An Update on SARS-CoV-2 Clinical Trial Results—What We Can Learn for the Next Pandemic

The coronavirus disease 2019 (COVID-19) pandemic has claimed over 7 million lives worldwide, providing a stark reminder of the importance of pandemic preparedness. Due to the lack of approved antiviral drugs effective against coronaviruses at the start of the pandemic, the world largely relied on repurposed efforts. Here, we summarise results from randomised controlled trials to date, as well as selected in vitro data of directly acting antivirals, host-targeting antivirals, and immunomodulatory drugs. Overall, repurposing efforts evaluating directly acting antivirals targeting other viral families were largely unsuccessful, whereas several immunomodulatory drugs led to clinical improvement in hospitalised patients with severe disease. In addition, accelerated drug discovery efforts during the pandemic progressed to multiple novel directly acting antivirals with clinical efficacy, including small molecule inhibitors and monoclonal antibodies. We argue that large-scale investment is required to prepare for future pandemics; both to develop an arsenal of broad-spectrum antivirals beyond coronaviruses and build worldwide clinical trial networks that can be rapidly utilised.


Introduction
The coronavirus disease 2019 (COVID-19) pandemic highlighted the world's underpreparedness when faced with a newly emerging viral pathogen, for which no specific antiviral therapy was available [1].Coronaviruses that cause human infections include alphacoronaviruses human coronavirus (HCoV) 229E and HCoV NL63, and betacoronaviruses HCoV OC43, HCoV HKU1, severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2 (Table 1) [2][3][4].However, over the past two decades, the world has witnessed the emergence of several highly pathogenic betacoronaviruses, with mortality rates ranging from 10% for SARS-CoV-1 in 2002, 34% for MERS-CoV in 2012, and 2% for SARS-CoV-2 in 2019, respectively [5,6].While the spread of SARS-CoV-1 and MERS-CoV was contained through public health measures, SARS-CoV-2 caused a global pandemic with more than 771.68 million cases recorded and 6.98 million confirmed deaths (Our World in Data, https://ourworldindata.org/grapher/cumulative-deaths-and-cases-covid-19, as of 2 November 2023).SARS-CoV-2 is highly transmissible and can cause asymptomatic or mild symptoms, which makes preventing its spread more difficult [6][7][8].New variants of SARS-CoV-2 keep emerging, with Omicron being up to 70% more transmissible than previously circulating virus variants [9,10].At the start of the 2019 pandemic, no therapeutics against SARS-CoV-2 were available.The SARS-CoV-2 life cycle provides insight into antiviral drug targets (Figure 1), with key targets that were exploited for the development of directly acting antivirals (DAA) and host-targeting antivirals (HTA) marked.DAAs target essential viral proteins, such as the main protease (M Pro ) and RNA-dependent RNA polymerase (RdRp), while host-targeting antivirals inhibit human proteins that the virus utilises for its entry, replication, or assembly.
SARS-CoV-2 keep emerging, with Omicron being up to 70% more transmissible than previously circulating virus variants [9,10].At the start of the 2019 pandemic, no therapeutics against SARS-CoV-2 were available.The SARS-CoV-2 life cycle provides insight into antiviral drug targets (Figure 1), with key targets that were exploited for the development of directly acting antivirals (DAA) and host-targeting antivirals (HTA) marked.DAAs target essential viral proteins, such as the main protease (M Pro ) and RNA-dependent RNA polymerase (RdRp), while host-targeting antivirals inhibit human proteins that the virus utilises for its entry, replication, or assembly.The coronavirus life cycle and the mechanistic actions of antiviral drugs within the viral replication process, using SARS-CoV-2 as an example.The virus-cell membrane fusion was induced by the binding of spike protein to the host cellular receptor angiotensin-converting enzyme 2 (ACE2), together with the cell surface transmembrane serine protease 2 (TMPRSS2).Following viral entry, the release of the viral genome is followed by the immediate translation of viral proteins and the formation of the viral replication and transcription complex.The 3-chymotrypsin-like protease (CL pro )/main protease (M pro ) and papain-like protease (PL pro ) cleave the virus polypeptide into 16 non-structural proteins.Structural glycoproteins are synthesised in the endoplasmic reticulum (ER) membrane for transit through the endoplasmic reticulum-to-Golgi intermediate compartment (ERGIC).Newly synthesised genomic RNA is encapsulated and buds into the ERGIC to form a virion.New virions leave the cell via lysosomes and are then able to infect new susceptible cells.SARS-CoV-2 infection activates the acid sphingomyelinase/ceramide system, resulting in the formation of ceramide-enriched membrane domains that serve viral entry and infection by clustering ACE2.The directly acting antivirals (DAA) mechanisms include the monoclonal antibodies that target the spike protein of the virus, M pro inhibitor, nucleoside analogues, and RNA-dependent RNA polymerase (RdRp) inhibitor.The host-targeting antivirals (HTA) include the inhibitors of viral entry, functional inhibitors of acid sphingomyelinase activity (FIASMA), and inhibitors of viral glycoprotein processing.The immunomodulatory drugs modify the negative effects of an overreacting immune system, such as the interleukins and JAK ½ inhibitors.Adapted from "Coronavirus Replication Cycle", by BioRender.com (2023).Retrieved from https://app.biorender.com/biorender-templates,accessed on 20 April 2023.
The choice of drug Is closely linked to the course of natural infection (Figure 2) [11].DAA and HTA are most suited for use within the first week of infection as they target viral replication.This leads to a short window of opportunity for treatment, as patients often only seek medical attention a few days after symptoms develop [12,13].In contrast, during later stages of disease, the immune system rather than viral replication as such is the main driver of disease progression, and here, immunomodulatory drugs were shown to be effective [14].
(ACE2), together with the cell surface transmembrane serine protease 2 (TMPRSS2).Following viral entry, the release of the viral genome is followed by the immediate translation of viral proteins and the formation of the viral replication and transcription complex.The 3-chymotrypsin-like protease (CL pro )/main protease (M pro ) and papain-like protease (PL pro ) cleave the virus polypeptide into 16 non-structural proteins.Structural glycoproteins are synthesised in the endoplasmic reticulum (ER) membrane for transit through the endoplasmic reticulum-to-Golgi intermediate compartment (ER-GIC).Newly synthesised genomic RNA is encapsulated and buds into the ERGIC to form a virion.New virions leave the cell via lysosomes and are then able to infect new susceptible cells.SARS-CoV-2 infection activates the acid sphingomyelinase/ceramide system, resulting in the formation of ceramide-enriched membrane domains that serve viral entry and infection by clustering ACE2.The directly acting antivirals (DAA) mechanisms include the monoclonal antibodies that target the spike protein of the virus, M pro inhibitor, nucleoside analogues, and RNA-dependent RNA polymerase (RdRp) inhibitor.The host-targeting antivirals (HTA) include the inhibitors of viral entry, functional inhibitors of acid sphingomyelinase activity (FIASMA), and inhibitors of viral glycoprotein processing.The immunomodulatory drugs modify the negative effects of an overreacting immune system, such as the interleukins and JAK ½ inhibitors.Adapted from "Coronavirus Replication Cycle", by BioRender.com (2023).Retrieved from https://app.biorender.com/biorender-templates,accessed on 20 April 2023.
The choice of drug Is closely linked to the course of natural infection (Figure 2) [11].DAA and HTA are most suited for use within the first week of infection as they target viral replication.This leads to a short window of opportunity for treatment, as patients often only seek medical attention a few days after symptoms develop [12,13].In contrast, during later stages of disease, the immune system rather than viral replication as such is the main driver of disease progression, and here, immunomodulatory drugs were shown to be effective [14].Here, we summarise recent results from randomised controlled trials (RCTs) with linked in vitro results that were performed during the COVID-19 pandemic.We present Here, we summarise recent results from randomised controlled trials (RCTs) with linked in vitro results that were performed during the COVID-19 pandemic.We present data on key candidate compounds with a direct impact on virally encoded proteins (DAA), indirect mechanisms of action depriving the virus of essential host factors needed for replication and spread (HTA), and immunomodulatory compounds (Figure 3).An overview of compounds that are now fully approved or under emergency use authorisation (EUA) for COVID-19 by the Food and Drug Administration (FDA) is shown in Table 2. Further, the World Health Organization (WHO) is releasing a living update on COVID-19 therapeutics [15].In this review, we will summarise clinical trial results of now authorised compounds and highlight other drugs that were shown to be ineffective.
data on key candidate compounds with a direct impact on virally encoded proteins (DAA), indirect mechanisms of action depriving the virus of essential host factors needed for replication and spread (HTA), and immunomodulatory compounds (Figure 3).An overview of compounds that are now fully approved or under emergency use authorisation (EUA) for COVID-19 by the Food and Drug Administration (FDA) is shown in Table 2. Further, the World Health Organization (WHO) is releasing a living update on COVID-19 therapeutics [15].In this review, we will summarise clinical trial results of now authorised compounds and highlight other drugs that were shown to be ineffective.than HTA [39].However, by targeting the virus directly, DAA are known to cause mutations that can lead to reduced drug efficacy and drug resistance [40,41].
Rapid SARS-CoV-2 small molecule drug discovery efforts were carried out during the 2019 SARS-CoV-2 pandemic, targeting the viral polymerase (remdesivir, Gilead; molnupiravir, Merck, and others) and main protease (nirmatrelvir, Pfizer; ensitrelvir, Shionogi; and others).The pandemic also gave rise to innovative and collaborative approaches such as collaborative alliances across biopharmaceutical companies creating public-private partnerships such as the Innovative Medicines Initiative Corona Accelerated R&D in Europe (https://www.imi-care.eu/,accessed on 8 November 2023), close-knit consortia of companies, and even completely open science consortia with company participation such as the COVID Moonshot [42,43].

