Screening of Phytochemicals from Medicinal Plants for WNK-1 Inhibition In silico: Potential Anti-cancer Therapeutics

Asma Saqib³, Kokila S⁴, Sandhya BC⁵, Ramachandra Kini K², Shailasree Sekhar¹*

¹Division of Biochemistry, School of Life Sciences, Mysuru, JSSAHER, Mysuru-570015, Karnataka, India.

²Department of Studies in Biotechnology, University of Mysore, Mysuru-570006, Karnataka, India.

³Department of Biochemistry, Maharani Cluster University, Bangalore-560001, Karnataka, India.

⁴Department of Biochemistry, Oxford Science College, Bangalore-560001, Karnataka, India.

⁵Department of Biochemistry, Maharani Cluster University, Bangalore-560001, Karnataka, India.

*Corresponding Author E-mail: shailasreesekhar@jssuni.edu.in

ABSTRACT:

Phytochemicals for cancer reversal has been known for several millennia.  With WNK-1 close link to cancer pathology has rendered it attractive target in anticancer drug discovery.  Molecular in silico docking of phytochemicals to target WNK-1 protein paves way to identifying lead entities for clinical applications.  This computational method to investigate phytochemicals as potential WNK-1 inhibitors was attempted to validate their anticancer activity.  WNK-1 (PDB ID: 5TF9) 3D structure from RSCB Protein Data Bank with 2.50Å resolution was retrieved.  PyMOL was used to stabilize and adjust it to human physiology adding required hydrogen entities at appropriate positions.  Identified phytochemical 3D structure was downloaded from PubChem for docking via PyRx software in 213, 163, 177 Å grid box.  The best inhibitor was one with strong ligand binding to WNK-1 forming stable complex. Swiss ADME analysis applied identified those with < 5 H-bond donors, < 500 Dalton molecular weight, < 5 H acceptors and < 5 Q Plog Po/w.  The two amino acids Asp465 and Asn463 of protein WNK-1 contributed to binding with ligands.  Oleuropein aglycone and Dioscin with strong binding to WNK-1 at energies of -18.3 and -14.3 respectively with highest WNK-1 inhibitory potential are reported.  They were followed by Epigallocatechin gallate, Naringenin and Quercetin as inhibitors with binding energy of -8.4 kcal/mol, forming a stable complex to WNK-1.  The standard inhibitor WNK463 recorded ∆G of -8.9 kcal/mol.  Oleuropein aglycone and Dioscin exhibited strong binding characteristics to WNK-1 and so these molecules could be repurposed for cancer treatment.

KEYWORDS: Molecular docking, WNK-1 inhibition, Phytochemicals, WNK463, Oleuropein aglycone, Dioscin.

INTRODUCTION:

The intracellular metabolic pathway small intermediates produced in minute quantities during several enzyme catalyzed reactions are the metabolites with critical function find themselves priotized for research¹. These major plant metabolites include phenolic compounds, alkaloids, flavonoids, terpenoids, terpenes, coumarins, nitrogen containing compounds and a few polysaccharides among several others².  Bioactives are also sourced from wide range of microbes including bacteria, fungi, broad spectrum of plants and marine species like sponges, corals, tunicates and snails³.  A total of 2 million specialized metabolites have been reported, classified and identified for their role in host protection under challenging environmental conditions, cellular signaling and from several millennia in general health care⁴.

About 428 reported protein kinases with a capacity of protein phosphorylation fall into seven major families named TK, CAMK, AGC, CMGC, STE, TKL and CK1⁵. There are some about 63 kinases with minor sequence differences in catalytic domains and hence termed ‘Other’. They are so named due to absence of a highly conserved lysine-233 in subdomain II^6,7. They include the phylogenetically distinct WNK kinases with a close relationship to Saccharomyces cerevisiae STE, serine/threonine kinases and TKL; tyrosine kinases-like enzymes.  The sequence variation that identified WNK kinases is the absence of the highly conserved catalytic lysine in subdomain II leading to term with no K kinases^{8, 9}.  Several studies have reported progression of cancer dependence to components of WNK pathways. The influence of WNKs on the three major signaling pathways associated with cancer like PI₃-AKT, NF-kB and TGF-a leading to angiogenesis at the heart of progression of cancer as reported^{10, 11}.

