Current Updates on Naturally Occurring Compounds Recognizing SARS-Cov-2 Druggable Targets

molecules

Review Current Updates on Naturally Occurring Compounds Recognizing SARS-CoV-2 Druggable Targets

Isabella Romeo 1,2, Francesco Mesiti 2 , Antonio Lupia 2 and Stefano Alcaro 1,2,*

1 Dipartimento di Scienze della Salute, Università “Magna Græcia” di Catanzaro, Campus “S. Venuta”, Viale Europa, 88100 Catanzaro, Italy; [email protected] 2 Net4Science Academic Spin-Off, Università “Magna Græcia” di Catanzaro, Campus “S. Venuta”, Viale Europa, 88100 Catanzaro, Italy; [email protected] (F.M.); [email protected] (A.L.) * Correspondence: [email protected]; Tel.: +39-0961-3694198

Abstract: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified in China as the etiologic agent of the recent COVID-19 pandemic outbreak. Due to its high transmissibility, this virus quickly spread throughout the world, causing considerable health issues. The scientific community exerted noteworthy efforts to obtain therapeutic solutions for COVID-19, and new scientific networks were constituted. No certified drugs to efficiently inhibit the virus were identified, and the development of de-novo medicines requires approximately ten years of research. Therefore, the repurposing of natural products could be an effective strategy to handle SARS-CoV-2 infection. This review aims to update on current status of the natural occurring compounds recognizing SARS-

CoV-2 druggable targets. Among the clinical trials actually recruited, some natural compounds are ongoing to examine their potential role to prevent and to treat the COVID-19 infection. Many

Citation: Romeo, I.; Mesiti, F.; Lupia, natural scaffolds, including alkaloids, terpenes, flavonoids, and benzoquinones, were investigated by A.; Alcaro, S. Current Updates on in-silico, in-vitro, and in-vivo approaches. Despite the large data set obtained by a computational Naturally Occurring Compounds approach, experimental evidences in most cases are not available. To fill this gap, further efforts to Recognizing SARS-CoV-2 Druggable validate these results are required. We believe that an accurate investigation of naturally occurring Targets. Molecules 2021, 26, 632. compounds may provide insights for the potential treatment of COVID-19 patients. https://doi.org/10.3390/ molecules26030632 Keywords: SARS-CoV-2; natural products; drug repurposing; multi-targeting; COVID-19; in silico; in vitro; in vivo; clinical trials Academic Editors: Diego Muñoz-Torrero and Beatriz De Pascual-Teresa Received: 31 December 2020 1. Introduction Accepted: 21 January 2021 Published: 26 January 2021 Coronaviruses are a diverse panel of viruses capable of producing infection in many animals and are responsible for respiratory infections in humans. After facing the fatal respiratory illness, caused by the Severe Acute Respiratory Syndrome Coronavirus (SARS- Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in CoV) and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV), the end of published maps and institutional affil- 2019 marked the start of one of the worse global pandemics [1]. More precisely, the first iations. outbreak was beheld in Wuhan city, probably in the local seafood market, where there was witnessed a wave of noteworthy suspected pneumonia disease that began with progressive respiratory failure due to alveolar damage and even death. Successively, this “enemy” has been identified in SARS-CoV-2 as an etiologic agent of coronavirus disease 2019 [2]. SARS-CoV-2 is still a global health problem today. Due to its high transmissibility, the virus Copyright: © 2021 by the authors. was able to spread quickly around the world [3]. Licensee MDPI, Basel, Switzerland. This article is an open access article On 11 March 2020, the World Health Organization (WHO) declared the coronavirus distributed under the terms and disease, namely COVID-19, a pandemic and the scientific community shifted its focus conditions of the Creative Commons towards this fight. Today, there is no proven effective and specific treatment for COVID-19. Attribution (CC BY) license (https:// However, a sign of the extraordinary commitment carried out by researchers around the creativecommons.org/licenses/by/ world is provided by recently published scientific evidences. Suffice it to say that the 4.0/). word “SARS-CoV-2” is reported on PubMed in 45,860 scientific articles, with reference

Molecules 2021, 26, 632. https://doi.org/10.3390/molecules26030632 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 632 2 of 27

date as the end of December 2020, demonstrating how the scientific community has conducted intensive research on this emergent topic. However, it is also well known that the development of specific de-novo antiviral therapies generally takes from 10 to 17 years, while for particular vaccine production it should be necessary to wait for a timeframe of approximately 12–18 months. In December 2020, the Food and Drug Administration (FDA) issued an Emergency Use Authorization (EUA) for the Pfizer-BioNTech COVID-19 (BNT162b2) vaccine (Pfizer, Inc; Philadelphia, Pennsylvania). This vaccine is a lipid nanoparticle-formulated nucleoside- modified mRNA vaccine encoding the prefusion Spike glycoprotein of SARS-CoV-2. Despite the ultracold-chain storage and requirements for handling and administration, this vaccine will be feasible to implement. Still, these requirements could limit its availability to some populations, thereby negatively impacting health equity. In this perspective, scientific efforts should be made to overcome these challenges and advance health equity [4]. An issue to emphasize is the importance of the network between experts in com- plementary disciplines involved in COVID-19 investigation. In this connection, a trans- European Cooperation in Science and Technology (COST) action network is currently promoting scientific collaborations to speed up the achievements against COVID-19 [5]. Another main point is represented by the evolution and the adaptive mutations of the virus [6]. Indeed, the presence of SARS-CoV-2 Spike mutations can affect the interaction with the human epithelial cell receptors Angiotensin Converting Enzyme 2 (ACE2). The question of whether this would correspond to a novel and more potent COVID-19 specifici- ties eventually requiring the development of wider-spectrum anti-SARS-CoV-2 strategies, such as vaccines or antiviral drugs, remains open [7–9]. Hence, the repurposing of clinically evaluated drugs is one of the most promising strategies to address the immense landscape of diseases, including infectious ones such as COVID-19. Remarkably, while most drug repurposing occurrences are based on synthetic compounds, naturally occurring molecules may offer significant opportunities. In fact, nature itself may be considered a magic bullet providing molecules with promising or still unexplored pharmacological properties [10,11]. It was reported that around 80% of the human population draws on traditional plants in the developing world for health quality and requirements [12,13]. Moreover, natural products are considered an essential source of successful drug leads and provide unique structural diversity with respect to standard combinatorial chemistry. Since more than 95% of the world’s biodiversity has not been evaluated for potential biological activity, the main challenge involves how to efficiently access and enhance this natural chemical diversity [14–16]. Therefore, the different chemical space structural, affordability, lack of substantial toxic effects and inherent biologic properties of natural products allow to consider them good candidates for new therapeutics [17,18]. As a result of the current COVID-19 outbreak, the repurposing of natural products could be an effective strategy to handle the SARS-CoV-2 infection. Currently, several studies have highlighted the antiviral properties of several natural compounds [12,19,20]. Therefore, naturally occurring molecules with broad antiviral spectrum may offer a safe, effective, and inexpensive platform for identifying novel treatment of SARS-CoV-2. To prepare this review article, the PubMed database (www.ncbi.nlm.nih.gov/pubmed) was used to analyze scientific manuscripts matching the queries “SARS-CoV-2” and “natural products”. After analyzing the compelling articles dealing with the antiviral activity of natural compounds against SARS-CoV-2, we dedicated our attention to natural compounds recognizing SARS-CoV-2 druggable targets. Particularly, natural compounds currently under clinical trials were extensively herein discussed. Due to the amount of data from scientific literature, in our opinion sometimes inaccurate and superficial, our main goal is to give a vision on the possible role of the natural compounds against SARS-CoV-2. In this regard, based on the recent analysis gathered on SARS-CoV-2 targets [21], we focused on natural compounds under clinical trials in the first section. Meanwhile, in the second section, we provided in-vitro, in-vivo and in-silico results of the natural derivatives Molecules 2021, 26, x FOR PEER REVIEW 3 of 26

In this regard, based on the recent analysis gathered on SARS-CoV-2 targets [21], we focused on natural compounds under clinical trials in the first section. Meanwhile, in the second section, we provided in-vitro, in-vivo and in-silico results of the natural derivatives against SARS-CoV-2. This analysis may help to figure out the most promising natu- Molecules 2021, 26, 632 ral products which lead to the development of drugs for the treatment or prophylaxis3 of 27 of the emergent pandemic. Since the drug repurposing is a time-saving medicinal chemistry strategy, it could offer an excellent opportunity to obtain useful and non-toxic drugs to against SARS-CoV-2. This analysis may help to ﬁgure out the most promising natural fight COVID-19. products which lead to the development of drugs for the treatment or prophylaxis of the emergent pandemic. Since the drug repurposing is a time-saving medicinal chemistry strategy,2. Results it could offer an excellent opportunity to obtain useful and non-toxic drugs to ﬁght2.1. Natural COVID-19. Occurring Compounds Undergoing Clinical Trials

2. ResultsThe clinical severity of COVID-19 varies from asymptomatic infection to mortal dis- 2.1.ease. Natural Despite Occurring the academic Compounds and Undergoing industrial Clinical efforts Trials for fighting SARS-CoV-2, nowadays thereThe are clinical no drugs severity for the of treatment COVID-19 of varies this fromepidemic asymptomatic viral infection. infection Hence, to mortal the develop- disease.ment of Despite effective the and academic safe drugs and industrial is urgent efforts to control for fighting or prevent SARS-CoV-2, the spread nowadays of SARS-CoV- there2. Natural are no drugsproducts, for the which treatment have of thishistorically epidemic been viral infection.used to Hence,avoid theor alleviate development several dis- ofeases, effective are andstill safe offering drugs is molecules urgent to control with or promising prevent the or spread yet ofunexplored SARS-CoV-2. pharmacological Natural products,properties which from have the historicallyancient time. been Natural used to avoidoccurring or alleviate compounds several diseases,are among are stillthe options offeringbeing considered molecules with for promisingthe treatment or yet of unexplored SARS-CoV-2 pharmacological infection as propertiesthey are commonly from the inex- ancientpensive, time. available, Natural and occurring rarely compoundshave shown are undesirable among the optionsside effe beingcts [22]. considered In this forsection, we the treatment of SARS-CoV-2 infection as they are commonly inexpensive, available, and will discuss the natural compounds actually under clinical trials. rarely have shown undesirable side effects [22]. In this section, we will discuss the natural compounds actually under clinical trials. 2.1.1. Vitamin D 2.1.1.In Vitamin striving D to determine a COVID-19 potential treatment, the effectiveness of vitamin D towardsIn striving this to endemic determine health a COVID-19 issue potential was eval treatment,uated [23]. the effectivenessIn ancient times, of vitamin food-enriched D towardsvitamin thisD, including endemic health codfish, issue was was used evaluated to prevent [23]. In or ancient treat acute times, respiratory food-enriched syndrome vitamin[24]. Vitamin D, including D is well-known codfish, was usedto play to prevent an essential or treat role acute in respiratorythe maintenance syndrome of [bone24]. health Vitamin D is well-known to play an essential role in the maintenance of bone health and to and to regulate calcium-phosphorus metabolism [23]. Moreover, this vitamin is also re- regulate calcium-phosphorus metabolism [23]. Moreover, this vitamin is also responsible forsponsible the modulation for the ofmodulation the immune of response the immune in infectious response and in autoimmune infectious diseaseand autoimmune [25,26]. dis- Inease particular, [25,26]. the In active particular, form of the vitamin active D, calcitriolform of vitaminhas a defined D, calcitriol role in immune has a function, defined role in withimmune anti-inflammatory function, with activity, anti-inflammatory inhibiting the overexpression activity, inhibiting of inflammatory the overexpression cytokines of in- (e.g.,flammatory IL-1α, IL-1 cytokinesβ, TNF-α (e.g.,) (Figure IL-11)[α,27 IL-1]. β, TNF-α) (Figure 1) [27].

