5. Multi-Stage Inhibitors, Their Proposed Mechanisms, and Their Potency against SARS-CoV-2
Antiviral compounds that can inhibit early and late stages of virus infection are preferable as they have the potential to target multiple stages of the virus life cycle, thus preventing viral adaptation and the emergence of drug-resistant viruses. SARS-CoV-2 is highly mutagenic due to the inherent error-prone nature of RNA replication, and many novel variants of SARS-CoV-2 have emerged. These new variants might confer resistance to antivirals that target a single stage of the virus life cycle. Therefore, the search for SARS-CoV-2 inhibitors that target both the pre- and post-infection stages is crucial in reducing the likelihood of drug resistance.
Chingguan Yihau (NRICM101) is a TCM formulated in Taiwan and that was used in clinical settings for COVID-19 patients
[98][59]. Patients were administered 100 mL three times daily 30 min after a meal. Clinical data showed that patients with the underlying disease who showed no improvement after 21 days of hospitalizations had benefited from NRICM101 treatment without adverse effects
[98][59]. A large-scale observational study involving 51,000 participants was carried out to evaluate the outcome of NRICM101 on SARS-CoV-2 infection. Participants were subjected to 2–4 times intervention of NRISM101 daily, and the clinical trial’s primary endpoint (NCT04664049) was achieving a negative COVID-19 test result and being free from COVID-19 symptoms within 2 months. The trial has been completed, but the outcomes are still pending. In vitro findings from surface plasmon resonance (SPR) analysis indicated that NRICM101 could bind to the RBD protein dose-dependently. At the same time, ELISA showed that NRICM101 inhibited S protein from binding to the ACE2 receptor with an IC
50 value of 0.41 mg/mL. Additionally, NRICM101 could also inhibit the 3CL
pro enzymatic activity with an IC
50 value of 0.22 mg/mL. In addition, NRICM101 treatment also reduced the growth of SARS-CoV-2 in terms of viral protein expression (IC
50 = 0.28 mg/mL) and plaque formation. Besides its promising antiviral activity, NRICM101 could also reduce the production of pro-inflammatory cytokines, IL-6, and TNF-α in lipopolysaccharide (LPS)-stimulated alveolar macrophages with an IC
50 value of 0.42 and 1.18 mg/mL, respectively
[98][59]. When antiviral studies were performed on the individual herb of NRICM101, researchers reported that the
Scutellaria baicalensis component was responsible for its effective anti-3CL
pro activity (100%). Other components, such as
Schizonepeta tenuifolia,
Morus alba,
Magnolia officinalis, and
Mata and
Mentha haplocalyx in NRICM101, effectively blocked the binding of the S protein to ACE2 (>70%). In addition,
Scutellaria baicalensis and
Houttuynia cordata with 20-fold dilution could inhibit IL-6 and TNF-α production, and
Scutellaria baicalensis was able to inhibit cytokine production at 40-fold dilution
[98][59]. Another component of NRICM101,
Mentha haplocalyx, the natural source of Chinese peppermint, has gained some attention as an antiviral agent against SARS-CoV-2. It was used as traditional medicine for minor ailments
[99][119]. The essential oil extracted from peppermint leaves could improve muscle pain and itching or be used as a fragrance
[100][120]. Using a cell-based HTS assay to screen 190 traditional herbal medicines, researchers identify
Mentha haplocalyx extract as an effective anti-SARS-CoV-2 compound in vitro and in vivo. Along with
Mentha haplocalyx,
Ganoderma lucidum extracts could also reduce the CPE of SARS-CoV-2 in Vero cells at 960-fold dilution. In the hamster model, oral administration of
M. haplocalyx extracts at 200 mg/kg/day for three consecutive days after viral infection showed promising antiviral activity and significantly reduced lung viral titres
[101][60].
Isorhamnetin is a flavonoid extracted from sea buckthorn or
Hippophae rhamnoides L. The extract from the berries of this plant was reported to have anticancer, antiviral, anti-diabetic, and immune regulatory activities
[102][103][104][105][121,122,123,124]. In one of the studies, the active compound of sea buckthorn, isorhamnetin, was tested against the SARS-CoV-2 S pseudotyped virus in vitro. It was observed that isorhamnetin could block SARS-CoV-2 S pseudotyped virus entry into the cells expressing ACE by 47.7% at a non-toxic concentration of 50 μM. Quercetin, another component of sea buckthorn, was similarly evaluated, but there was no significant reduction in viral entry. Using SPR analysis, isorhamnetin showed an affinity towards the immobilized ACE recombinant protein, and molecular docking analysis further proved that isorhamnetin could bind to ACE2 at K353, E37, and H34 residues
[106][76]. In addition, high throughput virtual screening (HTVS) of naturally occurring phytochemicals also showed that, along with other flavonoids, isorhamnetin could interact with the S2 domain of the SARS-CoV-2 S protein with the binding energy of −8.3 Kcal/mol
[107][78]. On the other hand, various derivatives of isorhamnetin from
Salvadora persica were reported to have a strong binding affinity towards SARS-CoV-2 M
pro with a binding orientation similar to the positive control N3, which binds to the Cys–His catalytic dyad located between domains I and II of SARS-CoV-2 M
pro. Structure–activity relationship analysis revealed the presence of disaccharide rutinose (α-L-rhamnopyranosyl-(1-6)-β-D-glucopyranose) at position carbon no. 3 flavonoids helped to enhance the binding stability in the N3 binding site of SARS-CoV-2 M
pro [108][77]. Since these studies showed that isorhamnetin could interact with host protein (ACE2 receptor), viral structural protein (S protein), and a viral non-structural protein (SARS-CoV-2 M
pro), isorhamnetin should be considered for further study to develop it into a potent antiviral drug that could target multiple stages of the viral life-cycle, thus preventing the replication of SARS-CoV-2.
