Kaempferol as a Potential PAK4 Inhibitor in Triple Negative Breast Cancer: Extra Precision Glide

Docking and Free Energy Calculation.

Michael A. Arowosegbe1,2,*, Oluwamuyiwa T. Amusan5, Segun A. Adeola2, Oluwatosin B. Adu2,

Israel A. Akinola5, Bimpe F. Ogungbe2, Olaposi I. Omotuyi1, Gbemisola M. Saibu2, Adewale J.
Ogunleye2, Ramon I. Kanmodi2, Nekabari E. Lugbe4, Oluwafemi J. Ogunmola6, Sedoten O. Ogun2,
Faith O. Oyende2,3, Hamed O, Bello2, Abudulmalik O. Ishola2, Patrick E. Obasieke2.
1
Centre for Biocomputing and Drug Development, Adekunle Ajasin University, Akungba-Akoko,
Ondo, Nigeria
2
Department of Biochemistry, Lagos State University, Ojo, Lagos, Nigeria
3
Department of Biochemistry, Ruhr University of Bochum, North Rhein, Westphalia, Germany
4
Department of Biochemistry, Niger Delta University, Wilberforce, Bayelsa, Nigeria
5
Department of Microbiology, Obafemi Awolowo University, Ile-Ife, Osun, Nigeria
6
Department of Biology (Storage Technology), Federal University of Technology, Akure, Ondo,
Nigeria
*
Corresponding author: Michael Aderibigbe Arowosegbe
Email: michael.a.arowosegbe@gmail.com. Telephone: +2347039739507.

Abstract
P-21 activating kinase 4 (PAK4) is implicated in poor prognosis of many human tumors,
particularly in triple negative breast cancer (TNBC) progression. Studies have revealed the crucial
role of PAK4 in cell proliferation, anchorage-independent growth and cell migration among other
hallmarks of cancer. Thus, PAK4 is an attractive target for anti-TNBC drug design and
development. In our research, we used in silico methods to investigate the inhibitory potentials of
kaempferol against PAK4 as compared with co-crystallized 4T6 and a standard PAK4 inhibitor-
KPT-9274. Firstly, the ligands were docked into the ATP-binding cleft of the enzyme. The analyses
of the docking poses showed a favorable interaction between kaempferol and the catalytic-
important aminoacyl residues, especially GLU396, LEU398 and ASP458 in the ATP-binding site of
PAK4 when compared what with was obtained in the 4T6-PAK4 complex. Consequently,
molecular mechanics based MM-GBSA was used to validate docking results. The free energy
calculations revealed that kaempferol may have a favorable biological activity. Furthermore, the
druggability of each ligand was assessed using the QikProp module and the SwissADME online
tool. Kaempferol possessed a propitious drug-like property when compared to the standard ligands.
We, therefore, put forward a logical argument that kaempferol can be further evaluated as a
potential PAK4 inhibitor in TNBC.

Keywords: PAK4, kaempferol, docking, MM-GBSA, cancer.

Introduction
Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer. It has conflicting
epidemiology, risk factors prognosis, and treatment regimens when compared to other types of
breast cancer (BC) [1, 2]. In TNBC, the expression of estrogen receptor (ER), progesterone
receptors (PR), Human epidermal growth factor (HER2/neu) at normal and significant levels are
lacking [3, 4]. TNBC is approximately 10%–20% of all BC, constituting about 80% of all ‘basal-
like tumors' [5], more frequently affecting younger and premenopausal women [6]. Among racial
populations, TNBC has not been similarly distributed. It is reported to be usual in women of
African ancestry than women of other ethnic origins [7, 8]. According to the study reported by
Kaplan et al [9] at the San Antonio Breast Cancer Symposium, 47% of tumors in African American
women were "triple-negative" as compared with 22% in white women. Implying that the incidence
of the disease among African American women is above twice that of white women. Also, in a
study carried out by Stark et al [10], of 1664 considered sample size, Ghanaian women had the
highest incidence of triple-negative tumors (82%), followed by African-Americans (33%) and white
Americans (10%). These high distributions of TNBC already reported in women of African
ancestry have led to suggestions that there may be some gene mutations which predispose these
women to TNBC [11]. With the absence of well-defined molecular targets, treatment options for
TNBC have been inadequate [5]. Thus, it is imperative to discover novel probable targeted therapies
to attenuate the aggressiveness of TNBC.

