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Design and synthesis of APN and 3CLpro dual-target inhibitors based on STSBPT with anticoronavirus activity

March 2024
Journal of Medical Virology 96(3):e29512

March 2024
96(3):e29512

Authors:

Coronaviruses (CoVs) have continuously posed a threat to human and animal health. However, existing antiviral drugs are still insufficient in overcoming the challenges caused by multiple strains of CoVs. And methods for developing multi‐target drugs are limited in terms of exploring drug targets with similar functions or structures. In this study, four rounds of structural design and modification on salinomycin were performed for novel antiviral compounds. It was based on the strategy of similar topological structure binding properties of protein targets (STSBPT), resulting in the high‐efficient synthesis of the optimal compound M1, which could bind to aminopeptidase N and 3C‐like protease from hosts and viruses, respectively, and exhibit a broad‐spectrum antiviral effect against severe acute respiratory syndrome CoV 2 pseudovirus, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, feline infectious peritonitis virus and mouse hepatitis virus. Furthermore, the drug‐binding domains of these proteins were found to be structurally similar based on the STSBPT strategy. The compounds screened and designed based on this region were expected to have broad‐spectrum and strong antiviral activities. The STSBPT strategy is expected to be a fundamental tool in accelerating the discovery of multiple targets with similar effects and drugs.

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Received: 7 December 2023

Accepted: 26 February 2024

DOI: 10.1002/jmv.29512

RESEARCH ARTICLE

Design and synthesis of APN and 3CLpro dual‐target

inhibitors based on STSBPT with anticoronavirus activity

Youle Zheng

|Jin Feng

|Yanbin Song

|Yixin Yu

|Min Ling

Mengjia Zhang

|Haijiao Xie

|Wentao Li

|Xu Wang

1,4

National Reference Laboratory of Veterinary Drug Residues (HZAU) and MAO Key Laboratory for Detection of Veterinary Drug Residues, Huazhong Agricultural

University, Wuhan, China

National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University; Hubei Hongshan Laboratory, Wuhan, China

Hangzhou Yanqu InformationTechnology Co., Ltd., Hangzhou, China

MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong Agricultural University, Wuhan, China

Correspondence

Xu Wang, National Reference Laboratory of

Veterinary Drug Residues (HZAU) and MAO

Key Laboratory for Detection of Veterinary

Drug Residues, Wuhan, Hubei 430070, China.

Email: wangxu@mail.hzau.edu.cn

Wentao Li, National Key Laboratory of

Agricultural Microbiology, College of

Veterinary Medicine, Huazhong Agricultural

University, Hubei Hongshan Laboratory,

Wuhan 430070, China.

Email: wentao@mail.hzau.edu.cn

Funding information

National Key Research and Development

Program of China, Grant/Award Number:

2023YFD1800801; National Natural Science

Foundation of China, Grant/Award Number:

32272990; Fundamental Research Funds for

the Central Universities, Grant/Award Number:

2662023DKPY004

Abstract

Coronaviruses (CoVs) have continuously posed a threat to human and animal health.

However, existing antiviral drugs are still insufficient in overcoming the challenges

caused by multiple strains of CoVs. And methods for developing multi‐target drugs

are limited in terms of exploring drug targets with similar functions or structures. In

this study, four rounds of structural design and modification on salinomycin were

performed for novel antiviral compounds. It was based on the strategy of similar

topological structure binding properties of protein targets (STSBPT), resulting in the

high‐efficient synthesis of the optimal compound M1, which could bind to

aminopeptidase N and 3C‐like protease from hosts and viruses, respectively, and

exhibit a broad‐spectrum antiviral effect against severe acute respiratory syndrome

CoV 2 pseudovirus, porcine epidemic diarrhea virus, transmissible gastroenteritis

virus, feline infectious peritonitis virus and mouse hepatitis virus. Furthermore, the

drug‐binding domains of these proteins were found to be structurally similar based

on the STSBPT strategy. The compounds screened and designed based on this

region were expected to have broad‐spectrum and strong antiviral activities. The

STSBPT strategy is expected to be a fundamental tool in accelerating the discovery

of multiple targets with similar effects and drugs.

KEYWORDS

3CLpro, APN, binding, coronavirus, multiple targets

1|INTRODUCTION

Coronaviruses (CoVs) can induce multiple diseases in a broad range

of hosts. Before the emergence of severe acute respiratory syndrome

(SARS), they were not deemed to pose a significant threat to human

health, which was induced by bat‐originated SARS‐CoV.

The host

range switches of CoVs from animals to humans have demonstrated

potential to cross the host‐species barrier, and they have led to the

emergence of novel and highly fatal diseases, such as Middle East

respiratory syndrome (MERS) and the recent outbreak of coronavirus

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https://doi.org/10.1002/jmv.29512

Youle Zheng and Jin Feng contributed equally to this work.

disease 2019 (COVID‐19). Of note, there have been over 772 million

confirmed cases of SARS‐CoV‐2 infection, including almost 7 million

deaths reported worldwide as of 19 December 2023 (https://www.

who.int/data#reportsWorld). To date, the emergence of several

mutated variants of SARS‐CoV‐2, notably the Delta and Omicron

strains, has exhibited higher transmissibility and a surge in infection

cases worldwide.

Both exhibit limited neutralization by sera after

vaccination.

3–5

Therefore, it is imperative to explore prospective

broad‐spectrum antiviral agents to effectively address the ongoing

challenges posed by viral outbreaks.

6,7

Remdesivir, the first Food and Drug Administration (FDA)‐approved

drug targeting RNA‐dependent RNA polymerase (RdRp) for COVID‐19

treatment,

had little effect on hospitalized patients.

9,10

Molnupiravir,

which targets RdRp, introduces mutations in the genome of SARS‐CoV‐2

during viral replication,

butitcouldalsocause“error disasters”in

mammalian cells.

Despite the recent FDA approval of Paxlovid,

reported cases of reinfection have emerged following completion of

the prescribed course involving this medication.

13,14

This could be

caused by rapid drug resistance due to the drug's limited targeting of

only 3C‐like protease (3CLpro).

15,16

To overcome these limitations,

multitarget drugs are gradually gaining recognition and becoming

increasingly important, as biological systems are typically unable to fully

compensate for the effects of multiple targets acting simultaneously.

Recent studies have demonstrated the antiviral activity of compounds

targeting both transmembrane protease serine 2 (TMPRSS2) and

cathepsin L/B against diverse SARS‐CoV‐2variants.

In addition, dual‐

targeting of the 3CLpro and papain‐like protease (PLpro) of the virus has

also demonstrated remarkable efficacy in combating CoVs.

These

advancements propel us into the challenging area of developing novel

antiviral drugs with host‐directed and virus‐directed mechanisms,

thereby offering broad‐spectrum performance and sustained effective-

ness. In recent years, significant progress has been made in the

computational prediction of drug–target interactions. These methods

utilize the “guilt‐by‐association”assumption, which suggests that similar

drugs may share similar targets and vice versa.

To further support this

idea, the concept of similar topological structure binding properties of

protein targets (STSBPT) was introduced and successfully applied to our

drug discovery and target identification efforts.

Aminopeptidase N (APN) and 3CLpro are pivotal for CoV

attachment and replication, respectively, making them enticing dual

targets for drug intervention.

21,22

The former acts as a cross‐genus

functional receptor for CoVs from humans, swine, dogs and cats,

such as porcine transmissible gastroenteritis virus (TGEV),

human

respiratory coronavirus 229E (HCoV‐229E),

canine coronavirus

(CCoV),

feline infectious peritonitis virus (FIPV)

and porcine delta

coronavirus (PDCoV).

Swine, dogs and cats have frequent contact

with humans, wildlife and livestock, which increases the likelihood of

cross‐species transmission.

27,28

However, there are few studies on

screening antiviral drugs targeting APN. The viral protein 3CLpro acts

as the main protease by cleaving 11 of the 13 individual proteins,

facilitating proper protein folding and subsequent assembly into the

active polymerase complex. This cleavage event makes the proteo-

lytic activity of 3CLpro essential for the virus.

Furthermore, the

proteinase has a high level of similarity of structure and function

among CoVs, which is supported by the remarkable degree of amino

acid sequence conservation among SARS‐CoV, HCoV‐229E and

TGEV 3CLpro and mediating the cleavage of a TGEV 3CLpro

substrate through SARS‐CoV‐derived recombinant 3CLpro.

While

inhibitors targeting 3CLpro have been extensively explored from

various sources, including plants, marine organisms, microbial origins,

and approved or commercially available drugs, there is a lack of

studies focused on designing APN/3CLpro dual‐target inhibitors.

30,31

The primary objective of this study is to develop multi‐targeted

antiviral compounds that can effectively combat multiple CoVs.

Additionally, the aim is to investigate the shared characteristics of

protein pockets targeted by a single compound to understand the

mechanism of multitargeting. The findings of this research could

potentially make a contribution to the development of future multi‐

targeted drugs and assist in the identification of new drug targets.

2|MATERIALS AND METHODS

2.1 |Cells and viruses

PK‐15, IPEC‐J2, Vero‐E6, CRFK, LR7 and ACE2‐overexpressing HEK‐

293T (Obio Technology) cells were maintained in Dulbecco's

Modified Eagle medium (DMEM) supplemented with 10% fetal

bovine serum (FBS) at 37°C in 5% CO

. The following viruses were

used and listed in the Supporting Information S1: Table S1: TGEV

strain WH‐1 (provided by Wentao Li), PEDV‐DR13 (provided by

Qigai He), MHV‐A59 (provided by Wentao Li), cell‐adapted serotype

II FIPV 79‐1146 (provided by Wentao Li) and COVID‐19‐Spike

Pseudovirus Omicron B.1.1.529 (11991ES50, Yeasen Biotech).

