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Signal Transduction and Targeted Therapy volume  7, Article number: 387 (2022 ) Cite this article

The outbreak of COVID-19 has become a global crisis, and brought severe disruptions to societies and economies. Until now, effective therapeutics against COVID-19 are in high demand. Along with our improved understanding of the structure, function, and pathogenic process of SARS-CoV-2, many small molecules with potential anti-COVID-19 effects have been developed. So far, several antiviral strategies were explored. Besides directly inhibition of viral proteins such as RdRp and Mpro, interference of host enzymes including ACE2 and proteases, and blocking relevant immunoregulatory pathways represented by JAK/STAT, BTK, NF-κB, and NLRP3 pathways, are regarded feasible in drug development. The development of small molecules to treat COVID-19 has been achieved by several strategies, including computer-aided lead compound design and screening, natural product discovery, drug repurposing, and combination therapy. Several small molecules representative by remdesivir and paxlovid have been proved or authorized emergency use in many countries. And many candidates have entered clinical-trial stage. Nevertheless, due to the epidemiological features and variability issues of SARS-CoV-2, it is necessary to continue exploring novel strategies against COVID-19. This review discusses the current findings in the development of small molecules for COVID-19 treatment. Moreover, their detailed mechanism of action, chemical structures, and preclinical and clinical efficacies are discussed.

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to more than 6 million deaths worldwide.1 SARS-CoV-2 is a betacoronavirus and possesses a positive-sense single-stranded RNA genome that contains 14 open reading frames (ORFs) (Fig. 1). Two ORFs encode polyproteins PP1a and PP1b.2 Four ORFs encode a series of structural proteins, including the spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins. In the SARS-CoV-2 lifecycle, S protein, which recognizes the human ACE2 receptor and is cleaved by host proteases, is responsible for virus binding and entry into host cells.3,4 Subsequently, Mpro and PLpro are necessary for the production and function of non-structural proteins (NSPs). The key NSP RNA-dependent RNA polymerase (RdRp, also known as NSP12) catalyzes the synthesis of viral RNA and plays a central role in the lifecycle of SARS-CoV-2.5,6,7 Therefore, targeting these functional proteins is a rational strategy to inhibit infection and the replication of SARS-CoV-2. Infection with SARS-CoV-2 activates the host immune system, which may elicit a dysfunctional inflammatory response and cause organ damage.8,9,10 Therefore, therapeutic interventions targeting the immune system are also potential approaches for COVID-19 therapy.

Schematic illustration of the genome of SARS-CoV-2 and its structure. The size of SARS-CoV-2 genome is close to 30 kb; it contains 14 open reading frames (ORFs) and encodes 29 proteins. Two ORFs, comprising approximately two-thirds of the genome, encode two polyproteins, which are digested by M protease (Mpro) and Papain-like protease (PLpro) into 16 nonstructural proteins (nsps). Four ORFs encode a series of structural proteins, including the spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins

Small molecules targeting specific signals and functions are widely applied in the treatment of diseases. Compared with biologics such as monoclonal antibodies and plasma products, small molecules are more flexible in binding with target molecules when acting as antagonist or agonist.11,12 Their lower production cost and higher stability also make them ideal therapeutic agents for both clinical and research applications. In parallel with the growing understanding of the pathogenic mechanisms of SARS-CoV-2 infection, small molecules from natural sources or those produced via chemical synthesis have demonstrated their immense therapeutic potential by intervening with various processes.13,14,15 The development of small molecules to treat COVID-19 has been achieved by several strategies, including computer-aided lead compound design and screening, natural product discovery, drug repurposing, and combination therapy. In this review, we present a comprehensive overview of the latest progress in the development of small molecule therapeutics for COVID-19 treatment. These therapeutic compounds are classified according to their chemical structures. The anti-COVID-19 molecular mechanisms are also discussed.

RdRp of SARS-CoV-2 is composed of NSP12 as the catalytic subunit and the NSP7–NSP8 complex as accessory subunits.16,17,18 RdRp is central to RNA transcription and viral replication, and may thus be an ideal target for anti-SARS-CoV-2 drugs (Fig. 2). The structural conformation of the SARS-CoV-2 RdRp complex is highly similar to that of SARS-CoV RdRp.17,19 NSP12 is classified into three domains: an N-terminal nidovirus RdRp-associated nucleotidyltransferase domain (residues 1–250), an interface region (residues 251–398), and the core RdRp domain (residues 399–932). NSP12 is formed by polymerase motifs A to G. These motifs are conserved in most RNA viruses.17 Studies of this RdRp domain have provided information on the role of these conserved motifs during RNA synthesis. Briefly, initial nucleotide recognition is mediated by positively charged Lys and Arg residues, which are located in motifs D and F of NSP12. The nucleotide flips into the active site through interaction with motifs A, B, and F to form a base pair with the template nucleotide, close to the active site. The incoming NTP forms a phosphodiester bond with the product RNA and after catalysis releases pyrophosphate. Then, the conformation of the active site immediately changes to an open state through a subtle rotation of motif A for the next nucleotide addition cycle.20,21,22 RdRp is the primary target of many existing antiviral nucleotide drugs. Based on its high conservation in diverse RNA viruses, repurposing of existing nucleotide drugs is an effective strategy that could shorten drug development time.18,19

Lifecycle of SARS-CoV-2. The SARS-CoV-2 S protein recognizes the ACE2 receptor while being cleaved by the host proteases and entering into the target cells. Then, the gRNA is released and translated into pp1a and pplb, thereby being digested into the NSPs necessary for viral replication. Under the catalyzation of RdRp, new gRNAs are produced and encode the structural proteins to assemble the progeny virus

The possible antiviral mechanism of nucleotide drugs is threefold; they can act as mutagens, as obligate chain terminators, and as non-obligate chain terminators (Fig. 3).23,24 Mutagens incorporated into RNA strands can cause permanent mutations.25,26 Obligate terminators lacking a 3-OH group will terminate RNA extensions immediately, while non-obligate chain termination usually proceeds when a drug contains both a natural base and a 3-OH on the sugar but has a modified ribose skeleton that disrupts translocation.27,28

Antiviral mechanisms of nucleotide drugs. The triphosphate form of remdesivir acts as a non-obligate chain terminator to exert an inhibition effect (Protein Data Bank entries 7VB219). The active form of molnupiravir can be directly incorporated into RNA as a substrate instead of cytidine triphosphate (C) or uridine triphosphate (U), thereby leading to mutated RNA products (Protein Data Bank entry 7OZU38). The triphosphate form of AT-527 (AT-9010) incorporates at the 3′ end of the RNA product, causing termination of RNA synthesis (Protein Data Bank entry 7ED551)

Remdesivir was first developed for the prevention of the Ebola virus infection.29,30,31 It is a non-obligate chain terminator of SARS-CoV-2.32 A study conducted by Yin et al. revealed that the triphosphate form of remdesivir (GS-441524) mimics a nucleotide and is covalently linked to the replicating RNA, thus blocking further synthesis of SARS-CoV-2 RNA.19 Kokic et al. reported that incorporation of remdesivir into the RNA product could stop RNA synthesis after the addition of three more nucleotides.33 They showed that the stalling is caused by the C1ʹ-cyano group in the remdesivir ribose moiety. Insight into this non-obligate chain termination mechanism may facilitate the search for compounds with potential to interfere with SARS-CoV-2 replication.16,34

Molnupiravir, an orally available antiviral drug, is a mutagen of SARS-CoV-2.35,36,37 According to research reported by Kabinger et al., the active form of molnupiravir, beta-D-N4-hydroxycytidine triphosphate, can be directly incorporated into RNA as a substrate instead of cytidine triphosphate or uridine triphosphate, leading to mutated RNA products.38 Structural analysis of RdRp-mutated RNA indicated that beta-D-N4-hydroxycytidine triphosphate formed a stable base pair with G or A in the RdRp active region, thus escaping proofreading and synthesizing mutated RNA. Like molnupiravir, ribavirin abrogates viral RNA synthesis by incorporation into nascent RNA strands.39,40,41,42 Cheung et al. confirmed it is a mutagen for influenza virus by increasing the G-to-A and C-to-T mutation rates in vitro.39 The molecular docking study of Bylehn et al. indicated that it binds strongly at the active site of SARS-CoV-2 RdRp.43 However, their results revealed that ribavirin does not bind the nucleotide on the complementary strand as effectively and seems to act by a different mechanism.

