Wnt/β‐catenin signaling pathway inhibitors, glycyrrhizic acid, solanine, polyphyllin I, crocin, hypericin, tubeimoside‐1, diosmin, and rutin in medicinal plants have better binding affinities and anticancer properties: Molecular docking and ADMET study

Abstract Wnt/β‐catenin signaling pathway plays a role in cancer development, organogenesis, and embryogenesis. The abnormal activation promotes cancer stem cell renewal, proliferation, and differentiation. In the present study, molecular docking simulation and ADMET studies were carried out on selected bioactive compounds in search of β‐catenin protein inhibitors for drug discovery against cancer. Blind docking simulation was performed using PyRx software on Autodock Vina. β‐catenin protein (PDB ID: 1jdh) and 313 bioactive compounds (from PubChem database) with selected standard anticancer drugs were used for molecular docking. The ADMET properties of the best‐performing compounds were calculated using SwissADME and pkCMS web servers. The results obtained from the molecular docking study showed that glycyrrhizic acid, solanine, polyphyllin I, crocin, hypericin, tubeimoside‐1, diosmin, and rutin had the best binding interactions with β‐catenin protein based on their binding affinities. Glycyrrhizic acid and solanine had the same and lowest binding energy of −8.5 kcal/mol. This was followed by polyphyllin I with −8.4 kcal/mol, and crocin, hypericin, and tubeimoside‐1 which all had a binding energy of 8.1 kcal/mol. Other top‐performing compounds include diosmin and rutin with binding energy of −8.0 kcal/mol. The ADMET study revealed that the following compounds glycyrrhizic acid, solanine, polyphyllin I, crocin, hypericin, tubeimoside‐1, diosmin, rutin, and baicalin all violated Lipinski's rule of 5 which implies poor oral bioavailability. However, based on the binding energy score, it was suggested that these pharmacologically active compounds are potential molecules to be tested against cancer.


| INTRODUC TI ON
The term Wnt is a portmanteau word formed from the terms Wingless and Int-1. In animals, they are extremely evolutionarily conserved, meaning that they have similarities in all species of animals (Catalano et al., 2020;Söderholm & Cantù, 2020;Steinhart & Angers, 2018). The abnormality in Wnt/β-catenin signaling pathway promotes cancer stem cell renewal, proliferation, and differentiation, playing important roles in carcinogenesis and therapeutic response (Zhang & Wang, 2020). Three Wnt signaling pathways are categorized, which include the "canonical Wnt signaling pathway," the "noncanonical Wnt/calcium pathway," and the "noncanonical planar cell polarity pathway." All these pathways are usually activated by Wnt-protein ligand binding to frizzled (Fz) family receptors, which transfer the biological signals to the disheveled proteins in the cells. The canonical Wnt pathway results in gene transcription regulation, and is believed to be partly regulated negatively by the SPATS1 gene (Steinhart & Angers, 2018). The pathway of noncanonical planar cell polarity is responsible for regulating the cytoskeleton that determines the cell shape. The pathway of noncanonical Wnt/calcium is responsible for regulating calcium in the cells. The canonical and noncanonical categories differ in that the noncanonical pathway operates independently of the protein β-catenin while the canonical pathway involves β-catenin (Chae & Bothwell, 2018;Söderholm & Cantù, 2020). The clinical significance of the pathway of noncanonical Wnt/calcium was shown by the mutations that resulted in several diseases, such as type II diabetes, glioblastoma, and breast and prostate cancer (Fatima et al., 2021). Recently, there is a report of the first successful use of the inhibitors of the Wnt pathway of disease in mouse models (Ng et al., 2019).
Wnt is made up of several secreted lipid-modified signaling glycoproteins family which are 350-to 400-amino-acid units in length.
Palmitoleoylation of a single completely conserved residue of serine is all Wnts' lipid modification (Hannoush, 2015). Palmitoleoylation is essential as it is a requirement for the binding of Wnt to its carrier protein WLS (Wntless) to facilitate its transportation to plasma membranes for secreting purposes; it makes protein of Wnt to bind its Fz Wnt protein receptors and undertake glycosylation, attaching a carbohydrate so as to ensure appropriate secretion (Hosseini et al., 2019;Janda et al., 2012;Nygaard et al., 2021;Yu et al., 2014).
"In the Wnt signaling, these proteins function as ligands that activate the various Wnt signaling pathways through autocrine and paracrine routes" (Fatima et al., 2021). The proteins are extremely conserved in all species. They are found in Drosophila, zebrafish, Xenopus, humans, mice, etc. (Hosseini et al., 2019;Janda et al., 2012;Yu et al., 2014). Wnt signaling starts when the "N-terminal cysteine-rich extracellular domain of a Fz family receptor" is bound by a Wnt protein (Chae & Bothwell, 2018). These Fz family receptors span the plasma membranes by sevenfold and constitute a different "G-protein coupled receptors" (GPCRs) family Kramer et al., 2017;Li et al., 2021;Takahashi et al., 2017). Nevertheless, co-receptors may be required to encourage Wnt signaling along with the interactions between the Fz receptor and the Wnt proteins. Examples include ROR2, receptor tyrosine kinase (RTK), and "lipoprotein receptor-related protein (LRP)-5/6." Upon the receptor activation, disheveled (Dsh), a cytoplasmic phosphoprotein, is sent a signal (Takahashi et al., 2017;Kramer et al., 2017). This signal sent to Dsh is transmitted through direct interaction between disheveled and Fz. Disheveled proteins occur in all organisms, which all have a share of highly conserved domains of protein; including a carboxyterminal DEP, a central PDZ, and amino-terminal DIX domains (Patel et al., 2019). "All the various domains are significant because after dishevelled, the Wnt signal may branch off into several pathways, with each pathway interacting with a separate combination of all domains" (Takahashi et al., 2017;Kramer et al., 2017).
In addition, Wnt signaling has been known to regulate several other signaling pathways which have no extensive elucidation yet.
One of the pathways includes the biological interactions between GSK3 and Wnt. Wnt could inhibit GSK3 during cell growth so as to activate mTOR without the presence of β-catenin. Also, Wnt can act as an mTOR-negative regulator through tumor suppressor TSC2 activation, which is upregulated through the interaction of GSK3 had the best binding interactions with β-catenin protein based on their binding affinities. Glycyrrhizic acid and solanine had the same and lowest binding energy of −8.5 kcal/mol. This was followed by polyphyllin I with −8.4 kcal/mol, and crocin, hypericin, and tubeimoside-1 which all had a binding energy of 8.1 kcal/mol. Other topperforming compounds include diosmin and rutin with binding energy of −8.0 kcal/ mol. The ADMET study revealed that the following compounds glycyrrhizic acid, solanine, polyphyllin I, crocin, hypericin, tubeimoside-1, diosmin, rutin, and baicalin all violated Lipinski's rule of 5 which implies poor oral bioavailability. However, based on the binding energy score, it was suggested that these pharmacologically active compounds are potential molecules to be tested against cancer.

