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Identification of novel c-Kit inhibitors from natural sources using virtual

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Identification of novel c-Kit inhibitors from natural

sources using virtual screening and molecular
dynamics simulations

Abdelbaset Mohamed Elasbali, Waleed Abu Al-Soud, Elyasa Mustafa Elfaki,

Hamad H. Alanazi, Bandar Alharbi, Salem Hussain Alharethi, Khalid Anwer,
Taj Mohammad & Md. Imtaiyaz Hassan

To cite this article: Abdelbaset Mohamed Elasbali, Waleed Abu Al-Soud, Elyasa Mustafa Elfaki,
Hamad H. Alanazi, Bandar Alharbi, Salem Hussain Alharethi, Khalid Anwer, Taj Mohammad &
Md. Imtaiyaz Hassan (2023): Identification of novel c-Kit inhibitors from natural sources using
virtual screening and molecular dynamics simulations, Journal of Biomolecular Structure and
Dynamics, DOI: 10.1080/07391102.2023.2231547

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JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
https://doi.org/10.1080/07391102.2023.2231547

Identification of novel c-Kit inhibitors from natural sources using virtual

screening and molecular dynamics simulations
Abdelbaset Mohamed Elasbalia,b, Waleed Abu Al-Soudc, Elyasa Mustafa Elfakia, Hamad H. Alanazia,
Bandar Alharbid, Salem Hussain Alharethie, Khalid Anwerf, Taj Mohammadg and Md. Imtaiyaz Hassang
a
Department of Clinical Laboratory Science, College of Applied Sciences-Qurayyat, Jouf University, Sakakah, Saudi Arabia; bHealth Sciences
Research Unit, Jouf University, Sakakah, Saudi Arabia; cDepartment of Clinical Laboratory Science, College of Applied Sciences-Sakaka, Jouf
University, Sakakah, Saudi Arabia; dDepartment of Clinical Laboratory, College of Applied Medical Sciences, University of Hail, Hail, Saudi
Arabia; eDepartment of Biological Science, College of Arts and Science, Najran University, Najran, Saudi Arabia; fDepartment of Botany,
C. M. Science College, L. N. Mithila University, Darbhanga, India; gCentre for Interdisciplinary Research in Basic Sciences, Jamia Millia
Islamia, New Delhi, India
Communicated by Ramaswamy H. Sarma

ABSTRACT ARTICLE HISTORY

The Mast/Stem cell growth factor receptor Kit (c-Kit), a Proto-oncogene c-Kit, is a tyrosine-protein kin- Received 9 April 2023
ase involved in cell differentiation, proliferation, migration, and survival. Its role in developing certain Accepted 24 June 2023
cancers, particularly gastrointestinal stromal tumors (GISTs) and acute myeloid leukemia (AML), makes
KEYWORDS
it an attractive therapeutic target. Several small molecule inhibitors targeting c-Kit have been devel-
Tyrosine-protein kinase c-
oped and approved for clinical use. Recent studies have focused on identifying and optimizing natural Kit; gastrointestinal stromal
compounds as c-Kit inhibitors employing virtual screening. Still, drug resistance, off-target side effects, tumors; plant-based
and variability in patient response remain significant challenges. From this perspective, phytochemicals compounds; anilinonaph-
could be an important resource for discovering novel c-Kit inhibitors with less toxicity, improved effi- thalene; licoflavonol; drug
cacy, and high specificity. This study aimed to uncover possible c-Kit inhibitors by utilizing a structure- discovery
based virtual screening of active phytoconstituents from Indian medicinal plants. Through the screen-
ing stages, two promising candidates, Anilinonaphthalene and Licoflavonol, were chosen based on
their drug-like features and ability to bind to c-Kit. These chosen candidates were subjected to all-
atom molecular dynamics (MD) simulations to evaluate their stability and interaction with c-Kit. The
selected compounds Anilinonaphthalene from Daucus carota and Licoflavonol from Glycyrrhiza glabra
showed their potential to act as selective binding partners of c-Kit. Our results suggest that the identi-
fied phytoconstituents could serve as a starting point to develop novel c-Kit inhibitors for developing
new and effective therapies against multiple cancers, including GISTs and AML. The use of virtual
screening and MD simulations provides a rational approach to discovering potential drug candidates
from natural sources.

1. Introduction targeting c-Kit has emerged as a promising therapeutic strat-

egy in drug discovery research (Pathania et al., 2021; Ritter &
Mast/stem cell growth factor receptor Kit (c-Kit), also known
Arteaga, 2003).
as CD117, is a transmembrane receptor tyrosine kinase pro-
Several small molecule inhibitors and monoclonal anti-
tein that plays a critical role in various cellular processes, bodies targeting c-Kit have been developed and approved
such as proliferation, differentiation, migration, and survival for clinical use, such as Pexidartinib, Dasatinib, and
(Foster et al., 2018). It is primarily expressed on the surface Omacetaxine Mepesuccinate (Abbaspour Babaei et al., 2016;
of hematopoietic stem cells, melanocytes, interstitial cells of Galanis & Levis, 2015; Zamecnıkova, 2014). A recent study
Cajal, and germ cells, among others (Gibson & Cooper, 2002). focuses on identifying and optimizing natural compounds as
The c-Kit receptor is activated by binding to its stem cell fac- c-Kit inhibitors employing virtual screening and identified
tor (SCF) ligand. This leads to the autophosphorylation of multiple hits with nanomolar inhibitory activity against c-Kit
tyrosine residues in the receptor’s cytoplasmic domain, trig- when tested in vitro (Nada et al., 2023). Another study syn-
gering downstream signaling cascades (Lennartsson & thesized a series of flavanol-based fisetin derivatives using
Ro€nnstrand, 2012). Dysregulation of c-Kit signaling has been inverse-docking and kinase activity and identify multiple
implicated in several diseases, including cancer, allergies, and potential c-KITs inhibitors (Roy et al., 2021). The study
mast cell disorders (Vliagoftis et al., 1997). As a result, showed that these compounds could induce apoptosis of

