3.7. Lipinski’s rule and in silico ADME prediction of the
designed compounds
The pharmacokinetic parameters of the proposed molecules were determined
utilizing ADME prediction by Schrodinger QikProp v4.3. The proposed
molecules were surveyed for their primary parameters of Lipinski’s rule
of 5 and other pharmacokinetic parameters. Table 2 showed the
results got from QikProp with their reasonable qualities. Orally dynamic
compounds should not violate the Lipinski rule multiple times. The
designed compounds in the current study did not reach maximum
permissible violations, indicating they have medication similarity
properties. Oral bioavailability of medication-like molecules is
constrained by the number of rotatable bonds (0–15) and polar surface
area (7–200 Å). The two compounds had a reasonable rotatable bond and
polar surface area compared to pazopanib.
Intestinal absorption and permeation are important factors for drug
absorption. They were assessed by the predicted Caco-2 cell permeability
(QPPCaco), which is used as a model for the gut-blood barrier by
non-active transport. Caco-2 cell permeability of the proposed compounds
were greater than 638% compared to pazopanib, indicating excellent drug
permeability. The oral absorption of the designed compounds is superior
to pazopanib. QPPMDCK descriptor was utilized to predict permeability of
the proposed compounds through the blood brain barrier. MDCK cells are
an excellent mimic of the blood-brain barrier. Both compounds showed
excellent BBB permeability (QPPMDCK>25) in comparison to
pazopanib. This factor should be considered in future to diminish side
effects. Human serum albumin binding capability was predicted with the
QPlogkhsa descriptor of QikProp. The proposed compounds exhibited good
binding capability to plasma proteins. The other physicochemical
properties of the current two structures are listed in Table 2.
DISCUSSION:
Despite the improved survival rates of breast cancer patients,
metastatic breast cancer is still considered a hazardous disease
(Eroles et al., 2012 ). The current study aimed to investigate
the effect of two novel heterocyclic compounds with a potential role in
the treatment of breast cancer. The higher dose of
benzoimidazoquinazoline (C1) and both doses of benzimidazotriazine (C2)
showed a marked decrease in the tumor weight with a significant
reduction of the angiogenic factor VGEF serum levels compared to the
control group. All treatment doses significantly reduced the expression
of VEGFR2 and CD34 in the tumor tissue. Our rational in this study is to
design novel drug-like molecules that competitively bind to ATP binding
pocket of VEGFR intracellular domain inhibiting its activity. The main
feature to achieve that aim is to modulate the central core adenine ring
of ATP or 2-aminopyrimidine of pazopanib, a strategy becomes common in
medicinal chemistry to find novel potent VEGFR inhibitor with promising
pharmacokinetic properties. A range of heterocyclic systems have been
accounted to provide alternatives to adenine such as thienopyrimidines,
quinoline, imidazopyridazine, and the pyrido pyrimidine (Fan-Wei
et al., 2017, Holmes et al., 2007) . Herein, imidazoquinazoline and
imidazotriazine were selected to mimic adenine and pyrimidine in hinge
region presenting hydrogen bond to the key amino acid, cysteine
(Fig 6 ). Moreover, the current ring system showed extra
hydrogen bonds to certain amino acids in the hinge region giving the
designed compounds tight binding to VEGFR. Pazopanib shows an amazing
binding mode to VEGFR, where the indazole ring reaches out to the
protein lipophilic pocket. In this study, we kept that feature by
modifying indazole to hydroxyphenyl and dichlorophenyl, groups occupy
the same pose of indazole forming van der Wall and hydrophobic
interactions with key aromatic amino acids. Hydroxyl group of compound-2
shows extra hydrogen bonding with THR 916 in hydrophobic pocket giving
this compound certain superiority. The proposed compounds, therefore,
could be considered lead compounds which disrupt cell proliferation in
solid tumor mediated by aberrant VEGF/VEGFR signaling pathway.
The VEGF and its receptor VEGFR2 play a major role in generating new
blood vessels around the tumor mass. Binding of VEGF to its receptor
induces endothelial cell proliferation and development. Inhibition of
VEGFR2 and tumor angiogenesis are necessary for treatment of breast
cancer (Cohen et al., 2001, Sebolt-Leopold et al., 2006 ). VEGF
is associated with poor prognosis in breast cancer as there is increased
synthesis in breast cancer tissues (Maae et al., 2006 ). VEGF
can stimulate proliferation and migration of naturally inert endothelial
cells during tumor angiogenesis and growth resulting in the formation of
new vessel structures. Hypoxia is considered as the main cause of tumor
angiogenesis, even though growth factors and a variety of
transcriptional regulators are able to stimulate VEGF production through
paracrine or autocrine mechanisms (Fox et al., 2007, Jia et al.,
2013 )The higher dose of benzoimidazoquinazoline (C1) and both doses of
benzimidazotriazine (C2) caused a marked decrease in the tumor weight
and reduction in serum levels of the angiogenic factor VGEF. All
treatment doses significantly reduced the expression of VEGFR2 and CD34
in the tumor tissue.