FigureFigure 1. 1.2D 2D structure structure of calcitriolof calcitriol (Vitamin (Vitamin D). D).

Therefore,Therefore, researchers researchers have have hypothesised hypothesised a potential a potential link between link this between compound this and compound SARS-CoV-2,and SARS-CoV-2, and vitamin and vitamin D underwent D underwent several studies several for evaluating studies for its activityevaluating against its activity COVID-19 [28]. against COVID-19 [28]. A possible mechanism of action was attributed to the binding between the active form ofA vitaminpossible D mechanism and S protein of of action SARS-CoV-2. was attributed Docking resultsto the showedbinding that between calcitriol the active recognisedform of vitamin the Receptor D and Binding S protein Domain of SARS-CoV (RBD) of Spike-2. Docking glycoprotein results with showed good binding that calcitriol affinity,recognised thus engagingthe Receptor hydrogen Binding bonds Domain with Gln493 (RBD) residue of Spike and glycoprotein hydrophobic interactionswith good binding withaffinity, Ser494, thus Tyr449, engaging Asn450, hydrogen Leu452, Tyr489, bonds Leu492, with Gln493 and Glu484 residue residues and [ 29hydrophobic]. According interac- totions Weir with et al., Ser494, who recentlyTyr449, reviewed Asn450, Leu452, vitamin DTy activitiesr489, Leu492, in the and COVID-19 Glu484 context, residues its [29]. Ac- supplementcording to Weir would et offeral., who an easy recently option review in fightinged vitamin against D SARS-CoV-2 activities in [30the]. COVID-19 Indeed, con- thetext, authors its supplement provided would several offer reports an demonstrating easy option in that fighting low levels against of vitaminSARS-CoV-2 D are [30]. In- associated with increased inflammatory cytokines, which contribute to worsening COVID- deed, the authors provided several reports demonstrating that low levels of vitamin D are 19 severity [30]. These reports raise the possibility that adequate vitamin D levels can reduce the severity of the cytokine storm, which can occur in COVID-19.

Molecules 2021, 26, x FOR PEER REVIEW 4 of 26

associated with increased inflammatory cytokines, which contribute to worsening COVID-19 severity [30]. These reports raise the possibility that adequate vitamin D levels can reduce the severity of the cytokine storm, which can occur in COVID-19. Interestingly, Jain and co-authors analysed the vitamin D level in both asymptomatic and critically ill COVID-19 patients and its correlation with inflammatory markers (IL-6; Molecules 2021, 26, 632 TNF-α, and serum ferritin) [31]. Vitamin D deficiency and serum level of 4inflammatory of 27 markers were higher in the patients with severe COVID-19 symptoms. Moreover, a positive correlation was found between the fatal rate and vitamin D deficiency [31]. Despite this evidence, Interestingly, Jain and co-authors analysed the vitamin D level in both asymptomatic andwhich critically encourages ill COVID-19 a mass patients administration and its correlation of vitamin with inflammatoryD supplements markers to populations (IL-6; at risk of TNF-COVID-19,α, and serum the study ferritin) suffers [31]. Vitamin some Ddrawbacks. deficiency and The serum current level ofanalysis inflammatory has been conducted markersin a single were centre higher inlocated the patients in India, with severe an area COVID-19 with a symptoms. high prevalence Moreover, of a positivevitamin D deficiency. correlationIt does not was consider found between comorbidities, the fatal rate su andch vitaminas diabetes D deficiency and hypertension [31]. Despite this [31]. evidence, which encourages a mass administration of vitamin D supplements to populations at riskTherefore, of COVID-19, keeping the study these suffers results some in drawbacks. mind, a well-drawn The current analysis multicentre has been study should be conductedconducted in ato single generate centre more located robust in India, conclusion an area withs. aIn high general, prevalence retrospective of vitamin D studies demon- deficiency.strated a Itdirect does notlink consider between comorbidities, vitamin D such and as COVID-19 diabetes and cases hypertension and outcomes. [31]. Therefore,On the other keeping hand, these additional results in mind, data a well-drawn did not prove multicentre this correlation study should [32–34]. be Random- conducted to generate more robust conclusions. In general, retrospective studies demon- ized-double-blind, large-scale, well-built studies are necessary to clarify the efficacy of strated a direct link between vitamin D and COVID-19 cases and outcomes. vitaminOn the D other for SARS-CoV-2. hand, additional As data far did as not our prove knowledge this correlation goes [32 on,–34 ].no Randomized- conclusive evidence un- double-blind,derwent from large-scale, clinical well-built trials (www.clinicaltrials.gov). studies are necessary to clarify the efficacy of vitamin D for SARS-CoV-2. As far as our knowledge goes on, no conclusive evidence underwent from2.1.2. clinical Vitamin trials C (www.clinicaltrials.gov ). 2.1.2. VitaminVitamin C C, known as ascorbic acid, is an essential vitamin principally abundant in the CitrusVitamin spp. C, known and commonly as ascorbic acid,sold is as an a essential dietary vitamin supplement principally (Figure abundant 2) [35]. in Nowadays, it theis underCitrus spp.investigationand commonly for soldits biological as a dietary effects supplement for COVID-19 (Figure2)[ 35 patients]. Nowadays, [36]. It has well-es- it is under investigation for its biological effects for COVID-19 patients [36]. It has well- tablished antioxidant properties by scavenging oxygen free radicals and represents a key established antioxidant properties by scavenging oxygen free radicals and represents a key cofactorcofactor for for the the production production of several of seve neurotransmittersral neurotransmitters and hormones. and hormones.

FigureFigure 2. 2.2D 2D structure structure of ascorbic of ascorbic acid (Vitamin acid (Vitamin C). C). Vitamin C supports several cellular functions of both innate and adaptive immune systemsVitamin [37,38]. C Furthermore, supports several upcoming cellular studies function demonstrateds of both its anti-inflammatory innate and adaptive immune properties,systems [37,38]. being able Furthermore, to inhibit pro-inflammatory upcoming mediatorsstudies production,demonstrated including its IL-6,anti-inflammatory TNF-properties,α, and the being granulocyte-macrophage able to inhibit pro-inflammatory colony-stimulating mediators factor (GM-CSF) production, signalling including IL-6, responsesTNF-α, and [39]. the Altogether, granulocyte-macrophage the biological effects shown colony-stimulating by vitamin C may factor help to inhibit(GM-CSF) signalling the “cytokine storm” observed in the SARS-CoV-2 patients and to improve the host’s immunity.responses A [39]. meta-analysis Altogether, of 29 the clinical biological trials reported effects shown that vitamin by vitamin C decreased C may the help to inhibit durationthe “cytokine of the commonstorm” observed cold by 8% in in the adults SARS-CoV and 14% in-2 childrenpatients [ 40and]. Furthermore, to improve the host’s im- vitaminmunity. C amelioratedA meta-analysis upper respiratory of 29 clinical tract infections. trials reported However, that its supplementation vitamin C decreased did the dura- nottion prevent of the the common incidence cold of colds, by indicating8% in adults that a and regular 14% assumption in children is not [40]. justified Furthermore, [40]. vitamin Besides, the same authors reported in a meta-analysis of 12 clinical trials that vitamin CC reducesameliorated the length upper of the respiratory intensive care tract unit stay infect by 7.8%ions. and However, the duration its of supplementation mechanical did not aidedprevent ventilation the incidence [41–43]. Emergingof colds, evidence, indicating carried that out a regular on a small assumption clinical trial withis not 54 justified [40]. criticalBesides, COVID-19 the patients, same demonstratedauthors reported that high in dosea meta-analysis vitamin C infusion of 12 decreased clinical thetrials that vitamin IL-6C reduces level, concluding the length that of thisthe therapeuticalintensive care regimen unit stay may provideby 7.8% a and protective the duration clinical of mechanical effect without any adverse events in critical COVID-19 patients [44]. Other outcomes were aided ventilation [41–43]. Emerging evidence, carried out on a small clinical trial with 54 considered in the same clinical trial, including organ failure score, intensive care unit length,critical and COVID-19 mortality. Accordingpatients, todemonstrated the authors, a significant that high decrease dose vitamin in intensive C infusion care unit decreased the stayIL-6 and level, hospital concluding mortality that was observedthis therapeutical in the vitamin regimen C treated may patients provide [44]. Overall, a protective clinical vitamineffect without C seemed any to possess adverse relevant events pharmacological in critical COVID-19 properties against patients severe [44]. respiratory Other outcomes were infections,considered including in the COVID-19 same clinical [45,46]. Severaltrial, including clinical trials, organ assessing failure the efficacy score, of intensive high care unit dose vitamin C infusion, are still ongoing and will provide more definitive evidence. length, and mortality. According to the authors, a significant decrease in intensive care unit stay and hospital mortality was observed in the vitamin C treated patients [44].

Molecules 2021, 26, x FOR PEER REVIEW 5 of 26

Overall, vitamin C seemed to possess relevant pharmacological properties against severe respiratory infections, including COVID-19 [45,46]. Several clinical trials, assessing the ef- Molecules 2021, 26, 632 ficacy of high dose vitamin C infusion, are still ongoing and will provide more definitive5 of 27 evidence.

2.1.3. Lactoferrin Lactoferrin (LF) (LF),, or or lactotransferrin, lactotransferrin, is isa cationic a cationic glycoprotein glycoprotein constituted constituted by 691 by ami- 691 aminoacidsnoacids folded folded into intotwo globular two globular lobes lobes(80 kDa), (80 kDa),connected connected by an α by-helix. an α LF-helix. can reversi- LF can reversiblybly chelate chelate two Fe two3+ cations Fe3+ cationsper molecule per molecule (Figure (Figure3) [47–49].3)[47 –49].

Figure 3. 3D crystal crystal structure structure human human lactoferrin lactoferrin (PDB: (PDB: 1B0L) 1B0L) [50]. [50]. Orange Orange spheres spheres represent represent Fe Fe3+ 3+ cation binding sites.

