Computational Drug Repurposing Approach to Identify Novel Inhibitors of ILK Protein for Treatment of Esophageal Squamous Cell Carcinoma

Purpose Esophageal squamous cell cancer (ESCC) is a deadly malignant tumor characterized by an overall 5-year survival rate below 20%, with China accounting for approximately 50% of all cases worldwide. Our previous studies have demonstrated that high integrin-linked kinase (ILK) expression plays a key role in development and progression of ESCC both in vitro and in vivo. Here, we employed the drug repurposing approach to identify a novel FDA-approved anticancer inhibitor against ILK-induced tumorigenesis and progression. Methods We screened the ZINC15 database and predicted the molecular docking ability among FDA-approved and publicly available drugs to ILK and then performed computational docking and visual inspection analyses of the top 10 ranked drugs. Two computer-based virtual screened drugs were evaluated in vitro for their ability to directly bind purified ILK by surface plasmon resonance. Cytotoxicity of the two candidate drugs was validated in vitro using CCK-8 and LDH assays. Results We initially selected the top 10 compounds, based on their minimum binding energy to the ILK crystal, after molecular docking and subjected them to further screening. Taking the binding energy of −10 kcal/mol as the threshold, we selected two drugs, namely, nilotinib and teniposide, for the wet-lab experiment. Surface plasmon resonance (SPR) revealed that nilotinib and teniposide had equilibrium dissociation constant (KD) values of 6.410E − 6 and 1.793E − 6, respectively, which were lower than 2.643E − 6 observed in ILK-IN-3 used as the positive control. The IC50 values for nilotinib and teniposide in ESCC cell lines were 40 μM and 200–400 nM, respectively. Results of the CCK-8 assay demonstrated that both nilotinib and teniposide significantly inhibited proliferation of cells (P < 0.01). LDH results revealed that both drugs significantly suppressed the rate of cell death (P < 0.01). Conclusion The drug repositioning procedure can effectively identify new therapeutic tools for ESCC. Our findings suggest that nilotinib and teniposide are efficacious inhibitors of ILK and thus have potential to target ILK-mediated signaling pathways for management of ESCC.


Background Information
Esophageal cancer is one of the most common malignant tumors that threaten human health and life. Te disease is ranked 7th and 6th with regards to morbidity and mortality among global malignant tumors, respectively. In China, esophageal cancer accounts for about 43.0% and 37.0% of all global new cases and deaths worldwide, respectively, with esophageal squamous cell cancer (ESCC) shown to be the main subtype [1,2]. ESCC has a 5-year survival rate of about 30%, due to its atypical early symptoms, high malignancy, aggressiveness, and more limited treatment options [3].
Despite extraordinary advances in the cancer biology feld, progress in the area of drug research and development (R&D) remains slower than expected [4]. A promising solution to the considerable drug development challenges of novel compounds is drug repurposing (also called drug repositioning, reprofling or retasking), a strategy for identifying new clinical indications for approved or investigational drugs [5]. Approved drugs have undergone all phases of clinical trials in order to reach the market and thus have known and accepted safety data, which substantially reduce the R&D risk, time, and cost [6]. Sildenafl, a phosphodiesterase type 5 (PDE5) inhibitor, is a successful drug repurposing example. Sildenafl was originally developed to treat hypertension but was later approved by the FDA for the treatment of erectile dysfunction and pulmonary hypertension for the same [7,8].
Te participation of computer-aided drug design (CADD) can minimize the failure rate of research and development and further consolidate the advantages of drug repositioning, which includes two major types of drug design techniques: ligand-based drug design (LBDD) and structure-based drug design (SBDD) [9]. As one of the most commonly used in in silico approaches, SBDD utilizes the structural information from a 3D protein structure to predict macromolecular binding sites and ligand afnity [10]. Molecular docking is a typical SBDD method used to evaluate binding afnities between proteins and ligands [11]. For example, with performing molecular ft computations on 3,671 FDA-approved drugs across 2,335 human protein crystal structures, Dakshanamurthy et al. screened that mebendazole, an antiparasitic drug, has the structural potential to inhibit the angiogenic medium vascular endothelial growth factor receptor 2 (VEGFR2), a mediator of angiogenesis, which was also confrmed by experiments [12].
Integrin-linked kinase (ILK), an intracellular protein kinase, is not only involved in several signal transduction processes but also regulates the cell cycle, migration, and growth among other biological processes [13]. Previous studies have shown that high ILK expression mediates regulation of cell proliferation, migration, and diferentiation in a variety of tumors and also afects therapeutic efects of tumors [14][15][16]. Meanwhile, we found that ILK was highly expressed in ESCC, while its interference using the lentivirus markedly inhibited occurrence and development of ESCCs [17]. Tis project aims to screen some small molecules to inhibit ILK with high selectivity and activity that have become drugs through computer-aided drug research and to conduct primary screening and validation of their pharmacological activities so as to provide experimental and theoretical support for the research of novel anticancer-led compounds.