Prominent among studies reporting inverse relationship of WNK1 to tumor reduction, was by Jaykumar et al¹². Inhibition of WNK1 resulting in reduced tumor growth with loss of metastatic cells involving reduced mRNAs encoding Slug and AXL proteins was observed¹². These studies along with suppressed expression of IgH/MYC multiple myeloma prompted the possibilities of WNK1 as therapeutic targets in controlling cancer¹³. Of the several molecules tested WNK463 (MW 463.46) was identified to inhibit all the four WNK family kinases with in vitro and IC₅₀ values for WNK-1, 5nM, WNK-2, 1nM, WNK-3, 6nM and WNK-4, 9nM were reported.   The mechanism of inhibition involved phosphorylation of the oxidative stress response-1 (OSR1) protein, natural WNK-1 substrate, in vitro. It also exhibited inhibition of exogenously expressed OSR1 in human embryonic kidney-293 cells curtailing cancer onset^14,15.

Cancer progression due to multidrug resistance (MDR) via overexpression of ATP-binding cassette (ABC) transporters has been reported as one of the major concerns in its treatments. ABC transporters expressed in various healthy cells translocate molecules of small size for excretion across intestinal epithelial cells, cells of epithelia of renal tubules of proxima and for clearance of cytotoxic entities across endothelial cells in blood capillaries¹⁶. However the efflux of anticancer drugs by them has been identified as one of the mechanisms of MDR. Due to their over expression and increased activity drug efflux in targeted cancer cells makes them resistance to chemotherapy. A broad spectrum of studies has reported the capacity of phytochemicals in reversing this MDR mitigating ABC transporter expression in cancer cells as encouraging¹⁷. Docking via in silico technologies^18-22 for predicting binding affinities of small molecules to vital protein ligands highlights the interactions aid in the vital role of drug discovery^23-27.

With this background, the current study was directed towards use of computational methods to report the possible anti-cancer properties of phytochemicals for inhibitory activity towards WNK-1. Phytochemicals binding with a strong binding to WNK-1 forming a stable complex could be used as potent anti-cancer molecules for further developing them as drug entities.

MATERIALS AND METHODS:

Ligand selection and retrieval:

Phytochemicals from plant sources:

The present study includes phytochemicals as ligands from medicinal plants and they were selected after a comprehensive survey that included searching in scientific databases of Scopus, Science Direct, Google Scholar, Web of Science and PubMed.  About 50% of anti-cancer drugs derived from natural products and phytochemicals with antitumor capacity has been cited²⁸. However the in silico studies via docking of phytochemicals to WNK-1 could be a new perspective of looking at their properties aiding developing them as drug entities. A set of 17 phytochemicals from medicinal plants were docked to WNK-1 and the results were compared to standard drug WNK-463 molecule (Table 1).

Pharmacokinetic profile:

Swiss ADME (http://www.swissadme.ch/index.php) was applied to study the pharmacokinetic profiles of identified phytochemicals.  The ligand retrieved from the PubChem database was initially entered in the simplified molecular input line entry system (SMILES).   For the next step Lipinski’s Rule of Five analysis was applied to report the phytochemical pharmacokinetic properties.

Ligand and protein preparation:

The 3D structure of each phytochemicals was retrieved from PubChem or ChemSpider (http://www.chemspider.com) for saving in SDF format (https://pubchem.ncbi.nlm.nih.gov/). For ideal bond interaction effect Open Babel was used to minimize the energy associated with ligands for docking. The compound WNK463 (N-tert-butyl-3-(1-{5-[5-(trifluoromethyl)-1,3,4-oxadiazol-2-yl]pyridin-2-yl}piperidin-4-yl)imidazole-4-arboxamide) (ChemID58172620) with specific WNK-1 inhibitory capacity was used as a reference inhibitor control in this study^14,15. Protein WNK-1 – 5TF9 of 2.50Å resolution was retrieved from Protein Data Bank web server (https://www.rcsb.org/). PyMOL software was used to stabilize the same to human body’s physiology wherein hydrogen atoms were replacing the water entities.