CONTACT Md. Imtaiyaz Hassan [email protected] Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi,
110025, India; Abdelbaset Mohamed Elasbali [email protected] Department of Clinical Laboratory Science, College of Applied Sciences-Qurayyat, Jouf
University, Sakakah, Saudi Arabia
Supplemental data for this article can be accessed online at https://doi.org/10.1080/07391102.2023.2231547
ß 2023 Informa UK Limited, trading as Taylor & Francis Group
2 A. M. ELASBALI ET AL.

A375 and A431 cells in a dose-dependent manner, which is a 2000), followed by interaction analysis and all-atom molecular
promising finding for potential cancer treatments (Roy et al., dynamics (MD) simulations for 100 nanoseconds (ns).
2021). The continuous advancements in c-Kit-targeting inhibi-
tors have significantly improved their efficacy. Nevertheless,
it is important to address the challenge of drug resistance 2. Methods and materials
that can arise over time. This highlights the ongoing need to 2.1. Computational resources
develop novel and enhanced c-Kit-targeting drugs to
enhance further their effectiveness in combating cancer In this study, we utilized several tools and methods to carry
(Alizadeh & Larmonier, 2014; Caksa et al., 2022). out virtual screening and interaction analysis. Specifically, we
With the state-of-the-art advancements in structural biol- used InstaDock v1.1 (Mohammad et al., 2021), PyMOL
ogy technology, discovering novel leads with improved effi- (DeLano, 2002), and Discovery Studio Visualizer (Biovia, 2017)
cacy has become easier than ever (Lionta et al., 2014; Naz tools for our structure-based virtual screening process and
et al., 2016; Rao et al., 2021; Roy et al., 2020; Shahbaaz et al., interaction analysis. Additionally, we used GROMACS 2020
2016). One crucial technique in drug discovery is structure- beta for all-atom MD simulation studies (Van Der Spoel et al.,
based drug design, known for its specificity and effectiveness 2005). To begin our virtual screening process, we obtained
(Gupta et al., 2019; Khan et al., 2018; Naqvi et al., 2018). In par- the structure of c-Kit from the RCSB Protein Data Bank with
ticular, virtual screening has emerged as the most efficient PDB ID: 7KHG. This is a co-crystallized structure of the kinase
method to identify potential leads using computer-based domain of c-Kit with a small molecule inhibitor, PLX3397
screening techniques (Mohammad et al., 2020; Yousuf et al., (Wagner et al., 2021). Before the screening, the structure
2022). Virtual screening can prevent undesirable compounds, required preprocessing, which involved adding hydrogen to
saving both time and money (Amir et al., 2020). Refinement polar atoms and assigning the appropriate atom types. We
and filtering techniques, such as pharmacophore modeling, then utilized a set of 12,000 phytoconstituents compounds
molecular docking, Lipinski filter, and ADMET, play a significant from the IMPPAT database in the processed arrangement for
role in the lead discovery process by providing insights into our virtual screening process. The compounds were filtered
potential compounds (Gupta et al., 2020; Jairajpuri et al., 2021; based on their physicochemical properties to narrow down
Khan et al., 2022; Mohammad et al., 2019; Shafie et al., 2021). our search. This filtration process allowed us to select the
The goal of this study is to identify novel c-Kit inhibitors most promising candidates for further analysis and refine-
that are both effective and specific. To achieve this, we ment. Overall, these tools and methods enabled us to carry
explored IMPPAT 2.0 database, which contains
12,000 bio- out a thorough virtual screening and analysis process in our
active phytochemicals from Indian medicinal plants that can search for novel c-Kit inhibitors. Figure 1 provides a visual
be used for virtual screening in drug discovery (Mohanraj representation of the workflow utilized in this study.
et al., 2018). The compounds were filtered using the Lipinski
rule of five and then underwent molecular docking screening.
2.2. Molecular docking
The top 10 compounds were selected based on binding affin-
ities and ligand efficiency from the initial screening and then Molecular docking is a widely used technique in drug discov-
subjected to PAINS (Baell, 2016) and ADMET filters (Hodgson, ery to predict the binding affinity, selectivity, and specificity
2001). Finally, we conducted PASS analysis (Lagunin et al., of a small molecule drug candidate and its protein target (Ali