Virtual
Screening. First, we obtained a threedimensional structure of ILK protein from the ILK/alphaparvin core complex crystal structure downloaded from the PDB database [18] and then extracted the ILK protein (A chain) using the PyMOL tool. Subsequently, the potential binding pockets of ILK protein were determined by using the DoGSiteScorer online tool [19], which was developed to predict the binding pockets and rank them based on the size, surface area, and druggability score. Te chosen binding pocket was further validated by fpocket [20] and CASTp [21] tools. Next, we obtained the list of 1615 FDA-approved drugs from the ZINC database [22] for virtual screening. Tese drugs were converted to PDB formats using MGLTools and then subjected to molecular docking using the AutoDock Vina docking program [23]. We ranked the drugs by their binding afnities when bound to binding pockets. Finally, we selected 2 drugs with minimum free energy for wet-lab validation.

In Vitro Assessment of Direct Drug Binding to ILK.
SPR experiments were carried out using a Biacore S200 instrument (Biacore, GE Healthcare, Boston, MA, USA) at 25°C. Summarily, ILK (ligand) was frst immobilized to the activated CM5 sensor chip by amine coupling with pH 5.0 and sodium acetate concentration of 46 μg/mL. Te nonreaction group was then blocked by injection of ethanolamine hydrochloride 1 M (35 μL). Te drug (analyte) was dissolved in 100% DMSO (to an initial concentration of 10 mM) and diluted to 200 μM (5% fnal DMSO concentration) with 20 mM HEPES, 150 mM NaCl, and 0.005% surfactant P20 bufer (HBSP). It was further diluted with HBSP + 5% DMSO (HBSP5%D). We then confrmed concentrations of compounds based on the solubility and the test results from sensor chips of samples to be measured. An increase in the RU value from the baseline indicates that formation is complex and that the plateau region represents a steady-state phase of interaction (RUeq), whereas a decrease in RU after 100 s indicates separation of the analyte from immobilized ILK after injection of HBSP5%D bufer. Finally, we performed sensorgram analysis using the Biacore S200 evaluation software.

CCK-8 Assay.
KYSE150 and TE-1 cells were, respectively, seeded into 96-well culture plates (5 × 10 4 cells per well) and incubated for 24 h at 37°C, 5% CO 2 , and 100 μL of diferent concentrations of the candidate drugs was prepared and added to 1640 medium, followed by 48h incubation. Te medium was refreshed; then, 100 μL of the medium with 10% CCK-8 was added to each test well, followed by 2 h of incubation at 37°C. A microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) was used to measure OD at 450 nm in each well, and changes in cell proliferation ability were detected.

Identifcation of Potential ILK Binding Drugs by Virtual Screening
3.1.1. ILK Protein and the 3D Crystal Structure. Te threedimensional structure of the ILK protein was obtained from the ILK/alpha-parvin core complex crystal structure in the PDB database (3KMU) according to ILK protein sequence (supplementary fle). Next, the ILK protein (A chain) was extracted using PyMOL, and its three-dimensional structure is shown in Figure 1.