Favipiravir is another inhibitor of RdRp with two possible mechanisms of action.44,45,46,47 Shannon et al. demonstrated its active form could result in SARS-CoV-2 lethal mutagenesis by incorporation into the nascent viral RNA by error-prone SARS-CoV-2 RdRp, provoking C-to-U and G-to-A mutations in the SARS-CoV-2 genome.48 This mutagen mechanism of favipiravir was also reported by Peng et al.49. A study conducted by Naydenova et al. indicated that favipiravir could suppress the replication of SARS-CoV-2 RNA in the presence of natural nucleotides by weak incorporation into the RNA prime strand.50 They revealed that favipiravir–RTP represents an unusual, non-productive binding mode at the catalytic site of SARS-CoV-2 RdRp, thus inducing non-obligate chain termination.

The obligate chain terminator AT-527 is a guanosine nucleotide analog that serves as an orally available prodrug with inhibitory effects on hepatitis C virus (HCV) RdRp.51,52 Shannon et al. reported a 2.98 Å cryo-EM structure of the SARS-CoV-2 RdRp–RNA complex, showing the triphosphate form of AT-527 (AT-9010) bound at three sites of NSP12.51 Their results showed that after AT-9010 is incorporated at the end of the RNA product strand, its modified ribose group will prevent correct alignment of incoming NTP, thereby causing obligate chain termination.

Due to the conserved structure of RdRp, the effects of several molecules interfering with other viral RdRps against RdRp of SARS-CoV-2 were also studied.17,53 For example, sofosbuvir is an oral nucleoside that is used to treat chronic HCV infection.54,55,56,57 Appleby et al. indicated that the metabolized form of sofosbuvir could be recognized by HCV RdRp (NS5B) and incorporated into the growing chain. The presence of fluoro and methyl modifications at the 2′ position promotes non-obligate chain termination of HCV RNA.58 Enzymatic assays demonstrated that sofosbuvir acts as a competitive inhibitor of SARS-CoV-2 RdRp,59 revealing it might act as a non-obligate terminator. Another molecule, galidesivir, was initially designed to inhibit filovirus RNA polymerase activity indirectly through non-obligate RNA chain termination.60,61,62 It exhibited activity against numerous viruses, including yellow fever virus, dengue virus, Japanese encephalitis virus, West Nile virus, zika virus, and tick-borne encephalitis virus, in cell cultures and animal models.63 Molecular docking assays also revealed galidesivir is attached to the catalytic center of SARS-CoV-2 RdRp, and its binding mechanism needs to be further studied.61

SARS-CoV-2 Mpro (also named NSP5 or 3C-like protease) is a key enzyme that plays a vital role in viral replication and transcription.64,65,66 After membrane fusion, genomic RNA (gRNA) of SARS-CoV-2 is released into the cytosol of the target cell (Fig. 2). The gRNA of SARS-CoV-2 contains two large replicase ORFs, ORF1a and ORF1b. These ORFs encode two N-terminal polyproteins, PP1a and PP1ab, respectively.67 Mpro mainly digests both polyproteins at more than 11 conserved sites, thus helping to release NSPs.68 These NSPs are involved in the production of subgenomic RNA, encoding four major structural proteins and other helper proteins.69,70,71 Since no human protease has a structure similar to that of Mpro, it is an attractive target for SARS-CoV-2 treatment.72 The SARS-CoV-2 Mpro crystal structure revealed it is a homodimer containing two protomers (promoters A and B), and each protomer is composed of three domains.68,73,74 The substrate binding site was located between (i) Domains I and II and (ii) Domain III. It regulates the dimerization of Mpro, which is necessary for its catalytic activity.72 The active sites of Mpro between Domains I and II are composed of four sites (S1′, S1, S2, and S4), which often accommodate four fragments (P1′, P1, P2, and P3, respectively) of inhibitors.68,73,74,75 Among them, covalent linkage with the Cys-145 residue in the S1′ site is beneficial for the activity of inhibitors.70,76 Non-covalent SARS-CoV-2 Mpro inhibitors binding with Mpro in different patterns have also became clinical candidates for treating SARS-CoV-2.77,78

Mpro always accommodates four fragments—P1′, P1, P2, and P3—which occupy the S1′, S1, S2, and S4 pockets of Mpro, respectively. Following this rule, novel molecules against SARS-CoV-2 Mpro were developed by structure-based design methods. For example, Dai et al. designed and synthesized two lead compounds (11a and 11b) targeting Mpro70 (Fig. 4). In their design, an aldehyde was selected as a new warhead along with an (S)-γ-lactam ring in order to form a covalent bond with cysteine. A cyclohexyl or 3-fluorophenyl was introduced in P2, while an indole group was introduced into P3. The resulting 11a and 11b were covalently bound to Cys-145 of Mpro according to the X-ray crystal structures of their complexes with SARS-CoV-2 Mpro. Qiao et al. designed new inhibitors by fixing P1 as an optimal fragment, using P2 that was derived from either boceprevir or telaprevir and allowing P3 to change.79 According to their results, one of the most potent compounds, MI-23, covalently bound to the catalytic residue Cys-145 of SARS-CoV-2 Mpro as expected. The binding pattern of the representative compound MI-23 with Mpro is consistent with its design concept. Based on the structure of ML188(R), a non-covalent inhibitor of SARS-CoV Mpro, Kitamura et al. proposed a strategy for designing the SARS-CoV-2 Mpro inhibitor and obtained a novel Mpro inhibitor 23R with high specificity to SARS-CoV-2 and SARS-CoV Mpro.77 Furthermore, they designed covalent SARS-CoV-2 Mpro inhibitors Jun9-62-2R and Jun9-57-3R using novel cysteine reactive warheads to improve the target specificity of aldehyde warhead.80 To optimize oral bioavailability of Mpro inhibitors, Quan et al. chose alpha-ketoamide as warhead P1’, and P1, P2, and P3 were fixed as pyridine, tert-butylbenzene, and tert-butyl, respectively, similar to the groups in ML188.81 The resulting compound Y180 showed high oral bioavailability in mice and efficiently protected transgene mice from SARS-CoV-2 and variant infection.

Different binding models of inhibitors in complex with SARS-CoV-2 Mpro. a Binding models of inhibitors 11a and 11b complexing with SARS-CoV-2 Mpro (the Protein Data Bank entries for SARS-CoV-2 Mpro complexing with 11a and 11b are 6LZE and 6M0K, respectively70) b Binding model of inhibitor nirmatrelvir in complex with SARS-CoV-2 Mpro (Protein Data Bank entry 7RFW).84 c Binding model of non-covalent inhibitor 23R in complex with SARS-CoV-2 Mpro (Protein Data Bank entry 7KX577)

Besides the rational design of novel compounds, several SARS-CoV-2 Mpro inhibitors were discovered by optimizing existing Mpro inhibitors through drug design.82,83 The drug PF-07321332, more commonly known as nirmatrelvir, was optimized from the SARS-CoV Mpro inhibitor PF-00835231.84 Meanwhile, Zhang et al. optimized the structure of the alpha-ketoamide Mpro inhibitor 11r to increase its half-life and solubility and reduce its interaction with plasma proteins.72 Then, the authors replaced the P2 cyclohexyl moiety with a small cyclopropyl to increase the antiviral activity by scarifying the broad-spectrum nature.65 The molecule 13b was located in the substrate binding cleft of Mpro and interacted with the Glu-166 residue, thus disturbing the correct shape of the S1 pocket and inactivating the enzyme.72,85 Kenller et al. presented the design and characterization of three hybrid reversible covalent SARS-CoV-2 Mpro inhibitors named BBH-1, BBH-2, and NBH-2 by splicing the SARS-CoV protease inhibitors boceprevir and narlaprevir.86 By substituting the ketoamide group of boceprevir with the keto-benzothiazole moiety or introducing the nitrile warhead, they directed the warhead into the oxyanion hole. Then, they substituted the P1 group of boceprevir and narlaprevir with a Gln-mimic γ-lactam, thereby synthesizing the hybrid reversible covalent inhibitors BBH-1, BBH-2, and NBH-2. A study by Amporndanai et al. indicated that ebselen and its derivative MR6-31-2 solely bind at the Mpro catalytic site by donating a selenium atom, forming a covalent bond and blocking the His-41 and Cys-145 catalytic dyad.87