Figure 1. The visual representation shows the virtual screening workflow applied in this study to identify leads against c-Kit.
 JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 3

et al., 2019; Mohammad et al., 2019; Naqvi et al., 2018). systems were placed centrally in a cubic box at 10 Å from
Structure-based molecular docking was performed to identify the edges. To model the aqueous environment and provide
potential compounds with high binding affinity values. We water molecules, we used the SPC216 solvent model. An
utilized InstaDock v1.1 for docking and set the grid size of appropriate number of counterions were added to neutralize
the blind search space to 69, 69, and 64 Å, centralized at the charge and stabilize the system. We energy-minimized all
23.84, 0.39, and 6.50 Å for X, Y, and Z coordinates, respect- the systems using the steepest descent algorithm with 1500
ively. InstaDock is a freely available docking platform steps. The NVT and NPT ensembles were used to achieve
designed to be fast and efficient, allowing users to perform equilibrium over the position of the systems. The final MD
large-scale virtual screening of potential drug candidates run was conducted for 100 ns for each system, where the
(Mohammad et al., 2021). The default spacing parameter of resultant trajectories were examined through the inbuilt gmx
1 Å was used for the search space grid (Huey et al., 2012). utilities.
We used the affinity score value to filter and obtain the
required dock conformers for interaction analysis. We then
utilized PyMOL and Discovery Studio Visualizer to identify 2.6. Principal component analysis and free energy
the ideal interactors for the c-Kit binding pocket. Specifically, landscape
we focused only on potential compounds interacting with Principal component analysis (PCA) is a widely utilized tech-
the c-Kit binding pocket. nique to analyze the conformational dynamics of proteins. In
drug discovery, PCA is often used to analyze the conform-
2.3. ADMET prediction ational changes induced by ligand binding to a protein tar-
get (David & Jacobs, 2014; Mohammad et al., 2020; Shafie
We used multiple criteria to identify potential lead com- et al., 2021; Umair et al., 2021). Using the simulated trajecto-
pounds with drug-like properties. Our primary criteria for ries, we employed PCA to investigate the essential dynamics
identifying the compounds were the efficacy and safety of of c-Kit. Briefly, the covariance matrix-based PCA approach
the elucidated hits. To evaluate the efficacy, we utilized the was utilized for analyzing the MD trajectory of
SwissADME (Daina et al., 2017) and pkCSM (Pires et al., 2015) Anilinonaphthalene and Licoflavonol. This allowed us to gen-
tools to estimate the ADMET properties of each compound erate the systems’ free energy landscapes (FELs), which pro-
selected from the docking analysis. These tools allowed us to vide insights into the conformational changes of c-Kit with
understand the compound’s efficacy and suitability as a lead Anilinonaphthalene and Licoflavonol. To generate FELs, we
compound. Additionally, we used the PAINS filter to elimin- utilized gmx covar and gmx anaeig modules on the simulated
ate any Pan-assay interference compounds. These filters trajectories.
allowed us to identify lead compounds with high efficacy
and safety, essential for successful drug discovery.
2.7. MM-PBSA calculation

2.4. PASS analysis Molecular Mechanics Poisson-Boltzmann Surface Area (MM-

PBSA) is a widely utilized computational methodology to cal-
Prediction of Activity Spectra for Substances (PASS) analysis culate the binding free energy of a protein-ligand complex
was utilized to identify the biological activity of chemical (Kuhn et al., 2005). MM-PBSA is based on MD simulations,
substances based on their chemical structure (Lagunin et al., which provide input trajectories for free energy calculations.
2000). The PASS server generated a forecast rating for bio- Here, we used a 10 ns frame from a stable region of the MD
logical characteristics based on Pa to Pi ratio, where Pa rep- trajectory to estimate MM-PBSA. The gmx mmpbsa module in
resents the likelihood of activity and Pi represents the GROMACS was used to estimate the binding free energy of
likelihood of inactivity. We focused on the Pa value of each the protein-ligand complex (Kumari et al., 2014).
biological property, which helped us evaluate the probability
of a compound having that property. A high Pa value for a
particular biological property suggested a strong possibility 3. Results and discussion
that the compound would have that property. By utilizing
3.1. Molecular docking
the PASS server in this way, we gained insight into the bio-
logical activity of the chemical substances. Before the molecular docking screening, we utilized the
Lipinski rule of five to filter the parent library of phytochemi-
cals based on their physicochemical properties. A total of
2.5. MD Simulations

12,000 compounds were filtered, and molecular docking
MD simulations of c-Kit were conducted using the GROMACS was conducted on the filtered library through InstaDock. The
2020 package on a high-performance HP Z840 machine at a resulting docking scores were used to identify the top 10
temperature of 300K. The Gromos force-field (Schmid et al., hits, as shown in Table 1. Our findings indicate that these
2011) was used to model the molecular mechanics of the compounds possess considerable affinities for c-Kit, ranging
simulation process for the ligand-bound states with from 9.6 to 12.1 kcal/mol. These results suggest that the
Anilinonaphthalene and Licoflavonol produced from the selected compounds have the potential to be further devel-
PRODRG server (Schu €ttelkopf & Van Aalten, 2004). The oped as c-Kit inhibitors for therapeutic applications.
4 A. M. ELASBALI ET AL.

