylaxis to reduce transmission of SARS-CoV-2.

Chemical composition and pharmacological mechanism of Qingfei Paidu Decoction and Ma Xing Shi Gan Decoction against Coronavirus Disease 2019 (COVID-19): In silico and experimental study.

The Coronavirus Disease 2019 (COVID-19) pandemic has become a huge threaten to global health, which raise urgent demand of developing efficient therapeutic strategy. The aim of the present study is to dissect the chemical composition and the pharmacological mechanism of Qingfei Paidu Decoction (QFPD), a clinically used Chinese medicine for treating COVID-19 patients in China. Through comprehensive analysis by liquid chromatography coupled with high resolution mass spectrometry (MS), a total of 129 compounds of QFPD were putatively identified. We also constructed molecular networking of mass spectrometry data to classify these compounds into 14 main clusters, in which exhibited specific patterns of flavonoids (45 %), glycosides (15 %), carboxylic acids (10 %), and saponins (5 %). The target network model of QFPD, established by predicting and collecting the targets of identified compounds, indicated a pivotal role of Ma Xing Shi Gan Decoction (MXSG) in the therapeutic efficacy of QFPD. Supportively, through transcriptomic analysis of gene expression after MXSG administration in rat model of LPS-induced pneumonia, the thrombin and Toll-like receptor (TLR) signaling pathway were suggested to be essential pathways for MXSG mediated anti-inflammatory effects. Besides, changes in content of major compounds in MXSG during decoction were found by the chemical analysis. We also validate that one major compound in MXSG, i.e. glycyrrhizic acid, inhibited TLR agonists induced IL-6 production in macrophage. In conclusion, the integration of in silico and experimental results indicated that the therapeutic effects of QFPD against COVID-19 may be attributed to the anti-inflammatory effects of MXSG, which supports the rationality of the compatibility of TCM.

Structure-based design and characterization of novel fusion-inhibitory lipopeptides against SARS-CoV-2 and emerging variants.

The ongoing pandemic of COVID-19, caused by SARS-CoV-2, has severely impacted the global public health and socio-economic stability, calling for effective vaccines and therapeutics. In this study, we continued our efforts to develop more efficient SARS-CoV-2 fusion inhibitors and achieved significant findings. First, we found that the membrane-proximal external region (MPER) sequence of SARS-CoV-2 spike fusion protein plays a critical role in viral infectivity and can serve as an ideal template for design of fusion-inhibitory peptides. Second, a panel of novel lipopeptides was generated with greatly improved activity in inhibiting SARS-CoV-2 fusion and infection. Third, we showed that the new inhibitors maintained the potent inhibitory activity against emerging SARS-CoV-2 variants, including those with the major mutations of the B.1.1.7 and B.1.351 strains circulating in the United Kingdom and South Africa, respectively. Fourth, the new inhibitors also cross-inhibited other human CoVs, including SARS-CoV, MERS-CoV, HCoV-229E, and HCoV-NL63. Fifth, the structural properties of the new inhibitors were characterized by circular dichroism (CD) spectroscopy and crystallographic approach, which revealed the mechanisms underlying the high binding and inhibition. Combined, our studies provide important information for understanding the mechanism of SARS-CoV-2 fusion and a framework for the development of peptide therapeutics for the treatment of SARS-CoV-2 and other CoVs.

Pan-coronavirus fusion inhibitors possess potent inhibitory activity against HIV-1, HIV-2, and simian immunodeficiency virus.

EK1 peptide is a membrane fusion inhibitor with broad-spectrum activity against human coronaviruses (CoVs). In the outbreak of COVID-19, we generated a lipopeptide EK1V1 by modifying EK1 with cholesterol, which exhibited significantly improved antiviral activity. In this study, we surprisingly found that EK1V1 also displayed potent cross-inhibitory activities against divergent HIV-1, HIV-2, and simian immunodeficiency virus (SIV) isolates. Consistently, the recently reported EK1 derivative EK1C4 and SARS-CoV-2 derived fusion inhibitor lipopeptides (IPB02 ∼ IPB09) also inhibited HIV-1 Env-mediated cell-cell fusion and infection efficiently. In the inhibition of a panel of HIV-1 mutants resistant to HIV-1 fusion inhibitors, EK1V1 and IPB02-based inhibitors exhibited significantly decreased or increased activities, suggesting the heptad repeat-1 region (HR1) of HIV-1 gp41 being their target. Furthermore, the sequence alignment and molecular docking analyses verified the target site and revealed the mechanism underlying the resistance. Combined, we conclude that this serendipitous discovery provides a proof-of-concept for a common mechanism of viral fusion and critical information for the development of broad-spectrum antivirals.

