SARS-CoV-2 surface targets were observed to preferentially interact with ACE2 network proteins but not ACE2 itself. This wasn’t surprising because the previous reports showing the interaction of ACE2 with coronavirus spike protein were performed in cell based assay systems using Vero E6 cells isolated from African green monkeys.[7, 8] While structural similarities of proteins are evident across different species, the functional molecular interactions may differ between different species and as well as between different individuals of same species. The Insilco molecular interaction analysis allows to overcome this limitations by facilitating species specific molecular interaction analysis. Further the short peptide sequences used to identify protein targets based on antibody-antigen interactions in cell based assay system may often cross react due to similarities between different protein sequence, resulting in false positive outcomes. ACE2 exists in soluble as well as membrane bound[7, 17] forms. It is likely that the reported affinity of SARS-CoV to soluble form of ACE2 may differ with the membrane bound form of ACE2, this together with the species specific differences highlighted above may explain the weak interactions observed between ACE2 and SARS-CoV-2. Considering the weak interactions between ACE2 and SARS-CoV-2, it is unlikely that approaches to upregulate ACE2 or using soluble forms of ACE2[7, 17] will be therapeutically beneficial in the clinical management of COVID-19.
Coronavirus are also reported to bind to DPP4 receptors on cell surface.[18, 19] Both protein M and 6YLA but not protein S, were observed to strongly bind with DPP4 (Figure 1b). Interestingly DPP4 was one of the network protein of hACE2 identified (Figure 1a). Hence a network analysis[9, 20] was performed using the STRING database to identify all the primary ACE2 network proteins. The SARS-CoV-2 protein S did not bind to hACE2 or any of its other five network proteins, hence it is unlikely that this spike protein is involved in attachment and entry of the virus into the host cells. Further the stronger binding of SARS-CoV-2 protein M and 6YLA to DPP4 compared to hACE2 (Figure 1b), suggests that SARS-CoV-2 may preferentially attach to DPP4 rather than hACE2 for attaching and entering into the host cell. Consistent with this study, another strain of human coronavirus was reported to preferentially use DPP4 rather than ACE2 as the host cell entry receptor.[18, 19] DPP4 is highly expressed in apical surfaces of human bronchiolar epithelial cell and lung tissue,[18, 19] hence this receptor may be involved in increasing the susceptibility of the respiratory system to human coronavirus infection.
The SARS-CoV-2 surface proteins were observed to effectively bind with all the network proteins of ACE2 with better efficiency (Figure 2a). Among the network proteins DPP4 and meprin A alpha were observed to bind to eight different SARS-CoV-2 surface proteins (Figure 2b). While the meprin A beta, MME, PRCP and XPNPEP2 were observed to be binding with at least 3-4 SARS-CoV-2 target proteins (Figure 2b). This observations is in contrast to current knowledge on specific strains of coronavirus binding to selective host cell receptors.[3, 6] It is likely that SARS-CoV-2 can either sequentially or preferentially interact with ACE2 network proteins (but not ACE2) for its attachment and entry into host cells. Currently the following three host cell membrane receptors; ACE2, aminopeptidase N (APN or CD13) or DPP4 are reported to be receptor for coronaviruses[8, 19]. However this is the first report of a single coronavirus strain potentially utilizing multiple host cell receptors for attachment and cell entry. Such wider choices in host cell receptors for SARS-CoV-2 attachment and entry may enhance the infectivity potential of this virus and probably also lead to poor development of immunity by the host. Although APN was not among the network protein of ACE2, it was evaluated for its interaction with the seventeen SARS-CoV-2 target proteins. APN did not bind to any of the SARS-CoV-2 target proteins, but was observed to bind with SARS-CoV-2 non-structural protein 10/16 (nsp10/16) (data not shown), which is a RNA methyltransferase involved in virus replication rather than virus attachment to host cell. Unlike the previously known strains of coronavirus, SARS-CoV-2 seem to specifically bind with ACE2 network proteins (preferentially DPP4 and meprin A alpha) for attachment and entry into host cells. This knowledge of SARS-CoV-2 using the ACE2 network proteins will be helpful in the development of novel therapeutics and repurposing existing therapeutics for the clinical management of COVID-19.
Among the host cell receptors for SARS-CoV-2 identified in this study, DPP4 was the only receptors for which FDA approved selective inhibitors (sitagliptin, vildagliptin, saxagliptin) are available. Hence the DPP4 inhibitors, saxagliptin and sitagliptin were selected to assess their pharmacological efficacy against SARS-CoV-2. To assess the efficacy of DPP4 inhibitors, saxagliptin and sitagliptin were docked against the selected SARS-CoV-2 surface proteins (protein M, 6YLA and 6M0J) in the AutoDock Vina software.[12-14] Molecular docking with DPP4 and 6Y2E (SARS-CoV-2 main protease) were used as positive and negative controls respectively. The dose response effect of saxagliptin and sitagliptin against their selected targets (Figure 3a) were simulated using the one standard deviation variation of the IC50 values. This new approach to model the dose response curves of ligand based on the IC50 values estimated from the data of molecular docking will be a valuable tool in the Insilco and network pharmacology[9, 20] for drug evaluation or repurposing.
Although DPP4 was reported as a selective receptor for hCoV-EMC,[18, 19] its inhibitors were unlikely to block hCoV-EMC infection due to irrelevance of the DPP4 mediated proteolytic activity in the host cell entry of the virus. However in this study DPP4 inhibitors were observed to have similar binding efficacy to both DDP4 and SARS-CoV-2 surface proteins (Figure 3 and 4). Hence the potential of DPP4 inhibitors in preventing SARS-CoV-2 attachment and entry into the host cells merits validation in clinical trials. Further the analysis of the binding regions of the SARS-CoV-2 surface protein with ACE2 network proteins may be valuable in development of effective therapeutic, vaccine or diagnostic molecules.