2.2 |Structure‐based virtual screening

The crystal structure of pAPN as the modeling template was obtained

from the Protein Data Bank (ID: 4FKE). The binding pockets were

confirmed using SYBYL‐X Suite with default parameters. A diverse

subset of the compound library (i.e., SPECS with 1,084,348 small‐

molecule compounds and a Topscience compound library with 16,594

natural products) was prepared to generate three‐dimensional configu-

rations by Surflex to search SYBYL‐X with all options set as default.

Docking projects were conducted using the Surflex‐Dock Screen and

Surflex‐Dock GeomX modules in the SYBYL‐X Suite. Scoring calculations

were calculated by CScore in the SYBYL‐XSuite.

2.3 |Virus inhibition assays

For the infection assays, cells were seeded at 1 × 10

and 5 × 10

cells per well in 24‐well and 6‐well plates, respectively, and infected

with viruses (MOI = 0.1) 1 h before adding various concentrations of

compounds. Cell supernatants were harvested for further use. The

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ZHENG ET AL.

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cells were lysed and harvested in radioimmunoprecipitation assay

(RIPA) lysis buffer (P0013B, Beyotime) after washing three times with

phosphate‐buffered saline (PBS).

2.4 |Drug time‐of‐addition assay

The antiviral activity of salinomycin (SAL) was evaluated by the time‐

of‐addition assay for pre‐,co‐and posttreatment. Prevention of viral

infection was identified by pretreatment with 5 µM SAL 1 h before

infection in cells. The co‐treatment of 5 µM SAL and viruses for

30 min was evaluated to observe the virucidal effect and the

interaction of cell receptors with viruses of the agent. The inhibition

of viral replication was evaluated by treatment with 5 µM SAL 1 h

after viral infection in cells. Cell suspensions and cell lysates were

collected for further use.

2.5 |Design and synthesis of SAL derivatives

The SAL sample (Qilu) with a purity of 24% was used as the starting

material, which was dissolved in dichloromethane (DCM), filtered and

extracted three times with acidified water (pH 1.5). The organic phase

was dried for at least 2 h with anhydrous Na

, and the solvent was

removed under reduced pressure. The synthesis work was performed

as follows: to a solution containing SAL (800 mg, 88.77 mM, 1.0 eq) in

N,N‐dimethylformamide (DMF, 12 mL), N‐hydroxysuccinimide (NHS,

245 mg, 177.40 mM, 2.0 eq) and 1‐(3‐dimethylaminopropyl)‐3‐ethyl

carbodiimide hydrochloride (EDC, 408 mg, 177.36 mM, 2.0 eq) were

added. The reaction mixture was stirred at 25°C for 6 h, and 2‐

thiophenecarboxaldehyde hydrazone (CAS: 31350‐01‐3, 269 mg,

177.66mM, 2 eq) was added. The reaction mixture was stirred at

25°C for 24 h. The output was monitored by thin‐layer chromatogra-

phy (TLC) (hexyl hydride/ethyl acetate [hexane/EtOAc] = 1/1,

retention factor [Rf] = 0.25) and visualized by a 5% vanillin sulfuric

acid/ethanol solution. The product was purified by column chroma-

tography on silica gel (hexane/EtOAc = 2/1). Other derivatives, such as

M5, M7, M8, M9, M10, M12 and M13, were synthesized with the

same procedure (Supplemental Information). The general chemistry

information such as eluant and percentage yield of the product are

listed in Supporting Information S1: Table S2. SAL derivatives were

characterized by various techniques, including MS,

H‐NMR, and

C‐NMR.

2.6 |Immunofluorescence assay (IFA)

Normal and virus‐infected cells were both subjected to fixation,

permeation and incubation with primary antibodies, followed by

staining with the corresponding fluorescence‐conjugated anti‐IgG

secondary antibodies and DAPI, and visualized with an inverted

fluorescence microscope (Olympus IX83, Olympus Co.). The anti-

bodies used for IFA are provided in Table S1.

2.7 |Cellular thermal shift assay (CETSA) and drug

affinity responsive target stability (DARTS) assay

The CETSA and DARTS procedures were carried out according to a

previously described method.

32,33

Briefly, PK‐15 and CRFK cells were

treated with compounds (30 µM) for 30 min. Then, the cells were

washed three times using PBS, scraped off and subjected to different

temperatures or pronase solution. The samples were subjected to

freeze–thaw cycling twice using liquid nitrogen and a water bath set

at 25°C and then centrifuged at 500 gfor 3 min at 25°C to pellet the

cell debris. Supernatants were subjected to SDS‐induced protein

denaturation and saved for subsequent analysis.

2.8 |Co‐immunoprecipitation (Co‐IP)

PK‐15 cells were treated with compounds (30 µM) for 30 min and

then scraped off and lysed in cell lysate buffer (50 mM Tris‐HCl, pH

7.4; 150 mM NaCl) with complete protease inhibitors. Anti‐APN

antibodies (ET1611‐61, HUABIO) and mouse control IgG (AC011,

ABclonal, China) were preincubated with rProtein A/G MagPoly

Beads (AM001‐02, Nanjing ACE). Then, the cell lysate and TGEV

were mixed and immunoprecipitated with the Sepharose‐antibody

complexes on a rotator at 4°C overnight. The beads were washed five

times with cell lysate buffer and collected by centrifugation at 1000 g,

and the immunoprecipitates were subjected to a western blot assay

with the corresponding antibodies. The antibodies used for immuno-

precipitation are provided in Supporting Information S1: Table S1.

2.9 |Quantitative reverse transcription PCR

(RT‐qPCR)

Total RNA from viral RNA from cell suspensions were extracted with

RNA Isolater Total RNA Extraction Reagent (R401‐01, Vazyme).

Complementary DNA (cDNA) was synthesized using HiScript II Q

Select RT SuperMix for qPCR with gDNA wiper (R233‐01, Vazyme).

Each RT‒qPCR was carried out using 2X Universal SYBR Green Fast

qPCR Mix (RK21203, ABclonal). The results were monitored using a

CFX96 Real‐Time PCR Detection System (Bio‐Rad). All primers used

for RT‐qPCR are listed in Table S3.

2.10 |Western blot

Cell lysates were obtained using RIPA lysis buffer (P0013B,

Beyotime), separated by 10% SDS‒PAGE, transferred to 0.45 µm

polyvinylidene difluoride membranes (Millipore) and blocked with 5%

nonfat milk. The membranes were incubated with primary antibodies

at 4°C overnight, followed by washing three times for 10 min each

with Tris‐buffered saline with 0.1% Tween 20 (TBST). The mem-

branes were then incubated with the corresponding fluorescence‐

conjugated anti‐IgG secondary antibodies for 1.5 h, followed by

ZHENG ET AL.

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washing three times with TBST. Signals were detected using the

ImageQuant LAS 4000 Mini (Cytiva) after ECL (RM00021, ABclonal)

treatment. The antibodies used for western blot are listed inTable S1.

2.11 |Protein expression and purification

3CL amino acids from TGEV (GenBank accession numbers:

ABG89303), PEDV (GenBank accession numbers: AGK89913) and

FIPV (GenBank accession numbers: AAK09095) were ordered from

Sangon (China) in the pET28a (+) vector with E. coli codon

optimization to generate the pET28a‐3CL plasmids. These plasmids

were amplified in DH5αcompetent cells and then used to transform

BL21 (DE3) competent cells. Single colonies were picked and

transferred to LB medium. The cultures were shaken at 37°C for

4–6 h before induction of fusion protein expression by 0.1 mM

isopropyl thio‐β‐D‐galactoside (IPTG) at 37°C for 5 h. Then, the cells

were harvested and purified. Briefly, the cells were sonicated,

collected and passed through a Ni‐NTA column by a peristaltic pump

at 4°C overnight and then passed through the binding buffer (50 mM

sodium phosphate buffer pH 8.0, 500 mM NaCl and 10 mM

imidazole). The bacterial proteins were then eliminated with elution

buffer A containing 30 mM imidazole (50 mM sodium phosphate

buffer pH 8.0, 500 mM NaCl and 30 mM imidazole). Next, the column

was eluted to obtain the target protein with elution buffer B with

200 mM imidazole (50 mM sodium phosphate buffer pH 8.0, 500 mM

NaCl and 200 mM imidazole). The crude product was washed with

PBS via a 10 kDa Millipore centrifugal ultrafiltration tube to remove

imidazole. The fractions of pure product, total protein, supernatant

and precipitate were then analysed by SDS‒PAGE and western blot

using an anti‐His tag antibody (AE003, ABclonal).

pCAGGS mammalian expression vectors encoding human Fc‐

tagged soluble ectodomains (i.e. not membrane anchored) of pAPN

(GenBank accession numbers: XP_005653580.1) were generated.

Then, 60% confluent HEK‐293T cells were transfected with plasmids

encoding APN‐hFc fusion proteins with polyethylenimine (PEI) for 6

h. The medium was replaced with 293 SFM II‐based expression

medium (Gibco Life Technologies) and the cells were incubated at

37°C in 5% CO

. Tissue culture supernatants were harvested 5–6

days after transfection, and expressed proteins were purified using

Protein A Sepharose beads (GE Healthcare) according to the

manufacturer's instructions. The purity and integrity of all purified

recombinant proteins were checked by SDS‒PAGE. Purified proteins

were stored at 4°C until further use.

2.12 |Enzyme assay

The enzyme assay was conducted to evaluate the impact of various

drugs on the activity of APN protease. In brief, 2 mM L‐leucine‐p‐

nitroanilide (L‐Leu‐pNA, CAS: 4178‐93‐2) was utilized as the

substrate, 10 µg/mL purified porcine APN protein was utilized as

the enzyme, 50−200 µM UBE, SAL, or M1 was utilized as drugs, and

PBS was utilized as the buffer solution. The drugs were individually

preincubated with the enzyme for 10 min, followed by the addition of

the substrate. Control groups included: samples without drugs,

samples with one type of drug, and samples with only the substrate.