K E Y W O R D S
ADMET, anticancer drugs, β-catenin, molecular docking, Wnt signaling pathways and disheveled (Kuroda et al., 2013). Wnt makes use of CREB and PA to activate Myf5 and MyoD genes during myogenesis (Kuroda et al., 2013). It functions together with Src and Ryk in order to allow neuron repulsion regulation in axonal guidance. Several other signaling pathways are regulated by the Wnt (Kuroda et al., 2013;Malinauskas & Jones, 2014).
For ensuring appropriate functioning, there is continuous regulation of Wnt signaling at multiple points in the Wnt signaling pathways (Kramer et al., 2017;Malinauskas & Jones, 2014;Takahashi et al., 2017;Zhan et al., 2017). For example, Wnt proteins are palmitoylated via a process mediated by porcupine (protein), meaning that it aids the regulation once there is secretion of Wnt ligand by determining its full formation (Klemm & Joyce, 2015;Kramer et al., 2017;Sales et al., 2016;Takahashi et al., 2017). There is further control of the secretion with proteins (e.g., GPR177) and evenness disrupted and complexes including retromer complex. Once secreted, there can be prevention of the ligand from being in contact with its receptor via the binding of proteins including the stabilizers glypican 3 (GPC3) and Dally that inhibit diffusion. Both the GPC3 core protein and the heparan sulfate chains play role in the regulation of Wnt activation and binding for proliferation of cells in cancer cells (Gao et al., 2014(Gao et al., , 2015(Gao et al., , 2016Ho & Kim, 2011;Li et al., 2018Li et al., , 2019. Wnt recognizes a structure of heparan sulfate on GPC3 that has GlcNS6S and IdoA2S, and the Wnt binding to the glypican of heparan sulfate is enhanced by the "3-O-sulfation in GlcNS6S3S" (Gao et al., 2016;Gordon & Gordon, 2016;Minde et al., 2013). A domain rich in cysteine at the GPC3 N-lobe has been acknowledged for forming a "hydrophobic groove" that binds to Wnt, such as phenylalanine-41, which has interaction with Wnt (Kolluri & Ho, 2019;Li et al., 2019). Blocking the binding domain of Wnt with a nanobody known as HN3 could inhibit the activation of Wnt (Li et al., 2019). In this study, molecular docking simulation was carried out to determine β-catenin inhibitors for drug discovery against cancer.