Ionophore antibiotics are a family of natural compounds produced by microorganisms, which are well known for their antibacterial activity against gram-positive bacteria
[109][110][125,126]. In addition, the antiviral activity of ionophore antibiotics against HIV, influenza virus, ZIKV, MERS-CoV, and SARS-CoV was also reported
[111][112][113][127,128,129]. Recently, researchers screened 11 different naturally occurring polyether ionophores for their potential to prevent the CPE caused by SARS-CoV-2 in Vero E6 cells that overexpressed TMPRSS2. Ionophore antibiotics such as narasin, salinomycin, and nanchangmycin exhibited >100-fold selectivity. Surprisingly, two compounds, maduramycin and X-206, showed a selective index of 313 and 586, respectively, higher than the remdesivir control (SI ≥ 67). Compound X-206 displayed significant antiviral activity against SARS-CoV-2 by reducing CPE (IC
50 = 14 nM), viral RNA copy number, viral S proteins, and viral plaque formation by SARS-CoV-2. Time-of-addition assays revealed that X-206 achieved approximately two-fold log reduction in viral progeny at all time points from 4 h pre-infection to 8 h post-infection. Based on morphological profiling, which provided bioactivity fingerprints, X-206 had a different mechanism of action than the control lysosomotropic hydroxychloroquine (HCQ). However, the molecular mechanism of this compound has not been elucidated, and further experiments should be conducted to discover the mode of action of X-206
[114][86]. The ionophore antibiotics, salinomycin and niclosamide, were observed to inhibit syncytia formation, which indicates that this is caused by the fusion of cells induced by viral infection. It was observed that niclosamide and salinomycin exhibited cell protection effects against SARS-CoV-2 with IC
50 of 0.34 and 0.22 μM, respectively. Additionally, salinomycin and niclosamide also inhibited viral replications in respiratory Calu-3 cells. Furthermore, a low concentration of 1 μM of niclosamide blocked the transmembrane member 16 (TMEM16) chloride channel. It significantly attenuated the effect of spontaneous calcium transients in the presence of the S protein, suggesting that ionophore antibiotics might block intracellular calcium release to prevent syncytia formation
[115][87]. Unfortunately, a small scale (73 participants) phase 2 randomized clinical trial (NCT04399356) reported that niclosamide intervention did not shorten the symptom duration of mild to moderate COVID-19 compared to the placebo group
[116][130]. A larger (1200 participants) phase 4 randomized clinical trial (NCT05087381) is currently ongoing in order to further evaluate the benefit of niclosamide for COVID-19 early treatment.
Oleandrin is one of the bioactive compounds found in
Nerium oleander extract and was identified as a unique lipid-soluble cardiac glycoside that acted on the Na/K ATPases pump to enhance heart contraction in heart failure patients. It was also reported to be used in treating dermatological diseases and cancers
[117][118][119][131,132,133]. Recently, oleandrin was reported to be a potent inhibitor of SARS-CoV-2. When oleandrin was present before (prophylactic) and during the whole viral life cycle, it could completely reduce the plaque formation of SARS-CoV-2 by 4 log
10, with IC
50 values of 11.98 and 7.07 ng/mL for 24 and 48 hpi, respectively. Additionally, when oleandrin was added 24 h post-infection, where extensive viral replication had already occurred, oleandrin could still maintain its therapeutic effect. In the hamster model, oleander extract did not cause any toxicity effects regarding body weight, organ lesions, alkaline phosphatase (ALP), and alanine aminotransferase (ALT) levels. In vivo, the prophylactic efficacy of oleander extract against SARS-CoV-2 showed a significant reduction in viral loads in nasal turbinates after 3 days post-infection. From day 3 onwards, the viral titre was below the detection limit and, after four days of treatment, the virus was completely cleared
[120][64]. Besides SARS-CoV-2, oleandrin was also reported to be a potent inhibitor of HCoV-OC43 when CPE (IC
50 = 26 mM) and viral titre (2–3 log10 reduction) were measured as the end point
[121][134].