LPD-12: a promising lipopeptide to control COVID-19.

INTRODUCTION: The recent pandemic outbreak of SARS-CoV-2 has been associated with a lethal atypical pneumonia, making COVID-19 an urgent public health issue with an increasing rate of mortality and morbidity. There are currently no vaccines or therapeutics available for COVID-19, which is causing an urgent search for a new drug to combat the COVID-19 pandemic. The lipid membrane alternation efficiency of small antimicrobial lipopeptides enables them to block viral membrane fusion to the host cell. Lipopeptides could serve as potential antiviral agents, by interacting or competing with viral fusion proteins. METHODS: This study screened seven different lipopeptides (tsushimycin, daptomycin, surfactin, bacillomycin, iturin, srfTE, and LPD-12) and docked them individually against the spike (S)-glycoprotein of SARS-CoV-2. RESULTS: Based on the maximum docked score and minimum atomic contact energy, LPD-12 (-1137.38 kcal) was the appropriate molecule for proper binding with the S-glycoprotein of SARS-CoV-2 and thus significantly interrupted its affinity of binding with angiotensin-converting enzyme-2 (ACE2), which is the only receptor molecule found to be facilitating disease development. The results confirmed a strong binding affinity of LPD-12 with ACE2, with a binding free energy of -1621.62 kcal, which could also reciprocally prevent the binding of S-protein. CONCLUSTION: It can be concluded that LPD-12 may act as a potential therapeutic drug, by reducing the entry of SARS-CoV-2 to the human cells via the ACE2 receptor and related infections.

Prophylactic intranasal administration of a TLR2/6 agonist reduces upper respiratory tract viral shedding in a SARS-CoV-2 challenge ferret model.

BACKGROUND: The novel human coronavirus SARS-CoV-2 is a major ongoing global threat with huge economic burden. Like all respiratory viruses, SARS-CoV-2 initiates infection in the upper respiratory tract (URT). Infected individuals are often asymptomatic, yet highly infectious and readily transmit virus. A therapy that restricts initial replication in the URT has the potential to prevent progression of severe lower respiratory tract disease as well as limiting person-to-person transmission. METHODS: SARS-CoV-2 Victoria/01/2020 was passaged in Vero/hSLAM cells and virus titre determined by plaque assay. Challenge virus was delivered by intranasal instillation to female ferrets at 5.0 × 106 pfu/ml. Treatment groups received intranasal INNA-051, developed by Ena Respiratory. SARS-CoV-2 RNA was detected using the 2019-nCoV CDC RUO Kit and QuantStudio™ 7 Flex Real-Time PCR System. Histopathological analysis was performed using cut tissues stained with haematoxylin and eosin (H&E). FINDINGS: We show that prophylactic intra-nasal administration of the TLR2/6 agonist INNA-051 in a SARS-CoV-2 ferret infection model effectively reduces levels of viral RNA in the nose and throat. After 5 days post-exposure to SARS-CoV-2, INNA-051 significantly reduced virus in throat swabs (p=<0.0001) by up to a 24 fold (96% reduction) and in nasal wash (p=0.0107) up to a 15 fold (93% reduction) in comparison to untreated animals. INTERPRETATION: The results of our study support clinical development of a therapy based on prophylactic TLR2/6 innate immune activation in the URT, to reduce SARS-CoV-2 transmission and provide protection against COVID-19. FUNDING: This work was funded by Ena Respiratory, Melbourne, Australia.