Table 1. The top 10 hits with the reference c-Kit inhibitor, Pexidartinib, and their docking score with c-Kit.
S. No. Molecule ID Binding free energy (kcal/mol) Ligand efficiency (kcal/mol/non-H atom)
1. IMPHY006927 12.1 0.52
2. IMPHY005568 12.0 0.46
3. IMPHY005114 10.8 0.45
4. IMPHY012046 10.0 0.38
5. IMPHY009808 10.0 0.43
6. IMPHY006471 9.9 0.41
7. IMPHY006894 9.9 0.32
8. IMPHY005893 9.8 0.38
9. IMPHY012102 9.7 0.57
10. IMPHY006882 9.6 0.34
11. Pexidartinib 8.6 0.30

Table 2. ADMET properties of the selected molecules.

Molecule ID ESOL class GI absorption BBB permeant CYP2D6 inhibitor PAINS #alerts OCT2 substrate AMES Hepatotoxicity
IMPHY006471 Moderate High Yes No 0 No No Yes
IMPHY006882 Moderate High Yes No 0 No No Yes
IMPHY006894 High High No No 0 No No Yes
IMPHY006927 Moderate High Yes No 0 No Yes No
IMPHY009808 Moderate High Yes Yes 0 Yes Yes Yes
IMPHY012046 High High Yes Yes 0 No Yes No
IMPHY012102 Moderate High Yes Yes 0 No No No
IMPHY005114 Moderate High Yes No 0 No No Yes
IMPHY005568 Moderate High No No 0 Yes No No
IMPHY005893 Moderate High No Yes 0 No No No
Pexidartinib Moderate High No Yes 0 No No Yes

Table 3. Elucidated molecules and their biological properties.

Compound ID Compound Pa Pi Biological activity
IMPHY012102 Anilinonaphthalene 0.722 0.007 Protein kinase inhibitor
0.708 0.053 Membrane integrity agonist
0.765 0.010 Apoptosis agonist
0.635 0.038 Antineoplastic
0.553 0.009 Tyrosine kinase inhibitor
0.507 0.019 Angiogenesis inhibitor
IMPHY005893 Licoflavonol 0.972 0.002 Membrane integrity agonist
0.905 0.003 Kinase inhibitor
0.889 0.002 Antimutagenic
0.890 0.004 Apoptosis agonist
0.877 0.006 TP53 expression enhancer
PLX3397 Pexidartinib 0.562 0.013 Angiogenesis inhibitor
0.369 0.034 Antineoplastic
0.259 0.051 Protein kinase inhibitor
0.255 0.207 Anti-inflammatory
0.232 0.216 Cell adhesion inhibitor
Pa: the probability of being active; Pi: probability of being inactive.

3.2. ADMET properties 3.3. PASS analysis

ADMET properties are essential for determining the clin- The PASS predictions can guide drug discovery and optimiza-
ical effectiveness of potential drug candidates. We used tion by predicting the potential activities of a compound. We
SwissADME and pkCSM webservers to calculate the utilized the PASS server that calculates various biological
ADMET properties of the selected hits from the docking properties, including apoptosis, TP53 expression, antineoplas-
study. The ADMET properties were evaluated based on tic, and more. After analyzing the elucidated compounds,
CaCO-2 permeability, water-solubility, and absorption Anilinonaphthalene and Licoflavonol, that passed our previ-
parameters (Supplementary Table S1). Out of 10 com- ous filtering steps, we found both compounds with desirable
pounds, three, IMPHY012102, IMPHY005568, and biological properties with appreciable Pa values (Table 3).
IMPHY005893, were identified without any toxic patterns The prediction shows that Anilinonaphthalene and
(Table 2). One of them, IMPHY005568, was OCT2 substrate Licoflavonol exhibit antineoplastic, TP53 expression enhancer,
and hence was eliminated from further studies. These and apoptosis agonist properties with Pa values ranging
results suggest that the remaining two compounds, from 0.507 to 0.972, where Pa > Pi. These results suggest
IMPHY012102 and IMPHY005893, have promising drug- that Anilinonaphthalene and Licoflavonol are potential drug
likeliness and could be explored additionally as potential candidates for treating various cancers and other complex
c-Kit inhibitors. diseases, warranting further investigation.
 JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 5

Figure 2. c-Kit in complex with Anilinonaphthalene and Licoflavonol. (A) Cartoon representation of c-Kit with, Anilinonaphthalene (magenta) and Licoflavonol (yel-
low). (B) Magnified c-Kit binding pocket residues interactions with Anilinonaphthalene and Licoflavonol (C) Binding pocket of c-Kit occupied by both compounds.