All samples were incubated at room temperature in a 96‐well plate

and the absorbance values at 405 nm were measured at different

time points using a fluorescence microplate reader (Enspire PE,

PerkinElmer).

To evaluate the impact of various drugs on the activity of 3CL

protease, the fluorescence resonance energy transfer (FRET) assay

was conducted. The FRET peptide Dabcyl‐VSVNSTLQ ↓SGLRKMA‐

E (Edans) was synthesized by GenScript (China). It can be cleaved by

3CL‐TGEV and 3CL‐FIPV proteins and easily monitored using a

fluorescence microplate reader (Enspire PE, PerkinElmer). Inhibition

of enzymatic activity was assayed by preincubating 0.2 µM protein

with varying concentrations of test compound (SAL and M1) in a total

volume of 200 µL in 20 mM Tris–HCl (pH 7.4) and 200 mM NaCl for

15 min at 25°C in 96‐well plates. Then, 20 µM FRET peptide was

added and the reaction incubated for another 15 min at 25°C. Finally,

the cleaved product was measured in a 96‐well plate using an

excitation wavelength of 320 nm and an emission wavelength of

425 nm. Control reactions were carried out using the same reaction

mixture with DMSO or without the protein (MOCK).

2.13 |Gaussian process

The theoretical calculations were performed via the Gaussian 16 suite of

programs. The structures of the studied compounds were fully optimized

at the B3LYP‐D3/6‐31 G (d) level of theory. The solvent (H

O) effect

was included in the calculations using the solvation model based on the

density (SMD) model. The vibrational frequencies of the optimized

structures were determined at the same level. The structures were

characterized as a local energy minimum on the potential energy surface

by verifying that all the vibrational frequencies were real. The affinity of

SAL and M1 towards Na

and Ca

were evaluated at the same level

with the following equation: [SAL/M1 –solution] + [Na

/Ca

O–

solution] →[SAL/M1 + Na

/Ca

O–solution]. The equations [SAL/

M1 –solution], [Na

/Ca

O–solution] and [SAL + Na

/Ca

O–solution] represent SAL/M1, Na

/Ca

coordinated with H

and complexes with Na

/Ca

containing one water molecule in solution,

respectively. The ΔG of the equation was used for the evaluation.

2.14 |Measurement of intracellular Ca

The calcium‐sensitive dyes Fluo‐4 AM (F8501, Solarbio) and Pluronic

F‐127 (P6791, Solarbio) were used to measure the intracellular

calcium ion concentration. IPEC‐J2 cells were incubated with 4 μM

Fluo‐4 AM for 20 min at 37°C, treated with five volumes of HBSS

containing 1% FBS and incubated for another 40 min. To assess cell

death, PI (C0080, Solarbio) was then added to the solution to obtain a

final concentration of 1 µg/mL. Then, DMSO, SAL or M1 was added

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ZHENG ET AL.

to a final concentration of 1 mM. The fluorescence intensity was

detected using fluorescence inverted microscopy (Olympus IX83,

Olympus Co.).

To test the change in intracellular calcium ions, IPEC‐J2 cells

were incubated with SAL, M1 and amlodipine (AML, a calcium

channel blocker) at the same concentration of 5 µM for 24 h and then

stained with the calcium indicator Fluo‐4 AM and Pluronic F‐127.

The fluorescence intensity was detected using an excitation

wavelength of 494 nm and an emission wavelength of 516 nm by a

fluorescence microplate reader. To explore whether the cytotoxicity

was related to the change in intracellular calcium ions induced by SAL

and M1. An inhibitor of the Na

/Ca

exchanger was used at a

concentration of 10 µM for co‐incubation with test compounds in

IPEC‐J2 cells. The CCK‐8 (A311‐01, Vazyme) was used to test cell

viability after drug treatment for 24 h. Subsequently, OD was

measured at 450 nm on a microplate reader.

2.15 |Flow cytometry

Cell apoptosis was assessed using the Annexin V‐fluorescein

isothiocyanate (FITC) apoptosis detection kit (KGA1014, KeyGen

Biotech, China). A total of 2 × 10

IPEC‐J2 cells/well were plated on a

6‐well plate, incubated overnight, and then treated with various

compounds for 24 h. Cells were then digested using 0.25% trypsin

without EDTA into a single‐cell suspension. According to the

manufacturer's protocols, Annexin V‐FITC and PI were used for cell

staining. Then, at least 2 × 10

double‐stained cells were measured

using a flow cytometer (Beckman CytoFLEX, USA). Finally, FlowJo

software was used to analyze the results.

2.16 |Isothermal titration calorimetry (ITC)

All ITC experiments were carried out and analysed using the Launch

NanoAnalyze Software. All titrations were performed at 25°C while

stirring at 300 rpm in PBS. A control experiment of titrant in buffer

was performed to account for the heat of dilution. All titrations were

repeated at least three times with similar results. An approximate

protein concentration of 50 µM was used for ligand‒protein

titrations. The ligand concentration is approximately 10 times higher

than that of the protein. For Ca

–compound titrations, 22 mM CaCl

and 1.1 mM compounds (SAL or M1) were used.

2.17 |Differential scanning fluorimetry (DSF)

For the DSF experiments, 20 μL of samples were prepared in

duplicate using 100 µM protein and a compound concentration of

50 µM. The samples were heated from 20°C to 95°C at 1°C/min

before incubation for 20 min; fluorescence was measured at each

step in nanoDSF (Nano Temper Prometheus NT.48). The change in

melting temperature (T

) was calculated and recorded by the

instrument. Data analysis and image generation were performed

using the PR ChemControl Software.

2.18 |Animals, grouping and treatments

Fifty‐two specific pathogen‐free (SPF) Kunming mice and 56 SPF

BALB/c mice (male, 6–7 weeks old, weighing approximately 25 g)

were purchased from the Center of Laboratory Animals of Hubei

Province (Wuhan, China). The animals were maintained under

standard conditions of humidity (50% ± 10%), temperature

(25 ± 2°C) and dark and light cycles (12 h each) with free access to

food and water. All experimental procedures were performed in

accordance with animal welfare guidelines and were approved by the

Animal Welfare and Ethics Committee of the Huazhong Agricultural

University Wuhan, China (approval permit number: HZAUMO‐2023‐

0126, HZAUMO‐2023‐0085 and HZAUMO‐2023‐0084).

For the toxicity study, mice were divided into three groups (n=6).

The groups received intragastric administration of the following

compounds for 21 days: group A, vehicle (CMC‐Na)‐treated control;

group B, SAL (Qilu) at 10 mg/kg body weight/day; group C, M1 at

10 mg/kg body weight/day. At autopsy, testes, epididymis, sciatic nerve,

mid‐thigh muscle, kidney, lung, liver, heart and small intestine were

removed and collected for further analysis. An acute toxicity study of

SAL and M1 was performed according to the up‐and‐down procedure

described by Zhao et al.

on 16 Kunming mice (n= 8 per compound).

The LD

of SAL and M1 was estimated by AOT425StatPgm version 1.0

(US Environmental Protection Agency, USA)

For the DSS‐induced colitis study, 18 Kunming mice were

randomly assigned to one of three groups (n= 6 per group): healthy

control, DSS and DSS + 10 mg/kg body weight/day M1. All mice

except for the healthy controls were administered 3% (w/v) DSS

(molecular mass, 30–50 kDa; YEASEN, China) dissolved in drinking

water. At the end of the DSS challenge, the mice were killed after

12 h of fasting. The colon and small intestine were removed and

collected for further evaluation.

To gain insight into the in vivo efficacy of drugs against the CoV,

vehicle‐, SAL‐and M1‐treated mice (n= 14) were subjected to

intraperitoneal injection with MHV‐A59 (5 × 10

plaque‐forming

units). The control group received an equal volume of DMEM via

the same strategy. Then, the control and vehicle‐treated mice were

administered CMC‐Na orally twice a day. SAL‐and M1‐treated mice

were given 5 mg/kg body weight via the same strategy. Six mice from

each group were killed for pathological examination 3 dpi, and the

remaining eight mice in each group were observed until all the mice in

the vehicle group died.

2.19 |Histology

After decapitation, testes, epididymis, mid‐thigh muscle, kidney, liver,

lung, heart, colon and small intestine were immediately fixed in 4%

paraformaldehyde in phosphate buffer and stored at 4°C. Sciatic

ZHENG ET AL.

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nerve samples from the mid‐thigh were obtained from all animals,

fixed in 2.5% glutaraldehyde and stained with osmium tetroxide

before embedding in solvent‐free, modified bisphenol A epoxy resin.

For paraffin embedding, organs were washed and dehydrated

through a series of graded ethanol baths, followed by embedding in

paraffin wax. Serial sections of 5 µm thickness were cut using a

microtome and stained with haematoxylin and eosin. The sections

were observed using a light microscope.

2.20 |Enzyme‐linked immunosorbent assay

(ELISA)

The liver tissues were weighed and homogenized in PBS. After

centrifugation at 3000 rpm for 30min, the supernatant was collected

to quantify MHV, MPO, IL‐6 and TNF‐αlevels by ELISA kits

(MSKBIO) according to the manufacturer's instructions.

2.21 |Quantification and statistical analysis

Each reaction was performed at least in triplicate, and the results are

expressed as the mean ± standard deviation (SD). All the group

differences were assessed using one‐way analysis of variance. A p‐

value ≤0.05 was considered statistically significant. Statistical signifi-

cance is expressed as follows: p> 0.05 (ns, not significant),

0.01 ≤p< 0.05 (*) and p< 0.01 (**). Statistical analyses and graphical

presentations were performed using GraphPad Prism 8.