Although TNBC has higher rates of clinical response to presurgical (neoadjuvant) chemotherapy,
patients still have a higher rate of distant recurrence and a poorer prognosis than women with other
BC subtypes [12, 13]. Less than 30% of women with metastatic TNBC survive 5 years [14], and
almost all die of the disease despite adjuvant chemotherapy, which is the primarily-established
option of treatment [15, 16]. Therefore, intensive research efforts are focused on identifying
targeted agents and optimizing treatment for TNBC [17]. Targeted agents currently under clinical
investigation in TNBC include poly ADP-ribose polymerase (PARP) inhibitors [18-20],
Phosphoinositide 3-kinase (PI3K) inhibitors [21], Mitogen-activated protein kinase (MEK)
inhibitors [22], heat shock protein 90 inhibitors [23], anti-androgen therapies [24], histone
deacetylase inhibitors [25], p21-Activated kinase 4 (PAK4) and their combinations [17, 26].

PAK4 is a member of the p21-activated kinase (PAK) family of serine/threonine kinases. The PAK
family consist of six members which are subdivided into two groups based on domain structure,
sequence homology and regulation. Group I PAKs (PAK1-3) and group II PAKs (PAK4–6) [27].
PAKs are downstream effectors of the Ras-related Rho-family GTPases Cdc42 and Rac [28, 29].
PAK4 plays a pivotal role in regulating cytoskeletal signaling pathways, cell proliferation,
migration, and nuclear signaling [30]. It also inhibits cell adhesion and promotes anchorage-
dependent growth [31]. Studies have shown that PAK4 is over-expressed and amplified in many
types of human tumors, including BC [32, 33]. Rane et al [34], as well as others, reported that
PAK4 protein and its mRNA are significantly over-produced in many breast cancer cells and PAK4
expression increase as the disease progresses. In a study of basal-like BC (usually triple negative),
DNA analysis revealed that PAK4 gene was frequently amplified [32, 34, 35]. Thus, PAK4 is
considered as an attractive target for anti-TNBC drug design and development. PAK4 consist of an
N-terminal p21-GTPase-binding domain (PBD) and a C-terminal catalytic serine/threonine kinase
domain which is highly conserved [36]. Contrary to its Group I counterparts, PAK4 has much less
sequence N-terminal to the PBD. It is distinct from other Group II PAKs because it (PAK4) does
not harbor a nuclear localization signal in this region [36].

So far, several families of PAK4 inhibitors have been reported. For instance, indolocarbazole-based
inhibitor (Staurosporine) [37], 5 aminopyrazole-based inhibitor (PF-3758309) "Pan-PAK inhibitor",
the first compound to advance to phase I trials (NCT00932126) [38], aminopyrimidine-based
inhibitor (FRAX486) [37], tri-substituted purine analogue (CGP74514A) [30], tri-substituted 1, 3,

5- triazine analogue (KY-04031) [39], LCH-7749944 [40], a quinazoline derivative and KPT-9274
[34]. KPT-9274 is currently in phase 1 clinical trials (NCT02702492) in patients with advanced
malignancies [41]. Most of these inhibitors lack selectivity for PAK4. Studies on some previously
promising PAK4 inhibitors have been discontinued due to poor physicochemical and
pharmacological properties required for druggability [37]. Therefore, there is a need for the
discovery and development of potent and selective PAK4 inhibitors that will scale clinical trials.

Annona muricata Linn commonly named "graviola" or "soursop" is an evergreen tropical plant
belonging to the Annonaceae family [42, 43]. This plant has been used as folklore medicine to treat
various ailments including cancer in South America and tropical Africa, especially Nigeria [43].
Recent studies have shown that A. muricata crude extract exhibits a different level of cytotoxicity
toward tumor cells, especially breast cancer cell lines [44-47]. Hence, this present study was aimed
to investigate the therapeutic properties of 147 bioactive constituents of A. muricata L as potential
PAK4 inhibitors through various in silico analyses, including molecular docking calculations. We
systematically identified kaempferol as a putative PAK4 inhibitor using characterized interactions
between the PAK4 catalytic residues and the phytochemicals as compared to the co-crystallized
4T6 ligands and the Phase 1 KPT-9274.

