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Research Article
Inhibition of MERS-CoV papain-like protease by sunitinib: In vitro and in silico investigations
expand article infoAhmed L. Alaofi‡, Mudassar Shahid‡, Mohd Abul Kalam‡
‡ King Saud University, Riyadh, Saudi Arabia

Corresponding author: Ahmed L. Alaofi ( ahmedofi@ksu.edu.sa )

Academic editor: Georgi Momekov
© 2025 Ahmed L. Alaofi, Mudassar Shahid, Mohd Abul Kalam.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Alaofi AL, Shahid M, Kalam MA (2025) Inhibition of MERS-CoV papain-like protease by sunitinib: In vitro and in silico investigations. Pharmacia 72: 1-12. https://doi.org/10.3897/pharmacia.72.e141634
Open Access

Abstract

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) remains a significant public health threat, with high mortality rates and no approved antiviral therapies. The papain-like protease (PLpro) of MERS-CoV plays a critical role in viral replication and immune evasion, making it a key target for drug discovery. This study evaluated the inhibitory effects of four anticancer drugs (sunitinib, olaparib, mitoxantrone, and bicalutamide) on recombinant MERS-CoV PLpro using a combination of in vitro and in silico techniques. Protease inhibition assays revealed that sunitinib displayed potent, dose-dependent inhibition of PLpro activity, with an IC50 of 1.75 µM, while olaparib, mitoxantrone, and bicalutamide exhibited negligible inhibition. Thermal shift assays confirmed the strong interaction of sunitinib with PLpro, showing a ΔTm of 26.64 °C, indicative of increased protein stability. Furthermore, molecular dynamics (MD) simulations and docking studies provided structural insights into the mechanism of inhibition. Sunitinib bound within the thumb domain of PLpro, forming stable interactions with residues such as D76, R82, and F79. Binding induced significant stabilization of PLpro’s structure, reducing flexibility in critical regions, including the thumb and catalytic domains, as indicated by a decreased radius of gyration and alterations in the free energy landscape. Importantly, the stabilization of PLpro by sunitinib was consistent between in vitro and in silico analyses, highlighting its robust inhibitory potential. These findings position sunitinib as a promising inhibitor of MERS-CoV PLpro, with strong binding affinity and the ability to disrupt enzymatic function. Further preclinical studies are warranted to explore its therapeutic potential against MERS-CoV. This study underscores the utility of repurposing existing drugs for emerging viral threats and contributes to the development of targeted antiviral strategies.

Keywords

Middle East respiratory syndrome coronavirus, molecular dynamics simulations, viral inhibition

Introduction

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is an epidemic and zoonotic virus that was first identified in Saudi Arabia in 2012 (Zaki et al. 2012). It belongs to the Coronaviridae family, which includes other well-known coronaviruses like SARS-CoV and SARS-CoV-2. MERS-CoV causes a severe respiratory illness with symptoms ranging from mild to critical, including fever, cough, and shortness of breath, and in severe cases, it can lead to pneumonia, organ failure, and death (Al Mutair and Ambani 2020). MERS-CoV primarily causes outbreaks in the Middle East, but cases have been reported in other regions, primarily due to travel-related transmission. The largest outbreak outside the Middle East occurred in South Korea in 2015, which demonstrated the virus’s potential for rapid human-to-human transmission, especially in healthcare settings. According to the World Health Organization (WHO), MERS-CoV has a case fatality rate of approximately 35%, making it one of the deadliest coronaviruses known (Chafekar and Fielding 2018). This high mortality rate, combined with its potential for causing nosocomial outbreaks, underscores the importance of developing effective antiviral therapies and preventive measures.

The virus is primarily transmitted from dromedary camels to humans, although human-to-human transmission, particularly in healthcare settings, is also possible and poses a significant risk (Peiris and Perlman 2022). MERS-CoV is classified as a betacoronavirus and has a positive-sense, single-stranded RNA genome. It encodes several structural proteins such as the spike (S) protein, envelope (E) protein, membrane (M) protein, and nucleocapsid (N) protein, which are essential for the virus’s life cycle, including entry into host cells, replication, and assembly (Wernery et al. 2016). Among these, the spike protein is of particular interest because it mediates the virus’s entry into host cells by binding to the dipeptidyl peptidase-4 (DPP4) receptor on human cells. This interaction is a critical step in the infection process and is a primary target for therapeutic interventions (Wernery et al. 2016; Memish et al. 2020; Peiris and Perlman 2022).

