3.1. MERS-CoV Entry Inhibitors
MERS-CoV S protein plays a key role in mediating virus entry into host target cells. This process includes binding to host receptors, viral fusion, and final entry into host cells. MERS-CoV pseudovirus expressing S protein, which allows for single-cycle infection in cells expressing receptor DPP4, can be used for screening MERS-CoV fusion/entry inhibitors.
HR2P, spanning residues 1251–1286 in the HR2 domain, with low or no toxic effect in vitro, can effectively inhibit MERS-CoV replication by interacting with the HR1 domain to block spike protein-mediated cell–cell fusion and MERS-CoV pseudovirus entry (
Table 1;
Figure 3) [
16]. To increase its stability, solubility, and anti-MERS-CoV activity, Lu et al. introduced a Glu, Lys, or Arg residue into HR2P, generating a new peptide, HR2P-M2 (
Table 1). HR2P-M2 was indeed found to be more stable and soluble than HR2P. It blocked fusion core formation between HR1 and HR2 peptides by binding to the viral S protein HR1 domain and inhibiting S protein-mediated membrane fusion with an EC
50 of 0.55 µM (
Figure 4) [
16,
23]. HR2P-M2 is highly effective in inhibiting MERS-CoV infection in both Calu-3 and Vero cells with an EC
50 of about 0.6 µM. Intranasal application of HR2P-M2 could significantly reduce the titers of MERS-CoV in the lung of Ad5-hDPP4 (adenovirus serotype-5–human dipeptidyl peptidase 4)-transduced mice [
16,
18]. Furthermore, intranasal administration of HR2P-M2 before viral challenge fully protected hDPP4-transgenic mice from MERS-CoV infection, whereas all untreated mice died 8 days after viral challenge [
24]. Furthermore, by combining HR2P-M2 with interferon β, protection was enhanced for Ad5-hDPP4-transduced mice against infection by MERS-CoV strains with or without mutations in the HR1 region of the S protein, with >1000-fold reduction of viral titers in lung [
18].
P21S10, the most effective fusion inhibitor of MERS-CoV, can inhibit MERS-CoV pseudovirus infection with an EC
50 of about 1 µM in Huh-7 cells and a CC
50 of >100 µM in Huh-7 cells by CCK8 (Cell Counting Kit-8) assay (
Table 1) [
20]. In addition, a series of synthesized stapled peptides, such as P21S10, P21S2, P21S4, P21S5, P21S8, P21S9, P21S8F, P21S8ZF, etc., could effectively inhibit infection by MERS-CoV pseudovirus and its spike protein-mediated cell fusion by blocking helix-mediated NHR (N-terminal heptad repeats) /CHR (C-terminal heptad repeats) interactions with a low EC
50 and a high CC
50 in Huh-7 cells [
20].
P9, a short peptide, exhibited potent and broad spectrum antiviral effects against multiple respiratory viruses in vitro and in vivo [
21,
25]. P9 inhibited MERS-CoV with an EC
50 of about 5 µg/mL in Madin-Darby canine kidney (MDCK) cells, obtained by plaque assay, and a CC
50 of 380 µg/mL in MDCK cells obtained by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay (
Table 1) [
21].
Lipopeptides are bioactive peptides that replicate the α-helical chain from the viral fusion machinery [
22]. All 12 lipopeptides inhibit cell−cell fusion mediated by MERS-CoV S protein with EC
50 values ranging from 0.1 to >10.0 µM in Huh-7 cells (
Table 1) [
22]. Among these lipopeptides, LLS and IIS were found to be the most potent MERS-CoV fusion inhibitors with EC
50 values of 0.24 µM and 0.1 µM, respectively [
22]. Other lipopeptides such as AAS, FFS, YYS, IIY, IIW, IIH, IIQ, IIK, and IIE can also inhibit cell−cell fusion mediated by MERS-CoV S protein with variable EC
50 values [
22].
Three neurotransmitter inhibitors, including chlorpromazine, fluphenazine, and promethazine, were moderate inhibitors of cell–cell fusion with EC
50 values of about 23, 15, and 17 µM, respectively (
Table 2;
Figure 5(5), (45), (46)) [
26]. They can also disrupt clathrin-mediated endocytosis to inhibit MERS-CoV [
26].
A small-molecule HIV entry inhibitor targeting gp41 ADS-J1 (
Figure 5(1)) at the concentration of 20 µM could inhibit >90% of MERS-CoV pseudovirus infection in NBL-7 and Huh-7 cells. ADS-J1 could interrupt the interactions between the HR1 and HR2 of MERS-CoV to form the six-helix bundle, thus inhibiting the entry of pseudotyped MERS-CoV with an EC
50 of 0.6 µM in the DPP4-expressing cell line and with a CC
50 of 26.9 µM in NBL-7 and Huh-7 cells by MTT assay (
Table 2) [
27].
