Fighting COVID-19 intranasallyMarch 6, 2021
Containing the COVID-19 pandemic calls for a reduction in viral transmission rates. It is well established that the infection of the SARS-CoV-2 virus is initiated by the fusion of the virus’ spike protein (S- protein) with the ACE-2 membrane protein of the human cell or cell surface or the endosomal membrane. Upon the attachment of the virus to the cell surface, a host of reactions are triggered, including conformational rearrangements leading to viral entry. There are 4 other betacoronavirus (HCoV-OC43, HCoV-HKU1, SARS-CoV and Middle East respiratory syndrome CoV or MERS-CoV) but the fifth and the latest, SARS-CoV-2, seems to have a 20 times higher affinity to bind to the ACE-2 receptor, making it more infectious and transmissible than the others. The S protein is made out of two subunits called S1 and S2, of which S1 plays a crucial role in the virus binding to the cell surface. The S1 subunit comprises a receptor-binding domain (RBD) which interacts with the ACE-2 receptor, while S2 consists of heptad repeat regions (HR1 and HR2) which play an important role in spike protein trimerizing. A functional S protein needs to be a trimer and this function is crucial for viral assembly. There are several research groups and commercial organisations working on a broad spectrum of peptide-based inhibitors of both S1 and S2 subunits of S protein.
Blocking the entry
In the natural condition, the S protein on the surface of the virus is inactive. Once bound to the ACE-2 receptor, the host cell proteases cleave the S protein and further fusion is triggered, exposing the S2 for membrane fusion. In all coronaviruses, a site called S2’ located upstream of fusion peptide (FP) of the S protein is cleaved by the host protease. At the boundary of S/S2 subunits, there is a furin cleavage site present in SARS-CoV-2, which is different from SARS-Co-V, but similar to MERS-CoV. Of the two possible mechanisvwms mentioned above for viral cell entry, the direct fusion on the cell surface and endocytosis is promoted by a low pH environment due to the cleavage of S protein by pH-dependent cysteine protease cathepsin L (CPL). For the direct fusion on the cell membrane, the transmembrane protease serine 2 (TMPRSS2) plays a role in cleavage and activation. Therefore, there are two possible mechanisms to block the virus — design molecules that can block the HR1 and 2 thus suppressing the viral protein trimerization, or block the receptor-binding domain from interacting with the host cell receptor.
Fusion inhibitors can be small molecules, antibodies, or peptides. Several antibodies targeting the S protein of coronavirus have been reported, such as SARS-CoV neutralising CR3022, m369 and S109.8, similar to m336 and SAB-301 for MERS. Two of the reported SARS-CoV2 antibodies are 31B5 and 32D4. However, these are not broad spectrum, and the targeting epitope was not RBD of the S protein. Therefore, this is a low probability of its clinical use. The recombinant ACE2-Ig fusion protein showed neutralising function with pseudotyped SARS-CoV-2 and SARS-CoV by utilising ACE2 as their receptor. The inhibitory concentration (IC50) values of these were 0.1 and 0.8 μg/mL, respectively. They inhibited SARS-CoV-2 and SARS-CoV S-mediated cell fusion with the IC50 of 0.65 and 0.85 μg/mL, respectively. A few studies have shown that the ACE-2 receptor has a negative regulatory role in the renin-angiotensin system (RAS). Coronavirus down-regulated the angiotensin II receptor (AT2), leading to the inactivation of RAS and the increase of ACE-2 suggesting the important role of ACE-2 and its potential use in therapy. Some studies have shown that SARS-CoV-RBD protein immunised mouse serum could neutralize SARS-CoV2. Another study used monoclonal antibody 47D11 raised against full-length spike protein for similar results. This antibody was studied using the plaque reduction neutralisation test (PRINT), in which it gave IC50 values of 0.19 and 0.57 μg/mL for SARS-CoV and SARS-CoV-2 respectively. However, lack of cross-reactivity and efficacy makes antibodies not a good choice for therapy in comparison to peptides.
Protease inhibitors play key
A few small molecules which inhibit ACE-2 receptors and interferon-inducible transmembrane (IFITM) proteins, such as N-(2-aminoethyl)-1 aziridine-ethanamine, are undergoing evaluation to understand their utility in therapy. Recent studies have also identified some inhibitors of proteases which play an important role in viral entry, as described above. Camostat mesylate or nafamostat mesylate, clinical drugs used for chronic pancreatitis in Japan, were seen to suppress TMPRSS2 activity. Another molecule, K11777 – an inhibitor of cathepsin-L-like protease, is also being studied for blocking viral entry. Currently, there are two major efforts which are being undertaken to use peptides for blocking viral binding to ACE-2 receptor and viral S protein trimerization, are gaining prominence. Companies in India have designed a group of peptides which are presently combined with lipid molecules to block the S protein–ACE-2 interaction as also the viral assembly. These peptides can truly help in preventing virus-host cell interaction and keep the virus from multiplying. Pre-clinical studies of these molecules are currently underway.