3|RESULTS

3.1 |Identification of SAL as an antiviral compound

The in‐silico experiments were performed to identify molecules with

antiviral effects. The targets were ranked by scores obtained from

SYBYL‐X. Out of the library with over 1 100 000 compounds, the top

four candidates –salvianolic acid B (SAB), epmedin B (EB), SAL and

icariin (ICA) –exhibited strong binding energy interacting with the

porcine aminopeptidase N (pAPN) protein (Table S4). Of them, SAL

received a top three rating from SYBYL‐X (score = 10.0). The PK‐15

cells were infected with TGEV at a multiplicity of infection (MOI) of

0.1 for 1 h and then treated them with 5 µM of SAL, ubenimex (UBE),

SAB, EB or ICA, for 24 or 36 h. SAL strikingly inhibited replication by

reducing viral copy numbers. SAB could also inhibit viral replication,

but it was not as effective as SAL. Other candidates did not show an

obvious antiviral effect at 5 µM (Figure 1A,B). SAL manifested anti‐

TGEV activity in a dose‐dependent manner (Figure 1C). The antiviral

activity of each agent was evaluated by the time‐of‐addition assay as

follows: pre‐,co‐and posttreatment. Co‐and pretreatment with

1 µM SAL exerted the most potent antiviral activity against TGEV

(Figure 1D and E). Furthermore, SAL downregulated APN protein

expression at 24–36 h posttreatment in both PK‐15 (Figure S1A,B)

and IPEC‐J2 (Figure S1C,D) cells based on western blot and

immunofluorescence assay (IFA). SAL at 5 µM was found to exert a

significantly more effective (Figure S1B,C) and sustained (Figure S1A)

inhibitory effect than 5 µM UBE (positive control) on APN protein

expression in vitro, and the effect was dose dependent (Figure S1D).

3.2 |Identification of APN as a target of SAL

The cellular thermal shift assay (CETSA) and drug affinity responsive

target stability (DARTS) were conducted to test whether the compound

coulddirectlybindtothepAPN.Therewasagoodcorrelationinligand‐

induced thermal stabilization potency. At 30 µM in the culture medium,

SAL and UBE (positive control) dramatically stabilized the pAPN protein

in the cell material at a temperature of 48–52°C compared with the

dimethyl sulphoxide (DMSO)‐treated group (Figure 1F and G). SAL

exhibited a dose‐dependent response (1–30µM)whentheproteinwas

challenged at the melting temperature (48°C) (Figure 1H,I). In the DARTS

experiment, the interaction between SAL and UBE (at 30 µM) and pAPN

stabilized the structure so that it became more resistant to proteases

diluted 1:300 to 1:1000 (pronase:protein) in solution (Figure 1J,K). In

addition, there was dose‐dependent stabilization of the APN structure

with 1–100 µM of the compound and proteases in 1:500 in solution

(Figure 1L and M). The relative intensities of bands in western blot were

quantified using ImageJ software.

3.3 |Design and synthesis of the optimal

compound M1 based on structure‐based drug

design (SBDD)

SAL exhibits robust antiviral efficacy despite its strong cellular

toxicity in PK‐15 (Figure S1E) and IPEC‐J2 (Figure S1F) cells, as

indicated by the selectivity index (SI). SAL derivatives were designed

and synthesized using the SBDD approach to alleviate toxicity

without changing their antiviral effectiveness and targeting propert-

ies. SBDD was conducted using the Surflex‐Dock Screen module of

SYBYL‐X (Table S5) and PyMOL software (Figure 2A and S2A–I).

Three small fragments without oxygen atoms–cyclopropylamine (to

generate M7), cyclopentylamine (to generate M8) or cyclobutylamine (to

generate M9)–were first used to introduce amide bonds by replacing

carboxyl groups to inhibit the formation of a hydrophilic cavity due to the

interaction (hydrogen bond formation) between the carboxylic and

hydroxyl groups placed on the opposite ends of the molecule. According

to docking simulations, ligands with larger introduced fragments had

higher Crash values (values that are close to 0 are favorable), which

revealed that a five‐membered ring structure in a suitable pocket cavity

could reduce the level of internal self‐clashing that the ligand would have

experienced and could make the binding more stable. However, the total

score and polarity of all three derivatives (7.3, 6.7 and 7.8) were lower

than those of the starting compound (8.6). This result indicates that

derivatives M7, M8 and M9 would probably have poor antiviral

performance accompanied by the risk of off‐target toxicities due to

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ZHENG ET AL.

FIGURE 1 SAL can inhibit TGEV infection and direct bind to pAPN in vitro. (A and B) Analysis of the anti‐TGEV effects of different compounds

(5 µM), including salinomycin (SAL), ubenimex (UBE), salvianolic acid B (SAB), epmedin B (EB) and icariin (ICA). The level of TGEV‐Nexpressionwas

measured by western blot (A) and RT‐qPCR (B). (C) Identification of the dose‐dependent anti‐TGEV activity of SAL. (D and E) Analysis of antiviral activity

of SAL evaluated by the time‐of‐addition assay. The level of TGEV‐N expression was measured by RT‐qPCR (D) and western blot (E). (F−M) The CETSA

(F−I) and DARTS (J−M) assays were conducted to test whether the compound could directly bind to pAPN. The CETSA assay was employed to assess the

protective effect of SAL (30 µM) against protein denaturation in the cell material at a temperature of 37–58°C (F and G). The CETSA assay was utilized to

detect the dose‐dependent thermal stabilization potency of SAL (1 −30 µM) when the protein was challenged at 48°C (H). The western blot results of

CETSA assay were quantified using ImageJ (I). The DARTS assay was employed to assess the protective effect of SAL (30 µM) on the protein when

treated with proteases diluted 1:300 to 1:1000 (pronase:protein) in solution (J and K). The DARTS assay was utilized to detect the dose‐dependent

protective effect of SAL (1 −100 µM) when the protein was treated with pronase solution (pronase:protein ratio = 1:500) (L). The western blot results of

DARTS assay were quantified using ImageJ (M). Data are represented as mean ± SD. p> 0.05 (ns, not significant), 0.01 ≤p<0.05(*) and p< 0.01 (**).

ZHENG ET AL.

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FIGURE 2 (See caption on next page).

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ZHENG ET AL.

poor binding specificity and affinity (polarity = 1.1, 1.9 and 2.4). A small

fragment with an oxygen atom, 3‐oxetanamine (to generate M10), was

further introduced to increase the polarity and affinity of the C1

terminus. However, SYBYL‐X simulation‐based indicators were not

significantly improved (score = 7.7, polarity = 2.4). The results of the

experiment also verified that the antiviral activity became stronger with

the size of the fragment among these four compounds. M7, with the

smallest fragment, had the worst antiviral effect. The SI value was 28.34

for porcine epidemic diarrhea virus (PEDV) and 9.55 for TGEV. While

M8, with the largest fragment, had the best antiviral effect among the

three molecules (SI = 83.44 for PEDV and 47.54 for TGEV).

Then, groups with five‐membered ring structures combined

with anti‐CoV activity were searched for application in the

synthesis of SAL derivatives. Thiophene‐based derivatives are

effective against various viruses, including CoVs.

35,36

Thus,

thiophene, including 2‐thiophenecarboxylic acid hydrazide (to

generate M5), 2‐thiophenemethylamine (to generate M12) and

thiophene‐2‐ethylamine (to generate M13), were attempted to

introduce to enhance binding stability and antiviral activity. The

docking simulations showed that M13 could extend to the deeper

site of the N‐terminus of APN. M13 had a longer and straighter

carbon chain than the other two derivatives, features that might

prove beneficial. SYBYL‐X also scored M13 (9.4) higher than M5

(8.1) and M12 (7.6). Next, the 2‐thiophenecarboxaldehyde

hydrazone (to generate M1) was applied to the carboxyl group

modification of SAL due to the non‐rotatable carbon‐nitrogen

double bond. Eventually, M1 tended to form shorter hydrogen

bonds with a higher total score (9.6) and polarity (3.6) than those

of SAL and other derivatives reported by SYBYL‐X. In total, eight

SAL derivatives were synthesized; information such as eluant and

percentage yield of the product are listed in Table S2.The

structures were confirmed by using liquid chromatography mass

spectrometry‐ion trap‐time of flight (LCMS‐IT‐TOF),

Hnuclear

magnetic resonance (

H‐NMR) and

Cnuclearmagneticreso-

nance (

C‐NMR) measurements (Supplemental Information).

3.4 |M1 exhibits low toxicity and efficient antiviral

properties in vitro

As expected, all SAL derivatives had lower cytotoxicity than SAL,

especially M1, with half maximal cytotoxic concentration (CC

)

values of 128.90 and 103.20 µM in Vero‐E6 and PK‐15 cells,

respectively (Supplemental Information). Some SAL derivatives still

had better or similar antiviral activities against TGEV, PEDV, FIPV and

mouse hepatitis virus (MHV). For TGEV, the results indicated that

SAL, M1 and M5 could significantly decrease the viral copy number

(Figure 2B) with low half maximal effective concentration (EC

)

values of 0.59, 0.63 and 0.68 µM, respectively (Table S6). M7 and

M10 exhibited no obvious antiviral activity against TGEV in vitro. The

other SAL modifications also significantly diminished the viral

inhibitory activity of the prototype drugs M8, M9, M12 and M13

(Figure 2B). M1 had a much better antiviral effect than UBE (APN

inhibitor) and melatonin (MEL, a reported anti‐TGEV and anti‐PEDV

agent). It exhibited evident antiviral activity comparable to that of

GC376 (3CL inhibitor) and GS441524 (RdRp inhibitor) at the same

concentration of 10 µM (Figure S3A). However, it was slightly weaker

than the effects of GC376 at low concentrations (0.1–2.5 µM) in

PK‐15 cells (Figure 2C).