The elucidation of MERS-CoV interaction with its host cell is critical to the development of antiviral interventions. In order to gain entry into host cells, MERS-CoV not only uses DPP4 as a functional virus receptor, but also utilizes certain cellular proteases, such as TMPRSS2 and members of the cathepsin family, as activators of the S glycoprotein [
9]. TMPRSS2 is expressed in epithelial cells of the human respiratory and gastrointestinal tracts [
28,
29,
30,
31]. The respective enzymes from host cells are also excellent targets for the identification of small-molecule MERS-CoV inhibitors. The serine protease inhibitor camostat mesylate (camostat) could completely block syncytium formation, but only partially block virus entry into TMPRSS2-expressing Vero cells (
Figure 5(2)) [
31].
K11777, a compound known to inhibit cruzain, a cathepsin-like protease from the protozoan parasite
Trypanosoma cruzi, can inhibit MERS-CoV with an EC
50 of 46 nM (
Figure 5(3)) [
32,
33].
Chloroquine inhibited MERS-CoV replication and blocked infection at an early step with an EC
50 of 3 µM and a CC
50 of 58 µM (
Table 2;
Figure 5(4)) [
34]. Chlorpromazine inhibited MERS-CoV replication at both early and post-entry stages with an EC
50 of about 5 µM and a CC
50 of 21 µM (
Table 2;
Figure 5(5)) [
34]. However, high cytotoxicity narrowed the therapeutic window in both monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs) [
34].
Ouabain and bufalin can inhibit MERS-CoV entry by blocking clathrin-mediated endocytosis (
Figure 5(6), (7)) [
25,
35]. The addition of small amounts of ouabain (50 nM) or bufalin (10 to 15 nM) inhibited infection with MERS-CoV and VSV (vesicular stomatitis virus) (
Table 2), but only when the drug was added prior to inoculation in Huh-7 cells [
35].
Dihydrotanshinone, a lipophilic compound, showed a decimal reduction at 0.5 µg/mL and excellent antiviral effects at ≥2 μg/mL with a reduction in titer from 6.5 Log to 1.8 Log TCID
50/mL by using a pseudovirus expressing MERS-CoV spike protein (
Figure 5(8)) [
36].
During the biosynthesis of MERS-CoV S protein, the furin inhibitor decanoyl-RVKR-chloromethylketone (dec-RVKR-CMK) at 75 µM can lead to a decrease of the 85-kDa cleaved product in MERS-CoV S wt and S2′ mutant (
Figure 5(9)) [
37].
3.3. Other Small-Molecule Inhibitors with Defined or Undefined Mechanisms of Action
Silvestrol, an eIF4A inhibitor, can inhibit MERS-CoV infection with an EC
50 of 1.3 nM, as shown by plaque assay in MRC-5 cells and CC
50 of 400 nM by MTT assay in peripheral blood mononuclear cells (PBMCs) (
Table 2;
Figure 5(23)) [
57]. Silvestrol has broad-spectrum antiviral activity via the inhibition of the expression of CoV structural and nonstructural proteins (N, nsp8) and the formation of viral replication/transcription complexes [
57].
The combination of interferon-α2b and ribavirin can effectively reduce MERS-CoV replication in vitro and in vivo (
Table 2;
Figure 5(24)) [
6]. Rhesus macaques treated with IFN-α2b and ribavirin 8 h after MERS-CoV infection showed improved clinical parameters with no or very mild radiographic evidence of pneumonia compared with untreated macaques [
6]. Moreover, treated macaques showed lower levels of systemic (serum) and local (lung) proinflammatory markers in addition to fewer viral genome copies, distinct gene expression, and less severe histopathological changes in the lungs [
6].
GS-5734 (Remdesivir), the monophosphoramidate prodrug of the C-adenosine nucleoside analogue GS-441524, can inhibit the replication of the model β-coronavirus murine hepatitis virus (MHV) and RNA synthesis in wild-type (WT) virus, while an nsp14 ExoN (-) mutant lacking proofreading demonstrated increased susceptibility to GS-5734 (
Figure 5(25)) [
58]. GS-5734 also inhibits MERS-CoV infection with an EC
50 of 0.074 ± 0.023 µM and a CC
50 of >10 µM in human amniotic epithelial (HAE) cells (
Table 2) [
58]. Furthermore, GS-5734 acts at the early post-infection stage to decrease viral RNA levels, whereas delaying the addition of GS-5734 until 24 h post-infection resulted in decreased viral titer in HAE cell cultures at 48 and 72 h post-infection [
58]. The nucleotide analogue GS-441524 also inhibits the infection of MERS-CoV with an EC
50 of 0.9 µM and a CC
50 of >100 µM in HAE cells (
Table 2;
Figure 5(26)) [
58].
Resveratrol was found to significantly inhibit MERS-CoV infection as well as prolong cellular survival after virus infection (
Figure 5. (27)) [
66]. It was found that resveratrol could reduce RNA levels and infection titers in Vero cells [
66]. Although resveratrol has minimal cytotoxicity, even at the high concentration of 250 μM, it can be ignored when compared to the much more severe toxicity of MERS-CoV infection [
66].
