Therapeutic Potential of Salicylamide Derivatives for Combating Viral Infections

Abstract

Since time immemorial human beings have constantly been fighting against viral infections. The ongoing and devastating COVID-19 pandemic represents one of the most severe and most significant public health emergencies in human history, highlighting an urgent need to develop broad-spectrum antiviral agents. Salicylamide (2-hydroxybenzamide) derivatives, represented by niclosamide and nitazoxanide, inhibit the replication of a broad range of RNA and DNA viruses such as flavivirus, influenza A virus and coronavirus. Moreover, nitazoxanide was effective in clinical trials against different viral infections including diarrhea caused by rotavirus and norovirus, uncomplicated influenza A and B, hepatitis B, and hepatitis C. In this review, we summarize the broad antiviral activities of salicylamide derivatives, the clinical progress, and the potential targets or mechanisms against different viral infections and highlight their therapeutic potential in combating the circulating and emerging viral infections in the future.

1. INTRODUCTION

There has been more than ten significant epidemics or pandemics of various viral diseases in the past two decades of the twenty-first century. This includes dengue, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), chikungunya, Zika fever, Ebola virus (EBOV) disease, H1N1 influenza, coronavirus disease 2019 (COVID-19), etc.¹ The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), represents one of the most challenging public health emergencies in human history². Since SARS-CoV-2 was first reported in December 2019 in Wuhan, a series of public health measures such as business closings, staying-at-home orders, mask-wearing, and social distance were taken to control its spread³. However, due to its ability for asymptomatic transmission, SARS-CoV-2 quickly raged worldwide and was declared a global pandemic on March 11, 2020, by the World Health Organization (WHO), causing millions of associated fatalities and enormous economic losses^4,5. Although it usually takes several years to develop new therapeutics, especially for novel emerging pathogens, the antiviral agent remdesivir was rapidly authorized emergence use by the FDA in May 2020 and finally approved in October 2020 for the treatment of COVID-19^6–8. Remdesivir was developed by Gilead initially to combat EBOV infection as an RNA-dependent RNA polymerase (RdRp) inhibitor and displayed broad-spectrum antiviral activities, highly effective against filoviruses, paramyxoviruses, respiratory syncytial virus (RSV), and pathogenic coronaviruses (CoVs)^9–11. After the outbreak of COVID-19, remdesivir was quickly found to potently inhibit SARS-CoV-2 replication and shorten the recovery time in hospitalized patients with COVID-19, which promoted its clinical use to benefit numerous COVID-19 patients^12,13. Molnupiravir, another broad-spectrum antiviral agent of an RdRp inhibitor developed by Emory University and Merck, showed robust anti-SARS-CoV-2 activity¹⁴. A phase 3, double-blind, randomized, placebo-controlled trial revealed that early treatment with molnupiravir could reduce the risk of hospitalization or in at-risk, unvaccinated COVID-19 patients, and it was authorized emergence use by the FDA on December 23, 2021 as an oral drug to treat mild-to-moderate COVID-19 in adults^15,16. The rest of the three FDA-approved anti-SARS-CoV-2 drugs is nirmatrelvir which is an oral 3C-like protease (3CLpro) inhibitor developed by Pfizer. In a phase 2–3 double-blind, randomized, controlled trial, treatment with nirmatrelvir plus ritonavir resulted in a lower risk of progression to severe COVID-19 than that with placebo (relative risk reduction, 89.1%)¹⁷. The combination of nirmatrelvir and ritonavir (the brand name Paxlovid) was authorized as the first oral antiviral by FDA on December 22, 2021, for the treatment of mild-to-moderate COVID-19 in patients (12 years of age and older)¹⁸. As highlighted by the successful examples of remdesivir and molnupiravir, it is imperative to develop broad-spectrum small molecule antiviral agents to fight the highly heterogeneous unknown viral infections that may emerge in the future.

Salicylamide (2-hydroxybenzamide) derivatives are a class of compounds that possess broad-spectrum antiviral activities, exemplified by niclosamide (1) and nitazoxanide (2) (Figure 1)^19–21. Niclosamide is an FDA-approved anthelminthic drug used to treat tapeworm infections in human for several decades. It works by suppressing oxidative phosphorylation and stimulating adenosine triphosphatase activity in the mitochondria. Niclosamide can modulate multiple signaling pathways and biological processes, including Wnt/β-catenin, STAT3, NF-κB, mTORC1, etc²². Nitazoxanide was initially developed as an oral antiparasitic agent and finally approved by FDA as an Orphan Drug to treat diarrhea caused by Cryptosporidium parvum and Giardia intestinalis in adults and children (≥ 12 months of age)²¹. Its mechanism of action against protozoa and anaerobic bacteria is related to inhibiting pyruvate:ferredoxin oxidoreductase (PFOR), an enzyme essential for anaerobic energy metabolism²¹. Nitazoxanide disrupts the membrane potential and pH homeostasis of Mycobacterium tuberculosis (Mtb), acting as an uncoupler²³. Accumulated studies indicated that niclosamide, nitazoxanide, and its active circulating metabolite, tizoxanide (3), inhibit the replication of a broad range of RNA and DNA viruses such as flavivirus, influenza A virus, and coronavirus^20,21,24. Moreover, nitazoxanide was effective in clinical trials against different viral infections, including diarrhea caused by rotavirus and norovirus, uncomplicated influenza A and B, hepatitis B, and hepatitis C^21,24. In recent years, structure-activity relationship (SAR) optimization on salicylamide derivatives has obtained more effective and safer antiviral agents against different viruses^24–31. This review summarizes the broad antiviral activities of salicylamide derivatives, the clinical progress, and the potential targets or mechanisms against different viral infections and highlights their therapeutic potential in combating the circulating and emerging viral infections in the future.

Figure 1.

The chemical structures of representative salicylamide derivatives niclosamide, nitazoxanide, and its metabolite tizoxanide.

2. ANTIVIRAL ACTIVITY OF SALICYLAMIDE DERIVATIVES

2.1. Anti-Coronavirus Activity

CoVs are a group of enveloped and positive-sense single-stranded RNA viruses which can be divided into four genera (α, β, γ, and δ) belonging to the family Coronaviridae within the order Nidovirales²⁰. Currently, seven human coronaviruses (HCoVs) have been identified, including two α-CoVs, HCoV-NL63 and HCoV-229E, and five β-CoVs, HCoV-HKU1, HCoV-OC43, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2^9,32. Among them, four human strains, HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1 usually produce mild symptoms like the common cold. In comparison, three strains, SARS-CoV, MERS-CoV, and SARS-CoV-2, can cause severe acute respiratory diseases (SARS, MERS, and COVID-19, respectively) with high morbidity and mortality⁹. The outbreak of SARS between November 2002 and July 2003 finally brought about 8098 confirmed cases and 774 associated deaths (case-fatality rate: 9.6%) in 29 countries and regions, while MERS-CoV, first reported in June 2012 in Saudi Arabia, has caused 2519 laboratory-confirmed cases in 27 countries, leading to 866 associated deaths (case-fatality rate: 34.3%) by the end of January 2020^33,34. SARS-CoV-2, as the third highly pathogenic HCoV, was first reported in Wuhan, China, in December 2019 and then fast spread worldwide due to its high transmissibility, leading to an ongoing COVID-19 pandemic^3,9. As of June 20, 2021, SARS-CoV-2 accounted for more than 177 million cases of infection and more than 3,857,972 associated deaths globally⁵. Although remdesivir, molnupiravir, and nirmatrelvir as well as three vaccines (Pfizer-BioNTech, Moderna, and Janssen) have been approved by FDA for the treatment or prevention of COVID-19, there is still an urgent need to develop more effective and safer small molecule antiviral therapeutics to cope with the emergence of various SARS-CoV-2 variants with increased transmissibility^8,35–39.

Niclosamide inhibits SARS-CoV and SARS-CoV-2 replication with EC₅₀s of < 0.1 μM and 0.28 μM, respectively, and suppresses MERS-CoV infection by up to 1000-fold at 48 h post-infection (p.i.) at a concentration of 10 μM (Table 1)^40–43. Niclosamide was reported to reduce MERS-CoV replication through S-phase kinase-associated protein 2 (SKP2) inhibition which increases the Benclin 1 (BECN1) level and enhances autophagy while it blocks SARS-CoV-2 spike-induced syncytia via inhibiting the activity of the calcium-activated TMEM16F (a.k.a. anoctamin 6), which acts as both a chloride channel and a lipid scramblase responsible for phosphatidylserine internalization on the cell surface^42,44. When human volunteers received a single oral dose of 2000 mg of niclosamide, the maximum serum concentration of niclosamide reached 0.25–6.0 μg/mL (0.76–18.3 μM)⁴⁵. To further improve the concentration of niclosamide in the lung, a significant site of SARS-CoV-2 infection, a new formulation for inhalation administration was developed and well-tolerated in healthy volunteers, warranting its further evaluation in COVID-19 patients⁴⁶. Currently, several clinical trials have been conducted to test the efficacy of niclosamide in COVID-19 treatment. Broad anti-CoV activities were also observed for nitazoxanide and tizoxanide, displaying EC₅₀ values of ~3 μM against MERS-CoV and SARS-CoV-2^13,47–49. In a phase 2b/3 clinical trial treating patients with nitazoxanide (600 mg controlled-release tablet) twice daily for 5 days, during the first 12 h after dosing on the morning of day 2, the plasma concentrations of tizoxanide ranged from 17.3 μM (4.6 μg/ml) to 3.0 μM (0.8 μg/ml), above the in vitro EC₅₀ value against SARS-CoV-2, supporting the repurposing of nitazoxanide for the treatment of COVID-19⁵⁰. Moreover, oral administration of one 500 mg nitazoxanide tablet with food could further increase the maximum plasma concentration of tizoxanide approximately to 37 μM (10 μg/mL)^51,52. Nitazoxanide is currently undergoing a series of clinical trials to evaluate its efficacy as a monotherapy or in combination with other antivirals to treat COVID-19. Shamim et al. reported niclosamide derivatives 4-9 (Figure 2), which showed potent inhibitory activities against SARS-CoV-2 infection with sub-micromolar EC₅₀ values, similar to niclosamide⁵³. Especially, compound 9 with 5-bromo substitution in the salicylic acid region also displayed better drug-like properties, offering a good candidate for further evaluation in in vivo models of COVID-19. Recently, our group found that compound 10 with 3´-Cl-4´-NO₂-aniline moiety was a potent allosteric inhibitor against SARS-CoV-2 3CLpro (IC₅₀ = 3.9 μM) and exhibited anti-SARS-CoV-2 activity (EC₅₀ = 1.75 μM) in A549-hACE2 cells⁵⁴. In addition, Juan et al. also reported a series of niclosamide derivatives including compounds 10-12 as potent inhibitors against SARS-CoV-2 (EC₅₀ = 0.057 μM, 0.39 μM, and 0.49μM, respectively). The phosphatidylserine externalization experiment through fluorescence microscopy demonstrated the inhibition of compound 10 towards TMEM16F, similar to niclosamide. Moreover, compounds 10-12 displayed higher stability in human plasma and liver S9 enzymes assay than niclosamide⁵⁵. Blake et al. screened their salicylanilide compound library and found that compound 13 inhibited SARS-CoV-2 infection with an EC₅₀ of 0.74 μM²⁹. Compound 13 displayed a good bioavailability of 76% in mice. The treatment of compound 13 (when administrated orally at 5 mg/kg twice daily for 5 days) led to 10-fold lower viral titers, reduced the induction of key cytokines, and mitigated the associated weight loss in a mouse model of SARS-CoV-2 infection^29,56. Considering its relatively high cytotoxicity in Vero cells (CC₅₀ = 1.22 μM), the in vivo safety of compound 13 needs to be further investigated.