Molecular docking:

The protein WNK-1 and the ligands were converted to pdb format by docking software PyRx 0.8 having built in Vina Wizard for specific docking of the phytochemicals to protein WNK-1.  A grid box size of 213, 163, 177 Å grid box was identified for presenting protein and ligand results.  Autogrid module was applied for atomic interactions and electrostatic maps.  The best ligand protein conformation with lowest energy of binding was exported to report the 2D plot generated via Ligplot+ and reported by PyMOL.  Specific docking was performed to ascertain the interaction between the protein WNK-1 and the ligands. The ligands and the protein were converted into pdb format using PyRx 0.8 docking tool with a built in Vina wizard. Vina, the AutoDock program doing docking within a space was defined by the coordinates and it provided the results in form of table but the coordinates were not a visual image.  The protein and ligands were docked with a grid box size of 213, 163, 177 Å. The atomic interactions and electrostatic maps of the ligands were calculated using the autogrid module. Molecular graphics laboratory (MGL) tools were used to analyze the results from Vina Wizard.   The best conformation with lowest binding energy was exported for 2D plot generation using Ligplot+. The docking conformation was represented using PyMOL.

RESULTS:

Ligand selection and retrieval:

With the main of identifying and reporting phytochemical(s) displaying a strong binding capacity to target WNK-1 protein forming a stable complex, a set of 15 phytochemicals with reported potent anti-cancer properties (Table 1) were incorporated in this study.

Table 1: Active ingredients in spices with reported anti-cancer property as ligands for docking to WNK-1

Sl. No.	Compound	Formula	Name of the molecule	Spice source	Reference
1	Curcumin (Pubchem CID: 839564)	C21H20O6	polyphenol	Curcuma longa L.,	(29)
2	Cinnamaldehyde (Pubchem CID: 637511)	C9H8O	aldehyde	Cinnamomum verum J. Presl.,	(30)
3	Carnosol (Pubchem CID: 442009)	C20H26O4	Phenolic diterpene	Salvia officinalis L. (Sage)	(31)
4	cis-Chlorogenic acid (Pubchem CID: 1794425)	C16H18O9	cinnamate ester	Solanum lycopersicum L.	(32)
5	Dioscin (Pubchem CID: 119245)	C45H72O16	Steroid saponins	Polygonatum  zanlanscianense Pamp.	(33,34)
6	Epigallocatechin Gallate (Pubchem CID 65064)	C22H18O11	ester of epigallocatechin and gallic acid	Camellia sinensis C. thea	(35)
7	Genistein (Pubchem CID 5280961)	C15H10O5	isoflavone	Genista tinctoria L.	(36)
8	Indole-3-Carbinol (Pubchem CID 3712)	C9H9NO	Plant hormone	Brassica oleracea var. italic	(37)
9	Naringenin (Pubchem CID 439246	C15H12O5	flavanone	Citrus grandis L.	(38)
10	Oleuropein aglycone (Pubchem CID 56842347)	C19H22O8	glycosylated seco-iridoid	Olea europaea L.	(39)
11	Paclitaxel (taxol) (Pubchem CID 36314)	C47H51NO14	semisynthetic taxoid	Taxus brevifolia Nutt.	(40)
12	Quercetin (Pubchem CID 5280343)	C15H10O7	flavonoid	Malus pumila P. Mill.	(41,42)
13	Resveratrol (Pubchem CID 445154)	C14H12O3	polyphenolic phytoalexin	Vitis vinifera L.	(43)
14	Rohitukine (Pubchem CID 13422573)	C16H19NO5	Chromone alkaloid	Amoora rohituka (Roxb.) Wight & Arn.	(44)
15	Rosmanol (Pubchem CID 13966122)	C20H26O5	diterpene	Rosmarinus officinalis L.	(45)
16	Sulforaphane (Pubchem CID 5350	C6H11NOS2	isothiocyanate organosulfur	Brassica oleracea var. capitata	(46)
17	Thymoquinone (Pubchem CID 10281)	C10H12O2	monoterpenoid benzoquinone,	Nigella sativa L.	(47)