Prediction of Binding Pockets.
We utilized the online tool DoGSiteScorer for prediction and description of potential binding pockets of the ILK protein. Te top 9 predicted binding pockets are listed in Table 1. We only considered the frst binding pocket during molecular docking, due to the fact that its contact surface area was much larger than that of others. Meanwhile, we used two other popular tools, fpocket and CASTp, to predict the binding pocket and found similar results.

Molecular Docking and Screening Results.
We screened the FDA-approved drug library, downloaded from the ZINC 15 database, for potential hits during our virtual screening. Te library contains 1615 FDA-approved drugs. Te drugs were retrieved in the MOL2 format and then converted to the .pdbqt format using MGLTools. During transformation, both Gasteiger charges and polar hydrogen atoms were added, and drugs were prepared for docking. Next, we utilized AutoDock Vina tools to dock FDAapproved drugs and ILK protein using the following molecular docking parameters: a binding site area of (28 * 30 * 32) angstrom with coordinates of (X: 6.292, Y: 18.998, and Z: 12.265). Te output format of the data was sorted by the minimum binding energy of these drugs to the ILK crystal. Next, we selected the top 10 hits from the molecular docking for further screening, as shown in Table 2. Taking the binding energy of −10 kcal/mol as the threshold, two drugs, namely, nilotinib (ZINC6716957) and teniposide (ZINC4099009), were selected for subsequent wet-lab experiments. For each drug, the ligand-target complex with the least binding energy pose is illustrated in Figure 2 and Figure 3, while information for their chemical formulas is listed in Table 3.

Assessment of Direct Drug Binding to ILK In Vitro.
To validate the virtual screening results, we carried out SPR in vitro to evaluate the biomolecular interaction of drugs.   Figure 4. ILK-IN-3 was included as a positive control [24]. Te SPR results showed that the top 2 drugs with strong afnities predicted by virtual screening could indeed bind to ILK. Biacore S200 software revealed that the drugs' dissociation constant (KD) values were in the micromolar range and were comparable to those of the control (Table 4). Interestingly, teniposide (KD � 1.793 μM) had a higher afnity for ILK than for ILK-IN-3 (KD � 2.643 μM).

Evaluation of Anticancer Activity in the Two Compounds.
Next, we used CCK-8 and LDH release assays to determine the function of the two compounds in proliferation and death of ESCC cells. To this end, we targeted KYSE-150 and TE-1 human esophagus squamous cancer cells, in which ILK was expressed at relatively higher levels than other ESCC cells according to our previous studies [17]. Notably, nilotinib had an IC50 value of 40 μM, while the IC50 value of teniposide ranged from 162.7 to 396.2 nM in these ESCC cell lines (Figures 5(a) and 5(b)). Tis suggested that teniposide has a relatively higher cytotoxic efect on ESCC cells than on nilotinib, consistent with the SPR results. Moreover, an