Despite its significant public health impact, there are currently no approved vaccines or specific antiviral treatments for MERS-CoV. Treatment options are primarily supportive, focusing on alleviating symptoms and providing respiratory support in severe cases (Dyall et al. 2014; Hart et al. 2014). Various therapeutic agents, including monoclonal antibodies, protease inhibitors, and antiviral drugs, have been studied for their potential efficacy against MERS-CoV. However, their effectiveness remains largely unproven in clinical settings (Hamada and Kiso 2016; Kumar et al. 2017; Galasiti Kankanamalage et al. 2018; Alaofi et al. 2022). Since there is no vaccine or specific treatment for the MERS-CoV, it remains a serious threat to global health due to its high mortality rate, zoonotic origin, and potential for human-to-human transmission. Continued research into therapeutic targets and preventive measures is essential to combat future outbreaks and reduce the impact of this deadly virus.

In targeting viral infections, protease inhibitors have proven to be a vital therapeutic class, particularly for viruses like HIV, Hepatitis C (HCV), and SARS-CoV-2. For instance, HIV protease inhibitors such as Ritonavir and Lopinavir disrupt the viral replication cycle by inhibiting the HIV protease enzyme (Majerová and Konvalinka 2022). Similarly, HCV therapies have evolved with protease inhibitors like Simeprevir and Paritaprevir enhancing treatment efficacy by targeting the HCV protease essential for viral proliferation (Gammeltoft et al. 2021). Notably, for Middle East Respiratory Syndrome Coronavirus (MERS-CoV), targeting the papain-like protease (PLpro) offers a strategic therapeutic approach. PLpro is critical not only for processing the viral polyproteins, facilitating viral replication, but also for modulating the host immune response by cleaving host cell proteins to evade the immune system. The recent introduction of Paxlovid (Nirmatrelvir + Ritonavir) against SARS-CoV-2 exemplifies the effectiveness of protease inhibitors in managing viral infections by targeting enzymes critical to viral life cycles, thereby blocking viral maturation and replication (Hashemian et al. 2023). Therefore, inhibition of key enzymes such as papain-like protease (PLpro) of MERS-CoV poses an important approach to treat the viral infections associated with the virus. Structural studies revealed Plpro’s catalytic domain is composed of finger, thumb, palm, and ubiquitin-like domains (Chou et al. 2012; Lei et al. 2014; Lee et al. 2015). The catalytic triad is composed of C111, H278, and D293 residues that orient a spatial region between the thumb and palm domain capable of clipping the motif sequence (L/I)XGG↓(A/D)X.

In literature, repurposing of small molecules as inhibitors for coronavirus enzymes is extensively studied. Sunitinib is a multi-targeted receptor tyrosine kinase (RTK) inhibitor that is primarily used in the treatment of renal cell carcinoma and gastrointestinal stromal tumors. It works by inhibiting multiple signaling pathways that promote tumor growth, angiogenesis, and metastasis (Hao and Sadek 2016). The consideration of sunitinib, a tyrosine kinase inhibitor, in the context of viral inhibition, specifically against targets like the papain-like protease (PLpro) of viruses such as MERS-CoV, is intriguing and potentially groundbreaking. Sunitinib primarily targets cellular signaling pathways to combat cancer by inhibiting multiple receptor tyrosine kinases (RTKs), which are crucial for tumor growth and angiogenesis (Christensen 2007). This mechanism is distinctly different from that of protease inhibitors, which specifically interrupt the viral replication process. However, exploring the mechanism of action of PLpro reveals why traditional tyrosine kinase inhibitors like sunitinib may be suitable for targeting such viral enzymes. PLpro is essential for the replication and maturation of coronaviruses as it cleaves the viral polyprotein at specific sites to produce functional viral proteins necessary for the viral life cycle (Durai et al. 2015). While there has been interest in exploring broad-spectrum antivirals that could target both viral and host factors, sunitinib has emerged as a promising broad-spectrum inhibitor of coronaviruses. Its antiviral properties extend beyond coronaviruses, as it also effectively blocks viral entry in other viruses such as Hepatitis C Virus (HCV), rabies virus, dengue virus, and Ebola virus. This wide range of activity underscores the potential of sunitinib in antiviral research and highlights the need for further exploration of its application across various viral infections, making it a critical candidate for drug repurposing efforts in combating emerging viral threats (Neveu et al. 2015; Bekerman et al. 2017; Wang et al. 2020). Olaparib, a PARP inhibitor, is particularly effective in treating cancers with BRCA1/2 mutations, including ovarian, breast, and prostate cancers. It also helps mitigate SARS-CoV-2-induced and inflammation-related cell death, promoting cell survival (Curtin et al. 2020). Mitoxantrone is an anthracenedione antineoplastic agent used in the treatment of certain types of cancer, including prostate cancer and acute myeloid leukemia (AML). Cellular and molecular docking studies have shown mitoxantrone to inhibit the SARS-CoV-2 coronavirus (Lokhande et al. 2021; Zhang et al. 2022). Bicalutamide is an anti-androgen drug used to treat prostate cancer by binding to androgen receptors and blocking the action of testosterone, which is essential for the growth of prostate cancer cells. However, clinical, epidemiological, and experimental evaluations have shown that anti-androgens like bicalutamide have no effect on SARS-CoV-2 infection (Welén et al. 2022).