3.4. Interaction analysis It is a well-established fact that point mutations can occur
within the kinase domain, leading to resistance against c-Kit
Protein-ligand interaction analysis plays a crucial role in drug
inhibitors and consequent drug-resistant relapse. One com-
discovery as it helps to understand the binding mechanism monly observed mutation in c-Kit resulting in V654A substi-
of small molecule ligands to the target proteins. A detailed tution has been documented in Imatinib-resistant GIST
interaction analysis of the elucidated compounds’ binding to patients (Roberts et al., 2007). To see the impact of this
the c-Kit binding pocket was conducted based on their con- mutation on Anilinonaphthalene and Licoflavonol binding,
formations. We selected conformers that fit well with the we also performed molecular docking of c-Kit mutant V654A
crystal structure by comparing them with the reference com- with these compounds (Supplementary Figure S1). Our single
pound Pexidartinib (PLX3397) (Wagner et al., 2021). Figure 2 point mutation docking study showed that the binding of
depicts the binding of Anilinonaphthalene and Licoflavonol Anilinonaphthalene and Licoflavonol was unaffected with a
with the c-Kit binding pocket. Our analysis revealed that minor decrease in binding affinity, i.e. 9.6 and 9.7 kcal/
Anilinonaphthalene and Licoflavonol interact with the ATP mol, respectively. These findings provide valuable insights
binding site Lys623, where the reference inhibitor of c-Kit into the molecular mechanism underlying the inhibitory
binds (Figure 2(B)). Conformational analysis showed that effects of Anilinonaphthalene and Licoflavonol on c-Kit and
Anilinonaphthalene and Licoflavonol interact similarly with c- suggest their potential as drug candidates for treating vari-
Kit (Figure 2(B)). Anilinonaphthalene and Licoflavonol bind ous diseases.
virtuously to the deep cavity, indicating their potential to
obstruct the ATP binding on c-Kit (Figure 2(C)). These find-
ings provide valuable insights into the mechanism of action 3.5. MD simulations
of Anilinonaphthalene and Licoflavonol, which can be further MD simulation of a protein-ligand complex can provide a
explored to develop novel therapeutics. more refined understanding of docking models (Hernandez-
To better understand the non-covalent interactions Rodrıguez et al., 2016). These simulations allow for a detailed
between Anilinonaphthalene, Licoflavonol, and the co-crystal- analysis of protein structure and dynamic behavior that is
lized reference inhibitor, Pexidartinib, with c-Kit, detailed not easily achievable through experimental approaches
interaction analyses were conducted. The selected interaction (Dahiya et al., 2019). MD simulations were conducted in an
modes of Anilinonaphthalene, Licoflavonol, and Pexidartinib aqueous solution to gain insights into the stability of the
were further examined for their detailed interactions with c- docking models of c-Kit with the identified compounds
Kit. Two-dimensional (2D) plots of all possible interactions Anilinonaphthalene and Licoflavonol. The selected conform-
were generated for Anilinonaphthalene, Licoflavonol, and ers of docked Anilinonaphthalene and Licoflavonol with c-Kit
Pexidartinib (Figure 3). The 2D plots showed that were used as starting points for the MD simulations run for
Anilinonaphthalene and Licoflavonol interact with the ATP 100 ns. The time evolution of various parameters was eval-
binding site Lys623 and share the same interactions with the uated to analyze how c-Kit interacts before and after the
known c-Kit inhibitor, Pexidartinib (Figures 3(A–C)). As a con- binding of Anilinonaphthalene and Licoflavonol. The results
sequence, Anilinonaphthalene and Licoflavonol may hamper of the MD simulations provided valuable information on the
c-Kit’s ATP availability, leading to the inhibition of its activity. stability of the protein-ligand complexes and further insights
6 A. M. ELASBALI ET AL.

Figure 3. The c-Kit’s residues interact with (A) Licoflavonol, (B) Anilinonaphthalene, and (C) Pexidartinib.

into the molecular mechanism underlying the inhibitory the c-Kit-Anilinonaphthalene complex. The probability distri-
effects of Anilinonaphthalene and Licoflavonol on c-Kit, as bution function (PDF) plot also showed a similar RMSD value
discussed in the ensuing section. with a minor increase in the RMSD of complexes. Based on
these findings, we conclude that the docked complexes were
stable during the simulation.
3.5.1. Structural changes and compactness
The root-mean-square fluctuation (RMSF) is a valuable
The conformational changes in protein structure upon bind-
tool for measuring the residual vibrations of proteins during
ing small molecules to the protein’s active binding site sig-
the simulation, indicating the flexibility of individual residues
nificantly impact the efficacy of drugs (Karplus &
in all three complexes (Hu €nenberger et al., 1995). The RMSF
McCammon, 2002). One widely used method for studying
these conformational changes is a root-mean-square devi- plot in Figure 4(B) shows that all systems display a similar
ation (RMSD). The study utilized RMSD to evaluate the back- pattern of RMSF. The average RMSF values for c-Kit, c-Kit-
bone movements of c-Kit protein before and after binding Anilinonaphthalene, and c-Kit-Licoflavonol complexes were
with Anilinonaphthalene and Licoflavonol. We computed the 0.12, 0.12, and 0.11 nm, respectively. The residual fluctuations
temporal fluctuations of RMSD values for the c-Kit backbone are minimized upon compound binding, especially
atoms pre- and post-Anilinonaphthalene and Licoflavonol Licoflavonol, indicating stable docked complexes. A break in
binding and depicted them graphically in Figure 4(A). The RMSFs values from the 694 to 752 residues is shown as the
average RMSD values for c-Kit, c-Kit-Anilinonaphthalene, and structural coordinates of cKIT at this place was missing and
c-Kit-Licoflavonol complexes were 0.23, 0.26, and 0.24 nm, joined from the original structure, 7KHG in PDB. The RMSF
respectively. Our analysis indicated that the simulation was analysis reveals that the residues interacting with
stable for up to 100 ns and that the changes in conformation Anilinonaphthalene and Licoflavonol were mostly stable with
over time were fairly minimal. The RMSD fluctuations of the some minor fluctuations. In summary, the RMSF analysis
c-Kit-Anilinonaphthalene complex were minor, indicating sys- shows that binding Anilinonaphthalene and Licoflavonol to
tem adjustment after 10 ns without any significant changes. c-Kit results in stable protein-ligand complexes with minimal
The c-Kit-Licoflavonol complex exhibited more stability than fluctuations in the residual vibrations of the protein.
 JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 7

