Punicalagin as an allosteric NSP13 helicase inhibitor potently suppresses SARS-CoV-2 replication in vitro.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) helicase NSP13 plays a conserved role in the replication of coronaviruses and has been identified as an ideal target for the development of antiviral drugs against SARS-CoV-2. Here, we identify a novel NSP13 helicase inhibitor punicalagin (PUG) through high-throughput screening. Surface plasmon resonance (SPR)-based analysis and molecular docking calculation reveal that PUG directly binds NSP13 on the interface of domains 1A and 2A, with a KD value of 21.6 nM. Further biochemical and structural analyses suggest that PUG inhibits NSP13 on ATP hydrolysis and prevents it binding to DNA substrates. Finally, the antiviral studies show that PUG effectively suppresses the SARS-CoV-2 replication in A549-ACE2 and Vero cells, with EC50 values of 347 nM and 196 nM, respectively. Our work demonstrates the potential application of PUG in the treatment of coronavirus disease 2019 (COVID-19) and identifies an allosteric inhibition mechanism for future drug design targeting the viral helicases.

Use of hyphenated analytical techniques to identify the bioactive constituents of Gunnera perpensa L., a South African medicinal plant, which potently inhibit SARS-CoV-2 spike glycoprotein-host ACE2 binding.

SARS-CoV-2, the causative agent of COVID-19, continues to cause global morbidity and mortality despite the increasing availability of vaccines. Alongside vaccines, antivirals are urgently needed to combat SARS-CoV-2 infection and spread, particularly in resource-limited regions which lack access to existing therapeutics. Small molecules isolated from medicinal plants may be able to block cellular entry by SARS-CoV-2 by antagonising the interaction of the viral spike glycoprotein receptor-binding domain (RBD) with the host angiotensin-converting enzyme II (ACE2) receptor. As the medicinal plant Gunnera perpensa L. is being used by some South African traditional healers for SARS-CoV-2/COVID-19 management, we hypothesised that it may contain chemical constituents that inhibit the RBD-ACE2 interaction. Using a previously described AlphaScreen-based protein interaction assay, we show here that the DCM:MeOH extract of G. perpensa readily disrupts RBD (USA-WA1/2020)-ACE2 interactions with a half-maximal inhibition concentration (IC50) of < 0.001 µg/mL, compared to an IC50 of 0.025 µg/mL for the control neutralising antibody REGN10987. Employing hyphenated analytical techniques like UPLC-IMS-HRMS (method developed and validated as per the International Conference on Harmonization guidelines), we identified two ellagitannins, punicalin (2.12% w/w) and punicalagin (1.51% w/w), as plant constituents in the DCM:MeOH extract of G. perpensa which antagonised RBD-ACE2 binding with respective IC50s of 9 and 29 nM. This good potency makes both compounds promising leads for development of future entry-based SARS-CoV-2 antivirals. The results also highlight the advantages of combining reverse pharmacology (based on medicinal plant use) with hyphenated analytical techniques to expedite identification of urgently needed antivirals.

Pomegranate peel extract polyphenols attenuate the SARS-CoV-2 S-glycoprotein binding ability to ACE2 Receptor: In silico and in vitro studies.

The novel coronavirus disease (Covid-19) has become a major health threat globally. The interaction of SARS-CoV-2 spike (S) glycoprotein receptor-binding domain (RBD) with ACE2 receptor on host cells was recognized as the first step of virus infection and therefore as one of the primary targets for novel therapeutics. Pomegranate extracts are rich sources of bioactive polyphenols that were already recognized for their beneficial health effects. In this study, both in silico and in vitro methods were employed for evaluation of pomegranate peel extract (PoPEx), their major polyphenols, as well as their major metabolite urolithin A, to attenuate the contact of S-glycoprotein RBD and ACE2. Our results showed that PoPEx, punicalin, punicalagin and urolithin A exerted significant potential to block the S-glycoprotein-ACE2 contact. These in vitro results strongly confirm the in silico predictions and provide a valuable insight in the potential of pomegranate polyphenols for application in SARS-CoV-2 infection.

Discovery of chebulagic acid and punicalagin as novel allosteric inhibitors of SARS-CoV-2 3CLpro.

The emerging SARS-CoV-2 infection is the cause of the global COVID-19 pandemic. To date, there are limited therapeutic options available to fight this disease. Here we examined the inhibitory abilities of two broad-spectrum antiviral natural products chebulagic acid (CHLA) and punicalagin (PUG) against SARS-CoV-2 viral replication. Both CHLA and PUG reduced virus-induced plaque formation in Vero-E6 monolayer at noncytotoxic concentrations, by targeting the enzymatic activity of viral 3-chymotrypsin-like cysteine protease (3CLpro) as allosteric regulators. Our study demonstrates the potential use of CHLA and PUG as novel COVID-19 therapies.

Computational study of pomegranate peel extract polyphenols as potential inhibitors of SARS-CoV-2 virus internalization.

The search for effective coronavirus disease (COVID-19) therapy has attracted a great deal of scientific interest due to its unprecedented health care system overload worldwide. We have carried out a study to investigate the in silico effects of the most abundant pomegranate peel extract constituents on the multi-step process of serious acute respiratory syndrome coronavirus 2 (SARS-CoV-2) internalization in the host cells. Binding affinities and interactions of ellagic acid, gallic acid, punicalagin and punicalin were studied on four selected protein targets with a significant and confirmed role in the process of the entry of virus into a host cell. The protein targets used in this study were: SARS-CoV-2 spike glycoprotein, angiotensin-converting enzyme 2, furin and transmembrane serine protease 2. The results showed that the constituents of pomegranate peel extracts, namely punicalagin and punicalin had very promising potential for significant interactions with the selected protein targets and were therefore deemed good candidates for further in vitro and in vivo evaluation.