This proteinprotein isis mainlymainly produced produced by by the the mammalian mammalian gland, gland, particularly particularly in mammalianin mamma- andlian and animal animal milk milk [51]. [51]. When When needed, needed, LF mayLF may also also be be taken taken as as a supplement, a supplement, acting acting as as a nutraceuticala nutraceutical or or functional functional food food [52 [52].]. Due to its iron-binding capabilities, this proteinprotein regulates thethe ironiron homeostasishomeostasis inin thethe body andand doesdoes notnot releaserelease iron iron even even at at pH pH 3.5 3.5 [53 [53].]. This This property property ensures ensures iron iron sequestering sequester- ining infected in infected body body compartments, compartments, where where the pH the is pH normally is normally acidic, acidic, decreasing decreasing the availability the avail- ofability essential of essential iron to iron microbes to microbes [53]. LF [53]. exert LF immune-modulatory exert immune-modulatory effects effects by increasing by increas- the expressioning the expression ability ofability B cells of B and cells by and regulating by regulating the function the function of T cells of T [cells54]. [54]. Further, Further, it is reportedit is reported that that LF has LF anti-inflammatoryhas anti-inflammatory properties. properties. This This protein protein can access can access inside inside host cellshost andcells shiftand inshift the in nucleus, the nucleus, thusregulating thus regulating pro-inflammatory pro-inflammatory gene expression gene expression [54,55]. [54,55]. As an example,As an example, the down-regulation the down-regulation of IL-6 of has IL-6 been has demonstrated been demonstrated in both in in-vitro both in-vitro and in-vivo and modelsin-vivo [models56,57]. [56,57]. LF is active against broad-spectrum viruses, including coronavirus [58,59]. [58,59]. It is be- lieved that that glycoprotein glycoprotein interacts interacts with with heparan heparan sulfate sulfate proteoglycans proteoglycans of the of host the host cells, cells, pre- preventingventing the theentry entry of viral of viral particles particles into into host host cells cells [60]. [60 Therefore,]. Therefore, LF LFhas has received received the the at- attentiontention of of the the scientific scientific community community in in the the prevention prevention of of SARS-CoV-2 SARS-CoV-2 infection. Exploring the possible mechanisms of action involving LF, aa computationalcomputational investigationinvestigation suggestedsuggested its possible effecteffect inin thethe earlyearly stages ofof thethe infection.infection. Indeed, by 2D-Zernike descriptorsdescriptors ofof molecular surfaces of sialic acid receptors on the host cell membrane, ACE2, S, M, and E molecular surfaces of sialic acid receptors on the host cell membrane, ACE2, S, M, and E proteins, it was highlighted that LF could affect the viral attachment to the host cell due to proteins, it was highlighted that LF could affect the viral attachment to the host cell due its probable competition with the virus in the binding to sialic acid [61–64]. Additionally, to its probable competition with the virus in the binding to sialic acid [61–64]. Addition- potential competition between ACE2 and LF in the binding site of SARS-CoV-2 Spike was ally, potential competition between ACE2 and LF in the binding site of SARS-CoV-2 Spike suggested. This is probably due to two high complementarity regions in the N- and C- was suggested. This is probably due to two high complementarity regions in the N- and terminal domains of the Spike protein to LF [65]. C- terminal domains of the Spike protein to LF [65]. Additional computational study sustained the idea of direct LF binding to the Spike S Additional computational study sustained the idea of direct LF binding to the Spike glycoprotein CDT1 domain, reducing the virus’s likelihood of entering cells. Moreover, S glycoprotein CDT1 domain, reducing the virus’s likelihood of entering cells. Moreover, after evaluating the human LF-Spike complex, it was observed that only two residues after evaluating the human LF-Spike complex, it was observed that only two residues (Thr500 and Tyr505) are involved in the interface recognition [66]. (Thr500 and Tyr505) are involved in the interface recognition [66]. From February 2020, the LF efficacy against SARS-CoV-2 has been reviewed since this protein showed promising antiviral and anti-inflammatory properties reducing the typical “cytokine storm” in COVID-19 patients [52,67,68]. Based on the encouraging preliminary results, several clinical trials have been registered but, the only clinical trial concluded was the one of Campione et al. [67]. According to the authors, LF might be used in asymptomatic Molecules 2021, 26, x FOR PEER REVIEW 6 of 26

From February 2020, the LF efficacy against SARS-CoV-2 has been reviewed since this protein showed promising antiviral and anti-inflammatory properties reducing the typical “cytokine storm” in COVID-19 patients [52,67,68]. Based on the encouraging preliminary results, several clinical trials have been registered but, the only clinical trial concluded was the one of Campione et al. [67]. According to the authors, LF might be used in Molecules 2021, 26, 632 asymptomatic or mildly symptomatic patients to prevent the worsening6 of 27of SARS-CoV-2. However, despite the courageous preliminary results, more adequate and well-drawn clinicalor mildly trials symptomatic should patients be carried to prevent out the to worsening evaluate of the SARS-CoV-2. useful, beneficial However, despite properties of LF for COVID-19the courageous disease. preliminary results, more adequate and well-drawn clinical trials should be carried out to evaluate the useful, beneﬁcial properties of LF for COVID-19 disease. 2.1.4. Quercetin 2.1.4. Quercetin QuercetinQuercetinis a is ﬂavonol a flavonol abundantly abundantly present inpresent vegetables in vegetables and fruits (Figure and 4fruits)[10]. (Figure 4) [10].

OH O OH

OH HO O

OH Figure 4. 4.2D 2D structure structure of quercetin. of quercetin. This compound gained attention for its pharmacological properties, such as antiviral, anti-inﬂammatory,This compound pro-metabolic gained andattention anticancer for [its10, pharmacological69,70]. In light of this, properties, quercetin was such as antiviral, anti-inflammatory,studied against COVID-19 pro-metabolic as well. and anticancer [10,69,70]. In light of this, quercetin was studiedIn particular, against theCOVID-19 capability as of quercetinwell. to inhibit the proteases of SARS-CoV-2 was discussed.In particular, Regarding the SARS-CoV-2 capability Mpro, of quercetin docking studies to inhibit highlighted the proteases quercetin asof able SARS-CoV-2 was to accommodate into this target binding site, establishing hydrogen bonds with Asn142, discussed.Ser144, and Met165.Regarding Also, SARS-CoV-2 hydrophobic contacts Mpro, weredock establisheding studies with highlighted Met49, Phe140, quercetin as able toLeu141, accommodate and electrostatic into this interactions target werebinding engaged site, withestablishing His164 and hydrogen Glu166 residues. bonds with Asn142, TheSer144, likelihood and Met165. to interfere Also, in the hydrophobic proteolytic activity contac of Mprots were could established be attributed with to the Met49, Phe140, Leu141,destabilizing and effect electrostatic of quercetin interactions into the catalytic were pocket.engaged It with occurs His164 due to itsand ability Glu166 to residues. The adopt multiple binding poses, resulting in a favourable entropy gain. Strong experimental likelihoodevidence validated to interfere this theoretical in the proteolytic hypothesis [ 71activity]. Concerning of Mpro the SARS-CoV-2could be attributed papain- to the desta- bilizinglike protease effect (PLpro), of quercetin quercetin didinto not the result catalytic as a good pocket. binder inIt aoccurs theoretical due study to its [72 ].ability to adopt multipleConversely, binding a computational poses, investigationresulting in evidenced a favour itable as able entropy to bind gain. at S protein: Strong ACE2 experimental evi- dencereceptor validated interface [73 this]. In theoretical vitro studies hypothesis demonstrated [71]. that quercetinConcerning inhibited the recombinantSARS-CoV-2 papain-like proteasehuman (rh) (PLpro), ACE2 activity quercetin [74,75]. did More not recently, result theas clinicala good trials binder (NCT04468139 in a theoretical and study [72]. NCT04377789) including quercetin, were involved in both prophylaxis and treatment of Conversely,COVID-19 [76– a78 computational]. investigation evidenced it as able to bind at S protein: ACE2 receptor interface [73]. In vitro studies demonstrated that quercetin inhibited recombinant human2.1.5. Resveratrol (rh) ACE2 activity [74,75]. More recently, the clinical trials (NCT04468139 and NCT04377789)Resveratrol is including the most extensively quercetin, studied were stilbene involved natural in compound both prophylaxis (Figure5). Stil- and treatment of benoids are phenolic molecules and due to their biosynthesis by the plants after ultraviolet COVID-19 [76–78]. Molecules 2021, 26, x FOR PEER REVIEWradiation, toxins and injures, are also classiﬁed as phytoalexins. Chemically, stilbenoids 7 of 26 show a common backbone including different types and position and substituents groups 2.1.5.on the Resveratrol aromatic rings. Resveratrol is the most extensively studied stilbene natural compound (Figure 5). Stilbenoids are phenolic moleculesOH and due to their biosynthesis by the plants after ultraviolet radiation, toxins and injures, are also classified as phytoalexins. Chemically, stil- benoidsHO show a common backbone including different types and position and substituents groups on the aromatic rings. This kind of natural compound is endowed with numerous biological activities; therefore, recent studies focused on possible resveratrol effect against COVID-19 [17,79]. OH Computational analysis showed its good binding property towards SARS-CoV-2 Spike proteinFigure 5. 5.2D and 2D structure structurehuman of resveratrol. ACE2of resveratrol. receptor interface. This kind of natural compound is endowed with numerous biological activities; therefore,Docking recent results studies focusedhighlighted on possible its strong resveratrol interactions effect against with COVID-19 crucial [residues17,79]. involved in the binding between S protein of SARS-CoV-2 and ACE2 receptor, establishing two hydrogen bonds with Lys353 of S protein and Gly496 of ACE2 receptor. The structural stability of resveratrol at its interface binding pose was confirmed by molecular dynamic simulations [80]. Furthermore, an in-vitro study reported a dose-dependent antiviral effect of resveratrol on SARS-CoV-2 infection in Vero E6 cells [81]. Currently, several clinical trials for resveratrol are undergoing (NCT04542993, NCT04536090, and NCT04377789).

2.1.6. Hanfangchin A According to Riva et al., hanfangchin A, also called tetrandrine (TET) (Figure 6), showed dose-dependent antiviral activities [82,83]. TET is a benzyltetrahydroisoquinoline alkaloid isolated from the root of the plant Stephania tetrandra with several pharmacological effects [84]. Structurally, two benzyl-tetrahydroisoquinolines are connected to ether linkages to form tetrandrine with tetrahydroisoquinoline N-methylation, bearing an irregular eighteen-member ring [85].

N O

O O O O

Figure 6. 2D structure of hanfangchin A (tetrandrine).

It was hypothesized that TET could work by blocking the two-pore channel 2 (TPC2) located in the membranes of host endolysosomal compartments by which SARS-CoV-2 can egress from these organelles and continue its replication [83,85]. TET was excluded by in silico studies because it violated three drug-likeness properties reported in Lipinski’s Rule of 5. In detail, it showed a LogP value of 6.4, and the increase of lipophilicity property can result in a probable lack of selectivity and attrition in drug development [86]. For this, further investigations are needed [87]. The promising pharmacological efficacy as anti-COVID-19 agent was demonstrated in Vero E6 (EC50 = 1.2 µM), Huh-7 (EC50 = 0.64 µM), and HEK293T (EC50 = 0.56 µM) cells transduced with ACE2. Furthermore, exceptional levels of synergy with remdesivir have been reported [88]. A clinical trial for TET has been designed (NCT04308317).