Table 1.

The activity of salicylamide derivatives against coronavirus^a

Compound	Activity against coronavirus	Ref.
Niclosamide (1)	SARS-CoV: EC50 < 0.1 μM, CC50 = 22.1 μM (Vero E6 cells) SARS-CoV-2: EC50 = 0.28 μM, CC50 > 50 μM (Vero cells) SARS-CoV-2: EC50 = 0.11 μM (Vero E6 cells/ USA_WA1/2020) Suppressed MERS-CoV infection by ~1000-fold at 48 h at 10 μM	40,42,43,53
Nitazoxanide (2)	Canine CoV S-378: EC50 = 3.3 μM (A72 cells) MERS-CoV: EC50 =3.0 μM (LLC-MK2 cells) SARS-CoV-2: EC50 = 2.12 μM, CC50 = 35.53 μM (Vero E6 cells)	13,47,49
Tizoxanide (3)	MERS-CoV: EC50 =3.1 μM (LLC-MK2 cells) SARS-CoV-2: EC50 = 3.16 μM (Vero E6 cells)	47,48
4	SARS-CoV-2: EC50 = 0.32 μM (Vero E6 cells/ USA_WA1/2020), cell viability: 61% at 30 μM in SNB-19 cells	53
5	SARS-CoV-2: EC50 = 0.38 μM (Vero E6 cells/ USA_WA1/2020), cell viability: 59% at 30 μM in SNB-19 cells	53
6	SARS-CoV-2: EC50 = 0.26 μM (Vero E6 cells/ USA_WA1/2020), cell viability: 63% at 30 μM in SNB-19 cells	53
7	SARS-CoV-2: EC50 = 0.13 μM (Vero E6 cells/ USA_WA1/2020), cell viability: 59% at 30 μM in SNB-19 cells; EC50 = 0.38 μM, CC50 = 1.18 μM (Vero E6 cells)	53,55
8	SARS-CoV-2: EC50 = 0.14 μM (Vero E6 cells/ USA_WA1/2020), cell viability: 55% at 30 μM in SNB-19 cells	53
9	SARS-CoV-2: EC50 = 0.10 μM (Vero E6 cells/ USA_WA1/2020), cell viability: 59% at 30 μM in SNB-19 cells	53
10	SARS-CoV-2: EC50 = 1.75 μM, CC50 = 4.75 μM (A549-hACE2 cells); EC50 = 0.057 μM, CC50 = 1.51 μM (Vero E6 cells);	54,55
11	SARS-CoV-2: EC50 = 0.39 μM, CC50 = 2.46 μM (Vero E6 cells)	55
12	SARS-CoV-2: EC50 = 0.49 μM, CC50 = 5.95 μM (Vero E6 cells)	55
13	SARS-CoV-2: EC50 = 0.74 μM, CC50 = 1.22 μM (Vero cells)	29

^aSARS-CoV, severe acute respiratory syndrome coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus.

Figure 2.

The chemical structures of salicylamide derivatives 4-13

2.2. Anti-Flavivirus Activity

The flaviviruses (genus Flaviviruses) are a diverse group of over 50 species of arthropod-borne viruses (arboviruses) in the family Flaviviridae, many of which are human pathogens of global importance⁵⁷. Among them, dengue virus four serotypes (DENV 1–4), Zika virus (ZIKV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV), and West Nile virus (WNV) are the best-known which can cause significant morbidity and mortality around the world⁵⁸. DENV infection can cause life-threatening diseases called severe dengue, including dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS)⁵⁹. Approximately 3.9 billion people in 128 countries are at risk of DNEV infection, and 390 million dengue infections are estimated to occur annually, leading to about 500,000 cases of severe dengue and 22,000 deaths worldwide⁶⁰. Several significant ZIKV outbreaks have occurred worldwide in recent years, resulting in devastating diseases such as congenital microcephaly and Guillain-Barré syndrome³¹. Currently, effective vaccines are available for YFV, JEV, and TBEV but do not exist for WNV and ZIKV⁵⁷; a DENV vaccine (Dengvaxia^®) was recently approved in a few countries for people aged 9 to 45 years and further recommended to those with confirmed previous DENV infection by WHO⁵⁹. However, there remains no clinically approved antiviral therapy to manage these viruses⁶¹.

Based on the previous finding that ZIKV infection of human neural progenitor cells (hNPCs) leads to an increased caspase-3 activation and subsequent cell death⁶², Xu et al. carried out a phenotypic screen via using the caspase-3 activity assay and identified niclosamide as an inhibitor of ZIKV infection that significantly suppressed production of infectious ZIKV particles with an IC₅₀ value of 0.28 μM⁶³. To improve the physicochemical properties, their group further conducted SAR studies focusing on the anilide and salicylic acid regions of niclosamide⁵³. Introducing various substituents in these two regions generated compounds 4–9 and 14-17 (Figures 2 & 3), which displayed sub-micromolar to low micromolar inhibitory activities against ZIKV (Table 2). Modifications of 2´-H (4), 2´-OMe (5), and 5-Br (9) substitutions are advantageous with increased aqueous solubility and permeability, compared to niclosamide⁵³. After screening 2816 approved and investigational drugs through a novel luciferase complementation (SLC)-based high-throughput screening (HTS) assay, our research team independently identified niclosamide and nitazoxanide as flavivirus N2B-NS3 interaction inhibitors which target the interface between viral protease NS3 and its cofactor NS2B and result in accumulation of nonfunctional viral polyprotein precursor⁶⁴. Niclosamide inhibited the NS2B-NS3 protease activity with an IC₅₀ value of 12.3 μM and showed similar in vitro efficacies for ZIKV, DENV-2, WNV, JEV, and YFV with EC₅₀ values of 0.48–1.02 μM⁶⁴. Nitazoxanide displayed an IC₅₀ value of 15.9 μM against the NS2B-NS3 protease and EC₅₀ values of 1.48 μM and 0.39 μM against ZIKV and JEV, respectively^64,65. In vivo studies showed that nitazoxanide treatment can protect mice from the challenge of JEV⁶⁵. Further screening a small library of niclosamide derivatives, we found compounds 18-20 maintained sub-micromolar potency against ZIKV and DENV-2, and 19 (JMX0207) with a 3-NO₂ group also exhibited improved efficacy in inhibition of NS2B-NS3 interaction. Intriguingly, JMX0207 displayed significantly improved pharmacokinetic (PK) properties and the in vivo efficacy in the A129 ZIKV mouse model³¹. The docking studies indicated that JMX0207 and niclosamide could be well docked into the NS3 pocket, termed as 2B53 pocket, in a similar manner using the induced fit docking (IFD) protocol (Figure 4)^31,64. JMX0207 and niclosamide were equally effective in reducing ZIKV titer even added at 24 h post-infection, suggesting that they act at a post-entry step^31,64. Besides N2B-NS3 interaction inhibition, niclosamide was found to neutralize the low-pH intracellular compartments, which affect multiple steps of the DENV life cycle, including viral and host membrane fusion and uncoating, viral RNA replication, and maturation of virus particles rendering it non-infectious^66,67.

Figure 3.

The chemical structures of salicylamide derivatives 14-20

Table 2.

The activity of salicylamide derivatives against flavivirus^a

Compound	Activity against flavivirus	Ref.
Niclosamide (1)	ZIKV: EC50 = 0.28 μM (SNB-19 cells); EC50 = 0.48 μM, CC50 = 4.8 μM (A549 cells/ PRV-ABC59) DENV-2: EC50 = 0.55 μM (A549 cells) IC50-pro = 12.3 μM (DENV-2 NS3-MBP fusion protein) WNV: EC50 = 0.54 μM (A549 cells) JEV: EC50 = 1.02 μM (A549 cells) YFV: EC50 = 0.84 μM (A549 cells)	63,64
Nitazoxanide (2)	ZIKV: EC50 = 1.48 μM, CC50 = 4.8 μM (A549 cells/ PRV-ABC59) IC50-pro = 15.9 μM (DENV-2 NS3-MBP fusion protein) JEV: EC50 = 0.39 μM, CC50 = 60.5 μM (BHK-21 cells)	64,65
Tizoxanide (3)	Completely abolished supernatant ZIKV proliferation at 10 μM	225
4	ZIKV: EC50 = 0.82 μM (SNB-19 cells/ viral titer reduction); EC50 = 2.5 μM (HEK293 cells/ NS1 assay), cell viability: 61% at 30 μM in SNB-19 cells	53
5	ZIKV: EC50 = 0.82 μM (SNB-19 cells/ viral titer reduction); EC50 = 2.5 μM (HEK293 cells/ NS1 assay), cell viability: 59% at 30 μM in SNB-19 cells	53
6	ZIKV: EC50 = 0.81 μM (SNB-19 cells/ viral titer reduction); EC50 = 1.7 μM (HEK293 cells/ NS1 assay), cell viability: 63% at 30 μM in SNB-19 cells	53
7	ZIKV: EC50 = 0.84 μM (SNB-19 cells/ viral titer reduction); EC50 = 0.8 μM (HEK293 cells/ NS1 assay), cell viability: 59% at 30 μM in SNB-19 cells	53
8	ZIKV: EC50 = 0.4 μM (SNB-19 cells/ viral titer reduction); EC50 = 1.6 μM (HEK293 cells/ NS1 assay), cell viability: 55% at 30 μM in SNB-19 cells	53
9	ZIKV: EC50 = 0.11 μM (SNB-19 cells/ viral titer reduction); EC50 = 0.84 μM (HEK293 cells/ NS1 assay), cell viability: 59% at 30 μM in SNB-19 cells	53
14	ZIKV: EC50 = 1.72 μM (HEK293 cells/ NS1 assay)	53
15	ZIKV: EC50 = 1.74 μM (HEK293 cells/ NS1 assay)	53
16	ZIKV: EC50 = 3.0 μM (HEK293 cells/ NS1 assay)	53
17	ZIKV: EC50 = 2.34 μM (HEK293 cells/ NS1 assay)	53
18	ZIKV: EC50 = 0.15 μM, CC50 = 50.2 μM (A549 cells/ PRV-ABC59) DENV-2: EC50 = 0.21 μM (A549 cells) IC50-pro = 44.5 μM (DENV-2 NS3-MBP fusion protein)	31
19	ZIKV: EC50 = 0.30 μM, CC50 = 31.9 μM (A549 cells/ PRV-ABC59) DENV-2: EC50 = 0.31 μM (A549 cells) IC50-pro = 8.2 μM (DENV-2 NS3-MBP fusion protein)	31
20	ZIKV: EC50 = 1.1 μM, CC50 = 241 μM (A549 cells/ PRV-ABC59) DENV-2: EC50 = 0.9 μM (A549 cells) IC50-pro = 20.3 μM (DENV-2 NS3-MBP fusion protein)	31

^aDENV, dengue virus; ZIKV, Zika virus; YFV, yellow fever virus; JEV, Japanese encephalitis virus; TBEV; tick-borne encephalitis virus; WNV, West Nile virus.