These drugs highlight the diversity in therapeutic strategies employed in oncology, ranging from targeted kinase inhibition and DNA repair disruption to androgen receptor antagonism and DNA intercalation. Each of these drugs plays a critical role in the tailored treatment of various cancers, reflecting the complexity and specificity required in modern cancer therapies. These drugs were shown to have promising antiviral activity against SARS-CoV-2 and have varying effects on inhibiting SARS-CoV-2 replication, but no study has been conducted on MERS-CoV. Therefore, we conducted experimental and in silico studies to evaluate the inhibitory activities of sunitinib, olaparib, mitoxantrone, and bicalutamide against MERS-CoV PLpro.

Materials and methods

Chemicals

Sunitinib, olaparib, mitoxantrone, and bicalutamide were purchased from Biosynth, Carbosynth Ltd. (United Kingdom). Z-Arg-Leu-Arg-Gly-Gly-AMC Acetate was purchased from Bachem (CA, United States). SYPROTM orange protein gel stain was purchased from Invitrogen by Thermo Fisher Scientific (Rockford, Illinois, United States). All other chemicals were analytical grade and were obtained from standard commercial suppliers.

Protease expression plasmids and protein purification

E. coli BL21(DE3)pLysS harboring MERS-CoV-Plpro (Genscript) was used to express Plpro as described in our previous work (Alaofi et al. 2022; Shahid et al. 2024). Briefly, recombinant MERS-CoV Plpro was purified using Ni-NTA pre-packed columns. Initially, cleared crude lysate was passed through the column pre-equilibrated with washing buffer, and bound protein was eluted with elution buffer (Alaofi and Shahid 2021).

MERS-CoV Plpro inhibition assay

MERS-CoV Plpro inhibition and IC50 values for anti-cancer drugs (sunitinib, olaparib, mitoxantrone, and bicalutamide) were determined using a fluorescence-based assay in a 384-well plate format. The hydrolysis of the fluorogenic peptide Z-RLRGG-AMC leads to increased fluorescence of the AMC moiety, allowing precise measurement of enzyme activity. The reactions were carried out in 50 µL total volume, consisting of 20 mM Tris buffer (pH 8.0), 30 µM Z-RLRGG-AMC, and various concentrations of inhibitors (0.78–100 µM). After the addition of 60 nM Plpro, the plates were shaken for 30 seconds, and fluorescence was monitored using a BiotekHT Microplate Reader at excitation 360 nm and emission 460 nm. IC50 values were calculated using the dose-response inhibition function in GraphPad Prism. The experiments were performed in triplicate for accuracy according to our previous methods (Alaofi et al. 2022).