2.1.7. Glycyrrhizin Glycyrrhizin, an active component of liquorice roots, was investigated against SARS- CoV-2 (Figure 7). Combining high throughput screening (HTS) and computational

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OH Figure 5. 2D structure of resveratrol. Molecules 2021, 26, 632 7 of 27 Docking results highlighted its strong interactions with crucial residues involved in Computationalthe binding analysisbetween showed S protein its good of SARS-CoV-2 binding property and towards ACE2 SARS-CoV-2 receptor, Spikeestablishing two hy- proteindrogen and bonds human with ACE2 Lys353 receptor of interface. S protein and Gly496 of ACE2 receptor. The structural sta- bilityDocking of resveratrol results highlighted at its interface its strong binding interactions pose with was crucial confirmed residues involvedby molecular dynamic in the binding between S protein of SARS-CoV-2 and ACE2 receptor, establishing two hydrogensimulations bonds [80]. with Furthermore, Lys353 of S protein an in-vitro and Gly496 study of ACE2 reported receptor. a dose-dependent The structural antiviral ef- stabilityfect of ofresveratrol resveratrol aton its SARS-CoV-2 interface binding infection pose was in conﬁrmedVero E6 cells by molecular [81]. Currently, dynamic several clinical simulationstrials for [resveratrol80]. Furthermore, are undergoing an in-vitro study (N reportedCT04542993, a dose-dependent NCT04536090, antiviral and effect NCT04377789). of resveratrol on SARS-CoV-2 infection in Vero E6 cells [81]. Currently, several clinical trials for2.1.6. resveratrol Hanfangchin are undergoing A (NCT04542993, NCT04536090, and NCT04377789). 2.1.6. HanfangchinAccording A to Riva et al., hanfangchin A, also called tetrandrine (TET) (Figure 6), showedAccording dose-dependent to Riva et al., hanfangchin antiviral activities A, also called[82,83]. tetrandrine TET is a (TET)benzyltetrahydroisoquinoline (Figure6), showedalkaloid dose-dependent isolated from antiviral the root activities of the [82 ,plant83]. TET Stephania is a benzyltetrahydroisoquinoline tetrandra with several pharmacolog- alkaloid isolated from the root of the plant Stephania tetrandra with several pharmacological effectsical effects [84]. Structurally, [84]. Structurally, two benzyl-tetrahydroisoquinolines two benzyl-tetrahydroisoquinolines are connected to ether are link- connected to ether ageslinkages to form to tetrandrine form tetrandrine with tetrahydroisoquinoline with tetrahydroisoquinolineN-methylation, bearing N-methylation, an irregular bearing an ir- eighteen-memberregular eighteen-member ring [85]. ring [85].

N O

O O O O

FigureFigure 6. 2D6. 2D structure structure of hanfangchin of hanfangchin A (tetrandrine). A (tetrandrine).

It was hypothesized that TET could work by blocking the two-pore channel 2 (TPC2) locatedIt in was the membranes hypothesized of host that endolysosomal TET could compartments work by blocking by which the SARS-CoV-2 two-pore can channel 2 (TPC2) egresslocated from in these the organellesmembranes and continueof host itsendolyso replicationsomal [83,85 ].compartments by which SARS-CoV-2 canTET egress was excludedfrom these by in organelles silico studies and because continue it violated its replication three drug-likeness [83,85]. properties reportedTET inwas Lipinski’s excluded Rule by of 5.in In silico detail, studies it showed because a LogP value it violated of 6.4, and three the increase drug-likeness proper- of lipophilicity property can result in a probable lack of selectivity and attrition in drug developmentties reported [86]. in For Lipinski’s this, further Rule investigations of 5. In deta are neededil, it showed [87]. The promisinga LogP value pharma- of 6.4, and the in- cologicalcrease of efﬁcacy lipophilicity as anti-COVID-19 property agent can was result demonstrated in a probable in Vero lack E6 (EC of 50selectivity= 1.2 µM), and attrition in Huh-7drug (ECdevelopment50 = 0.64 µM), [86].and HEK293TFor this, (EC further50 = 0.56 investigationsµM) cells transduced are needed with ACE2. [87]. The promising Furthermore,pharmacological exceptional efficacy levels as of synergyanti-COVID-19 with remdesivir agent havewas been demonstrated reported [88]. in A Vero E6 (EC50 = clinical trial for TET has been designed (NCT04308317). 1.2 µM), Huh-7 (EC50 = 0.64 µM), and HEK293T (EC50 = 0.56 µM) cells transduced with 2.1.7.ACE2. Glycyrrhizin Furthermore, exceptional levels of synergy with remdesivir have been reported [88].Glycyrrhizin A clinical, antrial active for componentTET has been of liquorice designed roots, (NCT04308317). was investigated against SARS- CoV-2 (Figure7). Combining high throughput screening (HTS) and computational approaches,2.1.7. Glycyrrhizin for terpene-based compounds, it was analyzed the likelihood of Glycyrrhizin to insert between SARS-CoV-2 RBD-ACE2 complex. In detail, it is worth noting that Gly- cyrrhizinGlycyrrhizin recognized Asn501,, an active Gln498, component Gly496, Tyr449, of liquorice Tyr453, and roots, Glu484 was by investigated means of hy- against SARS- drogenCoV-2 bond (Figure interactions 7). Combining and hydrophobic high contacts throug withhput Tyr489, screening Phe456, and (HTS) Leu455 and [89]. computational Other glycyrrhizin involvements could be summarized as follows: (1) targeting S-RBD- ACE2 complex, (2) controlling pro-inﬂammatory cytokines, (3) preventing the accumula-

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approaches, for terpene-based compounds, it was analyzed the likelihood of Glycyrrhizin to insert between SARS-CoV-2 RBD-ACE2 complex. In detail, it is worth noting that Glycyrrhizinapproaches, for recognized terpene-based Asn501, compounds, Gln498, it Gly496,was analyzed Tyr449, the likelihoodTyr453, and of Glycyrrhizin Glu484 by means to insert between SARS-CoV-2 RBD-ACE2 complex. In detail, it is worth noting that of hydrogen bond interactions and hydrophobic contacts with Tyr489, Phe456, and Molecules 2021, 26, 632 Glycyrrhizin recognized Asn501, Gln498, Gly496, Tyr449, Tyr453, and Glu484 by 8means of 27 Leu455of hydrogen [89]. Other bond glycyrrhizininteractions andinvolvements hydrophobic could contacts be summarized with Tyr489, as Phe456,follows: and (1) target- ingLeu455 S-RBD-ACE2 [89]. Other complex, glycyrrhizin (2) involvementscontrolling pro-inflammatory could be summarized cytokines, as follows: (3) (1) preventing target- the tionaccumulationing ofS-RBD-ACE2 the intracellular of thecomplex, intracellular ROS, (2) (4) controlling controlling ROS, (4) pro-inflammatory thrombi, controlling (5) preventing thrombi, cytokines, (5) the preventing(3) fast preventing production the the fast pro- ofductionaccumulation airway of exudates, airway of the and exudates,intracellular (6) persuading and ROS, (6) (4) endogenous persua controllingding interferon thrombi, endogenous (5) against preventing interferon SARS-CoV-2 the fastagainst [ 90pro-]. SARS- Recently,CoV-2duction [90]. glycyrrhizinof airwayRecently, exudates, was glycyrrhizin recruited and (6) in was apersua clinical recruitedding trial endogenous (NCT04487964).in a clinical interferon trial (NCT04487964). against SARS- CoV-2 [90]. Recently, glycyrrhizin was recruited in a clinical trial (NCT04487964). OOH O OH OOH O OH OH HO OH HO OO OO OH OHOH HOHO OO

HOHO O OO OO Figure 7. 2D structure of glycyrrhizin. Figure 7.7.2D 2D structure structure of of glycyrrhizin. glycyrrhizin.

2.1.8.2.1.8. ArtemisininArtemisinin Artemisinin TheThe anti-SARS-CoV-2 anti-SARS-CoV-2 activity activity was was also also investigated investigated for the for compounds the compounds extracted extracted from The anti-SARS-CoV-2 activity was also investigated for the compounds extracted Artemisiafrom Artemisia Annua. AmongAnnua. these,Amongartemisinin these, artemisinin(Figure8) the(Figure most abundant8) the most active abundant component, active isfromcomponent, an antimalarial Artemisia is an Annua agent antimalarial showing. Among agent a promisingthese, showing artemisinin broad-spectrum a promising (Figure broad-spectrum antiviral 8) the activity most antiviral [ 91abundant,92 ].activ- In- active silicocomponent,ity [91,92]. approaches In-silico is an helped antimalarial approaches to understand helpedagent theshowingto understand possible a promising mechanism the possible broad-spectrum of artemisinin mechanism to of prevent antiviralartemis- activ- theityinin [91,92]. interaction to prevent In-silico between the interaction approaches S protein between and helped ACE2 S protto receptor, understandein and due ACE2 to the its receptor, abilitypossible to due bindmechanism to Lys353its ability and of to artemis- Lys31ininbind to hotspotLys353 prevent and binding the Lys31 interaction region hotspot [93 ].binding between region S prot [93].ein and ACE2 receptor, due to its ability to bind Lys353 and Lys31 hotspot binding region [93].

FigureFigure 8. 8.2D 2D structure structure of of artemisinin. artemisinin.

FigureToTo 8. date, date,2D structure we we have have in-vitroof in-vitro artemisinin. evidence evidence for artemisininfor artemisinin and and its derivative its derivative arteannuin arteannuin B, and B, aand clinical a clinical trial is trial undergoing is undergoing (NCT04382040) (NCT04382040) [93,94 ].[93,94]. To date, we have in-vitro evidence for artemisinin and its derivative arteannuin B, 2.1.9.2.1.9. Colchicine Colchicine and a clinical trial is undergoing (NCT04382040) [93,94]. ColchicineColchicine(Figure (Figure9) extracted9) extracted from from the theColchicum Colchicum autumnale autumnaleis used is used for the for treatment the treat- and management of patients with gout. In recent years, this old drug was also proposed in 2.1.9.ment Colchicine and management of patients with gout. In recent years, this old drug was also pro- aposed variety in of a illnesses,variety of including illnesses, rheumaticincluding rheumatic and cardiovascular and cardiovascular diseases [95 diseases]. [95]. TheColchicineThe possible possible role(Figure role of of colchicine colchicine 9) extracted was was hypothesized hypothesizedfrom the Colchicumversus versusCOVID-19 COVID-19 autumnale infection. infection. is usedIto Ito for et et al. theal. treat- β highlightedmenthighlighted and management how how SARS-CoV SARS-CoV of andpatients and its its accessory accessory with gout. protein protein In recent could could activate years,activate this pro-IL-1 pro-IL-1 old drugβgene gene was tran- tran- also pro- scriptionposedscription in and aand variety protein protein of maturation, maturation,illnesses, including and and subsequently, subs equently,rheumatic NLRP3 NLRP3 and inflammasomecardiovascular inflammasome [ 96[96]. diseases]. Indeed, Indeed, [95]. thethe anti-inflammatory Theanti-inflammatory possible role activity ofactivity colchicine of colchicineof colchicine was mainly hypothesized mainly depends depends onversus the oninhibition COVID-19 the inhibition of theinfection. NLRP3 of the Ito et al. inflammasome [97]. Other anti-COVID-19 mechanism have been proposed [98]. highlightedNLRP3 inflammasome how SARS-CoV [97]. Other and anti-COVID-19its accessory protein mechanism could have activate been proposed pro-IL-1 [98].β gene tran- Therefore, this compound could be utilized for the treatment of COVID-19. To date, sev- eralscription clinical and trials protein are undergoing maturation, (NCT04527562, and subs NCT04392141,equently, NLRP3 NCT04375202, inflammasome NCT04355143, [96]. Indeed, andthe NCT04360980).anti-inflammatory activity of colchicine mainly depends on the inhibition of the NLRP3Scarsi inflammasome et al., report a 20% [97]. improvement Other anti-COVID-19 in the survival mechanism rate at 21 days have in patients been proposed treated [98]. with colchicine compared with the local standard of care [99]. However, Gendelman and co-authors did not detect a potential benefit of colchicine for COVID-19 treatment [100].