SAL showed poor anti‐PEDV activity at 24 h postinfection (hpi)

(Figure S3B), and up to 36 hpi (Figure 2D) it began to exhibit good

anti‐PEDV activity with an EC

value of 0.23 µM (Table S6). M1 and

M5 inhibited PEDV replication in vitro at both 24 and 36 hpi

(Figure 2D and S3B), with EC

values of 0.31 and 0.17 µM,

respectively (Supplemental Information). A similar antiviral effect

was noted for 10 µM M1, 25 µM GC376 and 25 µM GS441524 at 24

hpi (Figure S3C). M1 had a much better antiviral effect than UBE and

MEL. Of note, M1 had a significantly better antiviral effect than

GC376 at low concentrations of 0.1–2.5 µM at 36 hpi (Figure 2E).

PEDV infection was examined by IFA at 36 hpi, which also indicated

that M1 and SAL at 5 µM could effectively block PEDV infection in

vitro (Figure S3D).

The anti‐FIPV activity of these compounds was also tested in the

CRFK cell line. SAL and M1 at 5 µM significantly inhibited FIPV in

CRFK cells (Figure 2F). Treatment with SAL and M1 at 10 μM showed

detectable antiviral activity against MHV‐A59 in LR7 cells

(Figure 2G). The virus titration assays showed that the anti‐MHV

activity of M1 at 5 µM was better than that of SAL (Figure S3E).

Other derivatives of SAL in this study did not show obvious anti‐FIPV

or anti‐MHV activity, with >50% inhibition at specific concentrations

in vitro (data not shown). The pseudovirus of Omicron B.1.1.529 was

used to evaluate the antiviral efficacy of the compounds against

SARS‐CoV‐2. The relative light units (RLU) for luciferase activity were

recorded at 36 hpi. The assay with each compound was performed in

sextuplex. E64d (positive control) and SAL exhibited remarkable

antiviral activity (Figure 2H). Some SAL derivatives, such as M1, M5,

M12 and M13, exhibited ordinary antiviral effects with EC

values

ranging from 2.51 to 3.42 µM, which were worse than that of SAL

FIGURE 2 SAL and M1 exhibit antiviral activity. (A) The roadmap outlines the strategy for designing and synthesizing derivatives of SAL.

(B) Analysis of the anti‐TGEV activity of 5 µM SAL and its derivatives using western blot. (C) Comparison of anti‐TGEV activity between GC376

(3CL inhibitor) and M1 at low concentrations (0.1–2.5 µM) in PK‐15 cells. (D) Analysis of the anti‐PEDV activity of 5 µM SAL and its derivatives using

westernblot.(E)Comparisonofanti‐PEDV activity between GC376 and M1 at low concentrations (0.1–2.5 µM) in Vero cells. (F−H) Analysis of the anti‐

FIPV (F), anti‐MHV (G) and anti‐SARS‐CoV‐2 (H) activity using IFA. Identification of the recombinant FIPV with a green fluorescent protein (F) and

MHV‐A59 (G) using fluorescence microscopy. The MHV‐N protein was detected with specific MHV‐N mouse monoclonal antibody and fluorescein‐

conjugated goat anti‐mouse IgG (green). The cell nuclei were stained with DAPI (blue). Scale bar, 200 µm. Spike proteins of Omicron B.1.1.529

pseudovirus expressing luciferase was used to evaluate the antiviral efficacy of the compounds. The relative light units (RLU) were recorded for

luciferase activity (H). Data are represented as mean ± SD. p> 0.05 (ns, not significant), 0.01 ≤p<0.05 (*) and p<0.01(**).

ZHENG ET AL.

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FIGURE 3 (See caption on next page).

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ZHENG ET AL.

(0.69 µM) at 36 hpi in vitro. Most notably, due to the lowest

cytotoxicity (CC

= 128.90 µM) among all compounds, M1 showed

the highest SI of 37.69, surpassing SAL's SI of 12.28. M5 showed the

second‐best SI value of 13.04 in this study (Table S6).

3.5 |M1 targets the APN protein

SAL and M1 at 5 μM were found to inhibit the expression of APN in PK‐

15 (Figure 3A)andCRFK(Figure3B) cells. Oral administration of 10 mg/

kg body weight SAL or M1 could also effectively inhibit the expression of

APN in the small intestine of mice (Figure 3C). To test whether the

compoundcouldbindtoAPN,CETSA,DARTS,co‐immunoprecipitation

(Co‐IP) and differential scanning fluorimetry (DSF) were conducted. M1

presented a more obvious change than SAL at the same concentration of

30 µM in compound‐induced APN protein thermal stability at 48–50°C

in PK‐15 (Figure 3D)andCRFK(Figure3E) cells. In addition, M1 and M9

at 30 µM protected the target protein in PK‐15 cells from degradation

by a range of protease concentrations (from 1:300 to 1:1000

pronase:protein) markedly better than SAL at the same concentration

(Figure 3F). The Co‐IP experiment was conducted in PK‐15 cells. An

interaction between APN and TGEV was observed. SAL and M1 could

competitively interact with APN and therefore interfere with its

interactions with TGEV (Figure 3G). The pAPN was harvested and

detected as a single band on the gel (Figure 3H) and further identified by

western blot (Figure 3I). Then, DSF was carried out to detect the

compound‐induced protein thermal stability to assess the binding

interaction between compounds (SAL, M1 and M5) and the pAPN

protein. The results showed that SAL, M1 and M5 could markedly

enhance the thermal stability of pAPN, while tylosin (TYL, control group)

did not (Figure 3J).

While the catalytic activity of APN is not required for virus entry,

the enzyme assay was conducted to provide evidence supporting the

targeting of APN by the compound.

The results demonstrated

the remarkable cleavage ability of APN on L‐Leu‐pNA, resulting in the

formation of p‐nitroaniline as a product. The absorbance values

exhibited a linear increase throughout the 0 −60 min duration,

indicating a constant rate of substrate cleavage by APN. Notably,

UBE, SAL, and M1 exerted significant inhibitory effects on enzymatic

activity. Among them, UBE displayed the highest inhibitory potency

at an equivalent concentration of 50 µM (Figure S4A). Moreover, the

inhibitory activity of these compounds showed dose‐dependency

within the range of 50 −200 µM after 90 min of treatment. The half

maximal inhibitory concentration (IC

) value for UBE‐mediated

inhibition of APN activity was approximately 50 µM, while the IC

values for SAL and M1 were 100 −200 µM (Figure S4B).

3.6 |STSBPT revealed that 3CLpro is an additional

target of SAL and M1

In a previous study, the researchers considered SAL and its

derivatives to be a new class of multitarget “magic bullets”.

Other

candidate target proteins for SAL and its derivatives were attempted

to find in the study. Given that co‐treatment with SAL showed an

impressive inhibitory effect against TGEV in PK‐15 cells, viral proteins

rather than cellular proteins were considered to identify promising

new targets. The DBD was “unfolded”onto a planar space based on

the notion of dimensionality reduction to find a prospective target

protein with a similar DBD structure to pAPN (PDB: 4FKE).

Specifically, Surflex‐Dock Screen (SFXC) dockings were performed

using SYBYL‐X to find ligand‐proximal amino acids in the binding

pocket. The two‐dimensional interaction diagram was obtained by

using the Protein Contacts Atlas (http://pca.mbgroup.bio/index.html)

(Figure 4A). A contact was considered to have occurred if the

distance was less than 4 Å. It can be observed that the ligand

established 373 atomic contacts with its immediate neighbors

(Figure 4B).

In total, 22 amino acids (AGAERTVHESEYLGDRVFDSYS) of

pAPN were took from the first shell of immediate ligand contacts and

performed a Protein BLAST search (http://blast.ncbi.nlm.nih.gov/

Blast.cgi) to align them with proteins in the Protein Data Bank (PDB)

considering CoVs as the organism (taxid: 693996). 3CLpro, spike

glycoprotein, nonstructural protein 3 (nsp3) and nsp12 were found to

share high (46%–100%) residue percent identity with the query

sequence (Table S7). The mean root‐mean‐square deviations

(RMSDs) within 4 Å from the ligand were tentatively calculated to

determine the pocket similarity using PyMOL software. Only the

region of the binding sites of 3CLpro retained constructive similarity

with those of APN (RMSD < 3 Å). The values of other proteins

involved in the calculation were all above 3 Å, indicating significant

dissimilarity of the binding sites between the other proteins and APN.

The score calculated by SYBYL‐X was also used to evaluate the

affinity between SAL and these candidate targets. Most notably, the

DBD of 3CLpro of SARS‐CoV had both structural similarity (2.365 Å)

with APN and high affinity (score = 13.29) with SAL. Moreover, the

subject sequence of 3CLpro searched by BLAST was part of the

sequence of the DBD and participated in binding with SAL.

FIGURE 3 SAL and M1 can bind to APN. (A) The effect of 5 µM SAL and its derivatives on the expression of APN protein in PK‐15 cells.

(B) The effect of SAL and its derivatives on the expression of feline APN (fAPN) protein in CRFK cells. (C) The impact of orally administering 10 mg/kg

body weight of SAL or M1 on the expression of APN protein in the small intestine of mice (n= 3 per group). (D−F) Analysis of the binding ability of

compounds to pAPN (D) and fAPN (E) by CETSA, and to pAPN by DARTS assays (F). (G) Analysis of the interaction between APN and TGEV and

whether SAL and M1 could significantly block this interaction through the Co‐IP assay. The APN and TGEV‐N proteins were detected using western

blot with an APN rabbit monoclonal antibody and a TGEV‐N mouse monoclonal antibody. (H) Purified pAPN protein appeared as a single band in the

gel. (I) Identification of purified pAPN using western blot with an APN rabbit polyclonal antibody. (J) The DSF assay. The thermal shift (T

change) was

determined with DSF by incubating pAPN with DMSO (blank control), tylosin (TYL, negative control), SAL, M1 and M5.