Figure 4.

A) JMX0207 (magenta) docked into NS3pro of DENV2 (PDB Code: 2FOM). Binding site key interaction residues are highlighted in gray sticks. H-bonds are shown as purple dotted lines and π cation and π stacking are shown as cyan dotted lines. B) Niclosamide (green) docked into NS3pro of DENV2. H-bonds are shown as purple dotted lines. C) JMX0207 (magenta) and Niclosamide (green) superimposed at the predicted binding site in surface representation.

2.3. Anti-Hepatitis B Virus Activity

Hepatitis B virus (HBV) is a partially double-stranded DNA virus belonging to the genus Orthohepadnavirus of the family Hepadnaviridae, which can cause both acute (AHB) and chronic hepatitis B (CHB)^68–70. HBV is primarily transmitted through contact with infected blood and body fluids including vertical transmission from mother to child during delivery or sexual contact⁷⁰. HBV infection occurring in fancy or early childhood usually leads to lifelong chronic infection and carriage of the virus. In contrast, infection acquired later in life commonly develops into an acute, self-limiting illness with final successful control of the virus⁷¹. WHO estimated that in 2015, 257 million people had chronic HBV infection, 2.7 million people were coinfected with HBV and human immunodeficiency virus (HIV), and about 887,000 people died from long-term complications of HBV infection, mainly cirrhosis and hepatocellular carcinoma^72,73. Vaccination remains the most effective measure to prevent HBV infection. HBV treatment is limited to either a few nucleoside/nucleotide drugs that inhibit reverse transcription or immunomodulatory agents such as conventional or pegylated type I interferons (IFNs) and is recommended for chronically infected patients with persistently elevated HBV DNA levels and serum alanine aminotransferase (a marker of liver damage)^71,74. However, current anti-HBV drugs rarely attain complete viral elimination from patients (HBV cure), and a functional cure defined as the loss of detectable serum hepatitis B surface antigen (HBsAg) is a more practical goal for anti-HBV therapy, which is also not frequently achieved with currently available treatment⁷⁴.

Nitazoxanide and tizoxanide were reported to inhibit both intracellular HBV replication (EC₅₀ = 0.59 μM and 0.46 μM, respectively) and extracellular virus production (EC₅₀ = 0.12 μM and 0.15 μM, respectively) with low cytotoxicity and high selectivity index (Table 3)⁵¹. Nitazoxanide could efficiently suppress the interaction between HBV regulatory protein X (HBx) and the host protein damage-specific DNA-binding protein 1 (DDB1), leading to the restoration of structural maintenance of chromosomes 5 (Smc5) protein levels and inhibition of viral transcription and viral protein production in the HBV minicircle system as well as in human primary hepatocytes naturally infected with HBV⁷⁵. Two successful cases were reported that patients with CHB receiving nitazoxanide treatment or combination therapy of nitazoxanide with adefovir for two years developed undetectable HBV DNA as well as negative HBeAg and HBsAb⁷⁶. In one pilot clinical trial involving 12 adults with CHB receiving nitazoxanide treatment (500 mg, twice daily for 12 months), 7 of 8 patients with hepatitis B e antigen (HBeAg) negative initially became HBV DNA negative, and two of them became HBsAg negative; 3 of 4 patients with HBeAg positive initially became HBeAg negative, two of them became HBV DNA negative, and one patient became HBsAg negative⁷⁷. In another clinical trial, 8 of 9 patients with CHB displayed negative serum HBV DNA after nitazoxanide treatment for 4–20 weeks. Two patients with HBeAg positive initially became HBeAg negative, and 3 of 9 patients became HBsAg negative⁷⁸. These data provide clinical evidence of the therapeutic potential of nitazoxanide in treating patients with CHB, and the clinical trial (NCT03905655) has been initiated to evaluate the effectiveness of the combined use of nitazoxanide with existing regions for the treatment of CHB.

Table 3.

Activities of salicylamides derivatives against HBV replication^a

Compound	extracellular virion DNA		intracellular HBV RI		CC50 (μM)	SI
	EC50 (μM)	EC90 (μM)	EC50 (μM)	EC90 (μM)		virion	RI
Nitazoxanide (2)	0.12	0.83	0.59	2.10	>100	>121	>48
Tizoxanide (3)	0.15	0.58	0.46	1.20	>100	>172	>83
21	0.64	3.5	2.10	12.0	85	24	7.1
22	0.33	0.89	1.00	2.90	38	43	13
23	0.33	0.83	0.90	2.0	100	>120	>51
24	0.32	2.00	0.59	3.90	30	15	7.7
25	0.21	1.10	3.50	11.0	45.0	41	4.1
26	3.2	9.4	5.5	14.0	>100	>11	>7.1
27	3.8	11.0	9.1	27.0	>100	>9.1	>3.7
28	0.22	0.73	7.80	30.0	>100	>137	3.3
29	0.76	3.00	2.10	6.80	>100	>33	>15
30	0.20	1.20	0.73	3.30	>100	>83	>30
31	3.70	13.0			>100
32	0.28	0.73	0.66	1.7	89	122	52
Niclosamide (1)	>10.0	>10.0			>100

^aCC₅₀: drug concentration at which a 2-fold lower level of neutral red dye uptake is observed relative to the average level in untreated cultures. RI: replication intermediate. EC₅₀ or EC₉₀: drug concentration required to reduce extracellular virion HBV DNA or intracellular HBV DNA by 50% or 90% relative to untreated cells. SI (virion) or SI (RI): CC₅₀/EC₉₀ for extracellular or intracellular HBV.

The SAR studies on nitazoxanide derivatives as antiviral agents were conducted by Stachulski et al., and thiazolides 21-28 (Figure 5) with different substitutions exhibited sub-micromolar to micromolar EC₅₀ values against both intracellular HBV intermediates and extracellular HBV virions (Table 3)²⁷. The nitro group at the C-5 position of the thiazole ring is not essential for anti-HBV activity. It can be substituted with other electron-withdrawing groups as exemplified by compounds 22-27, while substituting the phenyl ring displayed varied effects on activity. As the nitro group is often classified as a structural alert or a toxicophore due to its potential toxicity issues⁷⁹, compound 22 with 5-Cl substitution excludes this liability and maintains the same level of potency against HBV, compared to nitazoxanide, thus selected for further development²⁷. In general, salicylanilide derivatives were less active and showed variable anti-HBV activities, depending on the exact substituents on the phenyl ring. The monosubstituted analogs 29, 30, and trisubstituted 32 showed suitable activities against HBV, while disubstituted 31 displayed moderate potency. Due to the poor oral bioavailability and limited aqueous solubility presented by niclosamide, which was close in structure and inactive against HBV up to 10 μM, further optimization efforts on this class of derivatives as anti-HBV agents were not pursued by Stachulski et al^27,80.

Figure 5.

The chemical structures of salicylamide derivatives 21–32

2.4. Anti-Hepatitis C Virus Activity

Hepatitis C virus (HCV) is a hepatotropic virus with a positive-sense single-stranded RNA genome which comprises at least 6 genotypes and more than 80 subtypes belonging to the genus Hepacivirus of the family Flaviviridae^81–83. HCV is transmitted mainly through direct blood-to-blood contacts between humans and can cause both acute (AHC) and chronic hepatitis C (CHC); 30% of patients infected with HCV can spontaneously clear the infection within 6 months without any treatment, while the remaining 70% of patients will develop CHC, triggering a chronic inflammatory disease process which can lead to cirrhosis and hepatocellular carcinoma if left untreated^84,85. WHO estimated that in 2015, 71 million persons were living with HCV infection globally, 2.3 million had HCV and HIV co-infection, and about 400,000 persons died from long-term complications of HCV infection, mainly cirrhosis and hepatocellular carcinoma like hepatitis B⁷³. Due to the genetic diversity of HCV and limited models for testing vaccines, developing a vaccine for hepatitis C remains a challenge, and no effective vaccine is currently available to prevent HCV infection⁸³. On the other hand, the emergence of direct-acting antivirals such as sofosbuvir led to a revolution in HCV therapy which is usually used in combination and can cure most people of their HCV infection, regardless of HCV genotype^85–87.

Nitazoxanide and tizoxanide also exhibited sub-micromolar potency against HCV genotype 1a (EC₅₀ = 0.33 μM and 0.25 μM, respectively) and genotype 1b (EC₅₀ = 0.21 μM and 0.15 μM, respectively)⁵¹. Nitazoxanide can activate protein kinase activated by double-stranded RNA (PKR), a key mediator of the innate immune response, which phosphorylates eukaryotic initiation factor-2α (eIF2α), leading to the inhibition of translation initiation and thereby affecting viral protein synthesis^88,89. In a study using bovine viral diarrhea virus (BVDV) as a surrogate model for HCV infection, nitazoxanide was shown to reduce both cytopathic and non-cytopathic BVDV replication, likely involving phosphorylation of PKR and eIF2α⁹⁰. Besides, nitazoxanide was also found to delete ATP-sensitive intracellular Ca²⁺ stores, leading to mild endoplasmic reticulum (ER) stress and consequent perturbation of N-linked glycosylation of BVDV structural proteins⁹⁰.

In a randomized clinical trial involving 47 adults with CHC genotype 4, 7 of 23 patients receiving nitazoxanide monotherapy achieved undetectable serum HCV RNA compared to 0 of 24 receiving placebo during therapy, and 4 of 6 virological responders, who were followed up for 24 weeks after treatment, suffered a sustained virological response (SVR)⁹¹. A pilot clinical trial (96 patients with CHC genotype 4) revealed that 36-week combination therapy of nitazoxanide plus PEG-IFN α−2a with (n = 28) or without (n = 28) ribavirin following a 12-week lead-in nitazoxanide treatment increased the percentages of patients with SVR (SVR rate = 79% and 61%, respectively), compared to that of patients (n = 40, SVR rate = 50%) treated with PEG-IFN plus ribavirin⁹². In another pilot, clinical trial enrolling 44 patients with CHC (40 with genotype 4; 3 with genotype 1; and 1 with genotype 2), an SVR rate of 80% was observed when patients received 36-week combination therapy of nitazoxanide plus PEG-IFN α−2a using a 4-week lead-in nitazoxanide monotherapy⁹³. In addition, coadministration of nitazoxanide with PEG-IFN α−2a and ribavirin also increased the SVR rate in CHC genotype 1 patients, showing SVR rates of 44% for naïve patients (n = 73) and 7% for patients with nonresponse to combination therapy of PEG-IFN plus ribavirin (n = 42), while the control groups (n = 37 and 21, respectively) receiving placebo plus PEG-IFN α−2a and ribavirin achieved SVR rates of 32% and 0%, respectively, in two randomized, double-blind, placebo-controlled clinical trials^94,95. Nevertheless, a conflicting result was obtained in a later clinical trial enrolling 100 patients with CHC genotype 4 that patients (n = 50) receiving combination therapy of nitazoxanide, PEG-IFN α−2a, and ribavirin showed no significant improvement in the virological or biochemical response rates as compared with those (n = 50) treated with PEG-IFN α−2a plus ribavirin⁹⁵. Due to the development of direct-acting antivirals, no more clinical trials were further conducted to validate the clinical benefit of nitazoxanide in HCV treatment.