Thermal shift assay (TSA)

To assess the interaction between anti-cancer drugs (sunitinib, olaparib, mitoxantrone, and bicalutamide) and the MERS-CoV PLpro protein, Differential Scanning Fluorimetry (DSF) was performed using the Applied Biosystems 7500 Real-Time PCR System. The assay involved incubating varying concentrations of tested drugs, ranging from 0.78 to 100 µM, with a reaction buffer containing a final concentration of 2 µM MERS-CoV PLpro. The buffer without the test compound acted as the control. After the mixtures were incubated for 30 minutes at 25 °C, the fluorescence signal was recorded using SYPRO Orange dye, which binds to hydrophobic regions exposed during protein unfolding. The fluorescence was monitored as the temperature was increased incrementally at a rate of 1 °C per minute, from 25 °C to 95 °C. This method allowed for the determination of the protein’s melting temperature (Tm), which is an indicator of protein stability (Huynh and Partch 2015). The difference in Tm (ΔTm) between the protein in the presence of drugs and the protein alone was calculated, which provided insights into the binding affinity and thermal stabilization effects of drugs on the MERS-CoV PLpro. A positive ΔTm indicated that drug binding increased the thermal stability of the protein, suggesting a potential interaction between the compound and the target protease. This method is effective in screening for small-molecule inhibitors by observing their effects on protein stability and folding (Lee et al. 2019; Alaofi et al. 2022).

MD simulations for the sunitinib-PLpro complex

Preparation of structures

The structure of PLpro of MERS-CoV was obtained from the Protein Data Bank (PDB ID 4RNA), while sunitinib was abstracted from a ligand-protein complex (PDB ID 3MIY). The small molecule, sunitinib, was prepared as follows: The Avogadro website was used to assign all hydrogen atoms, and the output file (.mol2) of the sunitinib ligand was obtained (Hanwell et al. 2012). Then the CGenFF server using the CHARMM force field generated accurate force field parameters for the ligand. Afterward, the topology file for the complex was prepared by combining the ligand and protein topologies, and the system, containing sunitinib and PLpro, was ready for MD simulations.

MD simulations

Molecular dynamics (MD) simulations were used to assess the binding interactions of sunitinib toward MERS-CoV PLpro. Sunitinib was placed with PLpro without predefined binding sites, i.e., before docking to thoroughly sample the PLpro’s conformational space, increasing the likelihood of identifying productive binding poses (Santos et al. 2019). Similar to our previous work, free PLpro and the complex sunitinib-PLpro systems were simulated for 100 ns in a box of water. The GROMACS 5.1.4 program with the CHARMM force field was used to conduct this MD simulation. First, free and bound systems were solvated with TIP3P water in a dodecahedron (Alaofi et al. 2017, 2022, 2022; Alaofi 2022, 2022, 2020; Alaofi and Shahid 2021; Shahid et al. 2024). The correct charges for titratable residues of free and complex systems of PLpro were set according to the pH 7.0 condition. The ionic strength for free and complex PLpro systems was simulated to be 0.15 M as a result of substituting equivalent amounts of water molecules with sodium and chloride ions. Energy minimization using the steepest descent approach was carried out, and each system was at energy minimum. Afterward, equilibration of both systems was done similarly in NVT followed by NPT to achieve well-equilibrated systems at the desired temperature and pressure. However, restraints to the ligand (sunitinib) were applied before equilibrating the ligand-PLpro complex system as well as proper treatment of temperature coupling groups. Finally, after the release of the position restraints, 100 ns-long production MD simulations were to be performed.

Docking sunitinib with MERS-CoV PLpro

Utilizing simulated structures takes into account the dynamics and flexible trajectories of both ligand and protein. Therefore, the trajectories at 100 ns produced from MD simulations of MERS-CoV PLpro and sunitinib (i.e., PDB files) were used to perform blind docking using the HADDOCK.2.4 server (Honorato et al. 2024). The docking parameter for sunitinib with PLpro was done using the center of mass restraints since no active residues were selected in the receptor (PLpro). The highest HADDOCK score model was chosen since it showed better van der Waals energy and electrostatic energy. Finally, visualization and conformational representation for the PLpro and sunitinib-PLpro complex as well as interaction residues were done using PyMol and SCHRODINGER maestro software (Schrödinger 2020). Interacting residues of PLpro with sunitinib were visualized and depicted using LigPlot software (Laskowski and Swindells et al. 2011).

Results

Plpro inhibition assays

To assess the in vitro inhibition potency of sunitinib, olaparib, mitoxantrone, and bicalutamide (Fig. 1A), their inhibition constants were estimated from the inhibition curves. Inhibition curves were obtained by measuring enzymatic activity using the previously described continuous FRET assay. Enzyme and substrate concentrations were fixed, while the compound concentration was varied (Fig. 1B). The enzymatic inhibition activity of sunitinib, olaparib, mitoxantrone, and bicalutamide was calculated at various concentrations using the initial slope of each curve. A dose-dependent effect of the compounds on Plpro enzyme activity was observed (Fig. 1B, C).