Methodology:
AutoDock tools (AutoDock 4.2 version, The Scripps Research Institute, La Jolla, CA, USA) and
Schrödinger suite 2017-2 (Schrödinger, LLC, New York, NY) were used to perform all the
computational analysis. The following steps were performed for molecular docking and post-
docking calculations:

Data Preparation:
147 previously identified and characterized compounds of Annona muricata L plant selected as
ligands and their 2D conformations were retrieved from PubChem (pubchem.ncbi.nlm.nih.gov), a
free and convenient database that contain about 54 million compounds. The X-ray crystal structure
of PAK4 with a resolution of 2.9Å was retrieved from RCSB (Research Collaboratory for Structural
Bioinformatics) Protein Data Bank (PDB) [48]. The PDB code of the protein is 5BMS with a Rfree
value of 0.23 which indicates the absence of structural defects [49, 50].

Molecular Docking Calculations Using AutoDock4.2 Software:

The crystallographic structures of PAK4 (PDB: 5BMS) [51] and ligands were processed with
AutoDock tools. Grids for docking evaluation with a spacing of 0.375 Å and 60x60x60 points,
centered in the ligand binding pocket of PAK4, were generated using AutoGrid 4.2 included in
Autodock 4.2 distribution. Docking runs were executed and the most favorable binding energies for
the co-crystallized 4T6 and Kaempferol were extrapolated from the docking results.

Molecular Docking Calculations Using Glide:

Ligand Preparation: Co-crystallized 4T6, Kaempferol and KPT-9274 were prepared for docking
using LigPrep module in maestro, Schrödinger suites. Low-energy 3D structures with correct
chiralities were generated. The possible ionization states for each ligand structure were generated at
a physiological pH of 7.2 ± 0.2. All other options were kept as default and the ligands were
minimized using an OPLS3 force field.

Protein Preparation: The PDB structure of PAK4 was processed using the Protein Preparation
Wizard in Maestro-v11.2 [52]. In the pre-process tab, bond orders were assigned, and possible
missing hydrogen atoms in the PBD structure were added. Also, disulfide bonds were created
between two sulfur atoms proximity and other options were left as default. PAK4 pre-processed
structure was further refined. The hydrogen bonding network was optimized through the
reorientation of the thiol and hydroxyl groups, water molecules, amide groups of Asn and Gln. The
imidazole ring in His was also reorientated and the predicting protonation states of important
residues were added. Full energetic optimization was performed in the final refinement step using
OPLS3 force field and the RMSD of heavy atoms was set at 0.3 Å.

Molecular Docking Study: The receptor grid was created around the binding site of PAK4 using
the "receptor grid generation" option in the glide-v7.5 programme of Maestro-v11.2 [53]. A grid of
10 Å was centred according to the position of the co-crystallized inhibitor. Afterward, the prepared
ligands were docked to the receptor grid using standard precision (SP) [53, 54] mode followed by
the extra precision (XP) [55] workflow module of the Schrödinger suite with default parameters.
XP docking applies a sophisticated scoring function that will remove the false positives and
penalize those ligands that could not fit well to the receptor. The results of the docking calculation
were then quantified using docking score, Glide Gscore, Glide energy, and Glide emodel.

Docking Pose Analysis:

This was done using pose-viewer. The H-bond interactions, as well as the bad and ugly van der
Waals contacts to the receptors, were visualized using default settings. This was to assess the
binding modes of the ligands in the site of the receptor.

Free Energy Calculation using Prime:

Prime molecular mechanics/generalized born surface area (MM-GBSA), also known as a post-
scoring approach, is a key and appealing computational method used to analyse the accuracy of
docking processes. The docked poses with the lowest Glide score (best docking score) were
minimized using MM-GBSA approach as implemented in the Prime module of the Schrödinger
suite with default settings [56, 57]. The final output obtained from the above process is ΔGbind, a
mixture of different kind of energies in which polar and non-polar energies contribute separately.
ΔGbind was computed as:

ΔGbind = ΔEMM + ΔGsolv + ΔGSA

Where ΔEMM is the difference in energy between the complex structure and the sum of the energies
of the ligand and un-ligand protein using the OPLS3 force field. ΔGsolv is the difference in the
GBSA solvation energy of the complex and the sum of the solvation energies for the ligand and
unliganded protein. Lastly, ΔGSA is the difference in the surface area energy for the complex and the
sum of the surface area energies for the ligand and uncomplexed protein.