Stachulski et al. evaluated the salicylamide derivatives they developed against HCV, and compounds 21 and 33–41 (Figures 5 & 6) displayed sub-micromolar to micromolar potency against HCV genotype 1a and/or 1b (Table 4)^28,51. Interestingly, the 5´-Cl analog 22 was basically inactive, in contrast to its sub-micromolar potency against HBV, while its O-acetate 33 displayed EC₅₀ values of <1 μM against both HCV genotype 1a and 1b. Contrarily, the m-fluoro derivative 40 inhibited HCV genotype 1b replication with sub-micromolar EC₅₀ value while its O-acetate 39 performed less well. Although niclosamide was inactive against HBV, it showed potent activity against HCV genotype 1b^27,28. The SAR studies showed the anti-HCV activities of these derivatives are jointly affected by the substituents on both aromatic rings and electron-withdrawing groups are preferred at the C-5 and C-4 positions of the thiazole ring.

Figure 6.

The chemical structures of salicylamide derivatives 33–41

Table 4.

Activities of salicylamides derivatives against HCV replication^a

Compound	Genotype 1b replicon (μM)				Genotype 1a replicon (μM)
	EC50	EC90	CC50	SI	EC50	EC90	CC50	SI
Nitazoxanide (2)	0.21	0.93	38.0	181	0.33	1.10	49.0	149
Tizoxanide (3)	0.15	0.81	15.0	100	0.25	1.00	14.0	56
21	0.36	6.4	5.0	14	0.39	2.30	12.0	31
22	10.0	10.0	15.0
33	0.23	1.10	4.3	19	0.40	1.90	5.7	14
34	2.0	5.9	>100	>50
35	0.59	23.0	12.0	20	1.00	3.80	14.0	14
36	4.2	14.0	45.0	11
37	3.5	10.0	26.0	7.4
38	2.2	7.0	16.0	7.3
39	3.8	11.0	>100	26
40	0.13	0.83	3.4	26
41	0.06	0.46	4.4	76
Niclosamide (1)	0.16	0.70	10.0	62

^aAntiviral activity was assessed in a 3-day assay using the stably expressed HCV replicon cell lines AVA5 (for genotype 1B) and H/FL-Neo (for genotype 1A). CC₅₀: drug concentration at which a 2-fold lower level of neutral red dye uptake is observed relative to the average level in untreated cultures. EC₅₀ and EC₉₀ were determined by measuring intracellular HCV RNA concentration in triplicate and with standard deviations of ±20% of the value quoted. SI: CC₅₀/EC₅₀

2.5. Anti-Influenza A Virus Activity

Influenza viruses are enveloped viruses of the family Orthomyxoviridae with a negative-sense, single-stranded, segmented RNA genome including four types termed influenza viruses A, B, C, and D (IAV, IBV, ICV, and IDV), which can infect various host species such as humans, birds, and pigs^96,97. IAV and IBV circulate and cause annual epidemics of seasonal influenza, characterized by a sudden onset of fever, cough, sore throat, headache, a runny nose, muscle, and joint pain, etc., which results in millions of human infections and 291,243~645,832 associated deaths estimated per year globally along with significant health and financial burdens^98–100. Among them, IAVs are especially important to public health due to their potential to cause flu pandemics⁹⁹. IAVs are further subtyped according to the combinations of two surface glycoproteins, haemagglutinin (HA) and neuraminidase (NA), which facilitate viral entry and release, respectively, served as the main targets for the protective antibodies induced by the influenza virus and vaccination⁹⁹. At present, there have been 16 antigenically different HA subtypes and 9 antigenically different NA subtypes identified in avian species, the main animal reservoir of influenza viruses¹⁰¹. IAV subtypes H1N1 and H3N2 are currently circulating in humans⁹⁶. When most humans lack pre-existing immunity for HA or NA proteins of the new emerged IAV, which are usually transmitted from reservoirs such as birds and pigs to humans directly or originated through reassortment of an avian influenza virus with a human influenza virus, IAV pandemics can occur and typically cause higher morbidity and mortality among humans, e.g., the 1918 H1N1 Spanish influenza and 2009 H1N1 swine-origin pandemic^102–104. Vaccines are the most effective way to prevent seasonal influenza and they need to be updated each year to match the expected circulating strains due to the constantly evolving state of influenza viruses¹⁰⁵. Currently, the NA inhibitors (NAIs), oseltamivir and zanamivir, are usually used to treat influenza virus infection in most countries. Still, the drug resistance remains a continuing challenge, highlighting an urgent need to develop a novel generation of anti-influenza agents^99,106.

Nitazoxanide and tizoxanide were reported to exhibit significant in vitro activities against a series of IAV and IBV strains, including oseltamivir-resistant and amantadine-resistant strains, with sub-micromolar to micromolar EC₅₀ values (Table 5)^107–110. Nitazoxanide affects the maturation of the viral hemagglutinin, impairs its intracellular transport and insertion into the host cell plasma membrane, and thus blocks the exit of mature virions from host cells¹⁰⁹. Nitazoxanide functions at a post-translational level, different from the currently available anti-influenza drugs, the NA-inhibitors oseltamivir, and zanamivir, which prevent the release of virus from the infected cell, and the M2 inhibitors amantadine and rimantadine, which affect virus uncoating¹⁰⁹. A synergistic effect was observed for the combination treatment of nitazoxanide with oseltamivir or zanamivir against H1N1 A/PR/8/34 (PR8) and H1N1 A/WSN/33 (WSN)¹⁰⁸. In addition, in the flu-stimulated peripheral blood mononuclear cells (PBMCs), nitazoxanide was found to potentiate the production of type I IFN (α and β) produced by the host’s fibroblasts, but the importance of this activity is not fully elucidated¹¹¹.

Table 5.

The activity of salicylamide derivatives against influenza viruses

Compound	Activity against influenza viruses	Ref.
Niclosamide (1)	H1N1 A/PR/8/34: EC50 = 0.83 μM, CC50 = 20.2 μM (A549 cells)	115
Nitazoxanide (2)	H1N1 A/PR/8/34 (PR8): EC50 = 3.3 μM, CC50 > 163 μM (MDCK cells) H1N1 A/WSN/33 (WSN): EC50 = 1.6 μM (MDCK cells) H5N9 A/Ck/It/9097/97 (A/Ck): EC50 = 3.3 μM (MDCK cells) H1N1 A/California/7/2009 (CA09): EC50 = 3.2 μM (MDCK cells) H1N1 oseltamivir-resistant A/Parma/24/2009 (OST-R): EC50 = 1.9 μM (MDCK cells) H3N2 amantadine-resistant A/Parma/06/2007 (AMD-R): EC50 = 1.0 μM (MDCK cells) H1N1 A/goose/Italy/296246/2003 (A/Tk): EC50 = 3.2 μM (MDCK cells) H7N1 A/turkey/Italy/RA5563/1999: EC50 = 1.6 μM (MDCK cells) H3N2v A/Ohio/83/2012: EC90 = 0.86 μM (MDCK cells) H3N2v A/Ohio/88/2012: EC90 = 1.09 μM (MDCK cells)	107–109
Tizoxanide (3)	H1N1 A/PR/8/34 (PR8): EC50 = 3.8 μM, CC50 > 189 μM (MDCK cells) H1N1 A/WSN/33 (WSN): EC50 = 1.9 μM (MDCK cells) H5N9 A/Ck/It/9097/97 (A/Ck): EC50 = 1.9 μM (MDCK cells) H1N1pdm09 (54 strains): EC50 = 0.1–1.23 μM (MDCK-SIAT cells) H3N2 (53 strains): EC50 = 0.22–1.14 μM (MDCK-SIAT cells) IBV Yamagata lineage (56 strains): EC50 = 0.34–0.88 μM (MDCK-SIAT cells) IBV Victoria lineage (47 strains): EC50 = 0.25–0.92 μM (MDCK-SIAT cells) NAI-resistant IAVs (10 strains): EC50 = 0.31–1.00 μM (MDCK-SIAT cells) NAI-resistant IBVs (4 strains): EC50 = 0.79–0.92 μM (MDCK-SIAT cells)	109,110
21	H1N1 A/PR/8/34 (PR8): EC50 = 7.2 μM, CC50 = 129 μM (MDCK cells)	30
26	H1N1 A/PR/8/34 (PR8): EC50 = 4.1 μM, CC50 > 204 μM (MDCK cells)	30
33	H1N1 A/PR/8/34 (PR8): EC50 = 3.4 μM, CC50 = 67 μM (MDCK cells)	30
42	H1N1 A/PR/8/34 (PR8): EC50 = 0.4 μM, CC50 > 189 μM (MDCK cells)	30
43	H1N1 A/PR/8/34 (PR8): EC50 = 0.29 μM, CC50 > 168 μM (MDCK cells)	30
44	H1N1 A/PR/8/34 (PR8): EC50 = 0.14 μM, CC50 > 141 μM (MDCK cells)	30
45	H1N1 A/WSN/33 (WSN): EC50 = 3.0–8.9 μM, CC50 > 297 μM (MDBK cells)	116
46	H1N1 A/WSN/33 (WSN): EC50 = 0.66–1.0 μM, CC50 > 332 μM (MDBK cells)	116
47	H1N1 A/WSN/33 (WSN): EC50 = 0.25 μM, CC50 = 110 μM (MDBK cells)	117
48	H1N1 A/WSN/33 (WSN): EC50 = 0.57 μM (MDBK cells)	118
49	H1N1 A/WSN/33 (WSN): EC50 = 0.18 μM, CC50 = 246 μM (MDCK cells)	119,120

In a double-blind, randomized, placebo-controlled, phase 2b/3 trial involving 624 patients aged 12–65 years with influenza-like illness (ILI) within 48 h of symptom onset, influenza-confirmed patients receiving nitazoxanide 600 mg (n = 79) or 300 mg (n = 89) twice daily for 5 days showed shorter times to alleviation of symptoms of 95.5 h (p = 0.0084) and 109.1 h (p = 0.52, not statistically significant), respectively, than those receiving placebo (116.7 h, n = 89)⁵⁰. Moreover, a significant decrease (p = 0.0006) in TCID₅₀ viral titers was shown by the nitazoxanide 600 mg group during treatment compared to the placebo group⁵⁰. Similar results were also observed for all treated participants that the nitazoxanide 600 mg group (n = 211) experienced a significantly shorter duration of symptoms (94.9 h, p = 0.0052) as compared with the control group (108.2 h, n = 212)⁵⁰. Besides, in two clinical trials, compared to the placebo group, nitazoxanide treatment reduced the time of symptom resolution by >3 and 3 days in children (1–11 years of age) and adults and adolescents (≥ 12 years of age) with a viral respiratory infection, respectively^112,113. However, another double-blind, placebo-controlled trial involving 257 patients with severe ILI revealed that there was no significant difference in duration of hospitalization between nitazoxanide treatment and placebo treatment in all anticipants (p = 0.56), children (p = 0.29), adults (p = 0.62), and influenza-confirmed patients (p = 0.32)¹¹⁴. Thus, more clinical trials are needed to evaluate the efficacy of nitazoxanide as monotherapy or in combination with NAIs, such as oseltamivir or zanamivir, for the treatment of influenza infection in otherwise healthy patients as well as those with severe illness or at risk of influenza complications.