The three-dimensional structure of SARS-CoV-2 Mpro is highly similar to that of SARS-CoV Mpro.72,88,89,90 Therefore, repurposing of drugs is a good strategy to develop drugs against SARS-CoV-2. Two SARS-CoV Mpro inhibitors, GRL-1720 and 5 h, have shown anti-SARS-CoV-2 activity.91,92,93 According to X-ray structural analysis, 5 h fully occupies all binding pockets and is stabilized by six direct hydrogen bonds with the residues inside the binding groove of SARS-CoV-2 Mpro, and covalent bonds are formed between 5 h and the Cys-145 residue.91 Su et al. reported that myricetin inhibits SARS-CoV-2 Mpro.94 According to a crystal structure of the SARS-CoV-2 Mpro–myricetin complex, an exact covalent bond can be observed between the sulfur atom of Cys-145 and the C6’ atom of the pyrogallol group of myricetin, revealing the potential of pyrogallol as an alternative warhead of an Mpro inhibitor. High-throughput screens were also applied to repurpose molecules with potential inhibitory effects on SARS-CoV-2 Mpro.95,96,97 For example, Günther et al. applied X-ray fragment screening experiments with approved drugs and drugs in clinical trials, and identified 37 compounds that bind to Mpro .88 Moreover, they obtained structural evidence for interaction of seven compounds at active and allosteric sites of Mpro, and identified two allosteric sites representing attractive targets for drug development. Another high-throughput screening study was conducted by Drayman et al. on a library of 1900 clinically safe drugs against OC43, which is also a betacoronavirus.98 As a result, they identified the most potent SARS-CoV-2 Mpro inhibitor, masitinib, and characterized the mechanism by X-ray crystallography. Virtual high-throughput screening methodology was also applied in identifying novel inhibitors from a large collection. Jin et al. assayed more than 10000 compounds through structure-based virtual screening and high-throughput screening, and identified ebselen as a promising inhibitor of SARS-CoV-2 Mpro.68

PLpro (NSP3) is an important coronavirus enzyme that digest polyproteins by recognizing the conserved sequence LXGG, thus generating a functional replicase complex which enables viral spread99,100,101 (Fig. 2). In addition, it is implicated in both the ubiquitination and inhibition of ISGylation on host proteins as an evasion mechanism against host antiviral immune responses.102,103,104 Shin et al. demonstrated that SARS-CoV-2 PLpro prefers to cleave the conserved LRGG motif at the C-terminus of interferon-stimulated gene 15 (ISG15), which attenuates type I interferon immune responses elicited by viral infection.105 This dual functionality of PLpro makes it an attractive antiviral target for SARS-CoV-2 treatment. PLpro has four subdomains: the ubiquitin-like domain, the Thumb domain, the Finger domain, and the Palm domain (Fig. 5).105 The substrate binding pockets are located at the interface of the Palm and Thumb domains, which include a conserved catalytic triad of Cys-111. The other two core residues, Phe-69 and Val-66, mediate interactions of PLpro with ISG15.105 Substrates accessing the active site are regulated by a flexible blocking loop 2 (BL2).99 The key Tyr-268 residue on BL2 is vital for regulating the function of the enzyme.102 In addition, the zinc finger domain comprises four cysteines which also contribute to the structural integrity and protease activity of PLpro.106,107,108 These sites are hotspots on PLpro, which have led to the discovery of drug leads with clinical potential for COVID-19 treatment.

Cartoon structure of SARS-CoV-2 PLpro in complex with GRL0617 (Protein Data Bank entry 7CJM)112 and the key residues in the PLpro domain (Protein Data Bank entry 7JRN)111

GRL-0617 is a non-covalent inhibitor of SARS-CoV PLpro, and it exhibited inhibitory effects against SARS-CoV-2 in vitro.103,109,110 Gao et al. demonstrated that GRL0617 not only occupies the substrate pockets, but also induces closure of the BL2 loop and narrows the substrate binding cleft, thus preventing binding of the LXGG motif of the substrate.99 This BL2 conformational change was also observed by Ma et al. through X-ray co-crystal analysis of PLpro complexed with GRL0617 (Fig. 5).111 Further, Shin et al. reported that GRL-0617 treatment of SARS-CoV-2-infected cells led to a marked increase in IRF3 ISGylation and significantly rescued the expression of IFN-responsive genes.105 According to Fu et al., GRL0617 blocks the binding of the ISG15 LRGG C-terminus to PLpro, thus interfering with cleavage of ISG15.112 Moreover, through a high-throughput screening and subsequent lead optimization, they identified two PLpro inhibitors, Jun9-72-2 and Jun9-75-4. Both inhibitors demonstrated improved enzymatic inhibition and antiviral activity compared to GRL0617. In addition, Zhao et al. identified SARS-CoV-2 PLpro inhibitors by high-throughput screening.108 They found that YM155, an anticancer drug candidate, efficiently inhibited the activity of SARS-CoV-2 PLpro. By analyzing crystal structures of SARS-CoV-2 PLpro and its complex with YM155, they found that YM155 simultaneously targets the substrate binding pocket, the ISG15 binding site, and the zinc finger motif of enzyme.

Based on substrate specificity and the structure of SARS-CoV-2 PLpro, rational design of compounds would greatly facilitate the development of novel PLpro inhibitors.100 For instance, by using a Hybrid Combinatorial Substrate Library, Rut et al. revealed the molecular rules governing PLpro substrate specificity, and designed and biochemically characterized potent inhibitors (VIR250 and VIR251) with high selectivity for SARS-CoV-2 PLpro.100 Further, they found that both inhibitors could selectively inhibit the activities of PLpro in both SARS-CoV and SARS-CoV-2. This revealed a high level of sequence and structural similarity between these PLpro in the substrate binding pocket. The crystal structures of VIR250 and VIR251 in complex with SARS-CoV-2 PLpro reveal they inhibit the enzyme by forming a covalent link with the Cys-111 residue and provide a structural basis for the observed substrate specificity profiles. Osipiuk et al. synthesized six naphthalene-based compounds derived from GRL0617. Five of them are further amine-functionalized derivatives of GRL0617, and one is a simplified variant of GRL0617 without a chirality center.112,113 All these compounds exhibited inhibition activities of PLpro, and the crystal structure indicated these inhibitors bind to protease S4/S3 sites, thus blocking peptide recognition. Shan et al. also synthesized a series of reported ScoV PLpro inhibitors (11–13) that partially resemble GRL0617 with a shared naphthyl subunit.114 Co-crystal structure analysis of SARS-CoV-2 PLpro-12 revealed 12 occupies a pocket between the S1 position and the catalytic position of SARS-CoV-2 PLpro, and the three hydrophobic rings of 12 are engaged simultaneously with the phenyl ring of Tyr-268, thus closing the binding pocket.114

SARS-CoV-2 virus entry into host cells depends on the viral S protein.115,116,117 In brief, the S protein recognizes the peptidase domain (PD) of the ACE2 receptor in host cells (Fig. 2). This initiates recognition of the virus and host cell receptor–viral membrane fusion.118,119,120 It was thought that targeting the virus entry process is more advantageous than targeting the subsequent stages of the SARS-CoV-2 lifecycle, thus many efforts have been made to find inhibitors blocking this process.121,122,123 Small molecules targeting the S protein, ACE2, and the S protein–ACE2 complex were found to potentially inhibit SARS-CoV-2 infection.124,125 The SARS-CoV-2 S protein consists of two subunits; S1 comprises the receptor binding domain (RBD) and S2 is responsible for viral membrane fusion.126,127,128,129,130 Previous studies revealed that the high affinity between the S protein RBD and the human ACE2 receptor could partially explain the efficient transmission of SARS-CoV-2 among humans.131,132,133 The structure of the SARS-CoV-2 RBD was found to have more ACE2-interacting residues than the SARS-CoV RBD.119 Compensating mutations in the S protein RBD of further variants (especially the Delta and Omicron variants) possibly account for their heightened transmissibility and immune evasion.134,135 Thus, interference with binding between them is beneficial for viral inhibition. A six-helical bundle (6-HB) structure of S2 conjuncts the viral and cell membranes for a fusion reaction.136 Blocking the 6-HB domain is considered effective for developing fusion inhibitors EK1 (Fig. 6).137,138 In human ACE2, Lys-31 and Lys-353 are sensitive to the RBD.139 Its glycosylation sites Asn-90 and Asn-322 also demonstrated the ability to interfere with S protein binding in a recent study.140 Glycosylation of asparagine residues within the RBD is an important mediator of ACE2 binding.141