| Ligand curation and preparation
To curate phytochemicals that have been previously asserted to have anticancer effects, a literature search was done. Phytochemistry: An in silico and in vitro Update, edited by Kumar and Egbuna, and

| Active site prediction
Due to the scarcity of information about the active site of β-catenin, blind docking was done on the protein surface in chain A using PyRx software by positioning the grid box as shown in Figure 2. The resolved center coordinate is as follows:

| Molecular docking studies
According to Dallakyan and Olson (2015), molecular docking simulations were carried out using PyRx software version 0.8 (https:// pyrx.sourc eforge.io). PyRx is a program for high-throughput virtual screening of compounds against protein targets using molecular docking simulations. By analyzing the binding energy of compounds in kcal/mol, it is possible to determine which substances have the F I G U R E 1 The 3D structure of β-catenin protein (PDB ID: 1jdh) before docking.
best chances of forming a strong bond with a protein. In this work, the 3D SDF-formatted ligands that had been generated and compressed were loaded into PyRx using the built-in OpenBabel graphical user interface. The conjugate gradient approach was used to minimize energy using the universal force field (UFF), with a total number of steps set at 200. One update step was specified, followed by a stop step if the energy difference was less than 0.1 kcal/mol.
Afterward, all ligands were changed into AutoDock ligands to reduce energy use (pdbqt). In order to prepare the protein for docking, it was loaded into PyRx and then changed to .pdbqt. Docking simulation was run with an exhaustiveness level of 8. The ligand with the highest binding affinity was identified as having the highest binding energy (which is most negative) (Prasanth et al., 2020). Using BIOVIA Discovery Studio, specific interactions of the optimum docking poses were shown. The docking protocol was validated.

| Theoretical prediction of ADMET parameters
Following the completion of the docking simulation, the top-ranked compounds (first 20 compounds plus common inhibitors) were transferred to SwissADME and pkCMS online servers in canonical SMILES format for toxicity and bioavailability prediction techniques such as Lipinski's rule of 5. SwissADME (http://www.swiss adme. ch/) and pkCMS (http://biosig.unime lb.edu.au/pkcsm/ predi ction) are free online resources for predicting the pharmacokinetics, drug likeness, and medicinal chemistry friendliness of small compounds (Daina et al., 2017;Pires et al., 2015). Table 1 lists the significance and normal distribution of a few ADMET parameters chosen for this investigation (Egbuna et al., 2021).

| Molecular docking
Wnt signaling pathway is a collection of signal transduction pathways that play a role in development, organogenesis, and embryogenesis as it concerns morphogenesis and cellular movements (Gruszka et al., 2019). Body axis patterning, cell fate determination, cell proliferation, and cell migration are among the embryonic activities it regulates. Wnt is implicated in carcinogenesis. It is found to be F I G U R E 2 Positioning of grid box for blind docking.

<6
Daina et al. (2017) Drug-likeness Lipinski's rule of 5 Lipinski et al. (2001) state that an oral medication candidate must not breach more than one of the following requirements (as stated above): 1. There should not be more than five hydrogen bond donors (the total number of nitrogen-hydrogen and oxygen-hydrogen bonds). 2. There should not be more than 10 hydrogen bond acceptors (all nitrogen or oxygen atoms). 3. The molecular weight should be <500 Daltons. 4. The octanol-water partition coefficient (log P) must not be more than 5. 5. TSPA should be no more than upregulated in acute myeloid leukemia and significant in the maintenance of leukemic stem cells (Gruszka et al., 2019). In solid and hematological tumors, Wnt signaling targeting is a strategy that is being investigated (Gruszka et al., 2019). The clinical significance of this pathway has also been shown by mutations that result in a number of disorders, including type II diabetes, glioma, breast and prostate cancer, and others (Logan & Nusse, 2004). In AML, there are a number of Wnt molecules with prognostic values. They include: β-catenin, LEF-1, phosphorylated-GSK3, AXIN2, PPARD, CXXC5, PCDH17, PSMD2, PTK7, XPNPEP, RUNX1, LLGL1, and sFRP2 (Gruszka et al., 2019). In AML, it was established that Wnt pathway activation leads to a high concentration of β-catenin, which causes significant effect on disease's prognosis. Poor event-free survival (EFS), shorter overall survival (OS), and increased clonogenic activity were shown to be predicted by high levels of β-catenin expression in AML revealed in Western blot analysis (Ysebaert et al., 2006). In other words, inhibiting β-catenin could be a life-saving measure in AML.
In this study, the result from β-catenin molecular docking study ( inhibition. However, one drawback of glycyrrhizic acid is that it is not readily absorbed in the intestine (Roohbakhsh et al., 2016). For solanine, many studies have linked it to having anticancer activity.
In Figure 4, the 2D view of β-catenin protein interactions with solanine after docking can be seen. The 2D view of β-catenin protein interactions with polyphyllin I after docking is shown in Figure 5. In Figure 6, the 2D view of the molecular interactions of β-catenin and standard inhibitors was presented.