Protease Inhibitors
SARS-CoV-2 M pro has been a drug development target from early in the pandemic, with numerous novel SARS-CoV-2 inhibitors in the clinical and preclinical pipeline (Table 3).Two SARS-CoV-2 M Pro inhibitors are now under EUA, including nirmatrelvir/ritonavir (brand name Paxlovid) and ensitrelvir (brand name Xocova).
The covalent SARS-CoV-2 M pro inhibitor nirmatrelvir (PF-07321332) developed by Pfizer is available in combination with ritonavir (available as Paxlovid) and has been evaluated in multiple RCTs to date (Table 3).Nirmatrelvir showed potent antiviral activity in in vitro assays, with a half-maximum effective concentration (EC 50 ) of 231 nM in Vero E6 cells [25].In RCTs, when nirmatrelvir/ritonavir was given within three days of symptoms onset, nirmatrelvir-treated individuals had a significantly reduced risk of severe COVID-19 in comparison to placebo-treated controls, alongside reduced viral loads at day five of treatment [24].A recent phase 2 RCT study result revealed that nirmatrelvir/ritonavir was more effective than the other leading oral antiviral drug for patients with COVID-19 [44].A drawback of the combination with the pharmacokinetic (PK) enhancer ritonavir are significant drug-drug interactions, preventing the use in patients with severe renal or hepatic impairment [45][46][47].A second-generation broad-spectrum protease inhibitor, PF-0781883 has been recently disclosed by Pfizer and is currently in Phase 2 clinical trials, with an initial human PK profile suggesting no further need to co-administer with ritonavir [48].
Ensitrelvir (S-217622) was developed by Shionogi as an oral noncovalent nonpeptidic M pro inhibitor of SARS-CoV-2 using virtual and biological screening of compound libraries and a structural-based drug design strategy of the hit compounds [49,50].In comparison to nirmatrelvir/ritonavir, ensitrelvir shows an optimized PK profile [50] and displays potent in vitro antiviral activity against SARS-CoV-2 variant of concern (VOC) including Delta (EC 50 = 34.8nM) and Omicron BA.1 (EC 50 = 23.9nM) variants, as well as other coronaviruses: SARS-CoV-1 (EC 50 = 0.21 µM), MERS-CoV (EC 50 = 1.4 µM), and HCoV OC43 (EC 90 = 0.074 µM) [50,51].Ensitrelvir is progressing to Phase 2/3 clinical trials.Results disclosed from the Phase 2a trial showed a significant reduction in viral titer and RNA on day four, a median time of negative RT-PCR conversion of two days, and acceptable adverse events [52].This drug recently received emergency regulatory approval from the Ministry of Health, Labour and Welfare of Japan, with ongoing Phase 3 clinical trials and plans to extend the approval for worldwide use.
Other oral main protease inhibitor candidates in clinical trials include PBI-0451 from Pardes Biosciences and EDP-235 from Enanta Pharmaceuticals [53].In addition, novel initiatives like the crowdsourced open-science effort COVID Moonshot brought together academic and industrial partners from across the world to develop SARS-CoV-2 M pro inhibitors [43,54].More than 2400 molecules were synthesised and rapidly shared to create a rich intellectual property-free dataset.The search for a potent covalent COVID-19 M pro inhibitor has also been aided by a computational pipeline to efficiently identify irreversible inhibitors [54].Due to its open access and free data-sharing nature, the Moonshot fragment dataset may have aided in the early development of the Shionogi M pro compound.
Due to its saliency as a target, repurposing efforts early in the SARS-CoV-2 pandemic focused on the M pro .Repurposed protease inhibitors evaluated in RCTs include lopinavir (with and without ritonavir), darunavir, and danoprevir; however, none of the tested inhibitors showed efficacy against SARS-CoV-2 (Table 3).
Lopinavir is a protease inhibitor that has been developed for the treatment of human immunodeficiency virus (HIV) and is used in combination with the PK enhancer ritonavir that blocks the enzyme cytochrome P450 3A [55].Lopinavir showed some in vitro activity against both SARS-CoV-1 and SARS-CoV-2 with half maximum inhibitory concentrations (IC 50 ) of 50 and 26 µM, respectively [55].Lopinavir/ritonavir was assessed in multiple COVID-19 RCTs, however, it failed to show any clinical benefit (Table 3) and is therefore not recommended as COVID-19 standard care for hospitalised patients [56,57].
Darunavir is another inhibitor of the HIV protease that is in clinical use in combination with the PK enhancer cobicistat [58].Darunavir was not active against SARS-CoV-2 in vitro at clinically relevant concentrations [59] and in line with these results, did not show clinical efficacy in COVID-19 RCTs (Table 3).
Danoprevir, a hepatitis C virus (HCV) protease (NS3/4A) inhibitor that is used in combination with ritonavir [60] showed cellular activity against SARS-CoV-2 with an EC 50 of 87 µM in Vero E6 cells [61].In hospitalised patients infected with SARS-CoV-2, danoprevir with ritonavir shows a good safety profile [60] with shorter times to PCR negativity and shorter hospital stays compared to the lopinavir/ritonavir group [62].Rapid antiviral response against SARS-CoV-2 shown by the higher proportion of patients who achieved negative PCR on Day 5 when treated using favipiravir compared to the control group. [79]