ADME/T Properties Calculations:

The QikProp module of Schrödinger is a fast, accurate, straight-forward absorption, distribution,
metabolism, and excretion (ADME) prediction programme. It is designed to predict the
physicochemical significant descriptors and pharmacokinetically relevant properties related to
ADME (Schrödinger Release 2017-2: QikProp, Schrödinger, LLC, New York, NY, 2017). We
predicted the drug-like properties of 4T6, Kaempferol and KPT-9274 using Qikprop-v5.2 of the
Schrodinger suite. The prepared ligands were used to calculate pharmacokinetic parameters,
physically significant descriptors and pharmaceutically-relevant properties following the Lipinski's
rule of five [58].

Druglikeness, Leadlikeness and Bioavailability Radar:

Computations were done using canonical smile notation in SwissADME web-based tool [59]. Also,
plots for the bioavailability radar for the co-crystallized 4T6, KPT-9472 and Kaempferol were
evaluated using the SwissADME platform.
Results:

Figure 1: workflow of the docking protocol and post-docking calculations.

Molecular Docking:
To screen for a probable PAK4 inhibitor with a very high level of accuracy, molecular docking
calculations were done using AutoDock tools (AutoDock 4.2 version) and Maestro-v11.2 of
Schrödinger suite [52]. The phytochemicals were initially screened using AutoDock4.2 docking
protocol. The benchmark binding energy for a promising and selective inhibitor was set at -
9.0kcal/mol. Docking-based virtual screening of 147 ligands into the binding pocket of the target
PAK4 revealed that only Kaempferol scaled through with a binding energy of -9.3kcal/mol as
shown in Table 1. Afterward, the co-crystallized 4T6, a standard inhibitor KPT-9274, and
Kaempferol were re-docked using Maestro-v11.2. Applying the SP mode and XP workflow module
of the Schrödinger suite, the SP docking scores, Glide Gscores and Glide energy for the 3 ligands
were reported in Table 2. In addition to the SP protocol, XP docking study was executed to
eliminate false positive results and determine accurate energy poses for the ligands. The optimum
docking calculations were evaluated through the XP Gscore, Glide energy Glide and emodel energy
as depicted in Table 3.

Table 1: docking calculations of hits using autodock4.2.

S/N Ligands (ID) Binding Energy (kcal/mol)

1 Co-crystallized 4T6 -8.4
2 kaempferol -9.3

Table 2: precision glide docking results of selected hits.

S/N Ligands (ID) Docking Score Glide Gscore Glide Energy (kcal/mol)
(kcal/mol) (kcal/mol)
1 Co-crystallized 4T6 -8.971 -9.952 -53.558
2 KPT-9274 -6.173 -8.125 -56.099
3 kaempferol -7.183 -7.194 -40.088

Table 3: Extra precision glide docking results with interacting amino acids.

S/N Ligands (ID) Docking XP glide glide H Interacting

score GScore energy emodel Bonds amino
acids
1 Co-crystallized -9.955 -10.936 -54.902 -80.759 5 ILE327,
4T6 GLU396,
LEU398,
ASP458.
2 KPT-9274 -5.458 -5.480 -58.937 -97.62 1 ASP440
3 kaempferol -8.528 -8.539 -37.656 -55.376 4 GLU396,
LEU398,
ASP458

Post-Docking Pose Analysis:

Li et al [35] revealed that there are five binding site pockets (A, B, C, D and P) and one water
molecule. Pocket A comprises seven residues (GLU399, GLY400, GLY401, ALA402, ILE327, and
VAL335). The hydrophobic pocket serves as the entrance for ligand interaction together with
another pocket B. The B strands and the N-lobe provide hydrophobic residues to form pocket B. In
the hinge region (GLU396, PHE397, LEU398), GLU396 is reportedly adjacent to the relatively
small and less flexible pocket C with hydrophobic residues (MET395, ALA348, VAL349, and
VAL379). Pocket D (GLY333, THR332, SER331, GLY330, GLU329, and GLY328) is located in
the highly flexible G-loop which can switch conformations depending on the state of the protein
kinase and the presence of a ligand. Pocket P is created by LYS442, SER443, ASP444, SER445,
and ASP458 in the active loop which is highly flexible, hydrophilic, and solvent-exposed as
explained by Li et al [35]. The binding mode of the co-crystallized 4T6, KPT-9274 and kaempferol
were analyzed using Pose Viewer module of Schrödinger suite.

Pose Orientation of Co-crystallized 4T6 in the ATP-binding Cleft of PAK4: 4T6 binds to the
PAK4 catalytic domain in the ATP-binding site between the N- and C-lobes, making important
interactions with key aminoacyl residues. The pyrazole ring of the 4T6 inhibitor aligns in the hinge
region of the target, forming a classic donor-acceptor-donor H-bonds with the hinge main-chain of
LEU398 (figure 2 & 3). Another H-bond interaction is formed between the amine moiety of the
pyrazole ring of 4T6 and carbonyl carbon of GLU396 in the hinge region. These interactions have
been previously established by different studies [51, 60]. The NH moiety on the
chlorobenzimidazole ring interacts via H-bond with side-chain carboxy oxygen of ASP458, which
is essential for potency [61]. To our surprise, 4T6 also form a new H-bond with the main-chain of
ILE327 as depicted in figure 2, a pocket A residue. Also, there was a -cation interaction between
the chloronezimidazole ring of 4T6 and the side-chain amino group of LYS350. Lastly, interrupted
hydrophobic patches were observed around the 4T6 inhibitor, including a key interaction with
MET395, the gatekeeper residue [60]. The inhibitor interacted with pocket A residue of the kinase
(ILE327 and VAL335) at the N-terminal lobe of PAK4. 4T6 entered into a less flexible pocket C
with hydrophobic residues (MET395, ALA348, and VAL379).
Figure 2: The 2D ligand interaction diagram of 4T6.
Figure 3: The 3D ligand interaction diagram of 4T6 complex showing the H-bonds and -cation
interaction.

Pose Orientation of KPT-9274 in the ATP-binding Pocket of PAK4: Unlike the significant H-
bond interactions with the hinge residues (GLU396 and LEU398) in the 4T6-PAK4 complex, the
amino scaffold of the pyridine ring of KPT-9274 donated a classic H-bond to the negatively-
charged carboxylic oxygen of ASP440. The fluorobenzene ring of the inhibitor extended into
pocket C (MET395, ALA348, and VAL379), adjacent to the hinge region formed by N- and C-
lobes. However, the hydrophobic interaction between KPT-9274 and the gatekeeper MET395 was
preserved (Figure 4).

Figure 4: The 2D ligand interaction diagram of KPT-9274.

Figure 5: The 3D ligand interaction diagram of KPT-9274, showing H-bond interaction as well as
bad ligand-receptor contacts/clashes.

Pose Orientation of Kaempferol in the ATP-binding Pocket of PAK4: Key H-bond interaction
between the inhibitor and the hinge region (GLU396, PHE 397, LEU398) was highly preserved as
seen in the 4T6-PAK4 binding modes. The OH group at position 7 of the flavone donated a classic
H-bond to the carboxylic oxygen of the side-chain of ASP458, important for potency as stated by
Murray et al [61] (figure 6 & 7). The same interaction was observed in the 4T6-PAK4 complex.
Also, the hydroxyl at position 5 of the inhibitor forms H-bond with the main-chain carbonyl oxygen
of GLU396, these H-bond interactions have been previously established in scientific articles (refs).
The flavone extended into the hydrophobic pocket C (MET395, ALA348, and VAL379) adjacent to
the hinge region as observed for 4T6 and KPT-9274. Kaempferol also interacted hydrophobically
with pocket A residues ILE327, VAL335, and ALA402.
Figure 6: The 2D ligand interaction diagram of kaempferol.