ZHENG ET AL.

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FIGURE 4 (See caption on next page).

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ZHENG ET AL.

The computer‐aided molecular binding analysis with multiple targets

based on the STSBPT was first conducted to explore whether SAL and

M1 could simultaneously target APN and 3CL proteins. First, the binding

sites of SAL and M1 on the pAPN and 3CL‐TGEV proteins were

predicted using SYBYL‐X and modeled them by PyMOL. Modification of

the carboxyl group of SAL gave rise to a significant relocation of the

compound to an alternative energy minimum, resulting in alteration of

the binding sites of amino acids and shortening of the distance between

the proton donor and acceptor. SAL was found to interact with the side

chains of GLU‐384, GLU‐413 and ARG‐437 of pAPN (Figure 4C)and

GLN‐8andARG‐294 of 3CL‐TGEV (Figure 4D). M1 interacts with ALA‐

346, GLU‐375, ARG‐376 and GLY‐433 of pAPN (Figure 4E)andGLN‐8,

GLU‐152 and ASN‐153 of 3CL‐TGEV (Figure 4F). There are more

hydrogen bonds participating in the ligand‒protein binding between M1

and both targets than between SAL and both targets. The increased

binding could directly improve binding affinity.

Then, the binding domains 4Å from the ligand of these target

proteins were extracted and superimposed to calculate the RMSD

values of superimposed proteins by PyMOL and the P‐min score

by PocketMatch version 2.0 (http://proline.physics.iisc.ernet.in/

pocketmatch/). The RMSD value is a measure of the average distance

between the superposed protein atoms. P‐min isthe value obtained by

dividing the number of matches by the total number of pairwise

distances of the smaller pocket. The results indicated that DBDs have

structural similarities between the host protein and viral proteins.

Furthermore, the 4 Å regions from M1 have an overall lower RMSD

and a higher P‐min score observed between pAPN and 3CL proteins

than those from SAL (Table S8).

Toexaminewhetherthecompoundscouldalsoinhibitandbindto

3CL proteins, three plasmids were constructed to express the 3CL fusion

proteins of TGEV, PEDV and FIPV in an Escherichia coli expression

system. The results of sodium dodecyl sulfate polyacrylamide gel

electrophoresis (SDS‐PAGE) indicated that the 3CL proteins were pure

with expected molecular masses of 35–40 kDa (Figure 5A,S5A,B). They

were expressed both in the supernatant and inclusion body as assessed

by western blot (Figure 5B,S5C,D). The peptide Dabcyl‐

VSVNSTLQ ↓SGLRKMA‐E(Edans) was used to identify the effect of

each inhibitory compound against the 3CL‐TGEV, 3CL‐FIPV proteins.

The peptide was obtained commercially from GenScript (Nanjing, China)

with >95% purity. The time‐dependent increase in fluorescence intensity

showed that SAL and M1 could inhibit the activity of the 3CL proteins of

TGEV and FIPV with similar efficiency. The percentage of inhibition of

SAL and M1 was 43.5% ± 4.0% and 56.6% ± 6.5% for 3CL‐TGEV,

respectively (Figure 5C), and 43.9% ± 7.2% and 44.6% ± 5.8% for 3CL‐

FIPV, respectively (Figure 5D), after 30 min of incubation. Moreover, SAL

and M1 also showed a dose‐dependent inhibitory activity against 3CL‐

TGEV (Figure S5E).

SAL and its eight derivatives were docked using SYBYL‐Xtofindkey

amino acid positions on 3CL proteins. All compounds bind to the GLN‐8

position of the 3CL‐TGEV protein via hydrogen bonds in the putative

substrate‐binding models (Figure 5E). The GLN‐8 residue was identified

as a highly conserved recognition site in the 3CL proteins of TGEV,

PEDV, FIPV and HCoV‐229E (Figure 5F), whereas the 3CL protein of

SARS‐CoV‐2 Omicron did not show conservation of this site

(Figure S5F). The prediction of interactions between compounds and

the active pocket of the 3CL proteins was confirmed using the

isothermal titration calorimetry (ITC) assay. SAL and M1 directly

interacted with the 3CL protein of TGEV with K

values of

0.14 ± 0.05 μM and 66.89 ± 0.02 nM, respectively, and N values of

1.62±0.05and1.58±0.04sites,respectively(Figure5G,H). Mutation of

GLN‐8(FigureS5G) altered the integrity of the binding pocket and

impaired the interaction between the small molecule and target protein,

as revealed by ITC, suggesting that GLN‐8intheN‐terminus might be

the key residue on 3CLpro (Figure S5H,I). DSF was subsequently carried

out to detect the compound‐induced variation of protein thermal

stability to assess the binding interaction. The thermal shift (T

change)

was introduced by incubating 3CLpro‐TGEV, 3CLpro‐PEDV and 3CLpro‐

FIPV with specific compounds compared with incubation with DMSO

(blank control) and TYL (negative control) (Figure 5I). The results

indicated that the incubation of SAL and its derivatives caused a shift

in the 3CL protein's T

value. The binding of compounds to the 3CL

protein resulted in decreased thermal stability, which is consistent with

previous reports.

These provides evidence that SAL, M1, M5, M8, M9,

M12andM13bindto3CLpro‐TGEV (Figure 5J); SAL, M1 and M5 bind

to 3CLpro‐PEDV (Figure 5K);andSAL,M1andM12bindto3CLpro‐

FIPV (Figure 5L).

3.7 |M1 attenuates intestinal damage and

inflammation

The gut serves as a reservoir for viruses.

Previous studies have

demonstrated the close relationship between CoVs and the digestive

system (including intestinal damage, intestinal inflammation, etc.).

cell model of virus‐induced cell junction protein deletion and a mouse

model of dextran sulfate sodium (DSS) injury were employed to test

the efficacy of drugs to treat symptoms. SAL and M1 at 5 µM

significantly reversed the TGEV‐induced low level of expression of

tight junction proteins, such as ZO‐1 and occludin, at 24 hpi

(Figure 6A). Cells infected with TGEV or PEDV were totally

FIGURE 4 STSBPT reveals that 3CLpro is possibly an additional target of SAL and M1. (A) The two‐dimensional diagrams of SAL and targets

interactions were generated based on the Protein Contacts Atlas. The inner ring indicates the first shell of immediate ligand contacts, and the

outer ring indicates the second shell of extended ligand contacts. The size of the circle is proportional to the total number of contacts between

the residue and any of the inner residues within the ring. (B) Statistics of the first shell of immediate ligand contacts. (C−F) The docking

simulation was conducted in SYBYL‐X and pictured by PyMOL, including simulated images of SAL in a complex with pAPN protein (PDB: 4FKE)

(C) and 3CL‐TGEV protein (PDB: 1P9U) (D), as well as M1 in complex with pAPN protein (E) and 3CL‐TGEV protein (F). Hydrogen bonds are

indicated by dotted lines. The name of the bound amino acid and the hydrogen bond distance are marked in the images. The modified part of

SAL is shown in magenta.

ZHENG ET AL.

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FIGURE 5 SAL and M1 can bind to 3CL proteins. (A) Purified 3CL protein (line 5) was detected as a single band in the gel, compared with the

negative control (line 1), the total protein of lysates (line 2) and the soluble (line 3) and insoluble (line 4) forms. (B) Detection of the protein of

interest by western blot with a mouse anti‐His‐tag monoclonal antibody. T, total protein; S, supernatant; P, precipitation. (C and D) The FRET

assay. The peptide Dabcyl‐VSVNSTLQ ↓SGLRKMA‐E (Edans) was used to detect the cleavage activity of 3CL proteins. Data are represented as

mean ± SD. (E) SAL and its eight derivatives share the same binding site with 3CL‐TGEV, namely GLN‐8 (colored in green). (F) A logo plot

indicates that GLN‐8 is a conserved amino acid residue in 3CL of TGEV, PEDV, FIPV and HCoV‐229E. (G and H) Detection of the ligand–target

binding affinity by the ITC assay. Data are represented as mean ± SD. (I–L) The DSF assay. The thermal shift (T

change) was introduced by

incubating 3CLpro‐TGEV (I and J), 3CLpro‐PEDV (K) and 3CLpro‐FIPV (L) with specific compounds compared with incubation with DMSO (blank

control) and tylosin (TYL, negative control).

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ZHENG ET AL.

FIGURE 6 (See caption on next page).

ZHENG ET AL.

15 of 21

morphologically restored after treatment with SAL or M1, and the

expression of ZO‐1 was changed to normal levels in IPEC‐J2

(Figure S6A) and Vero‐E6 (Figure 6B) cells at 24 hpi. Moreover,

SAL and M1 significantly reduced TGEV‐induced phosphorylation of

extracellular signal‐regulated kinase 1/2 (ERK1/2) and nuclear factor

kappa B (NF‐κB) in vitro, restoring virus‐challenged cells to the

normal levels as observed in virus‐free treatment at 24 hpi

(Figure 6C). IκBαprotein expression returned to normal. Some

inflammatory factors triggered by TGEV, such as interleukin 1beta (IL‐

1β) and tumor necrosis factor alpha (TNF‐α), were significantly

downregulated by SAL and M1 in IPEC‐J2 cells at 24 hpi (Figure 6D).