RNA-Dependent RNA Polymerase (RdRp) Inhibitor
The RNA-dependent RNA polymerase (RdRp) is essential for viral replication and is highly conserved among coronaviruses and positive-strand RNA viruses, and a clinically validated target across many viruses [108,109].Several existing RdRp inhibitors developed against other RNA viruses, including remdesivir [110,111], molnupiravir [112], and favipiravir [113], were in late-stage development or clinical use at the start of the pandemic and showed promising in vitro activity against the SARS-CoV-2.Subsequently, numerous clinical trials were conducted to explore their potential against SARS-CoV-2 infection (Table 3).
Remdesivir is a nucleotide analogue inhibitor of the RdRp that was originally developed for the Ebola virus (EBOV) [114].It shows potent broad-spectrum antiviral activity against pathogenic animal and human coronaviruses in vitro [110,111,115].However, remdesivir's intravenous administration limits its widespread use in the community.Based on multiple clinical trials, remdesivir was the first antiviral drug approved for the treatment of COVID-19 in adults and paediatric patients (≥28 days old and weighing ≥ 3 kg) with a positive SARS-CoV-2 test, for both hospitalised patients, and non-hospitalised individuals with mild-to-moderate COVID-19 that are at high risk for progression to severe COVID-19 [116].One randomised, double-blind, placebo-controlled clinical trial (ACTT-1 study) showed a shorter median time to recovery for remdesivir-treated patients compared to placebo [16].Two additional trials sponsored by Gilead Sciences informed the approval.
An open-label clinical trial of hospitalised adults with moderate COVID-19 showed an improvement in symptoms in patients receiving a five-day course of remdesivir, however, no improvement was demonstrated in those receiving a 10-day course of remdesivir [88].A third randomised, open-label trial in hospitalised adults with severe COVID-19 showed no statistically significant differences in recovery or mortality rates [87].However, it is important to note that with DAA, initiating treatment early whilst viral loads are high is crucial for compound efficacy (Figure 2), and variable treatment windows may explain conflicting clinical trial results [17].An oral pro-drug of remdesivir, obeldesivir, shows excellent cross-reactivity against multiple coronaviruses including MERS, in vitro [117].A recent Phase 3 RCT to evaluate the efficacy and safety for the treatment of COVID-19 in non-hospitalised patients with a high risk for disease progression was discontinued due to lower than expected incidence rates and related hospitalisations or all-cause death, whilst a Phase 3 RCT in hospitalised patients is still ongoing [118].
Molnupiravir is an orally available RdRp inhibitor with broad-spectrum antiviral activity [112] and was initially developed as an oral antiviral against Venezuelan equine encephalitis virus (VEEV) [108].It acts as a nucleoside analogue in the RNA elongation process where it incorporates mutation errors that accumulate into an "error catastrophe" and subsequent virus replication failure [22,108,112].Molnupiravir is authorised for emergency use for SARS-CoV-2 infection by the FDA (Table 2) in adults with confirmed mild-to-moderate SARS-CoV-2 infection, including those who are at high risk for progression to severe disease and those for whom alternative treatment options are not accessible or clinically appropriate.The FDA authorisation was based on the randomised, double-blind, placebo-controlled MOVe-OUT trial, demonstrating a reduction in hospitalization and mortality in the molnupiravir-treated group compared to the placebo [97].However, the European Medicines Agency refused market authorisation for molnupiravir [119], arguing that it was not possible to conclude that molnupiravir reduces the risk of hospitalisation or death in adults at risk of severe disease.
Several other RdRp inhibitors that have been developed for other viral infections have been investigated for clinical efficacy in COVID-19 as part of intensive repurposing efforts.Favipiravir is a guanine analogue that selectively inhibits the RdRp and has been developed as a novel antiviral compound against influenza by Toyama Chemical Co.Favipiravir has been evaluated against SARS-CoV-2 infection in multiple clinical trials (Table 3), with no significant effects on hospitalisation and mortality even when administered within 5 days of symptoms developing [84].Sofosbuvir, a potent inhibitor developed against the RdRp of HCV, has and is in clinical use in combination for the treatment of chronic HCV infection in combination with ribavirin, ledipasvir, and daclatasvir for different genotypes.In multiple RCTs conducted with sofosbuvir in SARS-CoV-2 infection (Table 3), no effect on endpoints was observed, apart from a small open-label study demonstrating an effect on median hospital stay [103].Azvudine, a nucleoside analogue RdRp inhibitor originally developed against HIV infection [120,121] has been approved for the treatment of COVID-19 by the Chinese regulatory agency in 2022, citing a phase 3 clinical trial showing "improved clinical symptoms", compared to a placebo [122,123].However, detailed clinical trial data has not been published to date.Publicly available data is limited, with studies available showing an impact on SARS-CoV-2 viral replication and a shortened time to viral clearance in patients with mild COVID-19, compared to the standard antiviral treatment [98], as well as an impact on disease progression outcome in retrospective studies (Table 3).