Stachulski et al. designed and synthesized the second-generation thiazolides as effective IAV inhibitors and, among these derivatives, 42 with para hydroxyl, 43 and 44 with 4´-sulfonyl displayed outstanding potency against IAV with low sub-micromolar EC₅₀ values, low cytotoxicity, and very high selectivity index (Figure 7 & Table 5)³⁰. The insertion of alkylsulfone groups at the C4 position led to a significant improvement in anti-IAV activity. Niclosamide was found to inhibit influenza H1N1 A/PR/8/34 virus with an EC₅₀ of 0.83 μM¹¹⁵. Researchers at Bristol Myers Squibb reported a series of N-alkyl substituted salicylamide derivatives as potent inhibitors against the H1 and H2 subtypes of IAV, represented by compounds 45-49^116–120. Compound 45 (BMY-27709) was found to be active against the H1 subtype influenza viruses A/WSN/33 (EC₅₀ = 3.0–8.9 μM) and A/PR/8/34 as well as against the H2 subtype influenza virus A/Japan/307/57, but it was inactive against H3 subtype viruses (A/HK/8/68 and A/Udorn/307/57) and influenza B/Lee/40 virus, partly limiting the significant utility of this series of compounds as anti-influenza agents¹²¹. Genetic analysis revealed that the mutation from Phe to Ser at position 110 of the HA2 subunit conferred resistance to BMY-27709 inhibition, indicating that this class of compounds likely target the hemagglutinin protein. BMY-27709 blocks viral replication at an early stage of the IAV life cycle and inhibits the virus-induced red blood cell hemolysis in a dose-dependent manner¹²¹. Further mechanistic studies suggested that BMY-27709 inhibits the HA-mediated endosomal fusion of virus and cell membrane through suppressing the essential low-pH-induced conformational change of the HA protein^118,122. Removing the 4-NH₂ substitution on the phenyl ring of BMY-27709 led to a significant increase in activity, as shown by compound 46¹¹⁶. Compounds 47–49 with diversified cycloalkyl or heterocycloalkyl groups all showed potent inhibitory activity against H1N1 A/WSN/33 virus with sub-micromolar EC₅₀ values^117–120.

Figure 7.

The chemical structures of salicylamide derivatives 42–49

2.6. Anti-Human Adenovirus Activity

Human adenoviruses (HAdVs) are non-enveloped viruses with a linear and double-stranded DNA genome of approximately 34–36 kb in size, which includes more than 100 different HAdV types classified into seven species (HAdV A-G)^123–127. HAdV infections are widespread in humans, primarily occurring in young children, which can cause respiratory disease, gastroenteritis, hepatitis, conjunctivitis, myocarditis, etc. In healthy individuals, HAdV infections are usually self-limiting with mild symptoms, providing long-lasting immunity to that infected type after recovery^128,129. However, in immunocompromised patients, such as solid-organ transplant (SOT) and allogenic hematopoietic stem cell transplant (allo-HSCT) recipients, who cannot easily clear the virus, HAdV infection can disseminate to multiple organ systems and cause life-threatening diseases^128,130. Currently, there is no antiviral specifically approved for treating HAdV infections, and the off-label use of intravenous cidofovir remains the major therapeutic choice to treat these infections, with unsatisfactory clinical responses and significant toxicity issues^125,131.

Our research team first found that niclosamide, oxyclozanide (50), and rafoxanide (51) exhibited significant anti-HAdV activity with low micromolar IC₅₀ values (Figure 8 & Table 6)¹³². The mechanistic studies indicated that niclosamide and rafoxanide inhibit the accessibility of the HAdV genome to the nucleus while oxyclozanide impacts the transcription of HAdV immediate early gene E1A¹³². To develop highly effective and safe anti-HAdV agents, we further conducted SAR optimization studies using niclosamide as a lead compound and discovered a series of salicylamide derivatives which showed equal or improved anti-HAdV activity with nanomolar to submicromolar IC₅₀ values and significantly increased selectivity index (SI > 100), as exemplified by compounds 52-57^25,133,134. Our mechanistic assays suggested different mechanisms of HAdV inhibition for these derivatives; compounds 52, 54, and 56 inhibit later steps after DNA replication, 53 likely targets the HAdV DNA replication, and 57 stabilizes the viral capsid to prevent the uncoating and blocks HAdV particle escape from the endosome^25,134. Moreover, compounds 54-56 displayed very low in vivo toxicity in the immunosuppressed Syrian hamster model, presenting maximum tolerated dose (MTD) values of 12.5 mg/kg, 150 mg/kg, and 50 mg/kg, respectively, as compared with niclosamide (MTD = 1 mg/kg), and thus were selected for further in vivo PK and efficacy evaluation for the treatment of HAdV infection¹³³.

Figure 8.

The chemical structures of salicylamide derivatives 50–57

Table 6.

The activity of salicylamide derivatives against human adenovirus^a

Compound	Activity against human adenovirus	Ref.
Niclosamide (1)	HAdV-5: IC50 = 0.6 ± 0.05 μM (293β5 cells); CC50 = 22.9 ± 9.8 μM (A549 cells); SI = 38.2 HAdV-16: IC50 = 0.45 ± 0.1 μM (293β5 cells) Virus yield reduction (HAdV-5 WT): 82 ± 35 folds at 5 μM Virus yield reduction (HAdV-16 WT): 21 ± 0 folds at 5 μM Maximum tolerated dose (MTD) = 1 mg/kg	132
Oxyclozanide (50)	HAdV-5: IC50 = 2.3 ± 0.7 μM (293β5 cells); CC50 = 76.1 ± 14.4 μM (A549 cells); SI = 33.0 HAdV-16: IC50 = 1.26 ± 0.0 μM (293β5 cells) Virus yield reduction (HAdV-5 WT): 10 ± 2 folds at 25 μM Virus yield reduction (HAdV-16 WT): 26 ± 7 folds at 25 μM	132
Rafoxanide (51)	HAdV-5: IC50 = 1.3 ± 0.1 μM (293β5 cells); CC50 = 80.6 ± 34.7 μM (A549 cells); SI = 62.0 HAdV-16: IC50 = 1.38 ± 0.2 μM (293β5 cells) Virus yield reduction (HAdV-5 WT): 186 ± 58 folds at 25 μM Virus yield reduction (HAdV-16 WT): 42 ± 6 folds at 25 μM	132
52	HAdV-5: IC50 = 0.05 ± 0.01 μM (293β5 cells); CC50 = 10.9 ± 0.5 μM (A549 cells); SI = 218.2 Virus yield reduction (HAdV-5 WT): 1.8 ± 0.3 folds at 2 μM	25
53	HAdV-5: IC50 = 0.08 ± 0.00 μM (293β5 cells); CC50 = 35.0 ± 3.7 μM (A549 cells); SI = 437.5 Virus yield reduction (HAdV-5 WT): 175 ± 33 folds at 10 μM	25
54	HAdV-5: IC50 = 0.18 ± 0.1 μM (293β5 cells); CC50 = 120.0 ± 33.6 μM (A549 cells); SI = 666.7 Virus yield reduction (HAdV-5 WT): 989 ± 361 folds at 50 μM MTD = 12.5 mg/kg	25
55	HAdV-5: IC50 = 0.27 ± 0.16 μM (293β5 cells); CC50 = 156.8 ± 11.0 μM (A549 cells); SI = 580.7 Virus yield reduction (HAdV-5 WT): 52.6 ± 6.2 folds at 2.7 μM MTD = 150 mg/kg	133
56	HAdV-5: IC50 = 0.45 ± 0.06 μM (293β5 cells); CC50 = 200.0 ± 1.9 μM (A549 cells); SI = 444.4 Virus yield reduction (HAdV-5 WT): 213 ± 78 folds at 50 μM MTD = 50 mg/kg	25
57	HAdV-5: IC50 = 0.78 ± 0.01 μM (293β5 cells); CC50 = 91.2 ± 18.4 μM (A549 cells); SI = 116.9 Virus yield reduction (HAdV-5 WT): 208 ± 108 folds at 7.8 μM	134

^aHAdV, human adenovirus; MTD, maximum tolerated dose; SI: CC₅₀/EC₅₀.

2.7. Anti-Respiratory Syncytial Virus Activity

RSV is an enveloped virus with a non-segmented, negative-sense, single-stranded RNA genome which consists of two major subgroups A and B, belonging to the Orthopneumovirus genus of the Pneumoviridae family¹³⁵. RSV is the most common cause of acute lower respiratory infection (ALRI) in infants, resulting in airway inflammation, bronchiolitis, pneumonia, and even respiratory failure¹³⁶. Virtually all children get an RSV infection by the age of two, and reinfection may occur in later life, especially during the first few years of life^137,138. It was estimated that globally in 2015, 33.1 million episodes of RSV-ALRI led to about 3.2 million hospital admissions and 59,600 in-hospital deaths in children younger than 5 years; about 45% of RSV-associated hospital admissions and in-hospital deaths occurred in children younger than 6 months¹³⁹. In addition, RSV infection can also be dangerous for the elderly, immunocompromised individuals, and patients with chronic medical conditions, with an increased chance of morbidity and mortality^140–142. Ribavirin is only approved antiviral as an inhaled formulation for RSV infection but is associated with limited efficacy and significant safety concerns¹⁴³. Palivizumab, a monoclonal antibody (mAb) licensed in 1998 for prophylactic use in high-risk infants, is not very cost-effective^144,145. Therefore, an urgent need remains to develop effective, safe, and specific antiviral therapies for RSV infection.

Niclosamide inhibited RSV infection in time- and dose-dependent manners and 6 h-pretreatment of niclosamide at a sub-micromolar concentration presented the highest anti-RSV activity, displaying an EC₅₀ value of 0.022 μM (Table 7)¹⁴⁶. Niclosamide significantly suppressed laboratory and clinical strains of RSV-A and -B in the human bronchial epithelial BEAS-2B cell at a concentration of 0.25 μM. Niclosamide was suggested to inhibit RSV by inactivating protein kinase B (AKT), which led to an increase of apoptotic protein markers (cleaved-caspase-3 and cleaved-PARP) and early apoptosis signal (AKT-apoptosis pathway), independent from pH and mTORC1 inhibition¹⁴⁶. Tizoxanide and thiazolide (22) were found to inhibit RSV with low micromolar EC₅₀ values⁴⁹. Our group identified a series of substituted N-(4-amino-2-chlorophenyl)-5-chloro-2-hydroxybenzamide analogs as potent RSV inhibitors²⁶. In our assay, niclosamide exhibited a 1.47 log reduction in RSV titer at 10 μM. Compounds 58-63 (Figure 9) effectively inhibited RSV replication with reduction in virus titer ranging from 1.74 to 2.59 log, while displaying relatively low cytotoxicity. Moreover, these derivatives suppressed RSV-induced interferon regulatory factor 3 (IRF3) and NF-κB activation and decreased replication-independent and -dependent cytokines/chemokines production, highlighting their great potential to ease RSV-associated symptoms as anti-inflammatory agents²⁶.

Table 7.