S2 subunit of SARS-CoV-2 S protein involves the HR1 and HR2 trimers to form a 6-HB domain. The binding model of the EK1 inhibitor in complex with the HR1 motif is presented (Protein Data Bank entry 7C53)679

The effects of molecules binding with S protein against SARS-CoV-2 were investigated. A previous study revealed that the RBD of the S protein of SARS-CoV-2 recognizes oligosaccharides containing sialic acid.142 Based on this, Petitjean et al. investigated the biophysical properties of S1 subunit binding to sialic acids or 9-O-acetylated sialic acid (9-AcSA) using force–distance (FD) curve-based atomic force microscopy.143 Then, they designed novel blocking molecules with various topologies and carrying multiple salic acid or 9-AcSA residues. They reported that 9-AcSA-derived porphyrin has strong inhibitory effects on SARS-CoV-2. Yi et al. searched for S protein RBD inhibitors by screening compounds from the Chinese herbal medicine licorice.144 They found that glycyrrhetinic acid (GA) and licorice saponin A3 target the S protein RBD, and Tyr-453 is a key residue for the affinity of triterpenoids with the S protein RBD. Another strategy to inhibit SARS-CoV-2 S protein is to disrupt the disulfide pairs of RBD.145,146 Disulfide bond formation is central to the dynamic structure of many viral receptor binding and entry/fusion proteins.147 The SARS-CoV-2 S protein RBD contains four disulfide pairs, which may interact with thiol-based reducing agents.146,148 Shi et al. reported that the preclinical thiol-based reducing agents P2110 and P2165 target a conserved hydrophobic binding pocket in the RBD, thus inhibiting SARS-CoV-2 infection.146 In detail, proteomic and reactive cysteine mapping showed that the disulfide pairs Cys-379–Cys-432 and Cys-391–Cys-525 are redox-sensitive and can be reduced by P2110 and P2165. A significant conformational change of the RBD was observed after reduction of both disulfide pairs. They also indicated that P2110 and P2165 could modulate the extracellular redox poise required for SARS-CoV-2 entry into cells, which is beneficial for preventing viral infection.

Besides finding molecules with inhibitory effects on the S protein, studies focused on finding molecules which can inhibit the RBD–ACE2 interaction.149,150,151 For example, Pei et al. applied a computer-aided approach based on the RBD binding residues on ACE2 to design ultrashort peptide inhibitors against SARS-CoV-2.152 Based on the critical residues of ACE2, they initially obtained the peptide inhibitor SI1. Then, using a “docking–activity test–molecular simulation–sequence improvement” scheme, they successfully obtained ultrashort peptides SI5α and SI5α-b, which had significantly higher activity. By analyzing the binding sites of ultrashort peptides to RBD, the residues from Glu-484 to Tyr-505 on the RBD were determined as the “binding pocket” in this study, which may be helpful for the design of RBD inhibitors or antibodies. A similar computer-aided strategy for the identification of novel inhibitors disrupting the RBD–ACE2 interaction was reported by Gupta et al. In their study, machine learning classifiers were applied for the prediction of new small molecular modulators of the SARS-CoV-2 S protein RBD–ACE2 interaction. Using this RBD: hACE2 predictor, they identified more than 300 novel small molecule scaffolds that can be repurposed for SARS-CoV-2. Panda et al. took the structure-based drug design approach for screening inhibitors with an affinity against Mpro and S protein.153 Molecular docking simulations indicated that the obtained molecule, PC786, has a binding affinity toward the RBDs of all the chains in the trimeric S protein. Their protein–protein interaction analysis revealed that conformational changes occur when PC786 interacts with the RBD–ACE2 complex, revealing that the binding of PC786 with S protein substantially affects S protein binding to the ACE2 domain. Lee et al. showed that both Etravirine and Dolutegravir preferentially bind to primary ACE2-interacting residues on the RBD domain, implying that these two drugs may inhibit attachment of SARS-CoV-2.154 Xiong et al. showed that the novel inhibitors DC-RA016 and DC-RA052 have the ability to interfere with the SARS-CoV-2 S protein RBD–ACE2 interaction, thus playing an anti-SARS-CoV-2 role.155

After binding to the ACE2 receptor of host cells, S protein needs to be activated by host protease at the putative cleavage site located at the boundary of the S1 and S2 subunits, thus exposing the S2 subunit for viral entry (Fig. 2).128,156,157 This cleavage is performed by host cells proteases, including serine protease transmembrane protease, serine 2 (TMPRSS2), cysteine protease cathepsin L (CTSL), and the arginine protease furin.54,121,158 TMPRSS2 was thought to play an essential role in SARS-CoV-2 viral entry.159,160,161 It enables rapid endosome-independent virus entry of SARS-CoV-2 into the cells (within 10 min).162 CTSL also enhances SARS-CoV-2 infection in both human cells and human ACE2 transgenic mice.163,164,165 CTSL is critical for SARS-CoV-2 entry via endocytosis during infection.157 The furin cleavage site also has a critical role in SARS-CoV-2 infection,164,166,167,168 since a study has revealed that its cleavage site at the S1/S2 boundary is essential for S-protein-mediated cell–cell fusion and entry into human lung cells.168 Based on these observations, inhibitors of TMPRSS2, CTSL, and furin were identified as promising therapeutical agents for COVID-19 treatment.169

The structure of TMPRSS2 is characterized by an N-terminal cytoplasmic domain, a transmembrane domain, a class A LDL receptor domain, a scavenger receptor cysteine-rich domain, and an activation domain linked to a serine protease domain via a disulfide bond.54,159,170 Since no crystal structure of TMPRSS2 is available, repurposing or optimizing inhibitors against well-known serine proteases may facilitate the discovery of effective TMPRSS2 inhibitors against SARS-CoV-2.170,171,172 For example, Sun et al. identified structurally similar serine proteases using a structure-based phylogenetic computational tool to find potential inhibitors of TMPRSS2.173 According to their computational results, six serine peptidases, including kallikrein-related B1, had a high structural similarity to the TMPRSS2 S1 protease domain. The kallikrein-related B1 inhibitor avoralstat with high potential to be repurposed for COVID-19 therapy was identified. In addition, based on a previously designed peptidomimetic tetrapeptide with inhibitory activity against matriptase, Shapira et al. developed a small library of peptidomimetic compounds to screen for inhibitors of TMPRSS2.174 Through the screening process, they found that N-0385, containing a ketobenzothiazole warhead, inhibits TMPRSS2. Then, by building a homology model of TMPRSS2 using the crystal structure of matriptase, they found that the catalytic Ser-441 residue of the enzyme forms a covalent bond with the warhead of N-0385. This contributes to its inhibitory activity against TMPRSS2. Rational structure-based drug design was also applied to discover TMPRSS2 inhibitors by Mahoney et al..175 Based on molecular docking studies using a published homology model of TMPRSS2 and substrate specificity data from PS-SCL, a set of ketobenzothiazole inhibitors of HGF-activating serine proteases (including HGF activator [HGFA], matriptase, and hepsin) were developed. After further optimization, they identified multiple potent inhibitors of TMPRSS2. Four of these analogs displayed activity at subnanomolar concentrations, both in the enzyme assay and in blocking the entry of VSV-SARS-CoV-2 chimeras into human Clau-3 epithelial lung cells. Besides blocking the cleavage function of TMPRSS2, molecules with the ability to reduce TMPRSS2 expression on host cells also drew attention for anti-COVID-19 research. A high-throughput screening using a library of 2560 FDA-approved or currently investigated clinical compounds was carried out by Chen et al. to identify small molecules that reduce TMPRSS2 expression.176 They found that halofuginone modulates TMPRSS2 levels through proteasomal-mediated degradation that involves the E3 ubiquitin ligase component DDB1- and CUL4-associated factor 1.