Figure 1. 

Chemical structures of sunitinib, olaparib, mitoxantrone, and bicalutamide (a). MERS protease inhibition by sunitinib, olaparib, mitoxantrone, and bicalutamide (b). Dose response curve B. Percent inhibition, dose response curve of MERS-CoV Plpro inhibition identified from the IC50 protease inhibition assay with dissociation of RLRGG-AMC substrate. Values represent the average ±standard deviation of three replicates.

Non-linear regression analysis employing a simple inhibition model allowed estimating apparent inhibition at different concentrations. Sunitinib can be assumed to bind to an active site on MERS-CoV Plpro, functioning as a competitive inhibitor. This apparent inhibition can be observed in the IC50 value of 1.75 μM, while there is no significant inhibition for olaparib, mitoxantrone, and bicalutamide (Table 1).

Table 1.
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IC50 of MERS-CoV Plpro inhibition identified by dissociation of RLRGG-AMC substrate against sunitinib, olaparib, mitoxantrone, and bicalutamide.

Sunitinib Olaparib Mitoxantrone Bicalutamide
IC50 (μM) 1.750 306162 6033 17896
R2 0.9842 0.7032 0.8825 0.8580

Thermal shift assay

While TSA is a widely accepted method for evaluating protein-ligand interactions by measuring shifts in protein melting temperature, it has limitations, including the potential for false negatives and not always correlating directly with inhibitory activity. However, methods measuring actual enzymatic activity offer more reliable evidence of inhibition. In our assay, olaparib (ΔTm 1.4), mitoxantrone (ΔTm 5.45), and bicalutamide (ΔTm 0.94) exhibited no binding affinity with MERS-CoV PLpro, and their presence did not significantly affect the melting temperature of the protein, indicating no major interaction (Table 2).

Table 2.
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Melting points (in Celsius; °C) and melting point differences ΔTm (ΔTm = Tmmolecule -TmPLpro) calculated for small molecules incubated with MERS-CoV PLpro.

Sunitinib Olaparib Mitoxantrone Bicalutamide MERS-CoV PLpro
Tm (°C) 69.6 41.56 48.41 42.02 42.96
ΔTm (°C) 26.64 1.4 5.45 0.94 0.00

Sunitinib, however, demonstrated consistent inhibition across a range of concentrations (0.78–100 µM), with a corresponding thermal shift in the TSA (ΔTm ranging from 16.26 °C to 23.4 °C) (Fig. 2). This significant shift suggests that sunitinib binds effectively with MERS-CoV PLpro, stabilizing the protein and altering its thermal properties. These findings highlight sunitinib’s potential as a promising candidate for further development, warranting additional preclinical trials to evaluate its therapeutic efficacy against MERS-CoV. Therefore, in silico studies were conducted for sunitinib with PLpro, as it was the only molecule that showed an inhibitory effect against MERS-CoV PLpro activity.

Figure 2. 

The melting temperature (Tm) of MERS-CoV-2 PLpro by thermal shift assay (TSA). Representative image of Tm profile of sunitinib (a), olaparib (b), mitoxantrone (c), bicalutamide (d), and MERS-CoV-2 PLpro (e). The raw and fitted data figure was plotted by TSA-CRAFT (Suppl. material 1: fig. S1).

MD simulations and docking

RMSD and RMSF

Cα-root mean square deviation (Cα-RMSD) of free and bound PLpro reached a plateau, indicating stable equilibrium systems (Fig. 3A). At later frames of the MD simulations, there were lower Cα-RMSD values for the complex system (i.e., sunitinib-PLpro) compared to the free PLpro system. Cα-root mean square fluctuation (Cα-RMSF) was used to assess flexibility per residue of proteins. In our studies, the complex system showed significant differences in important regions compared to free PLpro (Fig. 3A). Interestingly, the BL2 loop, known for its important role in PLpro catalytic activity, showed increased flexibility in the V275-H278 region in the complex system compared to the free PLpro. It is worth mentioning H278 residue is one of the catalytic triads for MERS-CoV PLpro activity along with C110 and D293 residues. Also, the M137-D142 region, in the thumb domain, showed a very significant rigidity in the complex system compared to the free PLpro (Fig. 3B). The diminishing of the M137-D142 region’s flexibility in the complex system might indicate stabilization of the bound PLpro structure. Moreover, a less flexible region in bound PLpro was located in the G50-D75 region (Fig. 3C). Finally, the finger domain flexibility was highly similar between bound and free PLpro, indicating that no conformational changes occurred in this domain.