The activity of salicylamide derivatives against respiratory syncytial virus (RSV)

Compound	Activity against respiratory syncytial virus	Ref.
Niclosamide (1)	EC50 = 0.022 μM, CC50 = 0.25 μM (Hep-2 cells) Virus titer reduction: 1.47 ± 0.04 log at 10 μM (A549 cells)	26,146
Tizoxanide (3)	EC50 = 1.9 μM, CC50 = 18.9 μM (Hela-ATCC)	49
22	EC50 = 1.6 μM, CC50 = 20.9 μM (Hela-ATCC)	49
58	Virus titer reduction: 1.88 ± 0.43 log at 10 μM; CC50 = 82.7 ± 1.6 μM (A549 cells)	26
59	Virus titer reduction: 1.82 ± 0.14 log at 10 μM; CC50 = 86.5 ± 3.7 μM (A549 cells)	26
60	Virus titer reduction: 2.59 ± 0.15 log at 10 μM; CC50 = 22.9 ± 0.3 μM (A549 cells)	26
61	Virus titer reduction: 2.13 ± 0.23 log at 10 μM; CC50 = 76.3 ± 2.3 μM (A549 cells)	26
62	Virus titer reduction: 1.91 ± 0.33 log at 10 μM; CC50 = 322.6 ± 45.8 μM (A549 cells)	26
63	Virus titer reduction: 1.74 ± 0.33 log at 10 μM; CC50 = 89.9 ± 4.7 μM (A549 cells)	26

Figure 9.

The chemical structures of salicylamide derivatives 58–63

2.8. Activity Against Viral Gastroenteritis

Viral gastroenteritis, a.k.a. stomach flu, is an inflammation of the stomach and intestine with symptoms characterized by watery diarrhea, abdominal pain, nausea or vomiting, and sometimes fever¹⁴⁷. Viral gastroenteritis can be caused by several viruses, including rotaviruses, noroviruses, sapoviruses, astroviruses, enteric adenoviruses, etc^147,148. This illness is commonly transmitted through close contact with an infected person or ingesting contaminated food or water. It can affect people of all ages, causing potentially severe complications in young children and elderly adults who are susceptible to dehydration^148–150. Viral gastroenteritis is typically self-limited within 2–5 days in immunocompetent individuals. However, in immunocompromised patients, this illness can last several weeks to months, even years. Currently, there is no specific antiviral therapy for viral gastroenteritis, and the primary treatment is maintaining adequate hydration¹⁴⁸.

2.8.1. Anti-rotavirus activity

Rotavirus is a genus of non-enveloped, segmented, double-stranded RNA viruses, which includes nine species (rotavirus A-I) belonging to the family Reoviridae^148,151. Rotavirus is the leading cause of severe diarrhea disease in infants and young children (≤ 5 years of age) worldwide, especially in developing countries, accounting for about 40% of diarrhea-related hospitalizations and half a million associated deaths per year^{148,152–155}. In 2006, two new rotavirus vaccines, which were demonstrated to be safe and effective in children, were licensed and recommended by WHO as routine vaccines included in all national immunization programs, significantly decreasing the incidence of severe rotavirus gastroenteritis in many countries^156–158. However, due to inherent limitations of vaccines such as availability and insufficient protection, there remains a need to develop safe and effective antiviral therapy to treat rotavirus infections.

Nitazoxanide and tizoxanide inhibit simian A/SA11-G3P and human WA-G1P rotavirus replication in a dose-dependent manner in MA-104 cells, with EC₅₀ values of 1.9–6.5 μM (Table 8)^159,160. Mechanistic studies indicated that tizoxanide alters rotavirus viroplasm formation by preventing the NSP5/NSP2 interaction, leading to a decrease of rotavirus double-stranded RNA (dsRNA) formation, not affecting viral entry and viral protein synthesis¹⁵⁹. In a double-blind placebo-controlled trial in 50 children with severe rotavirus diarrhea, nitazoxanide treatment (7.5 mg/kg twice for 3 days) showed a short median time of illness resolution of 31 h as compared with 75 h for the control group¹⁶⁰. In another two randomized control trials conducted in hospitalized children with acute rotavirus diarrhea, nitazoxanide treatment was reported to significantly reduce the median duration of illness and hospitalization compared to the standard treatment of diarrhea^161,162. In addition, a randomized, double-blind placebo-controlled clinical trial revealed that nitazoxanide treatment significantly reduced the median time of symptom resolution to 1.5 days in out-patients (≥ 12 years of age) with rotavirus or norovirus related gastroenteritis, compared to 2.5 days for the placebo group¹⁶³.

Table 8.

The activity of salicylamide derivatives against other viruses^a

Virus/strain	Cell line	IC50 or EC50 (μM)			Ref.
		Nitazoxanide	Tizoxanide	Niclosamide
Rotavirus/simian A/SA11-G3P	MA-104	3.3	1.9		159,160
Rotavirus/ human WA-G1P	MA-104	6.5	3.8		159
Human norovirus (HuNV)	HG23	Nitazoxanide and tizoxanide significantly inhibit HuNV replication at 1 μg/mL			171
Human astrovirus strain 1 (HAstV-1)	Caco-2	1.47			180
HIV-1	PBMCs/TZM-bl/ SupT1	87.8% at 10 μg/mL		0.119 (TZM-bl)/0.102 (SupT1)	183,188
EBOV	A549/ Vero 76	Virus titer reduction: 2.5 log at 40 μM		1.5	199,200
CHIKV	BHK-21	2.96		0.95	206
HRV/ 1A, 2, 14 and 16	Hela	>162		0.84–1.38	49,115
EBV	P3HR-1			0.13 (RI)/ 0.092 (virion)	213
KSHV	BCBL1			0.14 (RI)/ 0.17 (virion)	213
HCMV/AD169, TB40, VR1814, 759rD100, PFArD100	HFF	3.0 – 3.7			214

^aCHIKV, chikungunya virus; EBOV, Ebola virus; HIV, human immunodeficiency virus; HRV, human rhinovirus; EBV, Epstein–Barr virus; KSHV, Kaposi’s sarcoma-associated herpesvirus; HCMV, human cytomegalovirus. RI, intracellular replication intermediate.

2.8.2. Anti-norovirus activity

Noroviruses are a genetically diverse group of nonenveloped viruses with a linear, non-segmented, single-stranded, positive-sense RNA genome which can be divided into at least seven different genogroups with numerous genotypes within each genogroup belonging to the family Caliciviridae^164–166. Noroviruses affect people in all age groups and are the most common cause of acute viral gastroenteritis. It was estimated that norovirus resulted in about 699 million illnesses and 219,000 deaths per year worldwide, and children younger than 5 years are most often affected, accounting for about 43% of deaths¹⁶⁷. In addition, due to the suppressed immune system which facilitates viral infections, noroviruses gastroenteritis can result in long-term diarrhea and other complications in immunocompromised populations, causing considerable economic burden^168–170. At present, no vaccine or specific antiviral therapy is available for the treatment of norovirus infection.

Nitazoxanide and tizoxanide were reported to significantly suppress human norovirus (HuNV) replication at 1 μg/mL, in a dose-dependent manner¹⁷¹. These thiazolides inhibit HuNV replication, likely through activating cellular antiviral response by inducing the expression of a series of interferon-stimulated genes, especially IRF-1, independent of the JAK-STAT pathway¹⁷¹. Long-term treatment of nitazoxanide in combination with ribavirin showed a synergistic inhibitory effect against HuNV infection and completely cleared the replicons from host cells, supporting the potential of the combined use of nitazoxanide with ribavirin to treat norovirus gastroenteritis¹⁷¹.

Cases were reported that nitazoxanide successfully treated norovirus gastroenteritis in a pediatric kidney transplant recipient and a 43-year-old patient with relapsed refractory acute myelogenous leukemia (AML), respectively^172,173. Several retrospective reports revealed that nitazoxanide treatment was effective to cure norovirus gastroenteritis in immunocompromised patients after receiving chemotherapy and/or hematopoietic stem cell transplantation^174–176.

2.8.3. Anti-human astrovirus activity

Human astroviruses (HAstVs) are characterized by a non-segmented, positive-sense, single-stranded RNA genome within a non-enveloped icosahedral capsid which includes 8 classic serotypes in genotype MAstV 1 and other serotypes in genotypes MAstV 6, MAstV 8, and MAstV 9, belonging to the genera Mamastrovirus of the family Astroviridae¹⁷⁷. HAstVs are a leading cause of diarrhea, especially in children, the elderly, and immunocompromised individuals, and responsible for 2 to 9% of all acute nonbacterial gastroenteritis in children worldwide^177–179. So far, no vaccine or antiviral treatment exists for HAstV infections.

Nitazoxanide suppresses HAstV-1 replication in a dose-dependent manner, with an EC₅₀ value of approximately 1.47 μM¹⁸⁰. Nitazoxanide can significantly decrease dsRNA production and completely inhibit HAstV-1 replication when administered up to 8 h post-infection, indicating that it may act at an early stage of the viral life cycle after entry and uncoating but before structural protein synthesis¹⁸⁰. Further studies suggested this inhibition is dependent on IFN induction. Nitazoxanide was also effective against multiple classical HAstV serotypes and clinical isolates at 2.5 μM, displaying broad anti-HAstV activity. Moreover, nitazoxanide could reduce viral shedding and clinical disease in the symptomatic turkey poult model, suggesting its potential as a clinical therapy for astrovirus disease¹⁸⁰.

2.9. Activity Against Other Viruses

2.9.1. Anti-HIV activity

Human immunodeficiency virus (HIV) is a retrovirus that attacks the human immune system and causes acquired immunodeficiency syndrome (AIDS) if not treated, continuing to be a major global public health threat. It was estimated that 37.7 million people were living with HIV at the end of 2020, and about 680,000 people died from HIV-associated causes in 2020¹⁸¹. HIV infection can be managed by antiretroviral therapy (ART), which is usually composed of a combination of three or more antiretroviral drugs but cannot be cured¹⁸¹.

Nitazoxanide was shown to reduce HIV-1 replication in monocyte-derived macrophages (MDMs), acting at early and late stages of the HIV life cycle associated with downregulation of HIV-1 receptors CD4 and CCR5, and upregulation of two known type I IFN-induced anti-HIV host factors APOBEC 3A/3G and tetherin, which affect vial particle infectivity and release, respectively¹⁸². Nitazoxanide was also reported to inhibit HIV-1 replication in PBMCs with a percentage of 87.8% at 10 μg/mL, related to the activation of multifaceted innate immune responses and upregulation of several interferon-stimulated genes (ISGs), including those involved in cholesterol pathways, especially the cholesterol-25 hydroxylase (CH25H), an enzyme which converts cholesterol to 25-hydroxycholesterol (25HC) that broadly inhibits the growth of enveloped viruses by blocking virus-cell membrane fusion^183,184. Besides, nitazoxanide is highly synergized with glucocorticoids against HIV by targeting different steps of the HIV life cycle; nitazoxanide indirectly inhibits reverse transcription while glucocorticoids affect HIV-long terminal repeat (LTR)-driven transcription^185–187. Niclosamide inhibits HIV-1 replication in dose- and time-dependent manners, and pretreatment with niclosamide at 0.625 μM for 6 h displayed the most efficient inhibitory activity against HIV-1 infection, showing EC₅₀ values of 0.119 μM in TZM-bl cells and 0.102 μM in SupT1 cells¹⁸⁸. Niclosamide treatment downregulates mTORC1 via AMPK activation and reduces the synthesis of HIV-2 p24 protein¹⁸⁸. Our group found that niclosamide exhibits both antiviral and anti-mycobacterial activity in an in vitro model of HIV and MtB Beijing strain co-infection using human primary monocyte-derived macrophages, supporting the further exploration of niclosamide and its derivatives as dual-acting agents in HIV/TB co-infection¹⁸⁹.