CTSL is a lysosomal cysteine protease. It contains an L domain of alpha-helices and an R domain of beta-sheets.177,178,179 Gallinamide A is a potent covalent inhibitor of several parasite-derived cysteine proteases, as well as human CTSL.180,181 Ashhurst et al. demonstrated that Gallinamide A and analogs could directly interact with CTSL and potently inhibit SARS-CoV-2 infection in vitro.182 Structure-based design of CTSL inhibitors was carried out by Phan et al. According to their report, good peptidyl substrates can be converted into CTSL inhibitors that are active at submicromolar concentrations by a single thioamide substitution in the peptide backbone.169 By designing and scanning several thioamide-stabilized peptide scaffolds, they found that the peptide RS1A inhibits CTSL activity with >25-fold higher specificity compared to the other cathepsins. According to computational modeling analysis, the P1 thioamide N–H group of the peptide interacts with the His-163 catalytic triad of CTSL. In a recent preprint reported by Frueh et al., an orally available CTSL inhibitor K777 exhibited anti-viral ability and efficiently reduced COVID-19-related pulmonary pathology in African green monkeys.183 Despite these achievements, the ubiquitous expression of CTSL raises concern about the side effects of CTSL inhibitors.184 Combined use of a CTSL inhibitor and other protease inhibitors or development of a CTSL inhibitor with multiple functions might be effective in preventing viral infection at a lower dose and in reducing side effects. Thus, Hu et al. found that calpain inhibitors II and XII, and GC-376 have a dual mechanism of action by inhibiting both viral Mpro and host CTSL in vitro.185 In addition, Sacco et al. found that Mpro inhibitors targeting the hydrophobic methionine side chain in the S1 pocket are also active against CTSL, which paved the way for the design of dual inhibitors that target both viral Mpro and host CTSL.186

Furin recognizes and cleaves a polybasic stretch of an RRAR motif in the S1/S2 boundary of S protein. It is worth noting that the cleavage site of furin was only identified in SARS-CoV-2, and not in other lineages of betacoronaviruses.187,188,189,190 Even Papa et al. indicated that knockout of furin significantly suppressed but not abolished SARS-CoV-2 S-protein-mediated cell–cell fusion.191 Johnson et al. revealed that RRAR cleavage site mutation attenuates SARS-CoV-2 pathogenesis in both hamster and K18-hACE2 transgenic mouse models.167 Peacock et al. found that SARS-CoV-2 virus lacking the S1/S2 furin cleavage site was shed to lower titers from infected ferrets and was not transmitted to cohoused sentinel animals, unlike the wild-type virus.168 Thus, Cheng et al. reported that two molecular inhibitors of furin, decanoyl-RVKR-chloromethylketone (CMK) and naphthofluorescein, significantly inhibited syncytium formation in S-protein-expressing cells and cytopathic effects (CPEs) in SARS-CoV-2-infected cells.187 According to their results, CMK abolished CPEs and decreased virus titer in the preinfection treatment experiments, while it did not decrease virus production and infectivity but only decreased CPEs in postinfection treatment. This revealed that CMK affects the viral entry stage of SARS-CoV-2, and that it likely ameliorates viral virulence and pathogenicity. In addition, another furin inhibitor, naphthofluorescein, showed affinity at the replication stage when the virus entered the cell downstream.192,193 Authors speculated CMK and naphthofluorescein might act differently for furin substrates located in different compartments. It remains to be clarified whether naphthofluorescein’s function depends on furin activity or other new targets. Paszti-Gere et al. revealed that another furin inhibitor, MI-1851, could exert anti-SARS-CoV-2 effects on cells by suppressing the cleavage of S protein.194

SARS-CoV-2 infection activates both innate and adaptive immune responses, which may cause excessive inflammatory reactions and dysregulate the adaptive host immune response.9,195,196,197 Many studies have reported the influence of SARS-CoV-2 infection on the immune system of COVID-19 patients. In detail, lymphopenia was widely observed in patients with severe COVID-19.67,198 The proportion of lymphocytes is considered a reliable indicator of disease severity.199 In patients with severe COVID-19, the proportions of circulating CD4+ T cells, CD8+ T cells, B cells, and natural killer cells also decreased, while the proportions of immunosuppressive regulatory T cells were moderately increased in patients with mild COVID-19.200,201,202 Moreover, the levels of proinflammatory cytokines and chemokines (such as IL2, IL7, IL10, GSCF, IP10, MCP1, MIP1A, TNFα, and IL6) were significantly increased in severe patients.198,201,203 As a result of virus recognition, downstream immune-regulatory pathways such as nuclear factor κB (NF-κB), and Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) pathways are activated (Fig. 7). These pathways are crucial for the antiviral response.204,205,206 In fact, mortality of COVID-19 patients is often caused by acute respiratory distress syndrome (ARDS), and ARDS is the result of dysregulated hyperinflammation in response to viral infection.198,207,208 Thus, various immune regulators were developed or repurposed for COVID-19 treatment (Fig. 3). Most immune regulators, such as glucocorticoids, function as inflammatory extinguishers. Here, we present the immunomodulatory mechanism of these molecules against COVID-19.

Illustration of SARS-CoV-2-induced immune responses and pro-inflammatory signaling pathways

The JAK family consists of four non-receptor tyrosine protein kinases, JAK1, JAK2, JAK3, and TYK2.209,210 They are often activated when proinflammatory cytokines bind to their receptors, thus amplifying the inflammation caused by SARS-CoV-2 infection.211 So far, more than 50 cytokines that transmit their signals via JAK proteins have been identified.212,213,214 Based on this, it was recognized that JAK inhibitors could help to prevent the cytokine storm in severe COVID-19 patients.213,215 Baricitinib, a JAK1/JAK2 inhibitor, blocks the immune cascade and reduces SARS-CoV-2 replication in patients.216,217,218 According to a study conducted by Stebbing et al., type-1 interferons (IFNs), specifically IFN-α2, increased ACE2 expression in human liver cells could increase the viral load, and this induction is fully inhibited by the JAK inhibitor baricitinib.219 A study reported by Nystrom et al. indicated that baricitinib could block the cytokine-induced JAK/STAT/APOL1 signaling, which may rescue a severe kidney disease called COVID-19-associated nephropathy.220 Other JAK inhibitors, such as tofacitinib, ruxolitinib, and nezulcitinib, were also shown to exert effects against COVID-19 in clinical studies.221,222,223 According to a study of Yan et al., the JAK1/2 inhibitor ruxolitinib could normalize the SARS-CoV-2-induced complement hyperactivation in lung epithelial cells.224 Ruxolitinib was also clinically related to increased serum levels of inflammatory cytokines such as IL6 and the acute phase protein ferritin and cardiac improvement.225 Tofacitinib is a JAK1/JAK3 inhibitor known to be effective against cytokine signaling. It also inhibits JAK2 with a lower potency.226,227,228 Several studies indicated that it suppresses S-protein-potentiated STAT1 signaling and combats lung tissue-resident memory T cells which cause chronic inflammation and fibrosis when treating COVID-19.210,229,230

Bruton’s tyrosine kinase (BTK) is a cytoplasmic non-receptor tyrosine kinase (TK) expressed in all cells of the hematopoietic lineage, particularly B cells, mast cells, and macrophages.231,232 In addition, BTK-deficient macrophages are defective in expressing proinflammatory cytokines and preferentially polarize into anti-inflammatory M2 macrophages, even upon virus infection.233 A previous study indicated that inhibition of BTK attenuated neutrophil extracellular traps released into the lung with reduced levels of TNFα, IL1β, IL6, KC, and MCP-1 in mice after influenza A virus infection.233 Since cytokine release syndrome and resident macrophages may lead to pulmonary injury associated with COVID-19, Treon et al. reported that inhibitors of the BTK pathway may protect against pulmonary injury in COVID-19 patients.234 Chong et al. also suggested continuing BTK inhibitor treatment in patients who receive it for therapy of B cell malignancies with COVID-19, since the potential benefit of attenuation of M1 polarization to mitigate the immediate risk of COVID-19-related mortality outweighs the potential medium- to long-term risk of impaired humoral immunity.235 The BTK inhibitors ibrutinib, zanubrutinib, and acalabrutinib have been found to protect against pulmonary injury in a small group of participants infected with SARS-CoV-2.232,236,237

