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1. Interactions between heparin and SARS-CoV-2 spike glycoprotein RBD from omicron and other variants

   https://doi.org/10.3389/fmolb.2022.912887  

   Gelbach, Adrianne L.; Zhang, Fuming; Kwon, Seok-Joon; Bates, John T.; Farmer, Andrew P.; Dordick, Jonathan S.; Wang, Chunyu; Linhardt, Robert J. (August 2022, Frontiers in Molecular Biosciences)

   Heparan sulfate (HS) acts as a co-receptor of angiotensin-converting enzyme 2 (ACE2) by interacting with severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) spike glycoprotein (SGP) facilitating host cell entry of SARS-CoV-2 virus. Heparin, a highly sulfated version of heparan sulfate (HS), interacts with a variety of proteins playing key roles in many physiological and pathological processes. In this study, SARS-CoV-2 SGP receptor binding domain (RBD) wild type (WT), Delta and Omicron variants were expressed in Expi293F cells and used in the kinetic and structural analysis on their interactions with heparin. Surface plasmon resonance (SPR) analysis showed the binding kinetics of SGP RBD from WT and Delta variants were very similar while Omicron variant SGP showed a much higher association rate. The SGP from Delta and Omicron showed higher affinity ( K D ) to heparin than the WT SGP. Competition SPR studies using heparin oligosaccharides indicated that binding of SGP RBDs to heparin requires chain length greater than 18. Chemically modified heparin derivatives all showed reduced interactions in competition assays suggesting that all the sulfo groups in the heparin polysaccharide were critical for binding SGP RBDs with heparin. These interactions with heparin are pH sensitive. Acidic pH (pH 6.5, 5.5, 4.5) greatly increased the binding of WT and Delta SGP RBDs to heparin, while acidic pH slightly reduced the binding of Omicron SGP RBD to heparin compared to binding at pH 7.3. In contrast, basic pH (pH 8.5) greatly reduced the binding of Omicron SGP RBDs to heparin, with much less effects on WT or Delta. The pH dependence indicates different charged residues were present at the Omicron SGP-heparin interface. Detailed kinetic and structural analysis of the interactions of SARS-CoV-2 SGP RBDs with heparin provides important information for designing anti-SARS-CoV-2 molecules. 

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2. Potential Anti-SARS-CoV-2 Activity of Pentosan Polysulfate and Mucopolysaccharide Polysulfate

   https://doi.org/10.3390/ph15020258  

   Zhang, Fuming; He, Peng; Rodrigues, Andre L.; Jeske, Walter; Tandon, Ritesh; Bates, John T.; Bierdeman, Michael A.; Fareed, Jawed; Dordick, Jonathan; Linhardt, Robert J. (February 2022, Pharmaceuticals)

   With the increased prevalence of new SARS-CoV-2 variants of concern, such as Delta and Omicron, the COVID-19 pandemic has become an ongoing human health disaster, killing millions worldwide. SARS-CoV-2 invades its host through the interaction of its spike (S) protein with a host cell receptor, angiotensin-converting enzyme 2 (ACE2). In addition, heparan sulfate (HS) on the surface of host cells plays an important role as a co-receptor for this viral pathogen–host cell interaction. Our previous studies demonstrated that many sulfated glycans, such as heparin, fucoidans, and rhamnan sulfate have anti-SARS-CoV-2 activities. In the current study, a small library of sulfated glycans and highly negatively charged compounds, including pentosan polysulfate (PPS), mucopolysaccharide polysulfate (MPS), sulfated lactobionic acid, sulodexide, and defibrotide, was assembled and evaluated for binding to the S-proteins and inhibition of viral infectivity in vitro. These compounds inhibited the interaction of the S-protein receptor-binding domain (RBD) (wild type and different variants) with immobilized heparin, a highly sulfated HS, as determined using surface plasmon resonance (SPR). PPS and MPS showed the strongest inhibition of interaction of heparin and S-protein RBD. The competitive binding studies showed that the IC50 of PPS and MPS against the S-protein RBD binding to immobilized heparin was ~35 nM and ~9 nM, respectively, much lower than the IC50 for soluble heparin (IC50 = 56 nM). Both PPS and MPS showed stronger inhibition than heparin on the S-protein RBD or spike pseudotyped lentiviral particles binding to immobilized heparin. Finally, in an in vitro cell-based assay, PPS and MPS exhibited strong antiviral activities against pseudotyped viral particles of SARS-CoV-2 containing wild-type or Delta S-proteins. 

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3. Anti-SARS-CoV-2 Activity of Rhamnan Sulfate from Monostroma nitidum

   https://doi.org/10.3390/md19120685  

   Song, Yuefan; He, Peng; Rodrigues, Andre L.; Datta, Payel; Tandon, Ritesh; Bates, John T.; Bierdeman, Michael A.; Chen, Chen; Dordick, Jonathan; Zhang, Fuming; et al (December 2021, Marine Drugs)

   The COVID-19 pandemic is a major human health concern. The pathogen responsible for COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), invades its host through the interaction of its spike (S) protein with a host cell receptor, angiotensin-converting enzyme 2 (ACE2). In addition to ACE2, heparan sulfate (HS) on the surface of host cells also plays a significant role as a co-receptor. Our previous studies demonstrated that sulfated glycans, such as heparin and fucoidans, show anti-COVID-19 activities. In the current study, rhamnan sulfate (RS), a polysaccharide with a rhamnose backbone from a green seaweed, Monostroma nitidum, was evaluated for binding to the S-protein from SARS-CoV-2 and inhibition of viral infectivity in vitro. The structural characteristics of RS were investigated by determining its monosaccharide composition and performing two-dimensional nuclear magnetic resonance. RS inhibition of the interaction of heparin, a highly sulfated HS, with the SARS-CoV-2 spike protein (from wild type and different mutant variants) was studied using surface plasmon resonance (SPR). In competitive binding studies, the IC50 of RS against the S-protein receptor binding domain (RBD) binding to immobilized heparin was 1.6 ng/mL, which is much lower than the IC50 for heparin (~750 ng/mL). RS showed stronger inhibition than heparin on the S-protein RBD or pseudoviral particles binding to immobilized heparin. Finally, in an in vitro cell-based assay, RS showed strong antiviral activities against wild type SARS-CoV-2 and the delta variant. 