Pharmacokinetic profile:

The drug-like capacity and the docking score obtained after analysis has been provided the ADMET profiling was applied to identify one of the most favorable phytochemical as ligand applying the docking process. A good drug-likeness property was reported for the ligands (Table 2).  The analysis of the gastrointestinal (GI), LogP and MW logs identified them as having good membrane permeability, oral bioavailability and intestinal absorption.  A higher lipophilicity accounting for a good biological capacity to act as drug was also identified due to their capacity to be absorbed readily across biological membranes.  A suitable drug metabolism and pharmacokinetics (DMPK) facilitated via nHBDs and nRotb bond for the identified phytochemicals has been reported here. The ADMET analysis predicted complete solubility in aqueous media and suitable absorption by the gastrointestinal tract for the phytochemicals aiding for their optimal concentration in the blood for biological function.  The analysis reported their poor capacity to cross the blood brain barrier and thus a diminished capacity of toxicity to the central nervous system.

Table 2: Pharmacokinetic profile of phytochemicals reported in the current study

Compound	Formula	Number of hydrogen	No. of hydrogen donor	(Log P0/w)	(LogS)	GI Absorp-tion	Lipinski drug likeliness
Curcumin	C21H20O6	6	2	3.27	-3.94	High	Yes
Cinnamaldehyde	C9H8O	1	0	1.65	-4.83	High	Yes
Carnosol	C20H26O4	4	2	2.93	-4.77	High	Yes
cis-Chlorogenic acid	C16H18O9	9	6	0.69	-1.62	Low	Yes
Dioscin	C45H72O16	--	--	--	--	--	No
Epigallocatechin Gallate	C22H18O11	11	8	1.53	-3.56	Low	No
Genistein	C15H10O5	5	3	1.91	-3.72	High	Yes
Indole-3-Carbinol	C9H9NO	1	2	1.36	-1.96	High	Yes
Naringenin	C15H12O5	5	3	1.75	-3.49	High	Yes
Oleuropein aglycone	C19H22O8	8	3	2.55	-2.55	High	Yes
Paclitaxel	C47H51NO14	--	--	--	--	--	No
Quercetin	C15H10O7	7	5	1.63	-3.16	High	Yes
Resveratrol	C14H12O3	3	3	1.71	-3.62	High	Yes
Rohitukine	C16H19NO5	6	3	2.43	-2.86	High	Yes
Rosmanol	C20H26O5	5	3	2.52	-4.25	High	Yes
Sulporaphane	C6H11NOS2	2	0	2.11	-1.50	High	Yes
Thymoquinone	C10H12O2	2	0	1.99	-2.18	High	Yes

Molecular docking:

For the studies on drug likeliness capacity of the ligands based on Swiss ADME analysis their chemical properties as molecular weight <500 Daltons, <5 H-bond donors, <10 H-bond acceptors and QPlogPo/w<5 were looked into.  The partition coefficient (log P o/w) of the ligands in n-octanol/water systems, a key physicochemical parameter in drug discovery depicted lipophilicity index to be within the range of reference drug moieties.  PyRx software was used to perform docking of phytochemicals to protein WNK-1. Asp465 and Asn463 of protein WNK-1 served as binding residues in protein WNK-1. The binding energy profiles are reported in Table 3.