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Molecules 2021, 26, x FOR PEER REVIEW 9 of 26 Molecules 2021, 26, 632 9 of 27

Figure 9. 2D structure of colchicine.

Therefore, this compound could be utilized for the treatment of COVID-19. To date, several clinical trials are undergoing (NCT04527562, NCT04392141, NCT04375202, NCT04355143, and NCT04360980). Scarsi et al., report a 20% improvement in the survival rate at 21 days in patients treated with colchicine compared with the local standard of care [99]. However, Gendel- man and co-authors did not detect a potential benefit of colchicine for COVID-19 treatment [100].

2.1.10. Berberine FigureFigure 9.9.2D 2D structurestructure ofof colchicine.colchicine. Berberine (Figure 10) is an alkaloid extracted from Berberis vulgaris L. which was ex- 2.1.10.tensivelyTherefore, Berberine studied this compoundfor its pharmacological could be utilized activities for the treatment with applications of COVID-19. in To several date, therapeu- ticseveral areas,Berberine clinical including(Figure trials cancer10are) is undergoing an [101–103]. alkaloid extracted(NCT04527562, The antiviral from Berberis NCT04392141,activity vulgaris of berberineL. whichNCT04375202, was against ex- influenza tensivelyandNCT04355143, alphaviruses studied and for NCT04360980). itshas pharmacological been described activities [104]. with In applicationsthe SARS-CoV-2 in several context, therapeutic Pizzorno et al. areas,Scarsi including et al., cancer report [101 a –20%103 ].improvement The antiviral in activity the survival of berberine rate againstat 21 days influenza in patients and demonstrated the in vitro efficacy against SARS-CoV-2 in Vero E6 cells [105]. These find- alphavirusestreated with hascolchicine been described compared [104 with]. In the the loca SARS-CoV-2l standard context,of care [99]. Pizzorno However, et al. demon-Gendel- stratedingsman andagree the co-authorsin with vitro efficacyother did notstudies against detect SARS-CoV-2 [106,107]. a potential inAn benefit Vero in E6silico of cells colchicine study [105]. Thesereportedfor COVID-19 findings that agreetreat- berberine well- withrecognizedment other [100]. studies the [main106,107 protease]. An in silico by studyestablishing reported hydrogen that berberine bond well-recognized interactions the with Phe140 mainand proteaseAsn142 by[108]. establishing hydrogen bond interactions with Phe140 and Asn142 [108]. 2.1.10. Berberine Berberine (Figure 10) is an alkaloid extracted from Berberis vulgaris L. which was extensively studied for its pharmacological activities with applications in several therapeutic areas, including cancer [101–103]. The antiviral activity of berberine against influenza and alphaviruses has been described [104]. In the SARS-CoV-2 context, Pizzorno et al. demonstrated the in vitro efficacy against SARS-CoV-2 in Vero E6 cells [105]. These findings agree with other studies [106,107]. An in silico study reported that berberine well- recognized the main protease by establishing hydrogen bond interactions with Phe140 and Asn142 [108].

FigureFigure 10. 10.2D 2D structure structure of berberine. of berberine.

DueDue to to its its promising promising anti-COVID-19 anti-COVID-19 activities, activities, this compound this compound underwent underwent in clinical in clinical trial, but no results are available (NCT04479202). trial, but no results are available (NCT04479202). 2.2. Natural Compounds with Promising Activities Against SARS-CoV-2 Infection 2.2. ANatural few weeks Compounds later, after with the outbreakPromising of theActivities pandemic, Against natural SARS-CoV-2 occurring compounds Infection

derivingA few from weeks different later, chemical-diverse after the outbreak scaffolds of showed the pandemic, promising natural data against occurring SARS- compounds CoV-2derivingFigure druggable10. 2D from structure different targets. of berberine. However, chemical-diverse as most scientiﬁc scaffolds efforts showed provided promising computational data against SARS- studies without any experimental validation, the hope is that further in-vitro and in-vivo experimentsCoV-2Due druggable to its will promising be performed.targets. anti-COVID-19 However, In this perspective, activities, as most thisthis scientific compound section aimsefforts underwent to summarizeprovided in clinical thecomputational resultsstudiestrial, but extrapolated withoutno results any byare scientiﬁc availableexperimental literature (NCT04479202). validation, achieved so the far onhope the naturalis that chemicalfurther scaffoldsin-vitro and in-vivo withexperiments promising will activities be performed. against SARS-CoV-2 In this infection.perspective, this section aims to summarize the results2.2. Natural extrapolated Compounds withby scientificPromising Acliteraturetivities Against achieved SARS-CoV-2 so far Infectionon the natural chemical scaf- 2.2.1. Alkaloids foldsA with few weeks promising later, after activities the outbreak against of theSARS-CoV-2 pandemic, natural infection. occurring compounds derivingAlkaloids from different are fundamental chemical-diverse chemical sc componentsaffolds showed representing promising a richdata natural against source SARS- forCoV-2 exploitation druggable in humantargets. health.However, Their as chemical most scientific scaffolds efforts are characterized provided computational by nitrogen- containingstudies without groups, any often experimental within heterocyclic validation, the rings. hope Alkaloids is that further show in-vitro various and biological in-vivo propertiesexperiments in severalwill be diseasesperformed. [109 In]. this perspective, this section aims to summarize the resultsBased extrapolated on the known by scientific antiviral literature activity of achieved some alkaloids, so far on a computationalthe natural chemical study wasscaf- designedfolds with to promising investigate activities their effect against against SARS-CoV-2 SARS-CoV-2 infection. Mpro. Among them, thalimonine and sophaline well recognized the binding pocket of the protease (Figure 11).

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2.2.1. Alkaloids Alkaloids are fundamental chemical components representing a rich natural source for exploitation in human health. Their chemical scaffolds are characterized by nitrogen- containing groups, often within heterocyclic rings. Alkaloids show various biological properties in several diseases [109]. Based on the known antiviral activity of some alkaloids, a computational study was designed to investigate their effect against SARS-CoV-2 Mpro. Among them, thalimonine Molecules 2021, 26, 632 10 of 27 and sophaline well recognized the binding pocket of the protease (Figure 11).

Figure 11.11.2D 2D structures structures of of (a) ( thalimoninea) thalimonine and and (b) sophaline. (b) sophaline. Thalimonine engaged two hydrogen bonds with Cys145 and Ser144 and a π-cation interactionThalimonine with His41. engaged Instead, two sophaline hydrogen was bonds able to formwith aCys145 hydrogen and bond Ser144 with and His163 a π-cation interactionand hydrophobic with contactsHis41. Instead, with His41, sophaline Met49, Gly143, was able Cys145, to form Met165, a hydrogen and Gln189 bond residues. with His163 and Computationalhydrophobic contacts analysis with of alkaloids His41, fromMet49,Cryptolepis Gly143, sanguinolenta Cys145, Met165,was performed and Gln189 residues.against Mpro and RNA-dependent RNA–polymerase (RdRp), revealing that cryptomisrine, Computationalcryptospirolepine analysis, cryptoquindoline of alkaloids, and frombiscryptolepine Cryptolepis sanguinolenta(Figure 12) exhibited was performed againstrobust binding Mpro and afﬁnity RNA-dependent towards these twoRNA–polymerase SARS-CoV-2 targets. (RdRp), Overall, revealing all these that alka- cryptomis- rineloids, cryptospirolepine were able to recognize, cryptoquindoline the active site of the, and two biscryptolepine studied targets. Among(Figure these,12) exhibited for ro- SARS-CoV-2 Mpro, cryptospirolepine well interacted with the catalytic dyad; for the poly- bust binding affinity towards these two SARS-CoV-2 targets. Overall, all these alkaloids merase, the best one was the cryptomisrine which was involved in π-cation interactions werewith Arg553able to andrecognize Arg624 the residues active of site SARS-CoV-2 of the two RdRp studied [110 targets.]. Gyebi Among et al. studied these, the for SARS- CoV-2involvement Mpro, of cryptospirolepine these alkaloids in the well prevention interacted of SARS-CoV-2with the catalytic cell entry. dyad; In particular, for the polymerase,cryptospirolepine the best one waswas ablethe cryptomisrine to form several interactions which was with involved Phe40, Ala348,in π-cation Arg393, interactions His401 with Arg553in the subdomain and Arg624 I of residues ACE2. In of addition SARS-CoV-2 to the block RdRp access [110]. to Gyebi ACE2, etcryptospirolepine al. studied the involve- mentand cryptoquindoline of these alkaloidscould in inhibitthe prevention the cleavage of of SARS-CoV-2 Spike glycoprotein cell entry. by interacting In particular, with cryp- tospirolepineTMPRSS2 [111]. was able to form several interactions with Phe40, Ala348, Arg393, His401 in theAlso, subdomain tropane alkaloidsI of ACE2. from InSchizanthus addition to porrigens the blockhave access been to studied ACE2, as cryptospirolepine promising inhibitors of SARS-CoV-2 PLpro. Schizanthine Z and schizanthine Y (Figure 13) were and cryptoquindoline could inhibit the cleavage of Spike glycoprotein by interacting with able to recognize the protein through the interaction with Tyr164 and Tyr173 residues; TMPRSS2meanwhile, [111].schizanthine Y engaged an additional hydrogen bond with Tyr168. The in- vitroAlso, experiments tropane are alkaloids highly recommended from Schizanthus [112]. porrigens have been studied as promising inhibitorsAmong of the SARS-CoV-2 alkaloids from PLpro.Justicia Schizanthine adhatoda, anisotine Z and(Figure schizanthine 14a) could haveY (Figure promis- 13) were ableing inhibitory to recognize activity the against protein SARS-CoV-2 through Mprothe interaction due to its interaction with Tyr164 with the and key Tyr173 catalytic residues; meanwhile,residues (His41 schizanthine and Cys145) Y of engaged the Mpro pocketan additional using computational hydrogen bond approach with [ 113Tyr168.]. The in- vitro experiments are highly recommended [112]. Among the alkaloids from Justicia adhatoda, anisotine (Figure 14a) could have promising inhibitory activity against SARS-CoV-2 Mpro due to its interaction with the key catalytic residues (His41 and Cys145) of the Mpro pocket using computational approach [113].

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Figure 12. 2D structures of (a) cryptomisrine, (b) cryptospirolepine, (c) cryptoquindoline, and (d) FigureFigure 12. 12. 2D2D structures structures of of(a) (cryptomisrine,a) cryptomisrine, (b) cryptospirolepine, (b) cryptospirolepine, (c) cryptoquindoline, (c) cryptoquindoline, and (d and) biscryptolepine. Figurebiscryptolepine.(d) biscryptolepine. 12. 2D structures of (a) cryptomisrine, (b) cryptospirolepine, (c) cryptoquindoline, and (d) biscryptolepine.