For the DSS‐induced colitis study, the addition of 3% DSS in

drinking water led to body weight loss an increased diarrhea and

rectal bleeding. After the symptoms became obvious, each mice

began to receive oral administration of vehicle or 10 mg/kg body

weight M1 each day. Mice in the vehicle‐treated group died in 4

days, while mice in the M1‐treated group exhibited an 83.3%

survival rate; only one mouse died on Day 3 (Figure 6E). The colon

and small intestine were collected to evaluate histopathology

(Figure 6F). The vehicle‐treated mice showed serious colon

damage. The arrangement of intestinal epithelial cells and muscle

fibers appeared disordered. There was a large number of

inflammatory cells in the mucosa and submucosa. The muscle

fibers appeared dissolved and sloughed off. The gland, goblet cell

and crypt structures of the mucous membrane were absent. In

addition, there were fewer intestinal glands and goblet cells

compared with the healthy control group. In the M1‐treated

group, the colon texture was clear, the morphology was intact,

there was no congestion and the mucosal intestinal epithelial cells

were arranged neatly. The intestinal structure remained intact,

appearing essentially identical to that of the healthy control

group.

3.8 |SAL and M1 exhibit antiviral effects in vivo

MHV infection is regarded as a suitable animal model for studying the

mechanisms and pathology of CoV infections.

41–43

For the anti‐MHV

study, all mice died at 7 days postinfection (dpi) in the vehicle‐treated

group. MHV‐infected mice treated with M1 demonstrated significant

improvement with 75% survival. In contrast, there was 50% survival

in the SAL‐treated group (Figure 6G). At 3 dpi, six mice from each

group were killed for histopathological examination. Compared with

the vehicle‐treated group, SAL and M1 treatment significantly

alleviated liver tissue necrosis and damage caused by MHV infection

(Figure 6H). The liver‐to‐body weight ratios in the M1‐treated mice

were significantly decreased compared with those in the vehicle‐

treated mice (Figure S6B). Histopathological analysis showed that

MHV‐infected mice treated with vehicle and SAL exhibited signifi-

cant tissue necrosis and damage compared with the livers of the

MOCK group. Livers of the M1‐treated group showed relatively

normal morphology, similar to the MOCK group (Figure 6I). The

number of white blood cells (WBCs), lymphocytes (LYMs) and

platelets (PLTs) significantly decreased in MHV‐infected mice

compared with the MOCK group, and treatment with M1 restored

these blood indicators to the normal ranges (Figure 6J,K). The

enzyme‐linked immunosorbent assay (ELISA) results showed that the

levels of MHV, IL‐6, TNF‐α, and myeloperoxidase (MPO) in the livers

of the M1‐treated group were significantly lower than those in the

livers of the vehicle‐treated group. SAL treatment also significantly

reduced some inflammatory factors, such as MPO and TNF‐α,

compared with vehicle treatment (Figure 6L−O).

3.9 |Low toxicity of M1 in vitro and in vivo

M1 exhibited much lower cytotoxicity than SAL. A series of studies were

carried out to investigate this phenomenon. Flow cytometry showed that

M1 caused a less severe effect than SAL on early apoptosis of IPEC‐J2

cells at a low concentration (10 µM) (Figure 7A). M1 could not increase

the Ca

concentration of IPEC‐J2 cells, according to the fluorescence

intensity of cells preloaded with Fuo‐4 AM, whereas this did happen to

the cells treated with SAL. Treatment with 5 µM amlodipine (AML), a

calcium channel blocker, inhibited the initial influx of calcium and

reduced the fluorescence intensity of cells (Figure 7B). Then, 10 µM

CGP‐37157 (CGP, an inhibitor of the Na

/Ca

exchanger) was used to

reduce cytotoxicity triggered by Na

/Ca

exchange in response to

treatment with different concentrations of compounds. The cytotoxicity

induced by M1 could not be alleviated by 10 µM CGP. However, the

prevention of SAL‐induced cell death in response to treatment with CGP

was observed (Figure 7C). The theoretical calculations were performed

to evaluate the affinity of SAL and M1 towards Na

and Ca

via the

FIGURE 6 SAL and M1 exhibit antiviral and anti‐inflammatory effects. (A) SAL and M1 significantly reversed the TGEV‐induced reduction in

the expression of tight junction proteins. (B) The expression of ZO‐1 of Vero‐E6 cells infected with PEDV returned to the normal level after M1

treatment. The ZO‐1 protein was detected with ZO‐1 rabbit polyclonal antibody and fluorescein‐conjugated goat anti‐rabbit IgG (green). The cell

nuclei were stained with DAPI (blue). Scale bar, 10 µm. (C) SAL and M1 significantly reduce TGEV‐induced phosphorylation of ERK1/2 and NF‐

κB in vitro. (D) SAL and M1 significantly reduce TGEV‐induced expression of IL‐1βand TNF‐αin PK‐15 cells. (E) Survival curves of mice in the

DSS‐induced colitis study (n= 6 per group). (F) The colon and small intestine tissue stained with haematoxylin and eosin. Scale bar, 300 μm.

(G) Survival curves of mice in the anti‐MHV study (n= 8 per group). (H) Gross postmortem examination of liver tissue. (I) Liver sections from mice

killed at 3 dpi were stained with haematoxylin and eosin. Scale bar, 300 μm. (J and K) Routine blood tests were conducted in all 24 mice

(n= 6 per group). (L–O) The level of MHV, MPO, IL‐6 and TNF‐αin the liver of each group of mice. Data are represented as mean ± SD.

p> 0.05 (ns, not significant), 0.01 ≤p< 0.05 (*) and p< 0.01 (**).

16 of 21

ZHENG ET AL.

FIGURE 7 (See caption on next page).

ZHENG ET AL.

17 of 21

Gaussian 16 suite of programs. The simulated poses of complexes of

compounds and ions are shown in Figure 7D.ThesimulatedposeofSAL

is a macrocyclic structure closed by the head‐to‐tail hydrogen bridge.

Oxygen atoms are involved in interacting with the metal ion. However,

M1 cannot form the “head‐to‐tail”type of intramolecular hydrogen

bonds as a pseudocyclic structure. The Gibbs free energy change (ΔG)

shows that modification of the carboxyl group of SAL could downgrade

the carrying capacity of Na

and Ca

(Figure 7D and Table S9). An

increase in Fluo‐4 (green) staining that preceded the appearance of

propidium iodide (PI) signal (red) was found by approximately 5–10 min,

which supports that apoptosis could be triggered by SAL through

intracellular Ca

flux. However, there was no Fluo‐4signalevenafter

cellsbecamePIpositiveintheM1‐treated group (Figure 7E). The ITC

assay confirmed the complexation of Ca

by SAL. M1 had a much

weaker interaction with Ca

than SAL, which agrees with the results

obtained from theoretical calculations (Figure 7F,G).

The acute toxic effects of oral administration of SAL and M1

were examined in mice. The median lethal dose (LD

) was 55 mg/kg

body weight (95% confidence interval 11.81–79.9) for SAL and

1494 mg/kg body weight (95% confidence interval 1000–2000) for

M1 (Table S10). To evaluate the toxic effects of SAL and M1 on the

male reproductive system, muscle and the sciatic nerve, mice were

administered 10 mg/kg body weight of SAL or M1 once daily for 21

days. After 21 days, SAL had significantly reduced the body weight of

mice (by >14 g on average) compared with M1 and vehicle (CMC‐Na).

The histopathological results showed that SAL treatment induced

various structural changes in the seminiferous tubules of the testis

and epithelium of the epididymis. Seminiferous tubules in testes were

shrunken and disrupted in SAL‐treated mice. The cross‐section of the

epididymis showed the occurrence of vacuolization and necrosis in

SAL‐treated mice, with the disappearance of spermatozoa in the

lumen. Alveolar cavities of SAL‐treated mice were filled with red

blood cells. SAL‐induced sciatic neurodegeneration was visualized

with silver staining. Accumulation of silver grains were observed in

neurons from the substantia nigra pars compacta of SAL‐treated mice

(Figure 7H). Photomicrographs of the heart, liver, kidney, duodenum

and skeletal muscle did not show an obvious lesion in the SAL‐or

M1‐treated group (data not shown). The weight of the mice was

recorded on a daily basis during the experiment. There was no

significant difference in body weight between the M1‐and vehicle‐

treated groups (Figure 7I).

4|DISCUSSION

This study provides the first evidence that the drug‐binding domains

(DBDs) of pAPN and 3CLpro from hosts and viruses, respectively, are

structurally similar and could both be targeted by SAL and its

derivative M1, leading to the disruption of protein functions and the

inhibition of virus entry. It marks the successful application of the

STSBPT in both drug‐target discovery (DTD‐STSBPT) and multitarget

drug screening (DS‐STSBPT), providing new strategies to optimize

efficacy and to mitigate time and resource wastage resulting from

mechanical screening procedures.

The DTD‐STSBPT introduces a novel perspective to the

methodology of identifying secondary drug targets beyond the

primary target. Usually, drug–target discovery work is mainly based

on omics (including genomics, proteomics, metabolomics and

transcriptomics), mass spectrometry, CRISPR‐based gene editing

and computational approaches (including network‐based, machine

learning and molecular docking simulation approaches).

44–47

How-

ever, these methods have limitations in terms of exploring drug

targets with similar functions or structures. It is common for drugs to

have multiple targets in vivo, even if they were originally designed to

target a single specific target. Thus, it is difficult to anticipate other

potential targets during the initial design phase, which can result in

unforeseen side effects and a decrease in the overall success rate of

drug development. The DTD‐STSBPT proposed in this study provides

a valuable reference for guiding drug design and preventing off‐

target effects. On the other hand, multitargeting in drug development

opens up more possibilities for functionality, and the DTD‐STSBPT

can also be utilized to explore new applications for existing drugs or

to identify novel multitarget drugs.