A series of FDA-approved compounds were screened against MERS-CoV (
Table 2) by cell-based ELISA assay (
Figure 5(28–56)) [
7]. Pharmaceuticals that inhibit MERS-CoV include neurotransmitter inhibitors, estrogen receptor antagonists, kinase signaling inhibitors, inhibitors of lipid or sterol metabolism, protein processing inhibitors, inhibitors of DNA synthesis/repair, as well as inhibitors of ion transport, cytoskeleton (specifically tubulin), and apoptosis [
7]. Antiparasitics and antibacterials are two classes of pharmaceuticals, the functions of which are not obviously linked to coronaviruses, or viruses in general, but nonetheless show antiviral activity against MERS-CoV.
Nocodazole, targeting the cytoskeleton, specifically interferes with microtubule polymerization. It is an antimitotic drug developed for the treatment of cancer, but it was found to show high activity against MERS-CoV (
Figure 5(57)) [
67,
68]. Monensin and salinomycin sodium, two of the nine ion channel inhibitors, have inhibitory activity against MERS-CoV, indicating that MERS-CoV may be susceptible to ionophore activities (
Figure 5 (58), (59)). Chlorpromazine and chloroquine appear to target host factors, rather than viral proteins specifically, and the treatment of viral infections in patients aimed at host factors could reconfigure overt manifestations of viral pathogenesis into a less virulent subclinical infection and lower adverse disease outcome (
Figure 5(60), (29)) [
34,
69].
Loperamide, an antidiarrheal opioid receptor agonist that reduces intestinal motility, also inhibits the replication of MERS-CoV at low-micromolar concentrations (3.3–6.3 µM)
in vitro (
Table 2;
Figure 5(55)) [
34]. Lopinavir, the HIV-1 protease inhibitor, inhibits MERS-CoV replication with an EC
50 of 8 µM (
Table 2;
Figure 5(56)) [
34].
SSYA10-001 inhibits MERS-CoV replication with an EC
50 of ~25 μM in Vero E6 cells (
Table 2;
Figure 5(61)) [
70]. Molecular modeling data suggest that SSYA10-001 can be docked with a comparable “Glide” score [
70].
ESI-09 can reduce virus yield by inhibiting cAMP signaling in a cell type-independent manner (
Figure 5(62)) [
61]. The concentration of MERS-CoV inhibition by ESI-09 was found with an EC
50 of 5 to 10 µM and a CC
50 > 50 µM for both Calu-3 and Vero E6 cells by using the lactate dehydrogenase (LDH)-based cytotoxicity assay [
62]. In addition, the undetectable cytopathic effect (CPE) and minimal expression of viral antigen indicated that Calu-3 cells treated with ESI-09 were almost fully protected [
61].
Mycophenolic acid (MPA) can strongly reduce MERS-CoV replication by inhibiting inosine monophosphate dehydrogenase (IMPDH) and guanine monophosphate synthesis with an EC
50 of 2.87 µM by cell-based ELISA in Vero E6 cells (
Table 2;
Figure 5(63)) [
60].
K22 is a spectrum inhibitor which can inhibit MERS-CoV replication by reducing the formation of double membrane vesicles (DMVs) and by the near-complete inhibition of RNA synthesis (
Figure 5(64)) [
25,
71].
BCX4430, an adenosine analogue that acts as a non-obligate RNA chain terminator to inhibit viral RNA polymerase function, can inhibit MERS-CoV infection with EC
50 of 68.4 μM in Vero E6 cells by highly charged ions (HCIs)-based analysis and CC
50 of >100 μM by neutral-red uptake (
Table 2;
Figure 5(65)) [
25,
62].
Fleximer nucleoside analogues of acyclovir are doubly flexible nucleoside analogues based on the acyclic sugar scaffold of acyclovir and the flex-base moiety in fleximers responsible for inhibiting RNA-dependent RNA polymerase (RdRp) [
25,
63]. The target fleximer analogue 2 can inhibit MERS-CoV infection with EC
50 of 27 μM and CC
50 of 149 μM in Huh-7 cells, but EC
50 of 23 μM and CC
50 of 71 μM in Vero cells (
Table 2;
Figure 5(66)) [
63].
Interferon alpha1 (IFN-α1) and cyclosporine (CsA) have additive or synergistic effects in limiting MERS-CoV replication in ex vivo cultures of human bronchus (
Figure 5(67)) [
72]. In addition, the combined treatment of IFN-α1 and CsA has the most potent effect on inducing interferon-stimulated genes (ISGs) in both lung (24 hpi) and bronchial (56 hpi) tissues [
72].
Saracatinib, a potent inhibitor of the Src-family of tyrosine kinases (SFK), potently inhibits MERS-CoV with an EC
50 of about 3 μM in Huh-7 cells (
Table 2;
Figure 5(68)) [
64]. It possibly inhibits MERS-CoV replication through the suppression of SFK signaling pathways at the early stages of the viral life cycle [
64]. In addition, another seven compounds, primarily classified as antiprotozoal, anticancer, and antipsychotic, were also determined by complete dose-response analyses (
Table 2;
Figure 5(69–75)) [
64].
A spectrum-inhibitor, FA-613, can inhibit MERS-CoV with an EC
50 of ~10 μM in the interferon-competent cell line of Huh-7 cells, as shown by MTT assay (
Table 2;
Figure 5(76)) [
65].