Discussion
Te research on ESCC therapy progress is slow; therefore, there is an urgent need to identify efective antitumor therapeutic drug targets. Generally, drug development is a difcult task that takes a long period of time and is accompanied by high costs and high risk as well as low payofs.
In contrast, drug repurposing, which is an approach to discover a new efect for an already approved drug in novel disease types, ofers a higher success rate, lowers the risk ratio, and shortens the time taken before clinical use [6].
Notably, researchers are currently employing virtual screening (VS) based on target structures as an efective method for new drug discovery. Tis approach not only allows for rapid screening of a large number of compounds in drugs within a short period of time but is also a novel way to discover new drugs, as evidenced by recent successes [25,26]. Since most small molecules may have multiple molecular targets, the discovery of new therapeutic targets for a drug is essential for new indications [27]. ILK was originally thought to be a serine/threonine protein kinase that interacts with the cytoplasmic domain of β-1 integrin and mediates the connection between cells and the extracellular matrix. Functionally, it afects the transmission of extracellular signals and also plays a central role in regulating fundamental processes, such as cell morphology, motility, growth, survival, diferentiation, and gene expression [13]. To date, the exact molecular mechanism underlying ILK signal transduction has been quite controversial [28]. However, a recent study showed that ILK is a pseudokinase [29]. Subsequently, numerous studies have revealed that ILK plays a key role in epithelial-mesenchymal transition (EMT), invasion, and angiogenesis, suggesting that it could be an attractive target for tumor therapy [30][31][32]. In fact, some studies have shown that ILK is not only upregulated in a variety of malignancies but is also associated with a poor patient prognosis. At the same time, our research group previously demonstrated that interfering with ILK can signifcantly inhibit proliferation, invasion, and migration of ESCC cells and also improve patient prognosis [17,33]. ILK, as a therapeutic target, has attracted a lot of research attention. For instance, some researchers have designed small-molecule compounds targeting ILK to inhibit tumor growth, although these studies have neither clearly described whether these compounds directly bind to ILK nor demonstrated their specifcity, which necessitates further exploration before their clinical application [24,[34][35][36].
In the present study, we screened two known drug compounds with strong ILK binding afnity, nilotinib and teniposide, from the ZINC15 drug database, containing 1615 FDA-approved drugs, using the VS approach based on the ILK protein structure. In addition, we validated purifed ILK proteins using molecular biology techniques and measured the inhibitory efects of these compounds on ILK with a view of verifying reliability of the computerized virtual screening results. Our results showed that both nilotinib and teniposide had a stronger binding afnity to ILK than the positive reference compound ILK-IN-3, a phenomenon that initially demonstrated the feasibility of the protein structure-based drug virtual screening method. Subsequently, we evaluated the antitumor activity of the two inhibitors on ESCC TE-1 and KYSE150 cells and found that both inhibitors signifcantly promoted cell death and inhibited proliferation, consistent with drug virtual screening results.
Tese results suggest that the observed antitumor efects of nilotinib and teniposide on ESCC are partially related to ILK's binding ability coupled with their inhibitory efects. Nilotinib, which belongs to the second-generation tyrosine kinase inhibitor (TKI), has been used as an efective therapy in clinical treatment of chronic myeloid leukemia (CML) that is intolerant or resistant to imatinib (Gleevec) [37]. On the other hand, teniposide, a chemotherapeutic agent that targets DNA topoisomerase II, has been primarily used for treatment of acute lymphoblastic leukemia, lymphoma, and brain cancer in children [38]. However, the drug's efcacy has also been studied in other diferent types of solid tumors [39][40][41], with the resultant data suggesting that it may inhibit tumor growth through other targets. Tis also suggests that the drug might have multiple molecular targets, and the discovery of new therapeutic targets for the drug is essential to broaden new indications. Te results of the present study, albeit preliminary, demonstrated that the two drugs have antitumor activity. In the future, we intend to validate the observed antitumor efects and underlying mechanisms of the screened drugs using in vitro and in vivo experiments.

Conclusion
Tere is a need to continually screen for ILK inhibitors to identify broad antitumor activity and high selectivity because of the problems of selectivity and specifcity in the current research on ILK inhibitors. In this study, we employed VS, based on the ILK protein structure, to fnd out two potential antiesophageal squamous cell carcinoma drugs, which are nilotinib and teniposide, and verifed the binding ability of ILK protein with two compounds by surface plasmon resonance (SPR). In addition, we validated the antitumor activity of the two drugs in esophageal squamous cell carcinoma cell lines. All the above results confrmed that both nilotinib and teniposide are efective and selective ILK inhibitors, and this role merits further investigation.

Data Availability
Te datasets used to support the fndings of our study can be made available from the corresponding author on reasonable request.