Figure 4. Structural dynamics of c-Kit with Anilinonaphthalene and Licoflavonol. (A) RMSD plot of c-Kit with Anilinonaphthalene and Licoflavonol. (B) RMSF plot of
c-Kit with Anilinonaphthalene and Licoflavonol. The lower panels show the values’ probability distribution function (PDF) distribution.

Figure 5. Structural compactness of c-Kit with Anilinonaphthalene and Licoflavonol. (A) The Rg is a function of time. (B) SASA plot of c-Kit with
Anilinonaphthalene and Licoflavonol binding. Lower panels show the PDF distribution.

The radius of gyration (Rg) is a widely used parameter to Licoflavonol to investigate the folding and stability of the
investigate the conformational dynamics of a protein, such protein structure during the simulation. The SASA plot
as how it changes shape in response to a ligand binding. We showed no significant changes in the structure’s folding or
used Rg to measure the compactness and folding of the c- unfolding with time, similar to the Rg analysis. The pattern
Kit protein structure in free and ligand-bound states. The Rg of SASA throughput in all three systems was consistent, with
of c-Kit with Anilinonaphthalene and Licoflavonol ranged no drastic changes (Figure 5(B), upper panel). The PDF plot
from 1.98 nm to 2.06 nm, indicating that c-Kit remained con- also revealed a similar pattern of c-Kit distribution and com-
stantly stable with both compounds. The PDF plot also plexes with Anilinonaphthalene and Licoflavonol, with a
showed similar Rg distribution after the Anilinonaphthalene slight decrease in the area (Figure 5(B), lower panel). Based
and Licoflavonol binding to c-Kit (Figure 5(A), lower panel). on the SASA analysis, we can conclude that
Our findings suggest that both Anilinonaphthalene and Anilinonaphthalene and Licoflavonol in the c-Kit complex do
Licoflavonol can stabilize the c-Kit protein structure, poten- not significantly alter their folding and stability.
tially inhibiting its functionality. Overall, the Rg analysis pro-
vides valuable information on the stability and folding of the
c-Kit protein structure in the presence of ligands. 3.5.2. Dynamics of hydrogen bonds
The solvent-accessible surface area (SASA) is a significant The formation of hydrogen bonds is crucial for stabilizing
factor in protein stability and folding studies, representing protein structural conformation (Weiss et al., 2001). Studying
the area of a protein accessible to the surrounding solvent the dynamics of hydrogen bonds can provide valuable
(Ali et al., 2014; Durham et al., 2009). We analyzed the time insight into proteins’ compactness and conformational
evolution of SASA of c-Kit with Anilinonaphthalene and changes (Rose & Wolfenden, 1993). We evaluated the time
8 A. M. ELASBALI ET AL.

Figure 6. Hydrogen bonding. (A) Intramolecular hydrogen bonds formed within c-Kit. (B) The PDF distribution of the intramolecular hydrogen bonds within c-Kit.

Figure 7. Intermolecular hydrogen bonds of (A) Anilinonaphthalene and (B) Licoflavonol with c-Kit.

evolution of intramolecular bonds in c-Kit and its docked remain at their original docking site on c-Kit. The PDF plot fur-
complexes with Anilinonaphthalene and Licoflavonol, as ther supports this conclusion, indicating the highest value at
shown in Figure 6(A). The plot illustrates that hydrogen three and one hydrogen bonds, respectively (Figure 7, lower
bond formation by c-Kit and its complexes was consistent panel). This finding suggests that Anilinonaphthalene and
during the simulation, with a slightly varying distribution Licoflavonol would remain bound to c-Kit, maintaining the sta-
observed between the c-Kit-Anilinonaphthalene and c-Kit- bility of the protein-ligand complex.
Licoflavonol systems. This plot indicates that all three
systems were compact during the simulation, and the pro-
tein-ligand complexes were stable. The PDF plots of all three
3.5.3. Secondary structure dynamics
systems exhibit constancy, confirming the hydrogen bond
Protein conformational changes can be induced by variable
formation consistency throughout the simulation (Figure
degrees of dynamic residual quantity participating secondary
6(B)). Overall, the analysis of intramolecular hydrogen bond-
structure content (Atilgan et al., 2010). Changes in its sec-
ing demonstrated the stability of c-Kit and its docked com-
plexes with Anilinonaphthalene and Licoflavonol during the ondary structure composition can be studied to get insights
simulation. The results suggest that the binding of into a protein’s conformational behavior and folding process.
Anilinonaphthalene and Licoflavonol to c-Kit did not signifi- We analyzed the kinetics of c-Kit’s secondary structure com-
cantly alter the stability of the protein-ligand complexes. position before and after binding with Anilinonaphthalene
In addition to analyzing the intramolecular hydrogen bond- and Licoflavonol to examine its stability. The results demon-
ing in c-Kit, we also investigated the stability of the intermo- strate that c-Kit’s structural constituents in the free state
lecular hydrogen bonding between Anilinonaphthalene and remained persistent throughout (Figure 8). Upon binding
Licoflavonol with c-Kit. The average number of hydrogen with Anilinonaphthalene and Licoflavonol, there was a slight
bonds formed in the Anilinonaphthalene-c-Kit and increase in c-Kit’s overall secondary structure content (Figure
Licoflavonol-c-Kit complexes was three and two, respectively 8(B)). This increase in average secondary structure is primarily
(Figure 7, upper panel). This analysis confirms that the intermo- due to coil transformation into helix and b-bridge (Table 4).
lecular hydrogen bonding between Anilinonaphthalene and Our findings suggest that the secondary structure compos-
Licoflavonol with c-Kit is stable, and both compounds would ition of c-Kit did not exhibit significant fluctuations upon
 JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 9