Figure 3. 

The Cα-RMSD as a function of time (a) was plotted for free PLpro (black color) and bound PLpro (red color). Both systems reached a plateau indicated by the stabilities of the simulations. In (b), the Cα-RMSF as a function of residues was plotted for free PLpro (black color) and bound PLpro (red color). The thumb domain (A61-L178) showed a significant rigidity for bound PLpro in comparison to the free one. In (c), the left panel showed a surface representation of free PLpro (smudge color) and bound PLpro (violet color). In the right panel, the boxes showed cartoon representations of the alignment of free and bound PLpro. The upper box was rotated 180 °C in the lower box. Dark blue arrows showed the significant differences between bound and free PLpro.

Radius of gyration and free energy landscape

The use of radius of gyration (Rg) is an indicator of the conformational compactness of a protein. We assessed the conformational compactness using the Rg of the backbone over time for the free and sunitinib-bound PLpro. Sunitinib binding to MERS-CoV PLpro decreased the conformational compactness of the PLpro structure (Fig. 4A). The free energy landscape (FEL) was used to monitor the stability of protein conformations and favorable conformers. Based on FEL data, sunitinib clearly induced a stable conformation in comparison to MERS-CoV PLpro. This was observed as a dark blue region, indicating a favorable energy minimum for the complex compared to free PLpro (Fig. 4B).

Figure 4. 

In (a), the radius of gyration (Rg) values over time (ps) were monitored during the 100-ns run for free PLpro (black color) and bound PLpro (red color). The free energy landscape (FEL) was depicted for free and bound PLpro.

Docking results

The docking techniques are commonly used to identify binding sites for ligands in receptor pockets. Our docking results suggested sunitinib binding to the thumb domain of MERS-CoV PLpro (Fig. 5). Sunitinib was bound to R82, F79, D76, V75, P74, T159, C158, N157, Y155, A154, and M153 residues of PLpro (Fig. 5). The indolizidine ring of sunitinib was bound to the PLpro pocket composed of T159, C158, N157, Y155, A154, and M153. A strong hydrogen bond (less than 4.0 Å) was established between the hydroxyl group (-OH) of sunitinib and D76 of PLpro. The predicted binding affinity between sunitinib and PLpro was -7.92 kcal/mol. Three hydrogen bonds were observed between sunitinib and PLpro (Suppl. material 1: fig. S2). Specifically, two hydrogen bonds were formed between sunitinib and the backbone atoms of residues A154 and P74, while one hydrogen bond was formed between sunitinib and the side chain of residue D76. Hydrophobic interactions with sunitinib were observed for the following PLpro residues: V75, Y155, F79, L86, R83, C158, T159, and N157 (Suppl. material 1: fig. S2). Interestingly, the observed rigid region was in the thumb domain; this might be induced by sunitinib interaction with the PLpro thumb domain.

Figure 5. 

The left panel shows the docking results of sunitinib (stick representation; indicated by a small arrow) to the MERS-CoV PLpro (cartoon representation). MERS-CoV PLpro domains were labeled next to the corresponding domain. The right panel shows a surface representation of the binding pocket of sunitinib (indicated by a small arrow) in the upper box. The lower box shows the interaction residues of PLpro with the sunitinib molecule.

Discussion

The COVID-19 pandemic has underscored the urgency of developing effective pharmacological interventions against other coronaviruses, particularly Middle East Respiratory Syndrome Coronavirus (MERS-CoV), which poses a significant threat due to its high mortality rate. The primary challenge in developing treatments for MERS-CoV is the virus’s potential for rapid mutation and drug resistance. To address this, there is a need for a repository of active compounds with diverse mechanisms of action that maintain efficacy while minimizing susceptibility to resistance (Zumla et al. 2015; Chafekar and Fielding 2018; Jin et al. 2020). One promising approach is targeting conserved viral proteins, such as proteases, which play critical roles in viral replication and are less prone to mutation. Targeting these proteases can offer broad-spectrum antiviral activity across different strains (Anderson et al. 2009; Hilgenfeld 2014). Drug development efforts can start with small- or large-scale screenings using both experimental and computational methods. These pipelines have proven successful in the past for various viral infections, leading to the discovery or repurposing of antiviral drugs (Galasiti Kankanamalage et al. 2018; Alamri et al. 2020). In our study, we applied a similar strategy to screen anticancer drugs for their potential inhibitory activity against MERS-CoV’s papain-like protease (PLpro).