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4. A computationally designed ACE2 decoy has broad efficacy against SARS-CoV-2 omicron variants and related viruses in vitro and in vivo

   https://doi.org/10.1038/s42003-023-04860-9  

   Havranek, Brandon; Lindsey, Graeme Walker; Higuchi, Yusuke; Itoh, Yumi; Suzuki, Tatsuya; Okamoto, Toru; Hoshino, Atsushi; Procko, Erik; Islam, Shahidul M (December 2023, Communications Biology)

   Abstract SARS-CoV-2, especially B.1.1.529/omicron and its sublineages, continues to mutate to evade monoclonal antibodies and antibodies elicited by vaccination. Affinity-enhanced soluble ACE2 (sACE2) is an alternative strategy that works by binding the SARS-CoV-2 S protein, acting as a ‘decoy’ to block the interaction between the S and human ACE2. Using a computational design strategy, we designed an affinity-enhanced ACE2 decoy,FLIF, that exhibited tight binding to SARS-CoV-2 delta and omicron variants. Our computationally calculated absolute binding free energies (ABFE) between sACE2:SARS-CoV-2 S proteins and their variants showed excellent agreement to binding experiments.FLIFdisplayed robust therapeutic utility against a broad range of SARS-CoV-2 variants and sarbecoviruses, and neutralized omicron BA.5 in vitro and in vivo. Furthermore, we directly compared the in vivo therapeutic efficacy of wild-type ACE2 (non-affinity enhanced ACE2) againstFLIF. A few wild-type sACE2 decoys have shown to be effective against early circulating variants such as Wuhan in vivo. Our data suggest that moving forward, affinity-enhanced ACE2 decoys likeFLIFmay be required to combat evolving SARS-CoV-2 variants. The approach described herein emphasizes how computational methods have become sufficiently accurate for the design of therapeutics against viral protein targets. Affinity-enhanced ACE2 decoys remain highly effective at neutralizing omicron subvariants. 

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5. Computational and experimental identification of potential neutralizing peptides derived from human ACE2 against SARS-CoV-2 infection

   https://doi.org/10.1128/jvi.01468-25  

   Yao, Qiaobin; Mahase, Vidhyanand; Hou, Wangheng; Cruz-Cosme, Ruth; Tang, Qiyi; Teng, Shaolei (March 2026, Journal of Virology)

   Liu, Shan-Lu (Ed.)

   ABSTRACT The human angiotensin-converting enzyme 2 (hACE2) is the primary receptor for the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Some human alleles of ACE2 exhibit an improved affinity for the SARS-CoV-2 Spike protein. However, the impact of ACE2 polymorphisms on SARS-CoV-2 infection remains unclear. Our previous study predicted that G431 and S514 in the receptor-binding domain (RBD) of SARS-CoV-2 S1 domain are important for S protein stability, and that S protein residues G496 and F497 and ACE2 residues D355 and Y41 are critical for the RBD-ACE2 interaction. In this study, we explored the potential of hACE2-derived neutralizing peptides as a therapeutic strategy against SARS-CoV-2 and investigated how ACE2 polymorphisms affect RBD-ACE2 binding affinity. We applied computational saturation mutagenesis to systematically screen the binding affinity changes among all possible ACE2 missense mutations within the ACE2-Wuhan-S1 complex. Mutations at ACE2 residues D355 and Y41 were predicted to weaken binding affinity, whereas those at N330 and D30 enhanced it. We identified six ACE2 regions (19–49, 65–102, 320–333, 348–359, 378–395, and 552–563) to be vital for ACE2-RBD interaction. We synthesized peptides corresponding to these six regions and tested them using a pseudotyped viral particle system and dot blot assay. Three peptides were confirmed to bind with the S protein, and four exhibited inhibitory effects. We aligned ACE2-Wuhan-S1 and ACE2-Omicron-S1 complexes, conducted correlation analysis, and observed similar binding patterns, suggesting that these peptides also have the potential to neutralize Omicron strains.IMPORTANCESARS-CoV-2 continues its global spread. In this research, we identified six regions within ACE2 that are vital for interaction with the viral S receptor-binding domain and have the potential to neutralize SARS-CoV-2 infection. Among the six peptides derived from ACE2, three were confirmed to bind with the S protein of the Wuhan strain, and four exhibited inhibitory effects on the Wuhan strain SARS-CoV-2. We also found ACE2 residues D355 and Y41 as weakening affinity, and N330 and D30 as enhancing it. We also aligned this complex with the ACE2-Omicron-S1 complex, performed correlation analyses, and compared their patterns of stability changes upon mutations and obtained similar results, indicating that these peptides may also be effective against Omicron variants. These results provide insight into the role of ACE2 polymorphism in viral entry and suggest that hACE2-derived peptides may offer a promising therapeutic strategy against SARS-CoV-2, demonstrating strong consistency between our computational predictions and experimental outcomes. 

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