NF-κB is a proinflammatory transcription factor critically involved in both inflammatory and thrombotic responses.238,239 Its upregulation was widely observed in the development of SARS-CoV-2 infection.240,241,242,243 In addition, N protein and NSP5 of SARS-CoV-2 facilitate NF-κB hyperactivation, thus inducing inflammation.244,245,246 Therefore, NF-κB has become a potential immunotherapeutic target for COVID-19 treatment.247,248,249 Sharma et al. reported that curcumin could potently inhibit the inflammatory response elicited by SARS-CoV-2 S protein in cells by deactivating MAPK/NF-κB signaling.250 Lee et al. found that the NF-κB inhibitor pyrrolidine dithiocarbamate suppresses ACE2 protein expression in human lung cell lines, which indicates another potential mechanism by which NF-κB inhibitors may combat COVID-19.251

The Nod-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is activated when viral infection-associated pathogens are recognized by the innate immune system.252,253 Activation of the NLRP3 inflammasome pathway leads to release of the proinflammatory cytokines IL18 and IL1β, which mediate cytokine release and pyroptosis during lung injury and ARDS.254,255,256 Rodrigues et al. demonstrated that the NLRP3 inflammasome is activated in COVID-19 patients. Inflammasome-derived products such as IL18 in the serum were correlated with disease severity.257 A study of Pan et al. revealed that the N protein of SARS-CoV-2 promotes NLRP3 inflammasome activity and induces an excessive immune response.258 Therefore, inhibitors targeting the NLRP3 inflammasome might serve as drugs to treat COVID-19.259 A study conducted by Zeng et al. demonstrated that inhibition of the NLRP3 inflammasome by MCC950 alleviated excessive lung inflammation. Further, they showed that MCC950 could reduce COVID-19-like pathology in human ACE2 transgenic mice.260

Nucleoside/nucleotide analogs were investigated widely in the area of antiviral drugs (Fig. 8).261,262,263 Generally, nucleoside/nucleotide analogs resemble naturally occurring nucleosides, and act as normal nucleotides, being recognized by viral polymerases or cellular enzymes, and prevent virus replication.264,265 Various nucleoside/nucleotide analogs have been applied for clinical antiviral therapies. Besides the first anti-HSV drug, acyclovir,266 other nucleoside/nucleotide analogs such as zidovudine against HIV, telbivudine against HBV, and sofosbuvir against HCV also exhibited specific therapeutic effects.267,268,269 Although achievements have been made in the area of DNA virus application, these analogs are still facing challenges in the treatment of infections with RNA viruses with higher spread and mutation rates. In the area of SARS-CoV-2, looking for nucleoside/nucleotide analogs is the preferred strategy, as no homolog of RdRp has been found in human cells. Since the RdRp of SARS-CoV-2 is conserved, exploring the anti-SARS-CoV-2 effects of pre-existing antiviral nucleoside/nucleotide analogs against the virus has been shown to be an effective way.270,271 Nucleobase analogs and double-stranded RNA (dsRNA) compounds with anti-SARS-CoV-2 effects will also be discussed in this section. Although they do not function by imitating nucleosides, those analogs and compounds interfere with viral infection by various mechanisms.

Chemical structure of representative small molecules and their backbone (labeled in red)

As a constituent of ATP and cAMP, adenosine participates in numerous processes in the human body.272,273 Therefore, numerous adenosine analogs have been synthesized against various diseases, including COVID-19. Among the existing adenosine analogs against COVID-19, the most investigated one is remdesivir. It was developed by Gilead to combat the Ebola virus, and it bears the structure of an adenine c-nucleoside modified by monophosphoramide and cyano groups.274 As a nucleotide prodrug, remdesivir is metabolized by the host cell to the pharmacologically active triphosphate to inhibit the activation of RdRp.31 In a study reported by Pruijssers et al., remdesivir exhibited a potent in vitro inhibition ability against SARS-CoV-2 replication in human lung cells and primary human airway epithelial cells.275 Its in vivo effect was also confirmed in SARS-CoV-2-infected rhesus macaques. Remdesivir treatment in rhesus macaques with COVID-19 efficiently prevented progression to pneumonia. Holshue et al. first reported its clinical application, which described an immediate improvement in clinical symptoms in the first confirmed case of SARS-CoV-2 after receiving remdesivir administration.276,277 However, according to a recent study conducted by Stevens et al., remdesivir resistance was observed in SARS-CoV-2 after 13 passages of co-culturing with GS-441525.278 Although it is encouraging that natural variants did not propagate remdesivir resistance mutations, this study emphasized that the extended use of remdesivir might increase the possibility for SARS-CoV-2 to adapt to remdesivir. It is worth noting that remdesivir is a prodrug of GS-441524, which has also been proved to be effective against COVID-19.279,280 GS-441524 is also developed by Gilead, which is the dephosphoramidated ribonucleoside parent nucleus of remdesivir.281 Pharmacokinetic analysis showed that GS-441524 is the predominant metabolite of remdesivir reaching the lungs. Based on its easy synthesis and high lung loads, Yan et al. claimed it is superior to remdesivir for COVID-19 treatment.280 Li et al. reported that GS-441524 effectively inhibited SARS-CoV-2 in three cell lines (Vero E6, Calu-3, and Caco-2).282 In addition, remdesivir can only be given intravenously, and there is a pressing medical need for oral antivirals. Xie et al. performed an in vitro and in vivo drug metabolism and pharmacokinetics assessment to examine the potential of GS-441524 as an oral drug.283 In further in vivo studies in CD-1 mice, GS-441524 displayed a favorable oral bioavailability of 57%. Due to these advantages, the first study of orally administered GS-441524 for COVID-19 in humans was started on January 1, 2021, and conducted by Copycat Sciences. The clinical results suggested the high safety and low toxicity of orally administered GS-441524 in healthy people.284,285 Although further clinical studies of the compound remain to be implemented, GS-441524 has potential as an oral drug for treatment of COVID-19. Further, another prodrug of GS-441524 named VV116 was developed by the Shanghai Institute of Materia Medica. VV116 is derived from GS-441524 by esterification of all three hydroxyl groups and replacing a hydrogen atom on the basic group with a D atom.286 Wu et al. reported that VV116 is highly effective in inhibiting SARS-CoV-2 replication in cell-based and animal models.287 A clinical study of VV116 showed that it has good safety and efficacy.288 Moreover, studies have shown that VV116 exhibits antiviral activity against the Alpha, Beta, Delta, and Omicron variants with high oral bioavailability and good chemical stability.289 Two international phase II/III clinical trials of VV116 are underway. Besides remdesivir and its analogs, another adenosine analog, galidesivir, also is notable as an anti-SARS-CoV-2 drug. Galidesivir was developed by BioCryst Pharmaceuticals and was originally intended as a drug for HCV treatment.63 Unlike the pyrrolotriazine group in the abovementioned compounds, galidesivir bears a pyrrolopyrimidine group as its nucleobase. A molecular docking study conducted by Aftab et al. indicated that galidesivir binds effectively to SARS-CoV-2 RdRp, suggesting its potential use to treat COVID-19.290

Cytidine analogs have also been investigated for COVID-19 treatment. One of the cytidine analogs, molnupiravir, is the synthetic ribonucleoside derivative N4-hydroxycytidine developed by Merck and Ridgebace. It is a prodrug of β-D-N4-hydroxycytidine (EIDD-1931), which was originally developed for treating seasonal influenza.291 Unlike the abovementioned remdesivir, which terminates the elongation of viral genes, molnupiravir contains two forms of tautomers that can pair with A and T,35 thus causing large mutations in RNA products and preventing SARS-CoV-2 replication. According to the results reported by Sheahan et al., administration of molnupiravir improved pulmonary function and reduced virus titer and weight loss in mice infected with SARS-CoV-2.292 On November 4, 2021, it was first approved by the UK Medicines and Health Products Regulatory Agency (MHRA) for treating adults with mild to moderate COVID-19. Thus, molnupiravir was the word’s first orally administered anti-SARS-CoV-2 drug. A recent study revealed that the SARS-CoV-2 Omicron variant is highly sensitive to molnupiravir.293 However, the potential side effect of molnupiravir of eliciting mutation in mammalian cells has raised concern.294 Azvudine is a cytosine analog which was also found to be efficient to treat SARS-CoV-2.295 It was previously approved for HIV inhibition.296 Recently, Zhang et al. observed that azvudine significantly inhibited viral load, promoted lymphocyte subsets, protected histological structures, and reduced inflammation caused by SARS-CoV-2 infection.297