Monoclonal Antibodies
Neutralising monoclonal antibodies, mostly targeting the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, has been tested in several clinical trials [38] and are reviewed elsewhere [124] (Table 4).They offer a highly effective therapy against viral infections.However, their use is limited in clinical practice due to (i) the need for intravenous application which limits their use to hospital settings [125,126]; (ii) high costs that restrict access for large parts of the global population [127]; (iii) the reduced efficacy of some antibodies against VoCs [128].Specifically, some antibodies with good efficacy against earlier SARS-CoV-2 variants were less potent against Omicron and its sub-variants [129].Antibodies are effective in mild to moderate disease if given early.Further developments in the use and applicability of monoclonal antibodies for preventing COVID-19 and for early therapy are underway, including alternative applications such as nasal sprays [130].
The combination of bamlanivimab and etesevimab lowers COVID-19-related hospitalisation and mortality in mild to moderate COVID-19 cases and reduces SARS-CoV-2 viral load [134].The use of antibody cocktails that target different neutralisation epitopes may be one way to maintain their therapeutic efficacy [135].The non-competing monoclonal antibodies casirivimab and imdevimab bind to two different sites on the RBD of the SARS-CoV-2 spike protein resulting in the blockage of viral entry into host cells [125].This antibody combination therapy reduced mortality at 28 days in seronegative patients but not in seropositive patients at baseline [125].
The neutralising antibody sotrovimab, developed by GlaxoSmithKline and Vir Biotechnology neutralises SARS-CoV-2 by targeting a highly conserved epitope in the RBD of the spike protein [126].Treatment with sotrovimab reduced the risk of COVID-19 disease progression to hospitalisation or death in mild to moderate high-risk patients [126].Sotrovimab monotherapy retained activity against SARS-CoV-2 VOCs including Omicron [133], however, has been associated with the rapid development of spike gene mutations in vitro [128] and in vivo [133].
Bebtelovimab, developed by Eli Lilly, is a neutralising IgG1 monoclonal antibody that binds to an epitope within the RBD of the SARS-CoV-2 S protein with broad neutralising activity to all SARS-CoV-2 VOCs, including the Omicron variant [136].Bebtelovimab has been studied in the BLAZE-4 clinical trial and is associated with greater viral clearance.It is effective for the treatment of mild to moderate COVID-19 in adults and children above 12 years old who are at risk of disease progression and hospitalisation [137].
The long-lasting monoclonal antibody combination of tixagevimab and cilgavimab (brand name Evusheld from AstraZeneca) was derived from antibodies isolated from B cells of patients infected with SARS-CoV-2 [138,139].

Host-Targeting Antivirals (HTA)
Host-targeting antivirals (HTA) are drugs that modify host cell pathways required for viral replication [152].They often modulate virus-host interactions by targeting human proteins used by viruses.As they may target host cell pathways used by several viral families, HTAs may be suitable as a first-line antiviral drug when a novel virus emerges, provided the novel virus relies on the same host pathway.Conceptually, HTAs could be given early in a pandemic, prior to emerging viruses being characterised at the molecular level, and may carry a lower risk of drug resistance [40,41].Even though no HTA are approved for COVID-19 therapy due to the lack of efficacy in RCTs performed to date, we discuss results on a range of clinically tested compounds.