Figure 13. 2D structures of (a) schizanthine Z and (b) schizanthine Y. Figure 13. 2D structures of (a) schizanthine Z and (b) schizanthine Y. FigureFigure 13. 13. 2D 2D structures structures of of ( a()a )schizanthine schizanthine Z Zand and (b ()b schizanthine) schizanthine Y. Y.

Figure 14. 2D structures of (a) anisotine, (b) quinadoline B and (c) scedapin C. FigureFigureFigure 14. 14. 14. 2D 2D2D structures structures structures of of of ( a( ()aa )anisotine,) anisotine, anisotine, (b ( ()b quinadoline)) quinadoline B and B and (c )( cscedapin) scedapin C. C.

Molecules 2021, 26, 632 12 of 27

Quimque et al. virtually screened the fungal natural products against PLpro and Mpro, RdRp, nsp15, and the Spike binding domain to glucose-regulated protein 78 (GRP78). It was resulted that two fumiquinazoline alkaloids quinadoline B and scedapin C (Figure 14b,c) had strong in silico inhibitory activity towards the analyzed SARS-CoV-2 targets [114].

2.2.2. Terpenes Recently, the involvement of plant-based terpenes was also noticed for COVID-19 infection [115,116]. Chemically, they are formed by multiple isoprene units (C5) and based on their number they are classiﬁed as hemi-, mono-, sesqui-, di-, tri-, tetra-, and polyterpenes. This class of chemical compounds along with their derivatives, terpenoids, which contains steroids/sterols, saponines and meroterpenes, offer an extensive range of applications in human health [117,118]. Recent investigations reported that citronellol, geraniol, limonene, linalool, and neryl acetate contained in the geranium and lemon essential oils down-regulated ACE2 receptor activity in virus-host epithelial cells [119]. Kulkarni et al. evaluated the major

Molecules 2021, 26, x FOR PEER REVIEWcomponents of plant essential oils such as anethole, cinnamaldehyde, carvacrol, 13geraniol of 26 , cinnamyl acetate, L-4-terpineol, thymol, and pulegone as promising inhibitors of SARS- CoV-2 Spike protein by in-silico approach [120]. Among terpenoid derivatives, by molecular docking simulation, it was revealed that scaffoldlimonin of andthe scopadulcicursodeoxycholic acid Bacidshowed (Figure a good 16b) bindingis surrounded afﬁnity towardsby Lys378, RdRp, Thr376, Spike Phe377,protein, Tyr380, and ACE2 and Pro384 receptor and [119 formed,121]. a hydrogen bond with Cys379 [122]. The in vitro screeningCarino confirmed et al. identiﬁed that the ursodeoxycholic several triterpenoids acid andand steroidalits taurine molecules conjugate (Figure exerted 15 )a as limitedinhibitors inhibitory of Spike-ACE2 activity towards interactions Spike-ACE2 [122]. In complex. details, theseIn contrast, studies glyco-ursodeoxy- reported that gly- choliccyrrhetinic acid (Figure acid (Figure 16c), chenodeoxycholic15a), betulinic acid acid(Figure (Figure 15b), 16a) and,oleanolic and glyco-chenodeoxy- acid (Figure 15d), cholicwere acid able (Figure to well 16d) recognize reduce thethe Spike RBD’s binding pocket to 1. the Docking ACE2 receptor results underlined by at least 20% that in the a concentration-dependentsteroidal scaffold could interact manner. with Despite Trp436, thes Phe374,e promising Arg509, in-vitro and Leu441 results, residues the studies of the havehydrophobic several limitations; pocket of e.g., RBD. the In effects addition, of these glycyrrhetinic natural molecules acid engaged were hydrogen not tested bonds on viral with replicationAsn440, Ser375 [122]. and ionic contacts with Arg509.

FigureFigure 15. 15. 2D2D structures structures of of(a ()a glycyrrhetinic) glycyrrhetinic acid, acid, (b (b) )betulinic betulinic acid, acid, ( (c) ursolic acid,acid, ((dd)) oleanolicoleanolic acid. acid.

Figure 16. 2D structures of (a) chenodeoxycholic acid, (b) ursodeoxycholic acid, (c) Glyco-ursodeoxycholic acid, (d) glyco-chenodeoxycholic acid, (e) withanolide N, (f) dihydrowithaferin A, (g) ashwagandhanolide, (h) withanoside X, (i) withanoside V.

In addition to the activity against ACE2, the binding affinity of triterpene derivatives and steroid-based compounds against SARS-CoV-2 Mpro was explored. The ursodeoxycholic

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Molecules 2021, 26, 632 13 of 27 scaffold of the ursodeoxycholic acid (Figure 16b) is surrounded by Lys378, Thr376, Phe377, Tyr380, and Pro384 and formed a hydrogen bond with Cys379 [122]. The in vitro screeningThe confirmed experimental that resultsthe ursodeoxycholic found that the incubationacid and its of taurine the Spike-ACE2 conjugate complex exerted witha limitedthese inhibitory naturally occurringactivity towards triterpenoids Spike-ACE2 reduced complex. the Spike-ACE2 In contrast, binding glyco-ursodeoxy- in a concentration- dependent way. Among all the compounds mentioned, betulinic and oleanolic acid cholic acid (Figure 16c), chenodeoxycholic acid (Figure 16a), and glyco-chenodeoxy- showed significant inhibition at a concentration of 0.1 µM and 10 µM, respectively [122]. cholic acid (Figure 16d) reduce the Spike binding to the ACE2 receptor by at least 20% in Interestingly, the pre-incubation of oleanolic and glycyrrhetinic acid with Spike-ACE2 a concentration-dependent manner. Despite these promising in-vitro results, the studies complex exhibited a more remarkable ability to reduce the interaction between them and have several limitations; e.g., the effects of these natural molecules were not tested on viral biological targets [122]. replication [122]. In-silico study revealed that betulinic acid and glycyrrhetinic acid (triterpene derivatives) showed good binding energies against the protease SARS-CoV-2. In detail, these compounds could recognize the Mpro structure pocket forming an hydrogen bonds with Lys137 and hydrophobic contacts with Tyr237, Tyr239, Leu272, Leu286, and Leu287 [123]. Based on the structural similarity between oleanolic acid and ursolic acid (Figure 15c,d), and due to their comparable pharmacological activities, the possible binding mode of these compounds towards the catalytic pocket of SARS-CoV-2 Mpro was analyzed. Thus, it was observed that ursolic acid formed a hydrogen bond with Ser46 and several Van der Waals interactions with Thr24, Thr25, Thr26, Cys44, Thr45, Asn142, Gly143, Cys145, and Glu166 residues of the protease. Further, in silico results showed that oleanolic acid was involved with the catalytic dyad of the SARS-CoV-2 protease establishing both a hydrogen bond and several hydrophobic contacts with Gln189, Cys145 and His163 residues, respectively [124]. According to the idea that these triterpenoids are the natural substrates for two bile acid receptors such as Farnesoid-X-Receptor (FXR) and G-protein Bile Acid Recep- tor (GPBAR)-1, the behaviour of some natural bile acids (Figure 16) were also investigated against SARS-CoV-2 druggable targets. The analyzed steroidal compounds were able to fit into pocket 5 on the surface of the β-sheet core of Spike RBD. In detail, the steroidal scaffold of the ursodeoxycholic acid (Figure 16b) is surrounded by Lys378, Thr376, Phe377, Tyr380, and Pro384 and formed a hydrogen bond with Cys379 [122]. The in vitro screening confirmed that the ursodeoxycholic acid and its taurine conjugate exerted a lim- ited inhibitory activity towards Spike-ACE2 complex. In contrast, glyco-ursodeoxycholic acid (Figure 16c), chenodeoxycholic acid (Figure 16a), and glyco-chenodeoxycholic acid (Figure 16d) reduce the Spike binding to the ACE2 receptor by at least 20% in a concentration- Figuredependent 15. 2D structures manner. of Despite (a) glycyrrhetinic these promising acid, (b) in-vitro betulinic results, acid, (c) the ursolic studies acid, have (d) oleanolic several limi- acid.tations; e.g., the effects of these natural molecules were not tested on viral replication [122].

FigureFigure 16. 2D 16. structures2D structures of (a) chenodeoxycholic of (a) chenodeoxycholic acid, (b) acid,ursodeoxycholic (b) ursodeoxycholic acid, (c) Glyco-ursode- acid, (c) Glyco- oxycholicursodeoxycholic acid, (d) glyco-chenodeoxycholic acid, (d) glyco-chenodeoxycholic acid, (e) withanolide acid, (e) withanolide N, (f) dihydrowithaferin N, (f) dihydrowithaferin A, (g) A, ashwagandhanolide,(g) ashwagandhanolide, (h) withanoside (h) withanoside X, (i) withanoside X, (i) withanoside V. V.

In additionIn addition to the to theactivity activity against against ACE2, ACE2, the thebinding binding affinity afﬁnity of triterpene of triterpene derivatives derivatives andand steroid-based steroid-based compounds compounds against against SARS-CoV-2 SARS-CoV-2 Mpro was Mpro explored. was explored. The ursodeoxycholic The ursodeoxycholic acid formed two hydrogen bonds with Phe140 and Ser46 residues, while Met49,

Molecules 2021, 26, x FOR PEER REVIEW 14 of 26

acid formed two hydrogen bonds with Phe140 and Ser46 residues, while Met49, Met165, Cys145, and Leu141 residues participated in hydrophobic interactions that enhanced stability of the protein-ligand complex. These in silico results showed that ursodeoxycholic acid had potential viral inhibitory activity towards SARS-CoV-2 Mpro and human ACE2 protein, but further experimental validations are needed to confirm their biological activity [125]. Additionally, several natural constituents of Withania somnifera, also known as Ashwagandha and “Indian Ginseng”, were explored as possible inhibitors of Mpro SARS- CoV-2 protease. From the study, withanoside V (Figure 16i), a steroid-based compound, was the most potent natural inhibitor [126]. Others phytoconstituents from Withania somnifera such as quercetin-3-O-galactosyl- rhamnosyl-glucoside (Figure 17l), withanoside X (Figure 16h), ashwagandhanolide (Fig- ure 16g), dihydrowithaferin A (Figure 16f) and withanolide N (Figure 16e) were investigated as promising inhibitors of Spike glycoprotein and Nsp15 endoribonuclease [127]. Among phytoconstituents from Nigella sativa, campesterol, cycloeucalenol, apha- spinasterol, and beta-sitosterol showed strong affinities against viral N-terminal RNA binding domain (NRBD) and PLpro of SARS-CoV-2 [128]. Molecules 2021, 26, 632 14 of 27 2.2.3. Flavonoids Met165,Among Cys145, the naturally and Leu141 occurring residues compounds participated and in beyond, hydrophobic flavonoids interactions (Figure that 17) are en- hancedparticularly stability studied of the [129], protein-ligand including their complex. role versus These SARS-CoV-2 in silico results [130]. showed Many in-silico that ur- sodeoxycholicscreenings of medicinal acid had potentialplant databases viral inhibitory against SARS-CoV-2 activity towards druggable SARS-CoV-2 targets Mprohave been and humancarried out ACE2 [131,132]. protein, Indeed, but further analyzing experimental the potential validations role of the are flavonoids needed to against conﬁrm SARS- their biologicalCoV-2 main activity protease [125 [133],]. it was noticed that their inhibitory activity was more extensive than Additionally,that of the peptide-derived several natural inhibitors constituents [134]. of Withania Starting somnifera from this, also rationale known, a asthorough Ashwa- gandhainvestigation and “Indian was focused Ginseng”, on the were common explored pharmacophoric as possible inhibitors features of of Mpro flavonoids. SARS-CoV-2 protease.The presence From the of study, two phenylwithanoside groups V could(Figure be 16responsiblei), a steroid-based for inhibiting compound, the proteolytic was the mostactivity potent of SARS-CoV-2 natural inhibitor protease. [126 ].In particular, by analyzing docking and molecular dy- namicsOthers results, phytoconstituents the hydrogen frombond Withaniabetween somniferathe hydroxylsuch asgroupquercetin-3- of phenylO-galactosyl- moiety of rhamnosyl-glucosidekaempferol (Figure 17e)(Figure and Glu16617l), withanoside was formed; Xmeanwhile,(Figure 16 theh), chromen-4-oneashwagandhanolide scaffold (Figurewas able16 tog), accommodatedihydrowithaferin into the Ahydropho(Figure16bicf) site. and Additionally,withanolide the N (Figure complex16 e)was were fur- investigatedther stabilized as by promising two hydrogen inhibitors bonds of Spikebetween glycoprotein the hydroxyl and groups Nsp15 endoribonucle-and Ile188 and aseAsp142 [127 [134,135].].

