In this study, the high-content imaging system, coupled with the plaque assay, was utilized for the first time to identify anti-SARS-CoV–2 agents from the Thai medicinal plant library, consisting of 114 medicinal plant extracts and 8 purified compounds (details in Supplementary Table 1). Among the positive hits, the crude extract of B. rotunda and its purified compound, panduratin A, demonstrated the most potent inhibitory effect against SARS-CoV–2 replication and infectivity with the favorable cytotoxicity profile. Interestingly, panduratin A inhibited SARS-CoV–2 infectivity and replication at both pre-entry and post-infection phases, and its antiviral activity was even more potent than hydroxychloroquine FDA-approved drug currently used for COVID–19 treatment.8–10 The IC50, CC50, and the selectivity index of panduratin A and hydroxychloroquine were summarized in Table 1. This finding highlighted the potential implication of panduratin A as the novel anti-SARS-CoV–2 candidate for COVID–19 therapy. Nevertheless, in vivo study and the clinical trial are needed to assess the pharmacokinetic effect and the appropriate human dose of panduratin A before clinical use.
Table 1. A summary of anti-SARS-CoV-2 activity (IC50), cytotoxicity (CC50), and the selectivity index (SI) of panduratin A and hydroxychloroquine.
|
IC50
(mM)
|
CC50
(mM)
|
SI
(CC50/IC50)
|
Post-infection
|
|
|
|
Panduratin A
|
0.81
|
14.71
|
18.16
|
Hydroxychloroquine
|
5.08
|
>100
|
>19.68
|
Pre-entry
|
|
|
|
Panduratin A
|
5.30
|
43.47
|
8.20
|
Hydroxychloroquine
|
8.07
|
>100
|
>12.39
|
Boesenbergia rotunda (fingerroot) belongs to the ginger family (Zingiberaceae).. This herb is widely used culinarily in China and Southeast Asia. Extracts of fingerroot rhizomes are well-known for its various pharmacological effects such as anti-allergic,31 antibacterial,32,33 antioxidant,34 and anti-tumor activities.35,36 Among the major active ingredients found in fingerroot, panduratin A, a prenylated cyclohexenyl chalcone, has been reported to possibly exhibit the antiviral activity against HIV–1and dengue virus (DENV).37–40
Several molecular and cellular mechanisms might be employed by panduratin A to exert its effect on anti-SARS-CoV–2 activity. Using the biochemical approach, this phytochemical was demonstrated to physically bind and inhibit an HIV–1 protease37 and a DENV NS2B/NS3 protease.38 Also, the structure-based computational approach supported panduratin A potential as the competitive inhibitor of NS2B/NS3 of DENV2.39,40 Whether this compound interacts with those proteases in vivo is yet to be determined. In this view, panduratin A might act as the protease inhibitor to exhibit the anti-SARS-CoV–2 effect.
Another possible mechanism of panduratin A action might have occurred through its antioxidant activity. This compound itself is a potent reducing agent and can decrease levels of reactive oxygen species (ROS) in vitro.41,42 Whether the ROS scavenging mechanism facilitates the attenuation of SARS-CoV–2 infection by panduratin A, similar to that observed in Japanese Encephalitis virus (JEV),43 is yet to be deciphered. Further, this anti-oxidative stress might be coupled with anti-inflammatory responses widely reported for panduratin A. For example, panduratin A can reduce the expression of genes whose function is involved in inflammation.44–47 Undoubtedly, therapeutic strategies aiming at the modulation of inflammation has been proposed for COVID–19 as a mean to reduce the severity of the disease.48
Besides, panduratin A was found to induce autophagy, which is vital in restricting viral replication. Nonetheless, concerns have also been raised regarding the protective role of autophagy for the evasion of host innate immunity upon viral infection.49–51 Autophagic induction by panduratin A treatment in mammalian cells occurred through the activation of AMPK and inhibition of mTORC1.52,53 The small molecule compound has also been shown to induce PERK/eIF2α/ATF4/CHOP pathway pertinent to endoplasmic reticulum (ER) stress. Consequently, the induction of ER stress can further facilitate autophagy.54,55 Moreover, panduratin A can stimulate AMPK signaling leading to the activation of PPARα and PPARδ.56,57 The induction of these transcription factor machinery can, in turn, promote autophagy.58,59 Consistently, it was reported that MERS-CoV blocked the fusion of autophagosomes and lysosomes. As a result, the induction of autophagy attenuated the replication of this virus.60 Interestingly, the anti-helminthic and FDA-approved drug niclosamide has recently been proposed as a potential anti-SARS-CoV–2 agent,61,62 possibly through its autophagic induction mechanism.63 It has yet to be elucidated whether panduratin A suppresses SARS-CoV–2 infection via the induction of autophagy, and which pathway is a direct target for this compound.
Taken together, we identified B. rotunda extractand its active compound, panduratin A, as the promising anti-SARS-CoV–2 agents by using the high-content imaging system coupled with the plaque reduction assay. Importantly, B. rotunda extractandpanduratin A exhibited the potent antiviral efficacy when the treatment was performed after SARS-CoV–2 infection, with the optimal IC50 (3.62 μg/mL and 0.81 μM, respectively) and the favorable cytotoxicity profile (CC50 28.06 μg/mL and 14.71 μM, respectively). Panduratin A inhibited SARS-CoV–2 infectivity in the pre-entry phase as well. The information from this present study suggested the promise of panduratin A as a single therapy, and as the combinational therapeutic with other FDA-approved agents, for the effective treatment of COVID–19. The possibility of this rationale should be further evaluated. Since B. rotunda is the common plant affordable and available in tropical regions, a pharmaceutically active compound derived from B. rotunda offers a tremendous therapeutic opportunity to fight in this bloody COVID–19 battlefield. Accordingly, we suggested panduratin A as the novel natural candidate for anti-SARS-CoV–2 infection.