2.9.2. Anti-Ebola virus activity

EBOV, a.k.a. Zaire ebolavirus, is a negative-sense single-stranded RNA virus that is one of six known species within the genus Ebolavirus of the family Filoviridae^190–192. EBOV, and three other known ebolaviruses, can cause severe hemorrhagic fever, a.k.a. Ebola virus disease (EVD), with a high case-fatality rate varying from 25 to 90% in the past outbreaks^193–195. Currently, the vaccine Ervebo and two monoclonal antibodies, Inmazeb and Ebanga, have been approved by FDA to prevent or treat Zaire ebolavirus (EBOV) infections^196–198.

Nitazoxanide completely suppresses EBOV replication in A549 cells at a concentration of 20 μM. Mechanistic studies suggested that nitazoxanide could counteract EBOV VP35 protein’s ability to escape from retinoic-acid-inducible protein 1 (RIG-1) and PKR sensing of EBOV and broadly amplify the host innate immune response, including increasing RIG-1-like receptor (RLR) activation and enhancing mitochondrial antiviral signaling protein (MAVS), interferon regulatory factor 3 (IRF3), and IFN activities¹⁹⁹. Through an HTS campaign of FDA-approved drugs, niclosamide was found to potently inhibit EBOV infection with an EC₅₀ value of 1.5 μM²⁰⁰.

2.9.3. Anti-chikungunya virus activity

Chikungunya virus (CHIKV) is an arbovirus with a positive-sense single-stranded RNA genome of about 11.6 kb, which belongs to the genus Alphavirus, and the family Togaviridae^201,202. The most common symptoms of CHIKV infections are fever and severe joint pain, while other symptoms may include muscle pain, headache, joint swelling, nausea, fatigue, and rash^203,204. Sporadic outbreaks of chikungunya disease have been recorded in Africa, America, and Asia for decades^203,205. Currently, no vaccine or specific drug is available against CHIKV.

Through an HTS using a CHIKV 26S mediated insect cell fusion inhibition assay, niclosamide, and nitazoxanide were found to possess anti-CHIKV activities with EC₅₀ values of 0.95 μM and 2.96 μM, respectively²⁰⁶. Niclosamide and nitazoxanide were able to block CHIKV entry, disrupt the release of CHIKV virions, and significantly suppress cell-to-cell transmission of CHIKV infection²⁰⁶.

2.9.4. Anti-human rhinovirus activity

Human rhinoviruses (HRVs) are positive-sense, single-stranded RNA viruses which comprise over 100 different HRV serotypes divided into three species (HRV A−C), belonging to the genus Enterovirus in the family Picornaviridae²⁰⁷. HRV is the primary cause of the common cold and is associated with asthma exacerbations, severe bronchiolitis in infants and children, and fatal pneumonia in elderly and immunocompromised individuals^207,208. At present, no antiviral agents were approved to treat HRV infection.

Niclosamide was reported to protect Hela cells from pH-dependent HRV serotypes 1A, 2, 14, and 16 infections with EC₅₀ values of 0.84–1.38 μM¹¹⁵. Niclosamide inhibits HRV entry through neutralizing acidic membrane-bounded compartments acting as a protonophore. However, thiazolides (nitazoxanide and 19) showed no inhibitory activity against HRV serotype 2 up to 50 μg/mL⁴⁹.

2.9.5. Anti-herpesvirus activity

Herpesviruses are a large group of enveloped double-stranded DNA viruses that cause a range of diseases in animals and humans. So far there are more than 130 identified herpesvirus types classified into three subfamilies, Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae²⁰⁹. Among them, nine herpesviruses are known to infect humans, including herpes simplex virus 1 and 2 (HSV-1 and HSV-2, also known as HHV-1 and HHV-2), varicella-zoster virus (VZV or HHV-3), Epstein-Barr virus (EBV or HHV-4), human cytomegalovirus (HCMV or HHV-5), human herpesviruses 6A (HHV-6A), HHV6B, HHV-7, and Kaposi’s sarcoma-associated herpesvirus (KSHV or HHV-8)^209,210.

EBV and KSHV are two oncogenic γ-herpesviruses that can cause several types of severe malignances^211,212. Niclosamide was found to inhibit viral DNA lytic replication (EC₅₀ = 0.13 μM and 0.14 μM, respectively) and virion production (EC₅₀ = 0.092 μM and 0.17 μM, respectively) of EBV and KSHV in lymphoma cells, through disrupting mTOR activation²¹³. HMCV is an β-herpesvirus that is extremely widespread among humans and can cause life-threatening diseases in immunocompromised individuals such as organ transplant recipients, HIV-infected patients, and newborn infants²¹⁴. Nitazoxanide inhibits the replication of different strains of HCMV, including strains resistant to the licensed anti-HCMV drugs ganciclovir or foscarnet, which target the viral DNA polymerase, displaying EC₅₀ values of 3.0 – 3.7 μM. Nitazoxanide prevents viral DNA synthesis and early and late gene expression through inhibiting the viral transcription factor Immediate-Early 2 (IE2)-dependent transactivating activity²¹⁴.

3. CONCLUSIONS AND FUTURE DIRECTIONS

Despite various potential targets or mechanisms of salicylamide derivatives against different viral infections were suggested by previous studies (Table 9), their exact mechanisms of broad antiviral activity remain to be elucidated. Given the diversity of viruses that salicylamide derivatives are effective against, the broad-spectrum antiviral activities likely attribute to targeting some commonality in the viral life cycle rather than specific viral proteins. From viral entry and replication to virion assembly and release, viruses almost require host cell factors for every step of their life cycle. Identifying the most common host cell factors and pathways used by different viruses may be a feasible direction to figure out the exact mechanism of action of salicylamide derivatives, offering the opportunity for developing broad-spectrum antivirals. Host-based antivirals usually have a high barrier to resistance compared to direct-acting antivirals but may raise a concern of potential on-target toxicity. Actually, a number of salicylamide derivatives summarized in this review were found to exhibit relatively high cytotoxicity. The combinational use of host-based antivirals and direct-acting antivirals that target different processes involved in the viral life cycle may slow the development of drug resistance and reduce the potential side effects due to the decreased effective concentration of individual drugs. Nevertheless, for those chronic viral infections such as hepatitis B and hepatitis C, the potential toxicity caused by long-term antiviral treatment may make host-based antivirals less attractive. Although newly developed salicylamides were only assessed on one or several selected viruses, varied antiviral effects were observed for different chemical modifications; salicylanilides 4–9 potently suppress both SARS-CoV-2 and ZIKV infections, same as niclosamide, while thiazolide 22, unlike nitazoxanide, only displays good inhibitory activity against HBV, basically inactive against HCV. Thus, to get meaningful SAR and develop broad-spectrum antivirals of salicylamides derivatives, it is essential to build robust salicylamide libraries and conduct systematic screening against various viruses.

Table 9.

Summary of potential targets or mechanisms of salicylamide derivatives against different viral infections^a

Viral mode	Summary of potential targets or mechanisms of salicylamide derivatives against different viral infections	Ref.
MERS-CoV	Niclosamide inhibits SKP2, increases the BECN1 level, and enhances autophagy.	42
SARS-CoV-2	Niclosamide blocks SARS-CoV-2 spike-induced syncytia via inhibiting the activity of the calcium-activated TMEM16F.	44
ZIKV	Inhibits NS2B-NS3 interaction and results in accumulation of nonfunctional viral polyprotein precursor.	31,64
DENV	Niclosamide affects viral and host membrane fusion and uncoating, viral RNA replication, and maturation of virus particles rendering it non-infectious.	66,67
HBV	Nitazoxanide inhibits HBx-DDB1 interaction, restores Smc5 protein levels, and suppresses viral transcription and viral protein production.	75
HCV	Nitazoxanide increases PKR and eIF2α phosphorylation; it depletes ATP-sensitive intracellular Ca2+ stores and induces mild ER stress.	89,90
IAV	Nitazoxanide affects the maturation of the viral HA, impairs its intracellular trafficking and insertion into the host plasma membrane, and thus blocks the exit of mature virions from host cells. Nitazoxanide potentiates the production of type I IFN (α and β) in PBMCs. BMY-27709 inhibits the HA-mediated endosomal fusion of virus and cell membrane through suppressing the essential low-pH-induced conformational change of the HA.	109,111,118,121,122
RSV	Niclosamide reduces AKT pro-survival protein, activates cleaved-caspase-3 and cleaved-PARP, and induces early apoptosis.	146
Rotavirus	Tizoxanide alters rotavirus viroplasm formation through preventing the NSP5/NSP2 interaction and decreases rotavirus dsRNA formation.	159
Norovirus	Nitazoxanide activates cellular antiviral response by inducing the expression of a series of IFN-stimulated genes, especially IRF-1, independent of the JAK-STAT pathway.	171
HIV	Nitazoxanide downregulates HIV-1 receptors CD4 and CCR5 and upregulates anti-HIV host factors APOBEC 3A/3G and tetherin. Nitazoxanide activates multifaceted innate immune responses and upregulates several ISGs, especially CH25H. Niclosamide downregulates mTORC1 via AMPK activation and reduces the synthesis of HIV-2 p24 protein.	182,183,188
EBOV	Nitazoxanide counteracts EBOV VP35 protein’s ability to escape from RIG-1 and PKR sensing of EBOV and broadly amplify the host innate immune response, including increasing RLR activation and enhancing MAVS, IRF3, and IFN activities.	199
CHIKV	Niclosamide and nitazoxanide block viral entry, disrupt virion release, and significantly suppress cell-to-cell transmission of CHIKV infection.	206
HRV	Niclosamide inhibits HRV entry through neutralizing acidic membrane-bounded compartments acting as a protonophore.	115
EBV	Niclosamide disrupts mTOR activation.	213
HCMV	Nitazoxanide prevents viral DNA synthesis and early and late gene expression through inhibiting the viral transcription factor IE2-dependent transactivating activity.	214

^aMERS-CoV, Middle East respiratory syndrome coronavirus; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; ZIKV, Zika virus; DENV, dengue virus; HBV, Hepatitis B virus; HCV, Hepatitis C virus; IAV, influenza A virus; RSV, respiratory syncytial virus; HIV, human immunodeficiency virus; EBOV, Ebola virus; CHIKV, chikungunya virus; HRV, human rhinovirus; EBV, Epstein–Barr virus; HCMV, human cytomegalovirus; SKP2, S-phase kinase-associated protein 2; BECN1, Benclin 1; TMEM16F, anoctamin 6; HBx, HBV regulatory protein X; DDB1, the host protein damage-specific DNA-binding protein 1; Smc5, structural maintenance of chromosomes 5; PKR, protein kinase activated by double-stranded RNA; eIF2α, eukaryotic initiation factor-2α; ER, endoplasmic reticulum; HA, haemagglutinin; IFN, interferon; PBMC, peripheral blood mononuclear cell; AKT, protein kinase B; IRF, interferon regulatory factor; JAK, Janus kinase, STAT, signal transducer and activator of transcription protein; ISG, interferon-stimulated gene; CH25H, cholesterol-25 hydroxylase; mTORC1, the mammalian target of rapamycin complex 1; AMPK, 5’ AMP-activated protein kinase; RIG-1, retinoic-acid-inducible protein 1; RLR, RIG-1-like receptor; MAVS, mitochondrial antiviral signaling protein; mTOR, the mammalian target of rapamycin; IE2, Immediate-Early 2.