Figure 8. Secondary structure dynamics of (A) c-Kit, (B) c-Kit-Anilinonaphthalene, and (B) c-Kit-Licoflavonol. Structure ¼ a-helix þ b-sheet þ b-bridge þ turn.

Table 4. Amino acid residues participate in the secondary structure content of c-Kit.
Secondary structure content
System Structure Coil b-sheet b-bridge Bend Turn a-helix #
Other
c-Kit 200 77 55 6 41 42 97 10
c-Kit-Anilinonaphthalene 202 74 55 6 42 42 99 9
c-Kit-Licoflavonol 201 75 53 8 41 47 93 10
Structure ¼ a-helix þ b-sheet þ b-bridge þ Turn.
#
Other ¼ p-helix þ 310-Helix.

Figure 9. Conformational sampling of c-Kit and its docked complexes.

Anilinonaphthalene and Licoflavonol binding, indicating that not significantly affect the overall conformational behavior of
the complexes were stable during the simulation. c-Kit. Overall, the PCA analysis demonstrated the stability of
the c-Kit complexes and their similarity to the free c-Kit.

3.6. Principal component analysis

3.7. Free energy landscapes
PCA is a powerful computational technique that can provide
insights into the conformational sampling of a protein (David FEL is a widely utilized tool to understand protein folding
& Jacobs, 2014). We employed PCA to analyze the conform- and stability (Papaleo et al., 2009). During MD simulations,
ational dynamics of c-Kit, c-Kit-Anilinonaphthalene, and c-Kit- we generated FELs to assess the stability of c-Kit and its
Licoflavonol from the simulated trajectories. The results of complexes with Anilinonaphthalene and Licoflavonol. The
the PCA revealed that the c-Kit-Anilinonaphthalene and c-Kit- energy minima and conformational landscapes of c-Kit and
Licoflavonol have similar subspace as the free c-Kit, as its complexes were extracted using two principal compo-
illustrated in the 2D projection shown in Figure 9. The con- nents (PCs), and the resulting FELs are depicted in Figure 10.
formational sampling of c-Kit exhibited remarkable stability The dark blue regions indicate the global minima, represent-
during the simulation, indicating that the interactions ing the protein’s most stable conformational states. Our
between c-Kit and Anilinonaphthalene or Licoflavonol did results reveal that Anilinonaphthalene and Licoflavonol
10 A. M. ELASBALI ET AL.

Figure 10. Free energy landscape (FEL) plots of (A) free c-Kit, (B) c-Kit-Anilinonaphthalene, and (C) c-Kit-Licoflavonol.

Table 5. The binding free energy outcomes of anilinonaphthalene and licoflavonol obtained through MM-PBSA calculations.
Polar energies Non-polar energies
Complex Electrostatic Polar solvation van der Waal SASA Binding energy
c-Kit-Anilinonaphthalene 82.438 196.428 198.952 18.892 92.82
c-Kit-Licoflavonol 79.814 182.801 209.263 26.190 108.04
All energy values are expressed in units of kJ/mol.