Table 3: Results of docking scores of the phytochemicals to WNK-1

Sl. No.	Phytochemicals	Binding Energy
	Reference molecule
1	WNK-463	-8.9
	Phytochemicals
2	Curcumin	-7.5
3	Cinnamaldehyde	-6.2
4	Carnosol	-7.5
5	cis-Chlorogenic acid	-6.7
6	Dioscin	-14.3
7	Egigallocatechin gallate	-8.4
8	Genistein	-8.0
9	Indole-3-Carbinol	-6.6
10	Naringenin	-8.4
11	Oleuropein aglycone	-7.9
12	Paclitaxel	-18.3
13	Quercetin	-8.4
14	Resveratrol	-7.3
15	Rohitukine	-7.5
16	Rosmanol	-7.8
17	Sulforaphane	-3.4
18	Thymoquinone	-6.1

Figure 1: The molecular interactions and binding poses of WNK463 (A), Oleuropein aglycone (B), Dioscin (C), Epigallocatechin gallate (D), Naringenin (E), Quercetin (F)  to WNK-1. The two amino acids Asp465 and Asn463 of protein WNK-1 contributed to binding with ligands. 

Figure 2: The molecular interactions and binding poses of Curcumin (G), Cinnamaldehyde (H), Carnosol (I); cis-Chlorogenic acid (J); Genistein (K); Indole-3-carbinol (L) to WNK-1.  The two amino acids Asp465 and Asn463 of protein WNK-1 contributed to binding with ligands  

Oleuropein aglycone (Figure 1B) and Dioscin (Figure 1C) with strong binding energies of -18.3 and -14.3 respectively (Table 3) to WNK-1 with highest WNK-1 inhibitory potential are reported.  They were followed by Epigallocatechin gallate (Figure 1D), Naringenin (Figure 1E) and Quercetin (Figure 1F) with binding energy of -8.4 kcal/mol (Table 3) forming a stable complex with WNK-1.  The standard inhibitor WNK463 recorded ∆G of -8.9 kcal/mol.

The binding profiles of the other phytochemicals as ligands toWNK-1 have been provided in Figures 2 and 3.

Figure 3: The molecular interactions and binding poses of Paclitaxel (M); Resveratrol (N); Rohitukine (O); Rosmanol (P); Sulforaphane (Q); Thymoquinone (R)  to WNK-1.  The two amino acids Asp465 and Asn463 of protein WNK-1 contributed to binding with ligands

DISCUSSION:

The molecular docking of phytochemicals to WNK-1 has been attempted for the first time.  However there is one previous study for identification of entities with anti-cancer capacity inhibiting WNK-1 by docking studies were for non-peptide small-molecules⁴⁸.  The pharmacoinformatics and simulation studies indicated these non-peptide entities inhibit SPAK and OSR1 phosphorylation. A total of 11,870 molecules were screened in this study. Three molecules (Hit 1-3) were reported to have drug like properties possessing novel antihypertensive capacity⁴⁸.

CONCLUSION:

The current study is a in silico molecular docking strategy of phytochemicals with reported anti-cancer activity from known medicinal plants to WNK-1 that also includes WNK463 a known WNK-1 inhibitor.  Oleuropein aglycone and Dioscin with strong binding energies of -18.3 and -14.3 respectively to WNK-1 with highest WNK-1 inhibitory potential could find themselves as better inhibitors of WNK-1.  They were followed by Quercetin, Naringenin and epigallocatechin gallate with binding energy of -8.4 kcal/mol forming a stable complex with WNK-1.  The results of this study could provide a platform for further validation of the claims via advanced in vitro and in animal experimentation reversing cancer.  The findings open up strategies of their use in repurposing drugs for cancer treatment.

CONFLICT OF INTEREST:

The authors declare there is no conflict of interest with respect to this investigation.

ACKNOWLEDGEMENT:

The authors thank the authorities of JSS Academy of Higher Education and Research, Mysuru, India for infrastructural.

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Received on 30.04.2025      Revised on 03.08.2025

Accepted on 04.10.2025 Published on 20.05.2026

Available online from May 25, 2026

Research J. Pharmacy and Technology. 2026;19(5):2349-2357.

DOI: 10.52711/0974-360X.2026.00337

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