The DS‐STSBPT introduces evaluation criteria from a new

perspective for drug screening. In target‐based drug screening,

comparing the structural similarity between the DBDs of known

and new targets can aid in the identification of promising targeted

drugs. Specifically, this approach can help screen for drugs that are

more likely to target the new DBD effectively. In this study, the

structural similarity comparison between the binding pockets of

pAPN and hACE2 was conducted to forecast the different inhibitory

effects of SAL and M1 on SARS‐CoV‐2 pseudovirus. The 4 Å regions

from M1 were found to have an overall higher RMSD and a lower P‐

min score between the pAPN and hACE2 proteins than those from

FIGURE 7 The cytotoxicity of SAL and M1 is related to the ability to carry Na

and Ca

. (A) The results of flow cytometry of IPEC‐J2 cells

treated with SAL and M1 at 10 µM. (B) Analysis of the Ca

concentration of IPEC‐J2 cells according to the fluorescence intensity of cells

preloaded with Fuo‐4 AM. Amlodipine (AML), a calcium channel blocker, could inhibit the initial influx of calcium. Data are represented as

mean ± SD. p> 0.05 (ns, not significant), 0.01 ≤p< 0.05 (*) and p< 0.01 (**). (C) Analysis of whether CGP‐37157 (CGP, an inhibitor of Na

/Ca

exchanger) could alleviate the cytotoxicity caused by SAL or M1. Data are represented as mean ±SD. p> 0.05 (ns, not significant), 0.01 ≤p< 0.05

(*) and p< 0.01 (**). (D) The theoretical calculations of the affinity of SAL and M1 towards Na

and Ca

via the Gaussian 16 suite of programs.

(E) Detection of the relationship between cell apoptosis and accumulation of intracellular Ca

. (F and G) Binding isotherms for SAL and M1

titrated with calcium chloride solution by the ITC assay. (H) The tissue was stained with haematoxylin and eosin. SAL‐induced

neurodegeneration was visualized with silver staining. The scale bars in the slice images of sciatic nerve and other organs are 50 and 100 µM,

respectively. (I) The body weight of mice during the treatment of vehicle, SAL or M1 (n= 6 per group). Data are represented as mean ± SD.

18 of 21

ZHENG ET AL.

SAL (Table S11). Thus, SAL was more inclined to target the ACE2

protein than M1, with a stronger binding affinity. The subsequent

experiments unequivocally confirmed this prediction, providing

compelling evidence that SAL had a stronger inhibitory effect

(Figure 2F) on pseudovirus and ACE2 protein (Figure S6C,D). This

method provides new evaluation metrics to enhance the accuracy of

virtual screening.

The implementation of this dual evaluation system enhanced the

accuracy and reliability of the outcomes. These strategies ultimately

guided us to successfully identify an efficient antiviral compound

from a pool of only eight compounds that we had designed and

synthesized. In this experiment, M1 exhibited a broad‐spectrum

antiviral effect against PEDV, TGEV, FIPV, MHV and SARS‐CoV‐2

pseudovirus and could be applied to CoVs as novel therapeutic

approaches. To further highlight the superior antiviral effect of M1,

MEL (a reported anti‐TGEV and anti‐PEDV agent),

GC376 (a

3CLpro inhibitor)

and GS441524 (an RdRp inhibitor)

were

included as positive controls, which have exhibited robust antiviral

activity in previous studies. M1 had a much better antiviral effect

against PEDV and TGEV in vitro than MEL. A similar antiviral effect

among M1, GC376 and GS441524 against TGEV was observed at 24

hpi. Significantly, M1 showed a stronger anti‐PEDV effect than

GC376 at low concentrations of 0.1–2.5 µM at 36 hpi. Moreover, our

recent in vivo experiments have also demonstrated the effectiveness

of M1 at a dosage of 5 mg/kg body weight in treating FIPV infection

of cats (unpublished data).

The remarkable antiviral efficacy exhibited by these compounds

can be largely attributed to their exceptional capacity for dual

targeting. M1 demonstrated an impressive targeted affinity

= 6.7 × 10

−8

M) to 3CLpro of TGEV, surpassing the prototype

drug SAL (K

= 1.4 × 10

−7

M). It is believed to have equivalent or even

stronger affinity than GC376, which had a K

of 5.0 × 10

−7

M for

targeting 3CLpro of SARS‐CoV‐2.

Furthermore, 3CL‐TEGV was

employed as a model to successfully predict and identify GLN‐8asa

critical residue involved in the binding interaction between SAL and

its derivatives with 3CL proteins. Moreover, progressive nonlinear

curves were observed in the enzyme kinetic data for 3CLpro, which is

indicative of covalent inhibitors that irreversibly bind to and consume

enzymes. Considering the fact that the target protein 3CL is a viral

protein and that the host APN can be fully eliminated without

adverse effects on overall health,

52,53

it suggests that covalent

inhibitors may offer a more prolonged effect compared to non-

covalent inhibitors.

Further investigation is required to establish the

precise inhibitory mechanism of SAL and its derivatives.

Limitations arose due to the unavailability of purified mutant

APN protein with functional activity, preventing a thorough analysis

of the key binding residues between the ligand and APN protein.

Nonetheless, our molecular docking simulations revealed that SAL

binds to active pockets within APN, specifically interacting with GLU‐

384, GLU‐413, and ARG‐437 (Figure S2A), which are close to the

previously reported binding sites of ubenimex (UBE, a classical

enzyme inhibitor of APN) with APN (ALA‐348, GLU‐350, GLU‐384,

and TYR‐472).

These binding sites of UBE are considered crucial

for APN catalytic activity, elucidating the reason why SAL and M1

can inhibit APN catalytic activity but with less effectiveness

compared to UBE. The catalytic activity of APN is not required for

virus entry, as the active site of APN is distant from the CoV‐binding

sites. The CoV‐binding sites on APN consist of residues 728 −744 for

TGEV and residues 760 −784 for feline CoV and canine CoV. These

virus‐binding motifs are both α‐helix turns in the head domain of

APN,

positioned on the outer surface of the SAL and M1 binding

pockets. Compared to UBE, SAL and M1 exhibit larger chemical

structures and spatial occupancy within the active pocket, along with

closer proximity to the virus‐binding motifs, which may lead to a

more potent allosteric effect on virus attachment and entry. Further

research is needed to unravel the intricacies of the mechanisms

underlying the obstruction of virus‐receptor interactions by SAL

and M1.

The toxicity of SAL derivatives is significantly lower compared to

that of SAL. It has been reported that SAL has extraordinary

biological activity in many fields, but its target uncertainty and serious

dose‐dependent toxicity limit its applications. According to the

literature, the combination and transport ability of alkali metal ions

of SAL is related to its toxic effect.

Most SAL derivatives with a

single modification at C1 are much less biologically active than

unmodified SAL against cancers,

protozoa

and gram‐positive

bacteria.

59,60

These findings indicate that modifying the carbonyl

group at C1 may not be an effective approach, despite its ability to

reduce the toxicity of SAL by decreasing the transport capacity of

alkali metal ions. Improving the efficiency and reducing the toxicity of

SAL simultaneously seems to be a challenge. In this experiment, the

carboxyl group modification at C1 of SAL was designed based on

SBDD to enhance affinity for targets and to reduce toxicity caused by

off target or ion transport without sacrificing antiviral activity

(Figure 7D–G). M1 exhibits much lower toxicity, as evidenced by

its LD

value in mice being 27 times higher than SAL (1494–55 mg/

kg body weight). Moreover, oral administration of 50 mg/kg body

weight M1 did not have any adverse effects on cats, whereas a SAL

dose as low as 5 mg/kg body weight could be fatal to them. These

results revealed a new idea to design and synthesize host‐and virus‐

directed hits based on SBDD, which also contributed to the reference

for modification of more than 120 other compounds in the group of

polyether ionophore antibiotics.

5|CONCLUSION

In summary, our findings provide fresh insights into the DTD‐STSBPT

and DS‐STSBPT approaches for drug design. These strategies were

successfully applied in the discovery and modification of antiviral

drugs. The compound M1 that we successfully designed and

synthesized has highly efficient broad‐spectrum activity against

CoVs, as it can inhibit PEDV, TGEV, FIPV, MHV, and SARS‐CoV‐2

pseudovirus. Our work provides a series of promising drug candidates

for the treatment of CoVs, and paves the way for establishing a

scientific methodology to evaluate drug‐target interactions. The

ZHENG ET AL.

19 of 21

STSBPT is expected to be a fundamental tool to accelerate the

discovery of both targets and drugs.

AUTHOR CONTRIBUTIONS

Youle Zheng and Xu Wang planned the study. Youle Zheng performed

the research, analysed the data and wrote the initial draft. Jin Feng,

Yanbin Song, Yixin Yu, Min Ling and Mengjia Zhang participated in the

experiments, including cell culture, protein expression and animal

experiments. Haijiao Xie performed theoretical calculations via the

Gaussian 16 suite of programs. Wentao Li and Xu Wang supervised this

study, critically reviewed the final draft and obtained financial support.

All authors have read and approved the final manuscript.

ACKNOWLEDGMENTS

This work was supported by the National Key Research and Develop-

ment Program of China (2023YFD1800801), the National Natural

Science Foundation of China (32272990) and the Fundamental Research

Funds for the Central Universities (2662023DKPY004). We thank the

following investigators for contributing viral stocks: Prof. Qigai He

(PEDV‐DR13 strain).

CONFLICT OF INTEREST STATEMENT

The authors declare no competing interests.

DATA AVAILABILITY STATEMENT

Data are available upon a reasonable request.

ORCID

Wentao Li http://orcid.org/0000-0002-7114-762X

Xu Wang http://orcid.org/0000-0001-6476-0509

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SUPPORTING INFORMATION

Additional supporting information can be found online in the

Supporting Information section at the end of this article.

How to cite this article: Zheng Y, Feng J, Song Y, et al. Design

and synthesis of APN and 3CLpro dual‐target inhibitors based

on STSBPT with anticoronavirus activity. J Med Virol.

2024;96:e29512. doi:10.1002/jmv.29512

ZHENG ET AL.

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Design and synthesis of APN and 3CLpro dual-target inhibitors based on STSBPT with anticoronavirus activity

Abstract

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