Figure 17. 2D2D structures structures of of (a () abroussoflavonol) broussoflavonol F, (b F,) myricetin, (b) myricetin, (c) hesperetin, (c) hesperetin, (d) quercetin, (d) quercetin, (e) (kaempferol,e) kaempferol, (f) herbacetin, (f) herbacetin, (g) (tricetin,g) tricetin, (h) (nobiletin,h) nobiletin, (i) naringenin, (i) naringenin, (l) quercetin-3- (l) quercetin-3-O-galactosyl-O-galactosyl- rhamnosyl-glucoside, (m) rutin, (n) neohesperidin, (o) scutellarin, (p) quercetin-3-β-galactoside, rhamnosyl-glucoside, (m) rutin, (n) neohesperidin, (o) scutellarin, (p) quercetin-3-β-galactoside, (q) baicalin, (r) pectolinarin. (q) baicalin, (r) pectolinarin.

Among phytoconstituents from Nigella sativa, campesterol, cycloeucalenol, apha- spinasterol, and beta-sitosterol showed strong afﬁnities against viral N-terminal RNA

binding domain (NRBD) and PLpro of SARS-CoV-2 [128].

2.2.3. Flavonoids Among the naturally occurring compounds and beyond, flavonoids (Figure 17) are particularly studied [129], including their role versus SARS-CoV-2 [130]. Many in-silico screenings of medicinal plant databases against SARS-CoV-2 druggable targets have been carried out [131,132]. Indeed, analyzing the potential role of the flavonoids against SARS- CoV-2 main protease [133], it was noticed that their inhibitory activity was more extensive than that of the peptide-derived inhibitors [134]. Starting from this rationale, a thorough investigation was focused on the common pharmacophoric features of flavonoids. The presence of two phenyl groups could be responsible for inhibiting the proteolytic activity of SARS-CoV-2 protease. In particular, by analyzing docking and molecular dynamics results, the hydrogen bond between the hydroxyl group of phenyl moiety of kaempferol (Figure 17e) and Glu166 was formed; meanwhile, the chromen-4-one scaffold was able to accommodate into the hydrophobic site. Additionally, the complex was further stabilized by two hydrogen bonds between the hydroxyl groups and Ile188 and Asp142 [134,135]. Again, quercetin-3-β-galactoside (Figure 17p) a flavonoid-based compound, was Molecules 2021, 26, 632 15 of 27

found to bind SARS-CoV-2 Mpro through numerous interactions with the key residues of the catalytic pocket. Notably, the N atom of the main chain of Glu166 made two hydrogen bonds with the compound, while the Gln189 side chain was able to form other four similar interactions. The hydrophobic contacts between the compound and Leu141, Asn142, Met165, and Glu166 residues were evident too. Concentrating on their chemical scaffold, it is possible to rationalize that: (1) four hydroxyl groups of quercetin are crucial determinants of the bioactivity of this type of compounds; (2) the shape of sugar moiety and 7-hydroxy site of quercetin can be characterized by large structural modifications in order to enhance the binding interactions [136]. Other examples were represented by the computational analysis performed on baicalin (Figure 17q), herbacetin (Figure 17f), and pectolinarin (Figure 17r) towards SARS-CoV-2 Mpro. Therefore, herbacetin showed the same kaempferol binding mode. Pectolinarin showed its L-mannopyranosyl-β-D-glucopyranoside moiety accommodated into the S1 and S2 sites and the chromen-4-one scaffold located within the S2 and S3’ SARS-CoV Mpro domains. The baicalin glucuronate moiety interacted through two hydrogen bonds with Gly143 and Asn142 residues. In addition, the π-π stacking between His41 and its phenyl moiety was observed. In this case, Glu166 plays a pivotal role in the binding mode due to its interaction with the 6-hydroxyl group attached to the chromen-4-one moiety and the 5-hydroxyl group attached to the glucuronate moiety. Baicalin, herbacetin, and pectolinarin revealed the prominent inhibitory activity against SARS-CoV-2 Mpro. Indeed, the experimental IC50 values were 34.71, 53.90, and 51.64 µM, respectively [135]. In-silico study reported that broussoflavonol F (Figure 17a) could better recognize SARS-CoV-2 Mpro active site due to the interaction with the catalytic dyad Cys145 and His41 [137]. In addition to the estimated binding affinity towards the protease, flavonoids have also shown promising ACE2 inhibitory activities [138]. A study highlighted that quercetin (Figure 17d), myricetin (Figure 17b), hesperetin (Figure 17c), tricetin (Figure 17g), and rutin (Figure 17m) presented stable binding affinity against Spike-ACE2 protein [132]. It was suggested that these compounds formed interactions with Arg273, Thr371, His345, Pro346, Glu375, Glu402, and Tyr515 in the ACE2 binding site. With this in mind, it was hypothesized that these natural compounds could compete with the bind of SARS-CoV-2 to ACE2, thus preventing the virus entry into host cells [139,140]. Furthermore, other research team noticed that, in addition to the above-mentioned flavonoids, nobiletin (Figure 17h), naringenin (Figure 17i), neohesperidin (Figure 17n), and scutellarin (Figure 17o), were also able to interact with the Spike-ACE2 complex [138]. However, their IC50 values were not still calculated. Despite the encouraging results of these natural compounds, in vitro steps were halted. Some drawbacks probably related to the extraction of these compounds from plants or the chemical synthesis difficulties did not permit further biological investigations. Recently, in-vitro study revealed that neohesperidin could inhibit infected Vero E6 cell [141]. Baicalin interacted at the interface between ACE2 receptor and Spike protein through Asp67, Ala71, Lys74, and Asn448, Ala464, Val472 and Gly474 residues, respectively. These in silico results were validated by in vitro experiments, having an IC50 around 2.24 µM on inhibiting the Spike protein and ACE2 receptor binding [138].

2.2.4. Benzoquinones Quinones are a fascinating class of compounds with attractive chemistry. 1,4-benzoquinones reoccurring structural motif is present in various isolated natural products, and this be- stowing a broad range of biological activities [142]. Additionally, their derivatives also have shown promising ACE2 inhibitory activities [138]. In this regard, embelin, a naturally occurring para-benzoquinone extract from Embelia ribes plant, aroused particular interest (Figure 18a). This natural compound showed several pharmacological effects, and it was considered the second solid gold of India, preceded by curcumin [143,144]. The rationale is addressable to the quinone reactivity with cysteine Molecules 2021, 26, x FOR PEER REVIEW 16 of 26

Molecules 2021, 26, 632 16 of 27 preceded by curcumin [143,144]. The rationale is addressable to the quinone reactivity with cysteine protease [145]. Moreover, a density functional theory (DFT) computational ap- protease [145]. Moreover, a density functional theory (DFT) computational approach proach highlighted the mechanism of action of embelin into SARS-CoV-2 protease. The highlighted the mechanism of action of embelin into SARS-CoV-2 protease. The covalent covalent bond between the quinone carbonyl and the thiolate of Cys145 was hypothesized. bond between the quinone carbonyl and the thiolate of Cys145 was hypothesized. To help To help this reaction mechanism, His41 captured the hydrogen atom from Cys145 that became this reaction mechanism, His41 captured the hydrogen atom from Cys145 that became prone to release a proton to the carbonyl quinone engaged with the thiolate [146]. prone to release a proton to the carbonyl quinone engaged with the thiolate [146].

FigureFigure 18. 18. 2D2D structures structures of of ( (aa)) embelin embelin and and ( (bb)) emodin. emodin. emodin ByBy computational computational methods methods was was observed observed that that emodin (Figure(Figure 18b)18b) binds binds Asp67, Asp67, Ala71, and Lys74 residues of ACE2 receptor, and Asn448, Ala464, Val472, and Gly474 Ala71, and Lys74 residues of ACE2 receptor, and Asn448, Ala464, Val472, and Gly474 res- residues of Spike protein [147]. An experimental study has been shown that emodin, idues of Spike protein [147]. An experimental study has been shown that emodin, ex- extracted from Rheum officinale and Reynoutria multiflora, could act as an inhibitor of the tracted from Rheum officinale and Reynoutria multiflora, could act as an inhibitor of the viral viral Spike protein and human ACE2 receptor binding with IC50 value approximately of Spike protein and human ACE2 receptor binding with IC50 value approximately of 1– 1–10µM in other SARS coronavirus [148]. Thus, its promising theoretical mechanism and 10µM in other SARS coronavirus [148]. Thus, its promising theoretical mechanism and its its known experimental activity against other coronaviruses can stimulate to investigate known experimental activity against other coronaviruses can stimulate to investigate the the potential inhibitory effect against SARS-CoV-2. potential inhibitory effect against SARS-CoV-2. 2.2.5. Other Natural Compounds 2.2.5. Other Natural Compounds In-silico studies were also performed on natural compounds that are readily available in onion,In-silico garlic, studies ginger, were peppermint, also performed and fenugreekon natural [ 149compounds,150]. The that computational are readily availa- results blepointed in onion, out leucopelargonidin garlic, ginger, peppermint,and aronadendin and fenugreekare promising [149,150]. Mpro The inhibitors, computational while re-5S- sults5-Hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-3-heptanone pointed out leucopelargonidin and aronadendin are promising, contained Mpro in Ginger, inhibitors, inter- whileacted with5S-5-Hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-3-heptanone the SARS-CoV-2 Spike protein [111,132]. This study needs appropriate, contained biological in Ginger,experimental interacted validation with the against SARS-CoV-2 COVID-19 Spike [119 protein,121]. [111,132]. This study needs appro- priateAmong biological phenolic experimental compounds validation extracted against from COVID-19 honey and [119,121]. propolis, it was observed thatAmongellagic acidphenolicmade compounds hydrogen bonds extracted with from Gly808, honey Pro809, and His816,propolis, thr817 it was and observed Tyr831 thatresidues ellagic of SARS-CoV-2acid made hydrogen RdRp. Docking bonds resultswith Gly808, also showed Pro809, that His816, hydrogen thr817 bonds and between Tyr831 residuesellagic acid of andSARS-CoV-2 His41, Gly143, RdRp. Arg188 Docking were resu formed.lts also Furthermore, showed that Shaldam hydrogen et al. bonds proposed be- tweenartepillin ellagic C asacid a potentialand His41, inhibitor Gly143, ofArg188 the SARS-CoV-2 were formed. Mpro Furthermore, due to the Shaldam interactions et al. proposedbetween theartepillin phenolic C compoundas a potential and inhibitor Cys145, of Arg188, the SARS-CoV-2 Thr190 and Mpro Gln192 due [151 to]. the interactions between the phenolic compound and Cys145, Arg188, Thr190 and Gln192 [151]. 3. Discussion and Future Perspectives 3. DiscussionSince SARS-CoV-2 and Future has Perspectives become a pandemic infection with relevant health issues, the scientificSince community SARS-CoV-2 carried has become out much a pandemic effort for theinfection identification with relevant of potential health drug issues, targets the scientificto tackle community this virus infection.carried out Recently, much effort several for the articles identification have been of published,potential drug most tar- of getsthem to based tackle on this the virus virtual infection. screening Recently, of natural several occurring articles compounds have been towardspublished, viral most target of themproteins based [152 on–155 the]. virtual screening of natural occurring compounds towards viral target proteinsEspecially [152–155]. in emergent diseases, such as COVID-19, computer-assisted techniques are usefulEspecially tools to in find emergent out potential diseases, new such drugs. as COVID-19, By harnessing computer-assisted the innovative techniques technologies are usefulin rational tools to drug find design out potential in the new medicinal drugs. chemistryBy harnessing field, the it innovative has been possibletechnologies to obtain in ra- tionalpromising drug compoundsdesign in the fitted medicinal to recognize chemistry several field, targets it has ofbeen the possible other coronaviruses to obtain promising [21,156]. compoundsSeveral fitted chemical to recognize entities several have already targets been of the investigated other coronaviruses by computational [21,156]. approaches suchSeveral as docking, chemical DFT, entities molecular have dynamics already simulations; been investigated however, by no computational novel compounds ap- have been proposed until now, except for repurposed drugs. proaches such as docking, DFT, molecular dynamics simulations; however, no novel com- Considering this, drug repurposing represents the promising strategy to fight tricky pounds have been proposed until now, except for repurposed drugs. illness emerging in the future. Combined with polypharmacology, as a new therapeutic paradigm, the efforts of the scientific research towards the natural source compounds, though not new, could offer an opportunity to obtain the natural drugs scaffold useful