Inhibitors of Viral Cell Entry
SARS-CoV-2 enters host cells by attaching to the cell membrane and subsequently fusing in the endosome.Entry requires the spike glycoprotein, which is cleaved by the host protease furin into S1 and S2 subunits during viral release from an infected cell.The subunits remain non-covalently associated and are present on mature virions as trimeric spikes.The S1 subunits bind the obligate SARS-CoV-2 receptor ACE2 with their RBDs, while the S2 subunits anchor the spike protein to the membrane and contain the fusion peptide.When S1 binds ACE2, an S2 ′ site within S2 is exposed and triggers its cleavage by the transmembrane serine protease 2 (TMPRSS2) at the cell surface, or by cathepsin L in the endosome after receptor-mediated endocytosis [153].Every step in this intricate entry process is important and presents a potential DAA or HTA target.Clinical trials aimed at evaluating HTA are shown in Table 5.
Camostat mesylate, an inhibitor of TMPRSS2, has been reported as a potential entry inhibitor [154].Although considered safe and well-tolerated, no clinical benefit was observed from RCTs results in terms of reduction of the disease progression or mortality (Table 5).No statistical difference in viral clearance for any drug regimen, compared to the standard of care. [183] ACE2, angiotensin-converting enzyme 2; FIASMA, Functional inhibitors of acid sphingomyelinase; RBD, receptor binding domain.
Umifenovir (arbidol) was developed for the treatment of influenza virus as a hemagglutinin inhibitor preventing virus-mediated fusion with the cell membrane, blocking viral entry into target cells [184,185].Unsurprisingly, as SARS-CoV-2 viral entry does not depend on hemagglutinin, RCT results evaluating its activity against COVID-19 showed no favourable effect and limited efficacy of arbidol in treating COVID-19 [63,163] (Table 5, Figure 1).Some inhibitors work by interacting with dynamin, a GTPase responsible for clathrindependent endocytosis, which is essential for coronaviruses to enter the cell [186].Chlorpromazine (CPZ), an antipsychotic medication, has been repurposed as a COVID-19 therapeutic based on this mechanism and reported for its antiviral activity against MERS-CoV and SARS-CoV-1 [187].These findings led to a clinical trial that observed a lower incidence of symptomatic COVID-19 among patients after treatment with CPZ [188].
Disulfiram is a hepatic aldehyde dehydrogenase inhibitor that is used to treat chronic alcoholism [174,189].It reduced the incidence and severity of COVID-19 in a retrospective observational study (Table 5) [174], although no impact on viral load was shown [189].Further large-scale clinical trials are needed to assess the findings.
Fluvoxamine is a psychotropic medication that belongs to a group of functional inhibitors of acid sphingomyelinase (FIASMA) which were evaluated against COVID-19 [190].The rationale for using FIASMA is linked to the role of lipid rafts in viral entry.Sphingomyelinase activity is triggered by SARS-CoV-2 binding which in turn leads to the formation of ceramide-enriched membrane domains that help viral entry by clustering ACE2 [191].Treatment with fluvoxamine inhibits the sphingomyelinase activity formation of these domains in vitro [191,192].Fluvoxamine and other FIASMA drugs were associated with reduced mortality in COVID-19 patients were tested in a large cohort study [193].Although three RCT studies showed favourable clinical benefits of fluvoxamine (Table 5), clinical evidence was deemed insufficient to issue a treatment recommendation in the National Institutes of Health (NIH) treatment guidelines [35].

Inhibitor of Viral Glycoprotein Folding
Enveloped viruses require the glycoprotein-folding machinery in the host endoplasmic reticulum (ER) to correctly fold their glycosylated proteins [40].Pivotal players in this ER quality control (ERQC) pathway are the ER alpha glucosidases, which can be targeted for example by iminosugars [194][195][196].Partially inhibiting the ERQC prevents the proper folding and incorporation of viral glycoproteins into budding viruses, as shown previously for other enveloped viruses such as HIV [197], human papillomavirus [198], dengue [196,199,200], influenza [196,201], hepatitis B virus [202], HCV [203], Zika virus [204], Marburg virus [205] and EBOV [206], and could lead to a potential broadspectrum antiviral drug also against coronaviruses.The extensively glycosylated SARS-CoV-2 spike protein is essential for viral entry, and inhibition of its proper glycosylation leads to antiviral effects.The monocyclic UV-4 (N-(9-methoxynonyl)-1-deoxynojirimycin) or MON-DNJ prevented SARS-CoV-2-induced Vero cell death and reduced viral replication in vitro after 24 h of treatment [40,207].The results are encouraging and need to be further tested in vivo and in clinical trials.

Host-Targeting Antivirals with Unknown Mechanism
Some repurposed drugs work by targeting the host, although the exact antiviral mechanisms are unknown.Ivermectin, an FDA-approved anti-parasitic drug was reported to have an in vitro antiviral activity to SARS-CoV-2 [208].However, several adequately powered RCTs in Brazil [178], the US [209,210], and Malaysia [211] failed to report a clinical benefit from the use of ivermectin in COVID-19 outpatients and it is not approved or authorised by the FDA for the treatment of COVID-19.
Similar attention was given to the anti-malaria drug chloroquine and its derivatives [212].Chloroquine was reported to have efficacy and acceptable safety against COVID-19-associated pneumonia in multi-centre clinical trials conducted in China [213] and its use attracted disproportionate attention during the coronavirus pandemic, spurred by preliminary studies and endorsement from political leaders [214].The chloroquine derivative hydroxychloroquine was tested in RCTs with limited to no clinical benefit for COVID-19 (Table 5) [168].Based on sufficiently powered randomised trials [169,215] however, NIH treatment guidelines recommend against the use of both chloroquine and hydroxychloroquine for the treatment of COVID-19 [35].
The anti-protozoal drug nitazoxanide also showed in vitro activity against SARS-CoV-2 [216], and has been tested in multiple RCTs (Table 5).A possible mechanism of action may be linked to inhibition of SARS-CoV-2 spike-induced syncytia and its binding to TMEM16 [217].Nitazoxanide did not show efficacy in a number of RCTs when used at approved doses, and NIH treatment guidelines recommend against the use of nitazoxanide for the treatment of COVID-19 [35].However, nitazoxanide did not show serious adverse events when evaluated in a Phase 1 study at higher doses of 1500 mg twice daily, at which it may provide antiviral efficacy according to the pharmacokinetic modelling [218].