Several guanosine analogs were reported to be efficient for inhibiting SARS-CoV-2.298 The most investigated one is ribavirin. It is a broad-spectrum antiviral drug with triazole structure, whose conformation is similar to that of guanosine.299 In 1970, it was first synthesized by Joseph T. Witkowski of ICN Pharmaceuticals.300 In 2013, it was approved by the FDA for the treatment of chronic HCV infection.61 Eslami et al. showed that combination therapy with ribavirin can effectively improve disease symptoms in severe COVID-19 patients.301 Later, results of an open-label randomized phase II trial showed that this triple therapy in hospitalized patients with COVID-19 pneumonia can effectively alleviate symptoms and shorten the duration of viral shedding and hospital stay in patients.302 Combination treatment with ribavirin, which is currently clinically available and cheap, with other antiviral drugs may become the treatment of choice in COVID-19 patients. In addition to ribavirin, the guanosine analog thioguanosine potentially inhibits SARS-CoV-2 by binding to Mpro.303 The guanine analog triazavirin was reported to be a promising agent to treat SARS-CoV-2.304 A pilot trial by Wu et al. indicated that triazavirin can inhibit the tendency to bind to ACE2, and triazavirin showed a significantly better therapeutic effect and higher safety in the treatment of COVID-19 compared with a placebo or standard therapy.305

The uridylate analog sofosbuvir was also believed to play an anti-SARS-CoV-2 role.306 It was discovered in 2007 by Pharmasset (Gilead) and approved for HCV treatment.307 Previous studies have also shown that it can inhibit Zika virus replication.308,309 Sofosbuvir needs to be triphosphorylated to its active form (2’-F, Me-UTP) to be recognized by HCV polymerase, thereby preventing viral replication.310 A study by Chien et al. showed that the activated triphosphate form of Sofosbuvir can bind to RdRp of SARS-CoV-2.311 Currently, several clinical trials studying the effects of sofosbuvir on SARS-CoV-2 are being carried out.301 According to a multicenter Egyptian study involving 174 patients with COVID-19, patients receiving combination treatment with sofosbuvir/daclatasvir demonstrated shorter hospital stay, faster PCR negativity, and possibly reduced mortality.312 However, according to a meta-analysis by Kow et al., sofosbuvir-based direct-acting antiviral agents have no protective effects against the development of severe illness in patients with COVID-19 with the current dosing regimen.313 In a previous study, sofosbuvir demonstrated higher anti-viral efficiency against West Nile virus in hepatic cells than in lung cells.314 This liver-targeting characteristic of sofosbuvir raises concerns for its use in treating SARS-CoV-2. In this regard, future studies should be conducted to improve sofosbuvir’s targeting of the SARS-CoV-2-attacked organs by structural optimization or formulation improvement.

Favipiravir, a pyrazine analog with no nucleoside-like structure, can also be phosphorylated and acts as a nucleotide analog that selectively inhibits viral RdRp.315 It is being developed and manufactured by Toyama Chemical (a subsidiary of Fujifilm) and was approved for influenza virus treatment in Japan in 2014. An in vitro study showed that favipiravir exerts beneficial effects in Vero E6 cells infected with SARS-CoV-2 with a half-maximal effective concentration (EC50) of 61.88 μM and a half-cytotoxic concentration (CC50) of >400 μM.276 Many clinical trials proposed to use favipiravir in the treatment of COVID-19. Cai et al. reported that after favipiravir treatment, a significant improvement in chest CT of COVID-19 patients was observed, indicating that favipiravir is associated with better therapeutic responses in COVID-19 patients in terms of disease progression and viral clearance.316 In a multicenter randomized study, Dabbous et al. discovered that the patients who received favipiravir had a lower mean duration of hospitalization than patients in the chloroquine group.317 Thus, favipiravir has been recommended by Thailand’s Department of Disease Control for mild to moderate COVID-19 cases in both adults and children, while recommendations from India include mild COVID-19 patients with or without comorbidities.45 Furthermore, Rabie discovered a derivative of favipiravir named cyanorona-20 as a promising anti-SARS-CoV-2 compound.318 Pyrazine derivatives may serve as guides for further discovery of anti-SARS-CoV-2 agents.

As the basis of nucleotides, pyrimidines widely participate in viral metabolism. Thus, nucleobase analogs were found to effectively inhibit SARS-CoV-2 by various pathways. Among them, baricitinib, a pyrrolopyrimidine analog, is widely applied for treatment of severe COVID-19 in combination with remdesivir.319 Baricitinib is an oral selective inhibitor of JAK1 and JAK2.320 It was initially predicted by artificial intelligence algorithms as a potential treatment strategy against SARS-CoV-2. According to a study by Bronte et al., baricitinib improved the clinical outcomes of SARS-CoV-2 infection, affected the immune landscape in participants with COVID-19, and modified immune-suppressive features of myeloid cells.321 A study by Marconi et al. suggested that baricitinib reduces 28-day and 60-day mortality when used in addition to the current standard of care.322 As such, baricitinib plus standard of care could be a treatment option to reduce overall deaths globally. Another pyrrolopyrimidine analog, abivertinib, was found to depress cytokine production in patients with COVID-19.323 Several pyrimidine analogs have also been found to combat SARS-CoV-2. For example, according to a recent study conducted by Huntington et al., GLPG-0187, which bears a pyrimidin ring, effectively blocked SARS-CoV-2 pseudovirus infection across multiple viral variants, especially the Omicron and Delta pseudovirus variants, in a dose-dependent manner.324 Indu et al. reported that raltegravir combats SARS-CoV-2, because it demonstrated the highest interaction energy with Mpro and had high bioavailability among 65 FDA-approved small molecule antiviral drugs.325 Fostamatinib might be used to treat severe COVID-19.326 Other pyrimidine analogs, including ambrisentan and apilimod, were also reported to be promising agents for SARS-CoV-2 treatment.327,328

Another compound class, dsRNA, was also found to inhibit SARS-CoV-2. Rintatolimod, a Toll-like receptor 3 (TLR3) agonist, was reported to exert antiviral effects in human pancreatic cancer cells by activating the innate immune system, suggesting it could be used in the treatment of cancer patients who suffer from SARS-CoV-2 infection.329 Poly-ICLC is a synthetic complex of carboxymethylcellulose, polyinosinic-polycytidylic acid, and poly-L-lysine dsRNA.330 A phase I trial to study the safety and immunogenicity of poly-ICIC in healthy vaccinated COVID-19 adults is in its recruitment stage.

Flavonoids are a class of bioactive substances derived from plants. Chemically, flavonoids have a C6-C3-C6 skeleton structure, which consists of two phenyl rings and an oxygen heterocyclic ring.331 By regulating key enzymes participating in biological processes, flavonoids possess antioxidant, anticancer, anti-inflammatory, and antiviral properties.332 Due to their broad bioactivity, they may play complex roles to treat SARS-CoV-2 infection by blocking ACE2 receptor in host cells, directly inhibiting viral RdRp and Mpro, and affecting the activity of various inflammatory enzymes (such as phospholipase A2, cyclooxygenases [COXs], TK, and so on).333 These mechanisms make flavonoids an excellent supportive care strategy for patients suffering from chronic post-COVID-19 syndrome.