Inhibition of Coronavirus Entry In Vitro and Ex Vivo by a Lipid-Conjugated Peptide Derived from the SARS-CoV-2 Spike Glycoprotein HRC Domain.

The emergence of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), the etiological agent of the 2019 coronavirus disease (COVID-19), has erupted into a global pandemic that has led to tens of millions of infections and hundreds of thousands of deaths worldwide. The development of therapeutics to treat infection or as prophylactics to halt viral transmission and spread is urgently needed. SARS-CoV-2 relies on structural rearrangements within a spike (S) glycoprotein to mediate fusion of the viral and host cell membranes. Here, we describe the development of a lipopeptide that is derived from the C-terminal heptad repeat (HRC) domain of SARS-CoV-2 S that potently inhibits infection by SARS-CoV-2. The lipopeptide inhibits cell-cell fusion mediated by SARS-CoV-2 S and blocks infection by live SARS-CoV-2 in Vero E6 cell monolayers more effectively than previously described lipopeptides. The SARS-CoV-2 lipopeptide exhibits broad-spectrum activity by inhibiting cell-cell fusion mediated by SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV) and blocking infection by live MERS-CoV in cell monolayers. We also show that the SARS-CoV-2 HRC-derived lipopeptide potently blocks the spread of SARS-CoV-2 in human airway epithelial (HAE) cultures, an ex vivo model designed to mimic respiratory viral propagation in humans. While viral spread of SARS-CoV-2 infection was widespread in untreated airways, those treated with SARS-CoV-2 HRC lipopeptide showed no detectable evidence of viral spread. These data provide a framework for the development of peptide therapeutics for the treatment of or prophylaxis against SARS-CoV-2 as well as other coronaviruses.IMPORTANCE SARS-CoV-2, the causative agent of COVID-19, continues to spread globally, placing strain on health care systems and resulting in rapidly increasing numbers of cases and mortalities. Despite the growing need for medical intervention, no FDA-approved vaccines are yet available, and treatment has been limited to supportive therapy for the alleviation of symptoms. Entry inhibitors could fill the important role of preventing initial infection and preventing spread. Here, we describe the design, synthesis, and evaluation of a lipopeptide that is derived from the HRC domain of the SARS-CoV-2 S glycoprotein that potently inhibits fusion mediated by SARS-CoV-2 S glycoprotein and blocks infection by live SARS-CoV-2 in both cell monolayers (in vitro) and human airway tissues (ex vivo). Our results highlight the SARS-CoV-2 HRC-derived lipopeptide as a promising therapeutic candidate for SARS-CoV-2 infections.

Immune Modulation to Improve Survival of Viral Pneumonia in Mice.

Viral pneumonias remain global health threats, as exemplified in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, requiring novel treatment strategies both early and late in the disease process. We have reported that mice treated before or soon after infection with a combination of inhaled Toll-like receptor (TLR) 2/6 and 9 agonists (Pam2-ODN) are broadly protected against microbial pathogens including respiratory viruses, but the mechanisms remain incompletely understood. The objective of this study was to validate strategies for immune modulation in a preclinical model of viral pneumonia and determine their mechanisms. Mice were challenged with the Sendai paramyxovirus in the presence or absence of Pam2-ODN treatment. Virus burden and host immune responses were assessed to elucidate Pam2-ODN mechanisms of action and to identify additional opportunities for therapeutic intervention. Enhanced survival of Sendai virus pneumonia with Pam2-ODN treatment was associated with reductions in lung virus burden and with virus inactivation before internalization. We noted that mortality in sham-treated mice corresponded with CD8+ T-cell lung inflammation on days 11-12 after virus challenge, after the viral burden had declined. Pam2-ODN blocked this injurious inflammation by minimizing virus burden. As an alternative intervention, depleting CD8+ T cells 8 days after viral challenge also decreased mortality. Stimulation of local innate immunity within the lungs by TLR agonists early in disease or suppression of adaptive immunity by systemic CD8+ T-cell depletion late in disease improves outcomes of viral pneumonia in mice. These data reveal opportunities for targeted immunomodulation to protect susceptible human subjects.