Figure 7: The 3D ligand interaction diagram of kaempferol, showing H-bond interaction

MM-GBSA:
In this study, the co-crystallized 4T6, KPT-9274 and kaempferol were docked with PAK4. To
validate the docking scores, the free energy was calculated through the MM-GBSA post-docking
process and the result is displayed in table 4. Evaluation of the results for Kaemferol showed that
bond formation is made possible by van der Waals and non-polar energies. This is as opposed to the
coulombic energy (ΔG Coulomb) and polar solvation (SolvGB). Co-crystallized 4T6, KPT-9274,
and kaempferol were calculated to have van der Waals energies of -42.533, -30.081, -58.5 kcal/mol
respectively. This revealed that kaempferol may show favorable experimental biological activities.
The positive polar solvation energy term is also favorable. If this term is negative, the reaction will
be exothermic [62]. Our data indicate that although 4T6 exhibited very high polar solvation energy
of 48.998 kcal/mol. The value for kaempferol (41.794 kcal/mol) was far higher than the standard
KPT-9274 (24.472 kcal/mol). MM-GBSA/ΔGbind calculated values for 4T6, KPT-9274, and
kaempferol were -47.53 kcal/mol, -41.094 kcal/mol, and -43.65 kcal/mol respectively. The co-
crystallized 4T6 had the highest ΔGbind value. However, kaempferol still had an improved value than
KPT-9274 as depicted in table 4.

Table 4: Binding free energy calculation results for 4T6, KPT-9274 and kaempferol bound with
PAK4 (5BMS).
Ligand (ID) GBinda GBindb GBindc Lipo GBindd GBinde GBindf GBindg
Coulomb Covalent SolvGB vdW Hbond
Co- -42.727 1.835 -10.599 48.998 -42.533 -47.53 -2.448
crystallized
4T6
KPT-9274 -26.98 5.41 -11.85 24.472 -30.081 -41.094 -2.058
kaempferol -12.156 3.315 -20.929 41.794 -58.5 -43.65 -2.449

a
Contribution to the MM-GBSA free energy of binding (kcal/mol) from the Coulomb energy.
b
Contribution to the MM-GBSA free energy of binding (kcal/mol) from covalent binding.
c
Contribution to the MM-GBSA free energy of binding (kcal/mol) from lipophillic binding.
d
Contribution to the MM-GBSA free energy of binding (kcal/mol) from the generalized Born
electrostatic solvation energy.
e
Contribution to the MMGBSA free energy of binding (kcal/mol) from the van der Waals energy.
f
MM-GBSA free energy (kcal/mol) of binding.
g
Contribution to the MM-GBSA free energy of binding (kcal/mol) from hydrogen bonding.

Analysis of ADME Properties:

The prediction of the ADME/T properties for the hit molecules was done using the QikProp module
of Schrödinger interface. The pharmaceutically important properties such as drug-likeness of the
lead compounds were evaluated using the Lipinski rule of five. Lipinski rule is based on the
molecular weight, logP, the number of hydrogen bond acceptors and donors, percentage of human
oral absorption, and various other pharmacokinetic parameters needed to satisfy the ADME
properties [58]. The CNS descriptors for the ligands were -2 (inactive). Table 5 illustrates the
results of ADME/T of 4T6, KPT-9274, and kaempferol. These compounds were found to have high
human oral absorption (HOA) of 3. In addition, the ligand molecules were analyzed for their
QPlogKp, QPlogBB, QPlogS, QPlogPo/w, QPlogPw, QPlogPoct properties and were found to be in
the acceptable range. Thus, the three inhibitors were found to have good solubility, absorption
distribution and bioavailability.

Table 5: ADME/T properties of the screened ligands.