Flavonols, also named 3-hydroxyflavones, are the most abundant and widely distributed flavonoids in the nature. Chemically, these molecules differ from many other flavonoids due to the hydroxyl group at position 3 of the flavonol skeleton. Quercetin, the most abundant flavonoids in edible plants, is a flavonol with five hydroxy groups placed at the 3-, 3′-, 4′-, 5-, and 7-positions. It has broad-spectrum antiviral ability against a variety of viruses, including HIV, poliovirus, Sindbis virus, respiratory viruses, Mayarovirus, and Mengo virus.334,335 Pan et al. reported that quercetin may exert anti-SARS-CoV-2 effects by affecting the binding of viral S protein to the ACE2 receptor.336 Further, the anti-SARS-CoV-2 effect of quercetin was also thought to be achieved by (i) inhibiting Mpro and PLpro proteinase of SARS-CoV-2 and (ii) acting as a zinc ionophore.337 Currently, several clinical studies of quercetin are underway. A phase IV clinical study supported by the Ministry of Health of Saudi Arabia on quadruple therapy with quercetin, zinc, bromelain, and vitamin C for COVID-19 patients is in its recruitment stage (NCT04468139). Myricetin, a 7-hydroxyflavonol, has been isolated from the leaves of Myrica rubra and other plants. In research conducted by Su et al., myricetin inhibited Mpro at >90% at a concentration of 10 μM, and its EC50 value in Vero E6 cells infected with SARS-CoV-2 was 8.00 μM.94 The 3-hydroxyl group of flavonol can be glycosylated, thus forming flavonol glycosides, which are found in plants. As a quercetin O-glycoside, quercitrin is obtained by placing an alpha-L-rhamnosyl moiety at position 3 of quercetin via a glycosidic linkage. Several in silico studies have reported that quercitrin may be used against SARS-CoV-2 based on its affinity to the serine protease TMPRSS2, Mpro, and PLpro.338,339,340

There are a series of compounds whose backbone consists of a flavonol structure. They have also been found to be effective in combating COVID-19. For example, flavonolignans are a family of compounds containing a flavonol moiety linked together with coniferyl alcohol.341 Silymarin, extracted from the botanical source Silybum marianum, is a mixture of flavonolignans (silybin, isosilybin, silychristin, and siliandrin) and a flavonol (taxifolin).342 It is commonly known for its hepatoprotective potential.343 Its anti-SARS-CoV-2 effect was thought to be achieved by inhibiting the expression of the host cell surface receptor TMPRSS2.342 Hanafy et al. developed silymarin/curcumin dual-loaded BSA nanoparticles as an inhalable delivery system to treat pneumonia.344 According to their results, silymarin exhibited antiviral activity against SARS-CoV-2 at a concentration of 25 μg/mL in vitro. They reported that silymarin could protect the lungs during SARS-CoV-2 infection due to their anti-inflammatory and antioxidant effects, and it could inhibit the ACE2 receptor, thus preventing viral entry. As a natural-derived compound mixture, silymarin might be a good option for treating COVID-19 owing to its multifunction properties. A phase III clinical study of silymarin is in its recruitment stage, which is aimed at assessing the clinical outcome in adults with COVID-19 pneumonia under standard care plus placebo or oral silymarin (NCT04394208).

In addition to the abovementioned flavanols, the anti-SARS-CoV-2 effects of flavones, which have a 2-phenyl-1-benzopyran-4-one backbone, were also studied. Luteolin is the most investigated flavone compound. Luteolin is a flavone which bears four hydroxy groups located at the 3′-, 4′-, 5-, and 7-locations. It is obtained from the plant Reseda luteola. It was first isolated in pure form and named in 1829 by the French chemist Michel Eugène Chevreul.345 Results obtained from relaxed complex scheme analysis, classical molecular docking simulations, and metadynamics simulations suggest luteolin blocks SARS-CoV-2 entry into cells.346,347 A system pharmacology and bioinformatic analysis study conducted by Xie et al. indicated it has great potential to be used for treating COVID-19/asthma comorbidity due to its effects on viruses, regulating inflammation and immune responses, reducing oxidative stress, and regulating blood circulation.348 Luteolin was found to be safe for human use and showed good drug properties. Clinical results suggest that oral luteolin supplementation improves the recovery of olfactory function after COVID-19. Besides the above common flavones, amentoflavone, a hydroxyflavone and bioflavonoid, also has shown binding affinity with Mpro, RdRp, NSP13, NSP15, and ACE2 in several in silico surveys.349,350,351 Similar to flavanols, the hydroxy groups of flavones can be glycosylated, thus forming flavone glycosides. Baicalin, a 7-O-glucuronide of baicalein, is a biologically active flavonoid of natural origin obtained primarily from the roots of Scutellaria baicalensis Georgi. Zandi et al. have demonstrated that baicalein and its aglycon baicalein can directly inhibit the activity of SARS-CoV-2 RdRp and that it exhibits in vitro anti-SARS-CoV-2 activity with an EC50 of 4.5 µM and an EC90 of 7.6 µM.352 Su et al. also found its binding activity with Mpro and proved its anti-SARS-CoV-2 activity in vitro. Their further study revealed that baicalin and baicalein as two bioactive ingredients of Shuanghuanglian (a Chinese traditional medicine) provides supporting evidence for the antiviral activity of Shuanghuanglian. However, their exact antiviral ability has to be verified in animal models or clinical trials.

The effects of flavanols represented by epigallocatechin gallate (EGCG) against COVID-19 have also been studied. EGCG is a phenolic antioxidant found in a number of plants, including green and black tea, with reported antiviral effects against influenza virus, HIV, and HBV.353,354 Unlike other flavonoids with a chromone part, it bears a 2-phenyl-3,4-dihydro-2H-chromen-3-ol skeleton.355 EGCG exerts inhibitory effects on SARS-CoV-2 replication through its actions on ACE2, Mpro, and RdRp.356 Jang et al. demonstrated that EGCG inhibits SARS-CoV-2 Mpro activity in 293T cells in a dose-dependent manner without signs of cytotoxicity at any dose used.357 Chiou et al. conducted an in vitro study on the inhibitory effects of EGCG against SARS-CoV-2 Mpro. EGCG inhibited the activity of SARS-CoV-2 Mpro, thus suggesting its potential application in the treatment of SARS-CoV-2 infection.358 It is worth noting that a clinical phase II/III study of EGCG is underway to determine its chemoprophylactic effects on COVID-19 in healthy workers (NCT04446065). Other flavanols, including cianidanol,359 epicatechin gallate,360,361 and procyanidin,362 have also been found to have potential anti-SARS-CoV-2 effects in vitro.

Phenylpropanoids are a family of plant-derived compounds with a C6–C3 structure. In general, phenylpropanoids are derived from the shikimic acid pathway via phenylalanine and tyrosine. This phenylpropanoid metabolism pathway is a major anabolic pathway in plants, which plays a vital role in several processes, especially biotic and abiotic stress responses.363 Phenylpropanoids act as antioxidants and free radical scavengers. Their applications as antioxidant, anticancer, antiviral, anti-inflammatory, and antibacterial agents have attracted interest.364 Several phenylpropanoids were found to exert anti-SARS-CoV-2 effects. Some of them have demonstrated potential anti-SARS-CoV-2 effects in vitro or by computational analysis.

(S)-2-Amino-1-Phenylethanol Hydroxycinnamic acid derivatives belong to the basic phenylpropanoids. Based on the C6-C3 structure, they also possess an aromatic carboxylic acid substituted by phenolic hydroxyl groups. As a common derivative of hydroxycinnamic acid, caffeic acid possesses a phenyl ring substituted by hydroxy groups at the 3- and 4-positions.365 It is an orally bioavailable small molecule mainly found in Pavetta indica and Eupatorium cannabinum. Further studies have shown its potential antiviral activity against HBV366 and HPIV3.367 Several in silico molecular docking studies have revealed it could specifically bind to SARS-CoV-2 Mpro368 and Membrane protein.369 Chlorogenic acid, the ester of caffeic acid and quinic acid, is often found in coffee and black tea. Several studies have pointed out that chlorogenic acid and its derivatives have good antiviral activity against various types of viruses, including HIV, influenza A virus, herpes simplex virus (HSV), and hepatitis B virus (HBV).370 Its anti-SARS-CoV-2 ability was first predicted by Yu et al., whose molecular docking study revealed that chlorogenic acid could stably bind with ACE2, indicating it may inhibit SARS-CoV-2 entry into cells.371 Another molecular docking simulation conducted by Gizawy et al. suggested that chlorogenic acid can interact with the Asn-142, His-164, Arg-188, and Met-165 residues of the active site in Mpro of SARS-CoV-2.