Immunomodulatory Drugs
Immunomodulatory drugs are commonly used in autoimmune disease and include both monoclonal antibodies and small molecule inhibitors.They can either target cytokines directly, such as monoclonal antibodies against interleukins (ILs) or tumour necrosis factor (TNF)-alpha, inhibit proteins involved in inflammatory signalling pathways such as the janus kinase (JAK) inhibitors, or interfere with the hormonal regulation of inflammation such as corticosteroids [219,220].Multiple monoclonal antibodies and immunomodulatory compounds were evaluated in COVID-19 infection (Table 6), driven by the aim to impact on COVID-19 associated systemic inflammation that can be associated with heightened cytokine release, as indicated by elevated blood levels of IL-6, C-reactive protein (CRP), D-dimer and ferritin [221][222][223][224]. Crucial for the assessment of the effects of immunomodulatory drugs were large-scale platform trials initiated early in the pandemic.

Corticosteroids
Corticosteroids bind to the glucocorticoid receptor and inhibit the synthesis of multiple inflammatory proteins through the suppression of genes that encode them, as well as promoting anti-inflammatory signals [224,225].Coordinated from Oxford, the open-label RECOVERY trial recruited over 43,000 hospitalised patients with COVID-19 participants worldwide and randomly assigned patients to treatment groups that included dexamethasone, hydroxychloroquine, lopinavir-ritonavir, or azithromycin, and compared them to usual care, with the primary endpoint mortality at 28 days [226].Dexamethasone treatment significantly decreased mortality in patients who were receiving either invasive mechanical ventilation or oxygen alone at randomization [14].Another inhaled corticosteroid, budesonide, was assessed in the PRINCIPLE study in non-hospitalised patients with COVID-19, and improved recovery time with the potential to reduce hospital admissions or deaths [227].

Host-Targeting Monoclonal Antibodies
Multiple immunomodulatory monoclonal antibodies, directed against cytokines such as against tumour necrosis factor alpha (TNF-α), Interleukin (IL)-1, and IL-6, were assessed in COVID-19 patients [224].Tocilizumab is a recombinant humanised monoclonal antibody that binds to interleukin-6 receptors, thereby blocking the activity of the pro-inflammatory cytokine.IL-6 is produced by a variety of cell types including lymphocytes, monocytes, and fibroblasts, and has been shown to be induced by SARS-CoV-2 infection in bronchial epithelial cells.The IL-6 inhibitor tocilizumab is in clinical use for several inflammatory diseases such as rheumatoid arthritis, giant cell arteritis, polyarticular juvenile idiopathic arthritis, and systemic juvenile idiopathic arthritis.As of 24 June 2021, the FDA has authorised the use of tocilizumab under EUA for the treatment of COVID-19 in hospitalised adults who are receiving systemic corticosteroids and require supplemental oxygen, mechanical ventilation, or extracorporeal membrane oxygenation (ECMO) [228].The authorisation was based on the results of 4 clinical trials: RECOVERY, EMPACTA, COVACTA, and REM-DACTA (Table 6), with the decision to grant EUA mainly based on the positive results of RECOVERY and EMPACTA that demonstrated an impact on mortality and a composite readout of mechanical ventilation and mortality [30,229].
IL-1 inhibitors include the IL-1 receptor antagonist Kineret (brand name anakinra), the IL-1 "Trap" rilonacept, and the neutralising monoclonal antibody to IL-1β canakinumab [224,230].Anakinra was considered a safe and efficient treatment for severe forms of COVID-19 with a significant survival benefit in critically ill patients and features of macrophage activation-like syndrome [20,21], with most RCTs supporting the clinical benefit of this drug for COVID-19 (Table 6).Reduced mortality in patients compared to standard of care alone. [256]

Janus kinase inhibitor Prospective
A multi-centre randomised, double-blind, double placebo-controlled trial in hospitalised patients with COVID-19 requiring supplemental oxygen by low-flow, high-flow, or non-invasive ventilation (n = 1010) at 67 trial sites in the United States, South Korea, Mexico, Singapore, and Japan, between December 2020 and April 2021 (ACTT-4 study).
Similar mechanical ventilation-free survival by day 29 between groups, while dexamethasone was associated with significantly more adverse events, treatment-related adverse events, and severe or life-threatening adverse events.Lower risk of death or respiratory failure than placebo through day 28.

Janus kinase inhibitor Prospective
An open-labeled randomised control study in hospitalised adult patients with mild to moderate COVID-19 pneumonia (n = 100) in India in 2020.
Reduction of the overwhelming inflammatory response compared to the standard care alone. [258] TNF-inhibitors have been used in severe cases of autoimmune inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, or ankylosing spondylitis [259].Several formulations of TNF inhibitors are currently available, including adalimumab, etanercept, and infliximab [224].Elevated serum levels of TNF-α and soluble TNF-Receptor 1 have been detected in COVID-19 patients with severe infection, providing a rationale for the use of TNF-α inhibitors in SARS-CoV-2 infection [260].However, concerns regarding the potential suppression of antiviral immune responses have been raised by an observational study showing a negative impact on SARS-CoV-2 antibody levels following natural infection in patients with inflammatory bowel disease treated with infliximab [261,262].
In the CATALYST open-label phase 2 trial, infliximab showed no impact on CRP levels as a measure for inflammation in SARS-CoV-2 infection [245].Recently reported results of the ACTIV-1 trial, a placebo-controlled, masked, RCT among patients hospitalised for COVID-19, reported a significant benefit of infliximab on mortality [263], but no impact on the primary study endpoint length of pneumonia [246].
The latest addition to monoclonal antibodies under EUA is vilobelimab, a monoclonal antibody that specifically binds to the soluble human complement C5a, a product of complement activation.C5a activates the innate immune response, including the local release of histamines, contributing to inflammation and local tissue damage.In vivo studies demonstrated that an anti-C5a monoclonal antibody inhibited acute lung injury in a human C5a receptor knock-in mouse model [264].The phase 3 PANAMO RCT study recorded an improvement in invasive mechanically ventilated patients' survival that led to a decrease in mortality with the use of vilobelimab [19].

