2.1  Targeting the RBD
Structural investigations of the RBD‐ACE2 complex provided information about essential residues for viral entry. Hsiang et al. 35  reported a number of peptides that significantly blocked the interaction of the S protein with ACE2 with IC50 values as low as 1.88 nM. Michael et al. found charged residues between positions 22 and 57 crucial for SARS‐CoV‐1 viral entry. Based on this, they designed peptides P4 (IC50, 50 µM) and P5 (IC50, 6.0 µM) with significant inhibitory activity against SARS‐CoV‐1. The antiviral activity was further improved when they introduced the glycine binding linkage of peptide P4 (residues 22–47) with an ACE2‐derived peptide (residues 351–357) against a SARS‐CoV‐1 pseudovirus with an IC50 of 100 nM and devoid of cytotoxicity up to 200 µM. 36  It is worth highlighting that a similar strategy could work for the new SARS‐CoV‐2. The recently solved cryo‐EM structure of SARS‐CoV‐2 in complex with human ACE2 can provide a structural rationale for the peptide design. 29
For viral entry, MERS‐CoV uses its spike protein (S) to interact with the host‐receptor DPP4, 37 ,  38 ,  39  also known as adenosine deaminase‐complexing protein‐2 or CD26. 37  MERS‐CoV was also the first virus reported to use this particular path. 35 ,  37  DPP4 is a type II transmembrane glycoprotein, that forms homodimers on the cell surface, and it is involved in the cleavage of dipeptides. 37 ,  40  In humans, DPP4 is predominantly found on the bronchial epithelial and alveolar cells in the lower lungs. 40 ,  41
MERS‐4 and MERS‐27 are monoclonal antibodies targeting the RBD of MERS‐CoV S that were discovered in a nonimmune yeast‐display scFv library screening. The more active MERS‐4 potently blocked the infection of DPP4‐expressing Huh‐7 cells with pseudotyped MERS‐CoV (IC50, 0.056 μg/mL). It also prevented MERS‐CoV‐induced cytopathogenic effects in MERS‐infected Vero E6 cells (IC50, 0.5 μg/mL). 42
A heptad repeat (HR) is a repeating structural pattern of seven amino acids. A crucial membrane fusion framework of SARS‐CoV is the 6‐helix‐bundle (6‐HB) that is formed by HR1 and HR2 of the viral S protein. Enfuvirtide (T‐20) is an FDA approved HR2 peptide and the first HIV fusion inhibitor. It has opened up new avenues toward identifying and developing peptides as viral entry inhibitors. Such molecules represent a promising strategy against enveloped viruses with class 1 fusion proteins such as Nipah virus, Hendra virus, Ebola virus, and other paramyxoviruses, simian immunodeficiency virus, feline immunodeficiency virus, and respiratory syncytial virus. 43 ,  44 ,  45 ,  46  The HR regions of SARS‐CoV‐1 and SARS‐CoV‐2 S protein share a high degree of conservation, and such fusion inhibitors have potential applications in preventing SARS‐CoV‐2 entry.
Small molecule entry inhibitors, on the other hand, are reported to target the RBD. Compared to peptides, proteins, and biologics, small molecules have several advantages due to lower production costs, improved pharmacokinetics, stability, and dosage accuracy. Sarafianos et al. identified the oxazole‐carboxamide derivative SSAA09E2 (1; Figure 4) as an entry inhibitor against SARS‐CoV‐1 by screening a chemical library composed of 3000 compounds. 47  This inhibitor directly blocks ACE2 recognition by interfering with the RBD with an EC50 value of 3.1 µM and a 50% cytotoxic concentration (CC50) value of greater than 100 µM, not affecting ACE2 expression levels. 48
Figure 4  Inhibitors targeting the receptor‐binding domain Xu et al. 49  identified two small molecules, TGG (2; Figure 4) and luteolin (3; Figure 4), that can bind avidly to the SARS‐CoV‐1 S2 protein and inhibit its entry into Vero E6 cells (EC50: 4.5 µM, 10.6 µM; respectively). Compounds 2 and 3 showed cytotoxicity (CC50) of 1.08 and 0.155 mM, and the selectivity index (SI) values of 2 and 3 were 240.0 and 14.62, respectively. Further studies regarding acute toxicity revealed that the 50% lethal doses of 2 and 3 were ~456 and 232 mg/kg, respectively. These results indicate that these small molecules could be used at relatively high concentrations in mice. 49  Quercetin (4; Figure 4), an analog of 3, also showed antiviral activity against SARS‐CoV‐1, with an EC50 value of 83.4 µM and a CC50 value of 3.32 mM. 50
Ngai et al. reported ADS‐J1 (5; Figure 4) as a potential SARS‐CoV‐1 viral entry inhibitor with an EC50 of 3.89 µM. Molecular docking studies predicted that 5 can bind into a deep pocket of the SARS‐CoV‐1 S HR region and block viral entry into host cells. 51  Imatinib (6; Figure 4), an Abelson kinase inhibitor, could inhibit CoV S protein‐induced fusion with an EC50 value of 10 µM and showed no cytotoxic effects in Vero cells up to 100 µM concentration. 52 ,  53