To develop salicylamide derivatives as broad-spectrum antiviral agents, the PK properties and cytotoxicity of these agents are some critical features that need special attention and may be improved. The thiazolides, nitazoxanide and 33 (RM5038) are only absorbed from the gastrointestinal tract and quickly hydrolyzed into the active circulating metabolite, tizoxanide and 22 (RM4848), which are subsequently excreted from the body primarily as the O-glucuronides 64 and 65 (Figure 10)^27,215,216. Besides UGT1A1-mediated glucuronidation, niclosamide also undergoes efficient CYP1A2-medaited hydroxylation, and these two metabolic reactions generate the O-glucuronide 66 and the hydroxylated metabolite 67, respectively²¹⁷. Compared to niclosamide that has limited aqueous solubility and relatively low oral bioavailability (F = 10%), compound 19 (JMX0207) with a 3-NO₂ group instead of the 5-Cl group displayed significantly improved PK properties³¹; compound 13 exhibited a very high bioavailability of F = 76% in mice and equal anti-SARS-CoV-2 activity, in contrast to niclosamide, providing significant optimization directions for salicylamide derivatives to improve their PK properties^29,56. The ester or amino-acid ester prodrugs of salicylamide derivatives (e.g., 68–70) also effectively increase systemic drug exposure and extend the duration of exposure^218,219. However, it should be noted that such amino-acid esters of salicylanilide derivatives could rearrange to form pseudopeptide derivatives, structurally related to compound 56^220,221. In addition, it may be a fast and valuable approach to develop nano-based formulations of salicylamide derivatives to improve their PK properties and maximize their therapeutic potential, e.g., the inhaled niclosamide for COVID-19^222–224. As the nitro group may induce severe toxicity and is always classified as a metabolic alert, to remove this potential liability, replacing the nitro group of niclosamide or nitazoxanide with other substituents yielded derivatives 22, 43, 54, and 55, which also displays similar or improved antiviral activities and low cytotoxicity^24,79,133. Learning from the COVID-19 pandemic is vital to develop more effective, safer, and further drug-like salicylamide derivatives as broad-spectrum antiviral agents to ultimately fight the circulating viruses as well as the highly heterogeneous unknown zoonotic ones that may emerge in the future.

Figure 10.

The chemical structures of the metabolites (64–67) and prodrugs (68–70) of salicylamide derivatives

Abbreviations:

AHB

acute hepatitis B

AHC

acute hepatitis C

AIDS

acquired immunodeficiency syndrome

AKT

protein kinase B

allo-HSCT

allogenic hematopoietic stem cell transplant

ALRI

acute lower respiratory infection

AML

acute myelogenous leukemia

AMPK

5’ AMP-activated protein kinase

ART

antiretroviral therapy

BECN1

Benclin 1

BVDV

bovine viral diarrhoea virus

CH25H

cholesterol-25 hydroxylase

CHB

chronic hepatitis B

CHC

chronic hepatitis C

CHIKV

chikungunya virus

3CLpro

3C-like protease

CoV

coronavirus

COVID-19

coronavirus disease 2019

DDB1

the host protein damage-specific DNA-binding protein 1

DENV

dengue virus

DHF

dengue hemorrhagic fever

DSS

dengue shock syndrome

dsRNA

double-stranded RNA

EBOV

Ebola virus

EBV

Epstein–Barr virus

eIF2α

eukaryotic initiation factor-2α

ER

endoplasmic reticulum

EVD

Ebola virus disease

HA

haemagglutinin

HAdV

human adenovirus

HAstV

human astrovirus

HBeAg

hepatitis B e antigen

HBsAg

hepatitis B surface antigen

HBV

Hepatitis B virus

HBx

HBV regulatory protein X

HCMV

human cytomegalovirus

HCoV

human coronaviruses

HCV

Hepatitis C virus

HHV

human herpesviruses

HIV

human immunodeficiency virus

hNPCs

human neural progenitor cells

HRV

human rhinovirus

HSV

herpes simplex virus

HTS

high-throughput screening

HuNV

human norovirus

IAV

influenza A virus

IE2

Immediate-Early 2

IFN

interferon

ILI

influenza-like illness

IRF

interferon regulatory factor

ISG

interferon-stimulated gene

JAK

Janus kinase

JEV

Japanese encephalitis virus

KSHV

Kaposi’s sarcoma-associated herpesvirus

mTORC1

the mammalian target of rapamycin complex 1

mAb

monoclonal antibody

MAVS

mitochondrial antiviral signaling protein

MDM

monocyte-derived macrophage

MERS

Middle East respiratory syndrome

MERS-CoV

Middle East respiratory syndrome coronavirus

Mtb

Mycobacterium tuberculosis

MTD

maximum tolerated dose

mTOR

the mammalian target of rapamycin

NA

neuraminidase

NAIs

the neuraminidase inhibitor

PBMC

peripheral blood mononuclear cell

PFOR

pyruvate:ferredoxin oxidoreductase

p.i.

post-infection

PKR

protein kinase activated by double-stranded RNA

RdRp

RNA-dependent RNA polymerase

RIG-1

retinoic-acid-inducible protein 1

RLR

RIG-1-like receptor

RSV

respiratory syncytial virus

SAR

structure-activity relationship

SARS

severe acute respiratory syndrome

SARS-CoV-2

severe acute respiratory syndrome coronavirus 2

SKP2

S-phase kinase-associated protein 2

Smc5

structural maintenance of chromosomes 5

SOT

solid-organ transplant

STAT

signal transducer and activator of transcription protein

SVR

sustained virological response

TBEV

tick-borne encephalitis virus

TMEM16F

anoctamin 6

VZV

varicella–zoster virus

WHO

World Health Organization

WNV

West Nile virus

YFV

yellow fever virus

ZIKV

Zika virus

25HC

25-hydroxycholesterol

Biographies

Jimin Xu received his B.Sc. degree in Basic Pharmacy from China Pharmaceutical University in 2009 and his Ph.D. degree from Shanghai Institute of Materia Medica, Chinese Academy of Sciences in 2014 under the supervision of Professor Fajun Nan. He worked as a Research Scientist in pharmaceutical companies for two years. Dr. Xu has pursued his postdoctoral training and is continuing his drug discovery efforts as a Research Scientist in Professor Jia Zhou’s Chemical Biology Program at the University of Texas Medical Branch (UTMB). His research interests include the rational design and chemical synthesis of small molecules as novel pharmacological probes and therapeutics for infectious diseases and human cancers.

Yu Xue obtained his Ph.D. in Medicinal Chemistry from China Pharmaceutical University (CPU) in 2018 under the supervision of Professor Liping Sun at CPU and Professor Ao Zhang at Shanghai Institute of Materia Medica, Chinese Academy of Sciences. He is currently pursuing his postdoctoral training under the supervision of Professor Jia Zhou at the Chemical Biology Program, Department of Pharmacology and Toxicology at UTMB. His research interests focus on design and synthesis of novel small molecules as chemical probes and drug candidates for infectious diseases, cancer, and other human diseases.

Andrew A. Bolinger was born and raised in beautiful Colorado. He did his undergraduate research under the supervision of Dr. Joseph L. Richards and completed his BS degree in 2013 at Colorado Mesa University. He then joined Dr. Don Coltart’s group in 2014 where he studied organic methodologies related to umpolung alkylation via azo compounds at the University of Houston. He later completed his Ph.D. with a rotation in Dr. Jeremy May’s lab. After graduating in 2019 he did a postdoc at Rutgers - Newark in the Freundlich lab before joining Dr. Jia Zhou’s group at UTMB. His current research is focused on the rational design and synthesis of novel small molecules that target GPCR receptors related to substance use disorders and other human diseases.

Jun Li received his B.A. in Pharmacy from Ocean University of China in 2012. Afterwards, he stayed in the same university, and graduated in 2015 with a M.S. in Medicinal Chemistry. He then moved to the United States and completed his Ph.D. in 2021 in Organic Chemistry at University of Houston, under the supervision of Prof. Dr. Scott Gilbertson. After his graduation, he joined Prof. Dr. Jia Zhou’s research group at the University of Texas Medical Branch as a postdoctoral research fellow. His research interests include rational drug design, synthesis, and biological evaluation of a variety of bioactive small molecules as potential pharmacological tools and novel therapeutics.

Mingxiang Zhou received his Ph.D. in organic chemistry from Texas Tech University in 2021 under the supervision of Professors Andrew M. Harned and David M. Birney. After the graduation, he joined UTMB as a postdoctoral fellow under the supervision of Professor Jia Zhou. His research interests include design and synthesis of novel small molecules as chemical probes and drug candidates for infectious diseases, cancer, and other human diseases.

Haiying Chen obtained her BSc degree in Engineering from Tianjin University (Branch) in 1995. She then worked in Tianjin Research Institute of Construction Machinery as an engineer focusing on designing and programming computer testing systems. Since 2014, she has been conducting drug discovery and development research in Dr. Jia Zhou’s Chemical Biology Program as a Senior Research Associate by utilizing the computer-assisted rational drug design of novel target-based small molecules and the understanding of key interactions and binding modes of ligands with various drug targets for the treatment of human cancers, CNS disorders, and infectious diseases.

Hongmin Li obtained his Ph.D. in Molecular Biology from Institute of Biophysics, Chinese Academy of Sciences in 1995. Then he joined as a postdoctoral affiliate in Dr. Roy Mariuzza’s lab at the Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institutes. In 2000, Dr. Li became a faculty member in the Wadsworth Center, New York State Department of Health, where he held a Research Scientist 6 position (professor-equivalent) at the Wadsworth Center at Albany. Dr. Li is currently R. Ken and Donna Coit Endowed Chair Professor at the Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona. Dr. Li authored more than 100 scientific research articles and book chapters, as well as several patents.

Jia Zhou obtained his Ph.D. in organic chemistry from Nankai University in China in 1997. He then joined the chemistry faculty with the promotion to associate professor at the same university. He pursued his postdoctoral research training in organic and bioorganic chemistry with Dr. Sidney M. Hecht at the University of Virginia in 1999. After additional postdoctoral training in medicinal chemistry and drug discovery with Dr. Alan P. Kozikowski at Georgetown University Medical Center, he conducted research in the U.S. pharmaceuticals as a Senior Principal Scientist for seven years. He is currently a tenured full professor at UTMB, leading drug discovery research programs for the treatment of CNS disorders, human cancers, inflammatory and infectious diseases. He is an author of over 230 peer-reviewed articles and seven book chapters as well as an inventor of 32 patents. Dr. Zhou is a National Academy of Inventors (NAI) Fellow, and the Editor-in-Chief of Current Topics in Medicinal Chemistry.

DATA AVAILABILITY STATEMENT

No experimental data were generated for this review article.