Molecules 2021, 26, 632 17 of 27

against SARS-CoV-2 targets [157]. Naturally occurring compounds recognizing SARS-CoV- 2 druggable targets are reported in Table1.

Table 1. Naturally occurring compounds recognizing SARS-CoV-2 druggable targets under investigation.

Compound Name Natural Sources In Silico In Vitro Clinical Trials Ref Alpha-spinasterol Nigella sativa • [128] Fennel, anise, Star anise, Anethole • [120] Essential oils Anisotine Justicia adhatoda • [113] Aronadendin Onion • [132] Arteannuin B Artemisia annua • [94] Artemisinin Artemisia Annua ••• [93,94,158] Artepillin C Honey and propolis • [151] Ashwagandhanolide Withania somnifera • [127] Baicalin Scutellaria baicalensis •• [135,138] Berberine Berberis vulgaris L. ••• [101,102,104–108] Beta-sitosterol Nigella sativa • [128] Betulinic Acid Betula pubescens • [123] Biscryptolepine Cryptolepis sanguinolenta • [110] Broussoflavonol F Macaranga indica • [137] Campesterol Nigella sativa • [128] Carvacrol Origanum vulgare •• [120] Chenodeoxycholic acid Bile Acids •• [122] Cinnamaldehyde Cinnamomum sp. • [120] Cinnamyl Acetate Essential oils • [120] Cymbopogon winterianus, Citronellol •• [119] Geranium Colchicine Colchicum autumnale • [99,100] Cryptomisrine Cryptolepis sanguinolenta • [110] Cryptoquindoline Cryptolepis sanguinolenta • [110] Cryptospirolepine Cryptolepis sanguinolenta • [110] Cycloeucalenol Nigella sativa • [128] Dihydrowithaferin A Withania somnifera • [127] Ellagic acid Honey and propolis • [151] Embelin Embelia ribes plant • [146] Rheum officinale and Reynoutria Emodin •• [147,148] multiflora Geraniol Essential oils •• [119,120] Glico-chenodeoxycholic acid Bile Acids • [122] Glyco-ursodeoxycholic acid Bile Acids •• [122] Glycyrrhetinic Acid Glycyrrhiza glabra • [123] Glycyrrhizin Glycyrrhiza glabra ••• [89,90] Hanfangchin A Stephania tetrandra ••• [82–84,88,159] Herbacetin Rhodiola rosea •• [135] Hesperetin Citrus sp. • [140,141] Molecules 2021, 26, 632 18 of 27

Table 1. Cont.

Compound Name Natural Sources In Silico In Vitro Clinical Trials Ref Kaempferol Aloe vera • [134] L-4-terpineol Essential oils • [120] Lactoferrin Mammalian and Animal milk •• [56,57,67] Leucopelargonidin Onion • [111,132] Limonene Essential oils •• [119] Limonin Euodia rutaecarpa • [119–121] Linalool Essential oils •• [119] Myricetin Onion, Chilli • [132,139,140] Naringenin Citrus sp. • [138,139] Neohesperidin Citrus sp. •• [138,139,141] Neryl Acetate Citrus oils •• [119] Nobiletin Citrus sp. • [138,139] Oleanolic Acid Olea europaea L. •• [122] Pectolinarin Cirsium setidens •• [135] Pulegone Essential oils • [120] Onion, Garlic, Peppermint, Fenugreek, Origan Capers, Onions, Elderberries, Quercetin ••• [10,71,132,139] Kale, Okra, Apple Peels, Aronia Berries, Cranberries, Asparagus, Goji Berries Quercetin-3-O-galactosyl- Withania somnifera • [127] rhamnosyl-glucoside Quercetin-3-β-galactoside Artemisia capillaris • [136] Quinadoline B Fungi • [114] Red wine, Red grape juice, Peanuts, Fresh grapes, Pistachios, Peanut butter, Cocoa powder, Dark chocolate, Milk Resveratrol ••• [80,81] chocolate, Strawberries, Jackfruit skin, Blueberries, Bilberries, Red currants, Cranberries, Lingonberries, Mulberries Rutin Citrus sp. • [139] Scedapin C Fungi • [114] Schizanthine Y Schizanthus porrigens • [112] Schizanthine Z Schizanthus porrigens • [112] Scopadulcic Acid B Scoparia dulcis • Scutellarin Scutellaria barbata • [139] Sophaline Sophora alopecuriodes • [86] Thalimonine Thalictrum simplex • [86] Thymol Essential oils • [120] Ginger, Onion, ﬂowers of Tricetin • [132] pomegranate. Ursodeoxycholic acid Bile Acids •• [122] Molecules 2021, 26, 632 19 of 27

Table 1. Cont.

Compound Name Natural Sources In Silico In Vitro Clinical Trials Ref Ursolic acid Bile Acids •• [122] Citrus Fruit, Tropical Fruit, Vitamin C Peppers, Cruciferous Vegetables, • [36,40–46] Leafy Vegetables Vitamin D Human and Jay Jasmine plant •• [32–34] Withanolide N Withania somnifera • [127] Withanoside V Withania somnifera • [127] Withanoside X Withania somnifera • [127] 5S-5-Hydroxy-1,7-bis(4- hydroxy-3-methoxyphenyl)- Ginger • [132] 3-heptanone

Summarizing, numerous vitamin D clinical trials for COVID-19 could be useful to determine its effect on disease progression and post-exposure prophylaxis. One idea is that its supplementation can help patients to maintain sufficient serum levels of vitamin D as recommended by guidelines [160]. The known potential of vitamin D modulating the innate immune response may be auspicious to prevent acute respiratory infection [161]. Based on the knowledge that COVID-19 disproportionately affects race, ethnicity, minority groups, and the known vitamin D deficiency in the same groups, the use of natural derivatives may provide a chance for decreasing health disparities [162]. LF is the naturally occurring compound mostly differing from the others. Clinical trials are designed to evaluate the local treatment of nasal mucosa with LF by using a spray formulation. The rationale of the implication of LF for COVID-19 prevention has the following fundaments: reverting the iron disorders due to the viral colonization, modulating the immune response or down-regulating the pro-inflammatory cytokines released by the viral inflammation [67]. Among the flavonoid-based compounds, only quercetin is undergoing clinical trial due to its anti-inflammatory and scavenging activity. In detail, its dose scheduled is 500–1000 mg for prophylaxis and treatment, respectively [78]. Quercetin, in combination with zinc, bromelain and vitamin C, is recruited in clinical trials [77]. Several clinical trials on resveratrol, artemisinin, glycyrrhizin, colchicine, berberine, and tetrandrine have been designed and are going on. These compounds are characterized by well-known beneficial activities other than the antiviral implications against COVID-19 above-mentioned. Since these components are already taken differently in the daily diet, it could be challenging to attribute the antiviral properties to these natural compounds directly. Still, their assumption could be valuable for human health protection and to positively fill health equity. Herein, terpenes, anthraquinones, flavonoids, stilbenes, steroid-based scaffold, quinone derivatives, and polyphenols have been summarized based on their current update in the recognition and validation of the SARS-CoV-2 druggable targets. Although some of these have several drawbacks and lead to inconsistent results [2,163–170]. In this regard, we would like to provide critical thought. Undoubtedly, the computational tools are valid instruments for the drug identification process, but the support of these preliminary investigations with experimental biological data are strictly required. Although these feelings can appear obvious, we believe this concept should be stressed to avoid the spread of disinformation or to prevent equivocal scientific conclusions. Most investigations were carried out on therapeutic benefits of phytochemicals, according to their known antiviral, anti-inflammatory and immunoregulatory properties. Several research teams conclude by asserting that the use of polyphenolic compounds, anthraquinones and flavonoids, and steroid-based molecules for COVID-19 treatment can appear speculative. In fact, no clear Molecules 2021, 26, 632 20 of 27

evidence from well-drawn clinical trials has been reported so far. On the other hand, dietary use of these phytochemical derivatives is known to provide several benefits to humans. The safety and efficacy of the reported natural compounds in preclinical and clinical trials must be still assessed to establish their use and application in COVID-19 infection. In this perspective, the dietary nature and pleiotropic effects make natural occurring compounds a fascinating candidate for further investigation. Moreover, rational drug design highlights the possible binding mode of these compounds towards the key anti-SARS-CoV-2 targets. These data could be useful to suggest structural chemical modifications to improve the in-silico binding affinity. We expect scientists to perform relevant experimental studies to provide new derivatives within COVID-19 prevention and treatment, starting from the appropriate and most promising natural scaffolds.

Author Contributions: Conceptualisation, I.R., F.M., A.L., and S.A.; writing—original draft prepa- ration, I.R., F.M., and A.L.; writing—review and editing, I.R., F.M., and A.L.; supervision, S.A. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: The authors acknowledge the POR CALABRIA FESR-FSE 2014-2020 SISTABENE project. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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