F I G U R E 4
The 2D view of β-catenin protein interactions with solanine after docking.

| Drug-likeness and ADMET prediction
Absorption, distribution, metabolism, excretion, and toxicity collectively go by the abbreviation ADMET. To foretell a molecule's potential behavior both inside and outside the body, theoretical prediction of ADMET was performed. This test is helpful in providing medicinal chemists or drug developers with an insight into the drug-likeness of molecules in order to avoid end-stage test failures. The drug-likeness and ADMET features of the front-runners derived from the molecular docking result were determined using SwissADME and pkCMS web servers. The drug-likeness result (Table 3) shows that the first nine performing compounds (glycyrrhizic acid, solanine, polyphyllin I, crocin, hypericin, tubeimoside-1, diosmin, rutin, and baicalin) all violated the Lipinski's rule of 5. However, the following compounds, tomatidine, ginkgolide-C, myricetin, β-sitosterol, glabridin, ursolic acid, withanolide, and quercetin did not violate Lipinski's rule of 5.
According to the rule, a potential compound will be orally bioavailable if it did not violate more than one rule (Kumar & Egbuna, 2019;Lipinski et al., 2001;Rudrapal & Egbuna, 2022).
For the ADMET study (Table 4), under absorption, the result shows that some compounds exhibited excellent intestinal absorption which confer on them the desired drug-likeness properties of a compound for oral administration. Hypericin, ursolic acid, and venetoclax (standard anticancer drug) all had excellent intestinal absorption potentials. However, some compounds were found to exhibit poor intestinal absorption. For example, glycyrrhizic acid, crocin, and tubeimoside-1 all had 0% intestinal absorption potentials. This is a negative attribute if they are to be considered for oral administration. Under distribution, only β-sitosterol will be able to cross the blood-brain barrier (BBB) ( Table 4). This is because only compounds with logBB values greater than 0.3 will be able to cross the BBB. For the ability of compounds to cross the CNS, only compounds with logPS (permeability surface) greater than −2 will be able to do so (Pires et al., 2015). From the result, only β-sitosterol (−1.705), glabridin (−1.8), and ursolic acid (−1.187) had logPS greater than −2.
Under metabolism, it was found that none of the compounds will be a metabolite of CYP2D6 while many are metabolizable by CYP3A4.
Under toxicity, none of the compounds would be mutagenic. Finally, few compounds (solanine, tomatidine, and ursolic acid) were found to be hepatotoxic. The two standard Wnt/β-catenin pathway inhibitors IWP4 and cardionogen 1 are predicted to be hepatotoxic (Table 4). compounds with potentials to inhibit β-catenin or Wnt/β-catenin signaling through molecular docking simulation. Among 313 compounds docked, the following compounds glycyrrhizic acid, solanine, polyphyllin I, crocin, hypericin, tubeimoside-1, diosmin, and rutin came top with better binding energies compared to standard anticancer drugs which make them potential drug candidates.

| CON CLUS ION
However, from the ADMET study, a few of them were found to be very toxic which calls for caution when considering them as oral drug candidates. formal analysis (equal); methodology (equal); project administration (equal); software (equal); supervision (equal); validation (equal); visualization (equal); writing -review and editing (equal).

ACK N OWLED G M ENTS
The authors are thankful to the World Bank, the University of Port Harcourt, the National Universities Commission, the Association of African Universities, and the Kampala International University for their support.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare no conflict of interest whatsoever.

DATA AVA I L A B I L I T Y S TAT E M E N T
Additional data will be made available on request.

E TH I C S S TATEM ENT
The study does not involve humans or animals.

CO N S ENT FO R PU B LI C ATI O N
All the authors consent to the publication.