Janus Kinase (JAK) Inhibitors
The primary mechanism of action of Janus kinases (JAK) is the phosphorylating signal transducer and activator of transcription (STAT), a key player in signalling pathways involved signalling, growth, survival, inflammation, and immune activation.Inhibiting JAK prevents the phosphorylation of key signalling proteins involved in inflammation pathways, thereby blocking cytokine signalling [224,265].Tofacitinib and baricitinib are orally available small-molecule JAK inhibitors approved for the treatment of rheumatoid arthritis [265] and have been evaluated in multiple RCTs in patients with SARS-CoV-2 infection, leading to a treatment recommendation in hospitalised adults that require respiratory support [35].In brief, baricitinib had an impact on mortality [28,256], as well as reducing intensive care unit admissions, lowering the requirement for invasive mechanical ventilation, and improving patients' oxygenation index [27,29], effects that were maintained in a meta-analysis [266].Furthermore, baricitinib in combination with remdesivir showed superior results compared to remdesivir alone in reducing patients' recovery time and improving their clinical status [26].
In summary, for immunomodulatory drugs, the NIH issued guidelines based on the existing evidence, recommending dexamethasone, tocilizumab, and baricitinib for hospitalised patients with COVID-19, whilst the evidence for anakinra, inhaled corticosteroids, and vilobelimab was deemed inconclusive, despite all compounds receiving EUA from the FDA.

Discussion
The devastating effect of the COVID-19 pandemic was a stark reminder of the requirement for antiviral compounds that are ready to use against viruses of pandemic potential and emerging viral threads.
A major accomplishment during the pandemic was the rapid implementation of several large platform trials that facilitated the evaluation of various repurposed compounds on COVID-19 hospitalisation and mortality, including DAA, HTA, and immunomodulatory drugs (Figure 3).Due to the time required to develop novel small molecule inhibitors, initial trials focused on immunomodulatory drugs and known antivirals developed against other viruses, followed by rapidly developed monoclonal antibodies to deliver treatments quickly [42,66,246,267].Based on these RCTs, SARS-CoV-2 therapeutic management guidelines now include several repurposed drugs that were shown to be active against COVID-19.The success of initial repurposing efforts was highly variable for the different compound classes.Several immunomodulatory drugs such as baricitinib, tocilizumab, and dexamethasone improved SARS-CoV-2 disease severity and mortality and are now included in the treatment guidelines for hospitalised patients.Further, repurposing efforts of DAA-that were in the late stages of development but not yet approved at the start of the pandemic-shows the potential to rapidly translate in vivo antiviral activity findings into clinical trials [108].This holds true for remdesivir and molnupiravir which were developed against the RdRp of RNA viruses EBOV and VEEV, respectively and are also highly effective against the SARS-CoV-2 RdRp.In contrast, repurposed DAA approved for HIV, HCV, or influenza, such as lopinavir, favipiravir, darunavir, and danoprevir, showed limited or no clinical activity against SARS-CoV-2 across RCTs.Not surprisingly, repurposed inhibitors of viral proteins that are not expressed by coronaviruses, such as the neuraminidase targeted by oseltamivir, did not show clinical activity [268].Of note, comparably few repurposed HTA with a plausible mechanism of action were evaluated in large RCTs to date (Table 5).
Numerous in vitro high throughput screens have been conducted since early 2020 to identify potential candidates for repurposing, by screening approved and investigational drug collections [269,270].However, many trials included compounds based on insufficiently validated cellular screening results, with extensive resources wasted in unnecessary investigations aiming to translate screening hits with marginal cellular activity or false positive data in vitro.This is particularly poignant for compounds causing phospholipidosis, a phenomenon caused by cationic amphiphilic drugs such as Chloroquine or Amiodarone leading to false positive results in vitro due to lipid processing inhibition [271].Further, many compound libraries that were used in large in vitro screens, such as the ReFRAME library, are skewed towards human proteins, and therefore have, perhaps unsurprisingly, only yielded a few hits of compounds with previously known antiviral activity such as nelfinavir and MK-448 [272].We, therefore, conclude that in vitro hits should be translated into clinical trials with caution, thoroughly reviewing in vitro and in vivo efficacy in relevant animal models, and, for DAA, establishing a robust pharmacokinetic/pharmacodynamic relationship and demonstrating consistent exposure over 90% effective concentration/EC 90 prior to embarking on resource-demanding clinical trials.
With an abundance of in vitro screening data being generated, the sustainability and maintenance of data repositories is of particular importance, especially downstream for pandemic preparedness.The FAIR principles (Findable, Accessible, Interoperable, Reusable) provide a useful framework and should guide the development of data repositories that capture screening results against viruses of pandemic concern, which may aid pandemic response in the future.The maintenance of existing data repositories for cellular screening results, not only for SARS-CoV-2 but also for other viruses of pandemic concern, is of utmost importance to ensure an efficient pandemic response in the future.
In addition to repurposing efforts, the rapid discovery of novel, SARS-CoV-2 targeting DAA was unprecedented.This includes small molecule inhibitors such as M Pro inhibitors nirmatrelvir, based on existing chemical starting points from previous coronavirus protease targets [25], or ensitrelvir, developed through de novo high-throughput screening hits that were progressed using SARS-CoV-2 specific fragment hits [50].Similar to small molecule DAA efforts, highly efficient monoclonal antibodies designed to neutralise SARS-CoV-2 by binding to the spike protein on its surface were rapidly advanced early in the pandemic.However, many antibodies targeting spike lost their in vitro effectiveness against novel circulating variants such as Omicron and are no longer recommended for clinical use.
Drug resistance has always been the major challenge in drug development against viruses, including coronaviruses [273].Newly evolving viral strains are linked to recurring waves, with partial immune escape and waning immunity within the population [274][275][276].In addition, drug-induced viral mutations have been described for SARS-CoV-2 therapeutics, however, so far mostly in immunosuppressed individuals.Further, circulating viruses may harbour variants that are resistant to DAA, such as G15S and T21I that confer resistance to the M Pro inhibitor Paxlovid [277][278][279].The ongoing identification and characterisation of drug-resistant signatures within the SARS-CoV-2 genome will be crucial for clinical management and virus surveillance [280].
The accepted primary trial endpoints by the FDA include all-cause mortality, the need for hospitalisation and invasive mechanical ventilation, and a range of clinical signs such as sustained symptom alleviation [281].In addition, a virological measure is acceptable as a primary endpoint in a Phase 2 clinical trial, but only as a secondary endpoint in a Phase 3 trial.However, over the last 3 years, circulating viral strains have changed significantly, and both vaccinations and natural infection have led to an increased immunological memory against SARS-CoV-2.Therefore, these endpoints are now less common, leading to discontinued RCTs such as for obeldesivir, due to lower-than-expected hospitalisations or mortality.This also leads to ethical considerations, on whether it is acceptable to enroll patients into a placebo-controlled trial where there is a very low risk of the primary endpoints of death or hospitalisation (e.g., <1%).A promising approach to overcome these issues may include pharmacodynamic modelling of viral clearance [282].
For pandemic preparedness, the concept of "one drug, multiple viruses" carries more promise than a "one drug, one virus" paradigm.Broad-spectrum antiviral agents that inhibit a wide range of human viruses should therefore be the target of de novo pandemic preparedness drug discovery efforts, as well as a focus on HTA that could be deployed immediately [283,284].
The 100-Day Mission set ambitious goals to prepare us for "Disease X", aiming to generate safe, effective vaccines, therapeutics, and diagnostics within 100 days of the identification of a novel threat [285].As well as developing novel assets, their licensing to ensure global and equitable access to future assets remains a key consideration.In the COVID-19 pandemic, the existing international laws and intellectual property practices failed to ensure equitable access to vaccines and therapeutics globally [286].During the SARS-CoV-2 pandemic, companies appear to have financed their development efforts on the back of large procurement contracts with governments, rather than on the prospect of intellectual property, providing a useful case study for pandemic preparedness [287].
An urgent need remains [35,288] for drugs that can be easily stockpiled to ensure availability for pandemic preparedness.