The screening process involved assessing the efficacy of four drugs: sunitinib, olaparib, mitoxantrone, and bicalutamide against MERS-CoV PLpro. Using a peptidic FRET substrate (Z-RLRGG-AMC), we monitored the enzyme’s activity in a continuous enzymatic assay. Following initial evaluations of each drug’s ability to inhibit PLpro in a dose-dependent manner, we further examined their effects on the protease’s thermal stability using a fluorescence-based Thermal Shift Assay (TSA). Olaparib, mitoxantrone, and bicalutamide showed insignificant inhibition of MERS-CoV PLpro at any concentration. However, sunitinib demonstrated consistent inhibition across a range of concentrations (0.78–100 µM), with IC50 1.75 µM, indicating a significant inhibition of MERS-CoV PLpro. This shift in the protein,s unfolding temperature due to the drugs, precise and effective binding with the protease suggests their potential as candidates for further development and preclinical trials.

It is interesting that in silico studies were consistent with in vitro studies since sunitinib increased the MERS-CoV PLpro stability observed by elevated melting temperature and stable structural conformation of bound PLpro (i.e., sunitinib-PLpro complex). The interaction between active compounds or ligands and a protein can lead to changes in the protein’s stability, particularly in response to thermal or chemical denaturation. When a ligand preferentially binds to a specific conformational state of the protein, this interaction either stabilizes or destabilizes the protein depending on whether the native or non-native state is favored (Niesen et al. 2007). A Thermal Shift Assay (TSA), also known as differential scanning fluorimetry, is commonly used to detect these ligand-induced changes in protein stability through fluorescence measurements (Pantoliano et al. 2001). The process involves using an extrinsic fluorescent dye that tracks the unfolding of the protein as it is exposed to increasing temperatures. By assessing the melting temperature (Tm) of the protein in the presence of varying concentrations of a ligand, it is possible to determine how the compound affects the thermal stability of the protein (Ericsson et al. 2006; Cimmperman et al. 2008). Also, free energy landscape (FEL) is used to assess the stable minima of protein conformations. Both TSA and FEL showed sunitinib induced a stable structure of Plpro, suggesting a specific binding event between the ligand and protein. Olaparib, mitoxantrone, and bicalutamide had no major impact on the thermal stability of MERS-CoV PLpro. These compounds caused no stabilization or destabilization of the protein, as indicated by changes in the melting temperature (ΔTm). While it is more common for ligands to induce stabilizing effects on a protein, it is not uncommon to observe destabilizing effects, which can offer valuable insights into the protein-ligand interaction and potential inhibition mechanisms (Du et al. 2016; Stachowski and Fischer 2022). However, sunitinib interacted strongly with MERS-CoV PLpro, potentially guiding therapeutic development.

There are limited studies on the role of olaparib’s potential application against viral infections, including viruses like MERS-CoV or SARS-CoV-2, which may also counteract SARS‐CoV‐2‐induced and inflammation‐induced cell death and support cell survival (Curtin et al. 2020). DNA damage repair pathways targeted by PARPis could have implications in antiviral activity, particularly in viruses that rely on host DNA repair mechanisms for replication, like the Epstein-Barr virus (EBV) and human papillomavirus (HPV) (Pirotte et al. 2018). Similarly, some studies suggest that mitoxantrone inhibits viral replication by interfering with the DNA and RNA processes within cells. Specifically, its potential antiviral properties have been observed in inhibiting viruses that rely on DNA for replication, such as HIV-1 and certain DNA viruses (Deng et al. 2007). Additionally, studies have indicated that mitoxantrone inhibits the replication of cytomegalovirus (CMV) and Epstein-Barr virus (EBV), both of which are herpes viruses that rely on host DNA replication mechanisms (Huang et al. 2019; Soldan and Lieberman 2023). No studies have reported mitoxantrone activity against coronaviruses, including SARS and MERS. Moreover, there are no comprehensive studies directly exploring the antiviral activity of bicalutamide. However, its known role as an androgen receptor antagonist suggests that it may influence viral replication in diseases where androgens play a role in modulating immune response, such as in certain viral infections like COVID-19, where androgen-regulated TMPRSS2 is involved in viral entry. More research is needed to explore its potential against viruses like MERS-CoV or SARS-CoV-2 (Bhowmick et al. 2020; Cani et al. 2024).