Ligands MWa SAS FOS dH aH QPlogP QPlogP QPlogPo QPlo QPlog QPlog CN HOA
(ID) (Da) Ab Ac Bd Be octf wg /wh gSi BBj Kpk Sl m

Co- 380.8 602.6 167.4 4 5.5 21.584 13.963 2.696 - -1.12 -3.318 -2 3
crystalliz 93 92 68 4.392
ed 4T6

KPT- 610.6 507.5 0 3 4.5 17.548 12.337 1.042 - -1.893 -4.641 -2 3

9274 34 16 3.158

kaempf 286.2 962.1 215.0 3 7.5 30.882 16.151 6.801 - -1.746 -2.139 -2 3
erol 4 89 77 9.718

a
Molecular weight
b
Solvent accessible surface area
c
Hydrophobic solvent accessible surface area
d
Donor hydrogen bond
e
Acceptor hydrogen bond
f
Predicted octanol/gas partition coefficient
g
Predicted water/gas partition coefficient
h
Predicted octanol/water partition coefficient
i
Predicted aqueous solubility
j
Predicted brain/blood partition coefficient
k
Predicted skin permeability
l
Predicted central nervous system activity
m
Human oral absorption

Analysis of the Drug-likeness, Lead-likeness and Bioavailability Radar

The SwissADME drug-like properties for kaempferol is similar to what was obtained for the co-
crystallized 4T6 as shown in table 6. However, KPT-9274 is characterized by a poor drug-likeness
as depicted in table 6. The lead-likeness for kaempferol is more promising than the other screened
inhibitors (table 7). The bioavailability radar is plotted for a quick assessment of drug-likeness. Six
physicochemical properties are taken into account for the screened ligands. They were the
lipophilicity, size, polarity, solubility, flexibility and saturation of each ligands. The optimal range
was depicted as a pink area within the radar plot. In our study, we discovered that the saturation
parameter for kaempferol was lower than KPT-9274, but the co-crystallized 4T6 showed almost-
optimal saturation among the inhibitors. However, other bioavailability parameters for kaempferol
were better than the co-crystallized 4T6 and the drug standard KPT-9274.
Table 6: The drug-like properties of the screened ligands.
Ligands Lipinski Ghose Veber Egan Muegge Bioavailability
score
4T6 Yes Yes Yes Yes Yes 0.55
KPT-9274 Yes: 1 No: 4 Yes: No: 1 No: 2 0.55
violation: violations: violation: violations:
MW>500 MW>480, WLOGP>5.88 MW>600,
WLOGP>5.6, XLOGP3>5
MR>130,
atoms>70
Kaempferol Yes Yes Yes Yes Yes 0.55

Table 7: The medicinal chemistry of the screened ligands

Ligands PAINS Brenk Leadlikeness Synthetic Accessibility
4T6 0 alert 0 alert No: 1 violation: 3.09
MW>350
KPT-9274 0 alert 1 alert: michael No: 3 violations: 4.37
acceptor 1 MW>350,
Rotors>7,
XLOGP3>3.5
Kaempferol 0 alert 0 alert Yes 3.14

4T6 KPT-9274
 Kaempferol

Figure 8: The bioavailability radar for the screened molecules.

Discussion:
The quest for new anti-cancer therapies is known to require tremendous effort as regards cost and
manpower. This is because a large number of compounds will be synthesized and concurrently
evaluated in vitro/in vivo. Thus, pharmaceutical industries are initially applying theoretical and in
silico methods to enable the rational design of therapeutic chemical compounds [63-64]. For
decades, computer-aided drug designing strategies have greatly evolved. We now have uniquely-
developed and specialized softwares and web-based tools for drug discovery and development,
coupled with increased computer power. Therefore, we have applied sophisticated in silico methods
to predict the inhibitory capacity of kaempferol as anti-PAK4 agent with comparison with the co-
crystallized 4T6 and phase 1 KPT-9274.