binding to c-Kit impact the volume and location of regions (Roy et al., 2021). In this study, fisetin derivatives based on
constrained with 1–2 global minima (Figure 10). The plot flavanol were synthesized utilizing inverse-docking and kin-
also shows that c-Kit exhibits a large single global minimum ase activity. The study discovered several possible c-KIT
that expands to 2–3 before and after Anilinonaphthalene inhibitors. The results indicated that these compounds exhib-
and Licoflavonol binding. These findings suggest that c-Kit ited a dose-dependent ability to induce apoptosis of A375
and its complexes remain stable throughout the simulation. and A431 cells, which is a hopeful finding for cancer treat-
Overall, our FEL analysis provides valuable insights into the ment (Roy et al., 2021). The discovery of potential c-Kit inhib-
conformational sampling of c-Kit, which can aid in designing itors from Indian medicinal plants using structure-based
and developing novel therapeutics targeting c-Kit. virtual screening and MD simulations provides a good start-
ing point for developing new and effective therapies against
multiple cancers, including GISTs and AML. Phytochemicals
3.8. MM-PBSA analysis could offer a valuable resource for discovering novel c-Kit
The MM-PBSA approach was employed to assess the binding inhibitors with less toxicity and high specificity, paving the
free energy of C-KIT-Anilinonaphthalene and C-KIT- way for the development of safer and more effective cancer
Licoflavonol. A stable 10 ns frame of the MD trajectories was treatments.
chosen from a stable region, i.e. 40–50 ns to estimate the
MM-PBSA. The results demonstrated that the binding of the
4. Conclusions
ligands was favorable, as evidenced by the negative binding
free energy. Licoflavonol exhibited the most favorable binding This study utilized a structure-based virtual screening to dis-
energy (108.04 kJmol1) compared to Anilinonaphthalene, cover potent c-Kit inhibitors from bioactive phytoconstitu-
which had a binding energy of 92.82 kJmol1 (Table 5). The ents. The results led to the selection of two hits
negative binding energy was predominantly due to electro- (Anilinonaphthalene and Licoflavonol) with promising drug-
static interactions, non-polar solvation energy, and Van der like properties and binding affinity towards c-Kit. Further
Waals forces, while polar solvation energy contributed posi- analysis through all-atom MD simulations demonstrated that
tively to the overall binding energy. Based on these findings, these hits had stable interactions with c-Kit, making them
Licoflavonol appears to be the more promising inhibitor of potential binding partners. Using phytochemicals as c-Kit
the two. inhibitors present a promising alternative to conventional
Taken together, the results from this study suggested drugs, especially in the context of drug resistance and vari-
that the identified compounds, Anilinonaphthalene and able patient response. The hits identified in this study pro-
Licoflavonol, showed promising results in silico. Both com- vide a starting point for developing novel c-Kit inhibitors for
pounds showed appreciable binding potential towards c-Kit treating c-Kit-associated diseases, such as GISTs and AML.
and drug likeliness. MD simulations, PCA, and FELs further The combination of virtual screening and MD simulations
supported stability and time-evolution binding and folding proved an effective approach for identifying potential drug
mechanism c-Kit in the presence of Anilinonaphthalene and candidates from natural sources. This rational approach can
Licoflavonol. One of our elucidated compounds, Licoflavonol, help to reduce the time and cost involved in drug discovery
is very similar to the previously published Fisetin derivatives and provide a more sustainable and ethical approach to
 JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 11

drug development. In summary, this research establishes a targeting with anticancer agents. Cancer Research, 74(10), 2663–2668.
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Biovia, D. S. (2017). Discovery studio visualizer. San Diego, CA, USA, 936.
No potential conflict of interest was reported by the author(s).
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inhibitors in melanoma. Pharmacology & Therapeutics, 239, 108200.
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Funding Dahiya, R., Mohammad, T., Roy, S., Anwar, S., Gupta, P., Haque, A., Khan,
This work is supported by the Indian Council of Medical Research (Grant P., Kazim, S. N., Islam, A., Ahmad, F., & Hassan, M. I. (2019).
No. ISRM/12(22)/2020). Investigation of inhibitory potential of quercetin to the pyruvate
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Author’s contributions protein structure prediction. Journal of Molecular Modeling, 15(9),
1093–1108. https://doi.org/10.1007/s00894-009-0454-9
Abdelbaset Mohamed Elasbali: conceptualization, writing-review and Foster, B. M., Zaidi, D., Young, T. R., Mobley, M. E., & Kerr, B. A. (2018).
editing; Waleed Abu Al-Soud: visualization, data analysis, software, writ- CD117/c-kit in cancer stem cell-mediated progression and therapeutic
ing-review and editing, software; Elyasa Mustafa Elfaki: data validation, resistance. Biomedicines, 6(1), 31. https://doi.org/10.3390/
data analysis, writing-review and editing; Hamad H. Alanazi: conceptual- biomedicines6010031
ization, data analysis, editing, data curation, writing-original draft; Bandar Galanis, A., & Levis, M. (2015). Inhibition of c-Kit by tyrosine kinase inhib-
Alharbi: visualization, software, data validation, writing-review and edit- itors. Haematologica, 100(3), e77–e79. https://doi.org/10.3324/haema-
ing; Salem Hussain Alharethi: data validation, data analysis, writing- tol.2014.117028
review and editing; Khalid Anwer: data validation, software, project Gibson, P. C., & Cooper, K. (2002). CD117 (KIT): A diverse protein with
administration, writing-original draft, data duration, investigation; Taj selective applications in surgical pathology. Advances in Anatomic
Mohammad: methodology, investigation, writing-review and editing; Md. Pathology, 9(1), 65–69. https://doi.org/10.1097/00125480-200201000-
Imtaiyaz Hassan: data curation, supervision, visualization, writing-review 00007
and editing, formal analysis, project administration, writing-original draft. Gupta, P., Khan, S., Fakhar, Z., Hussain, A., Rehman, M. T., AlAjmi, M. F.,
Islam, A., Ahmad, F., & Hassan, M. I. (2020). Identification of potential
inhibitors of calcium/calmodulin-dependent protein kinase IV from
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