The study demonstrates a strong correlation between in silico and in vitro findings regarding sunitinib’s role as a PLpro inhibitor for MERS-CoV. In vitro assays, including protease inhibition and thermal shift analysis, revealed a dose-dependent inhibitory effect of sunitinib, with a substantial thermal stability increase (ΔTm of 26.64 °C) indicating a strong interaction with PLpro. Complementing this, in silico molecular docking and dynamic simulations identified sunitinib binding at the thumb domain of PLpro, stabilizing the protein structure by reducing flexibility in key regions such as the M137-D142 segment. This is consistent with previous findings where sunitinib has shown inhibitory interactions with key viral targets, including HCV NS5B polymerase virus envelope protein and spike protein, by stabilizing regions and disrupting their functional activity. These findings highlight sunitinib’s versatility in targeting conserved viral domains, supporting its repurposing for antiviral therapy (Wrasidlo et al. 2014; Chen et al. 2021; Afreen et al. 2022; Hicyilmaz et al. 2023).

Computational tools are extensively used in the literature for studying proteins and macromolecules. Our MD simulations and docking results were consistent with experimental data as sunitinib was able to inhibit the catalytic activity of the MERS-CoV PLpro enzyme. The MD simulation results indicate a significant stabilization of the PLpro structure in the complex form compared to free PLpro. Based on our observation, the overall flexibility of the PLpro was impacted by the presence of sunitinib, indicating a specific and potent binding of sunitinib to MERS-CoV PLpro. There was a clear stable structure of bound Plpro, suggesting favorable binding to the sunitinib molecule. This was consistent with the binding site of sunitinib, as the thumb domain showed higher rigidity than the palm and finger domains of PLpro. Computational analysis predicts a strong binding affinity between sunitinib and the thumb domain of MERS-CoV PLpro, mediated by both hydrophobic and hydrogen bond interactions. Experimental validation of this binding affinity would strengthen these findings.

Conclusion

MERS-CoV PLpro is a significant drug target with various inhibitors discovered through different approaches. However, quality science requires thorough validation, including detailed biochemical and binding studies. Drug candidates should show consistent IC50 values in FRET assays and align with the results of thermal shift assay results. This research aims to enhance understanding of PLpro inhibitors while ensuring scientific rigor. It supports further drug development without compromising quality in the rush for effective treatments. The identification of sunitinib as a potential inhibitor of MERS-CoV PLpro through enzymatic, thermal stability assays, and MD simulations underscores the importance of repurposing existing drugs and offers a promising avenue for therapeutic development. This approach not only addresses current treatment gaps but also enhances global preparedness for future coronavirus outbreaks.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statements

The authors declared that no clinical trials were used in the present study.

The authors declared that no experiments on humans or human tissues were performed for the present study.

The authors declared that no informed consent was obtained from the humans, donors or donors’ representatives participating in the study.

The authors declared that no experiments on animals were performed for the present study.

The authors declared that no commercially available immortalised human and animal cell lines were used in the present study.

Funding

This study was funded by the Research Supporting Project number (RSPD2025R560), King Saud University, Riyadh, Saudi Arabia.

Author contributions

Ahmed L. Alaofi: Conceptualization, Funding acquisition, Data curation, Writing – original draft, Writing – review & editing, Investigation, Validation, Formal analysis, Methodology, Supervision, Resources, Project administration, Software. Mudassar Shahid: Conceptualization, Data curation, Writing – original draft, Writing – review & editing, Visualization, Investigation, Validation, Formal analysis, Methodology, Software. Mohd Abul Kalam: Writing – review & editing, Methodology.

Author ORCIDs

Ahmed L. Alaof https://orcid.org/0000-0001-8967-173X

Mudassar Shahid https://orcid.org/0000-0003-3714-4772

Mohd Abul Kalam https://orcid.org/0000-0002-5713-8858

Data availability

All of the data that support the findings of this study are available in the main text or Supplementary Information.

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Supplementary material

Supplementary material 1 

Supplementary information

Ahmed L. Alaofi, Mudassar Shahid, Mohd Abul Kalam

Data type: docx

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
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