Kaempferol is a flavonoid mostly found in fruits and plants such as tomato, broccoli, apple, green
tea and A. muricata [65-66]. This phytoestrogen has been exhaustively investigated to reveal the
underlying mechanisms of its anticancer effect [67]. Kaempferol regulates some hallmarks of
cancer cells such as inflammation, cell cycle, apoptosis, cell migration and angiogenesis [65,67]. In
this study, we evaluated the inhibitory potency of kaempferol on PAK4, an interesting therapeutic
target in a number of tumours including breast cancer. PAK4 has implicated in many hallmarks of
carcinogenesis, including anchorage-independent growth, increased cell survival, migration and
invasion [30-31]. With the laudable advances in bioinformatics and computational biology, we
aligned this study to best practices using in silico methods. In the molecular docking results, had
higher affinity than the standard KPT-9274, however, the SP and XP docking scores for the co-
crystallized 4T6 were the highest. Analyzing the molecyular docking poses for the three ligands, we
discovered that hydrogen-bonding network for kaempferol was uniquely similar to that of 4T6. In
the ATP-binding site of the PAK4 target, we noticed that although the flavone extends into the less
flexible and hydrophobic pocket C of the enzyme, OH group at position 5 donates a hydrogen bond
to a hinge residue GLU396 which is adjacent to the pocket. This was similar to the same H-bond
interaction between the hydrogen in the amine moiety and the CO oxygen of GLU396. Unlike the
H-bond donor-acceptor interaction between 4T6 and another hinge residue LEU398 (figure 2 and
3), kaempferol-PAK4 complex revealed a acceptor-acceptor-donor H-bonds between the hydroxy-
carbonyl groups of the putative inhibitor and catalytic-important residue. Therefore, this interaction
may have contributed to the favorable docking scores obtained for the kaempferol compared to the
binding energy for KPT-9274. In order to validate our docking protocol, the prime molecular
mechanics/generalized born surface area (MM-GBSA) calculations were employed. With the van
der Waals energy for kaempferol, we can predict that it will possess some favourable biological
activities as compared to the standards inhibitors used in this study. The MM-GBSA/ΔGbind for
our putative inhibitor depicted improved values as against KPT-9274 which further corroborate our
results that kaempferol hold much promise as a PAK-4 selective inhibitor.

Early assessment of ADME/T properties extremely reduces pharmacokinetics-related failures in the

clinical phases [68-69]. Thus, we evaluated the pharmacokinetics of the three inhibitors using the
Lipinski rule of five and other drug-like properties such as Ghose, Veber, Egan, Muegge and the
bioavailability score [69]. Kaempferol had the same human oral absorption value with 4T6 and
KPT-9274 (Table 5). Also, kaempferol was seen to have the most favorable QPlogPo/w, QPlogPw
and QPlogPoct values. The octanol-water partition value (QPlogPo/w) is an important
physicochemical characteristic used to explain the hydrophobic/hydrophilic properties of drug-like
compounds [70]. The partition coefficients for kaempferol predicted that the inhibitor may possess
good solubility, absorption, distribution, and bioavailability characteristics. Also, the lead-likeness
for kaempferol was optimal when compared to the standard inhibitors. This feature shows that
kaempferol can still be chemically optimized to obtain a more potent and selective PAK4 inhibitor
since it has a very low molecular weight. The bioavailibility radar from the SwissADME web-based
tool ensures a first glance at the drug-likeness of a chemical compound. It accounts for the
lipophilicity, size, polarity, solubility, flexibility and saturation for each of the ligands under
investigation. Five of the six properties for kaempferol were within optimum ranges as compared to
4T6. However, the graph for the putative inhibitor depicted an increased saturation value as against
the other PAK4 inhibitors.

In the light of the molecular docking calculations, post-docking analysis, and the pharmacokinetics
and pharmacological properties, we conclude that kaempferol is a potential PAK4 inhibitor. We,
therefore, suggest that more work still needs to be done to preclinically/clinically assess kaempferol
in triple negative breast cancer therapy.

Conclusion:
In summary, we applied in silico strategies to identify kaempferol as a novel inhibitor of PAK4.
Based on docking studies, the binding energies for kaempferol in the catalytic site of PAK4 was
calculated using autodock4.2 and the SP mode and XP workflow module of the Schrödinger suite
2017-2. The results were -9.3kcal/mol, -7.194kcal/mol and -8.528kcal/mol respectively. Post-
docking analysis showed that kaempferol mimicked some important hydrogen bonding with
ASP458, LEU 398 and GLU396 as previously reported (refs). MM-GBSA validate our docking
calculations and it further emphasizes findings that kaempferol is a potential PAK4 inhibitor with
promising biological properties. Also, ADMET evaluation was used to grasp the physicochemical
properties and drug-likeness of the inhibitors. Kaempferol stood out to be very competitive with the
standard PAK4 inhibitors 4T6 and KPT-9274. Therefore, we are predicting that kaempferol can be
administered as an adjuvant therapy against cell proliferation, migration, and metastasis in triple-
negative breast cancer.

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