Figure 1 .
Figure 1.The coronavirus life cycle and the mechanistic actions of antiviral drugs within the viral replication process, using SARS-CoV-2 as an example.The virus-cell membrane fusion was induced by the binding of spike protein to the host cellular receptor angiotensin-converting enzyme 2

Figure 1 .
Figure 1.The coronavirus life cycle and the mechanistic actions of antiviral drugs within the viral replication process, using SARS-CoV-2 as an example.The virus-cell membrane fusion was induced by the binding of spike protein to the host cellular receptor angiotensin-converting enzyme 2 (ACE2), together with the cell surface transmembrane serine protease 2 (TMPRSS2).Following viral entry, the release of the viral genome is followed by the immediate translation of viral proteins and the formation of the viral replication and transcription complex.The 3-chymotrypsin-like protease (CL pro )/main protease (M pro ) and papain-like protease (PL pro ) cleave the virus polypeptide into 16 non-structural proteins.Structural glycoproteins are synthesised in the endoplasmic reticulum

Figure 2 .
Figure 2. COVID-19 disease progression and the windows of opportunities where antiviral drugs should be deployed.The directly acting antivirals (DAA) and host-targeting antivirals (HTA) are most effective for an intervention in the earlier course of the mild to moderate disease manifestation when viral load is increasing and detectable by RT-PCR.The immunomodulatory drugs are more potent in the later phase of the disease when the host immune response starts to develop as a response to the infection and the clinical manifestation starts to develop from severe to critical illness due to the risks of a cytokine storm.Adapted from "Time Course of COVID-19 Infection and Test Positivity", by BioRender.com(2023).Retrieved from https://app.biorender.com/biorender-templates,accessed on 20 April 2023.

Figure 2 .
Figure 2. COVID-19 disease progression and the windows of opportunities where antiviral drugs should be deployed.The directly acting antivirals (DAA) and host-targeting antivirals (HTA) are most effective for an intervention in the earlier course of the mild to moderate disease manifestation when viral load is increasing and detectable by RT-PCR.The immunomodulatory drugs are more potent in the later phase of the disease when the host immune response starts to develop as a response to the infection and the clinical manifestation starts to develop from severe to critical illness due to the risks of a cytokine storm.Adapted from "Time Course of COVID-19 Infection and Test Positivity", by BioRender.com(2023).Retrieved from https://app.biorender.com/biorender-templates,accessed on 20 April 2023.

Figure 3 .
Figure 3. Summary of compounds discussed in this review article, including directly acting antiviral (DAA), host-targeting antiviral (HTA), and immunomodulatory drugs.Therapeutics that received full approval and emergency use authorisation (EUA) for COVID-19 are marked in the table.FI-ASMA, Functional inhibitors of acid sphingomyelinase activity.
trial in patients hospitalised with COVID-19 (n = 289) at 15 sites in Brazil in 2020.

Table 1 .
Coronaviruses with a history of pathogenicity in humans and their respective cellular entry receptors.

Table 1 .
Coronaviruses with a history of pathogenicity in humans and their respective cellular entry receptors.