Assessment of tumoral micro-vessel density using immunohistochemical
endothelial markers like factor CD31, CD34, and the activated
endothelial cell marker CD105 is a commonly used procedure for measuring
tumor angiogenesis in breast cancer (Uzzan et al., 2004 ).
Benzimidazotriazine, compound C2-showed a promising activity in
suppressing CD34 expression in mice bearing EAC. It has been proposed
that tumor progression and metastasis in breast cancer is angiogenesis
dependent (Pyakurel et al., 2014 ) The relation between the VEGF
and CD34 expression has been reported in various malignant tumors. Thus,
measuring their expression is vital for deciding the prognosis of tumor
patients.
Reports showed that miRNA‑122 is dysregulated in several cancer types,
including liver, breast and lung cancers (Hopcraft et al., 2015,
Ahsani et al., 2017, Akuta et al., 2016 ) In hepatocellular carcinoma
(HCC), overexpression of miRNA‑122 stimulates cell cycle arrest and
apoptosis by inhibiting Cyclin G1 and Bcl‑2 like 2 expression
(Von Felden et al., 2017 ) On another hand, the overexpression
of miRNA‑122 in non- small cell lung cancers (NSCLC) may result in
reducing the number of invasion and migration cells (These data confirm
the hypothesis that miRNA‑122 acts as a tumor suppressor in various
types of cancer (Qin et al., 2015 ). The roles of specific miRNA
in regulating tumor-associated angiogenesis and lymphangiogenesis have
been reported., miRNA-20b is implicated in the regulation of VEGF in
breast cancer cells by targeting HIF-1 verified the role of miRNA-10b as
an angiogenic regulator (Cascio et al., 2010, Liu et al., 2017 )
miR-128 acts as a tumor suppressor through reducing the expression of
both VEGFA and VEGFC (Hu et al., 2014 ).
miRNA-122 is involved in various cancers where they function as
oncogene/tumor suppressor. Thus, it can serve as a diagnostic biomarker
in cancer (Duc Duy et al., 2014 ).
Perez‐Añorve
et al. reported that miRNA-122 is up‐regulated in breast cancer cell.
Functional analysis showed that miR‐122 plays dual function as a tumor
suppressor and oncomirR dependent on the tumor cells by diminishing
endurance and advancing radiosensitivity (Isidro et al., 2019 ).
Many studies have targeted miRNA-122 for disease management
(Emile et al., 2020 ). Benzoisoquinoline derivatives activated
miR-122 in hepatocarcinoma and function in the treatment of HCV
infection by inducing apoptosis and reducing cell viability viacaspase activation. These compounds are considered promising lead
molecules (Emile et al., 2020, Young et al., 2010 ) Expression
of miRNA-122 in breast cancer tissue was significantly upregulated on
treatment with both compounds, especially with benzimidazotriazine.
Benzoisoquinoline was replaced by benzoimidazoquinazoline and
benzimidazotriazin in an attempt to investigate the role of heterocyclic
systems in mir-122 modulation. Both systems kept the modulation activity
towards miR-122 activation. As a result, they may be a valuable tool for
exploring the regulation of miR-122 in breast cancer and can possibly
provide new targets and lead structures for the advancement of new
chemotherapeutics.
A large number of drug-like molecules cure infectious diseases and treat
cancer but have adverse effects on the host. In the current study,
groups treated with benzoimidazoquinazoline and benzimidazotriazin had
lower urea and creatinine levels compared to the cisplatin treated
group, indicating a minimal nephrotoxic effect. Physicochemical
properties also play an important role in deciding if a compound is
suitable for therapeutic use. Solubility directly affects absorption;
any therapeutic compound has to dissolve in biological fluids to
effectively pass through a biological membrane. The designed compounds
had the appropriate solubility as indicated by the partition coefficient
(QplogPO/W). Orally active medications must be absorbed in the GIT and
should penetrate the cellular membrane to reach their proposed targets.
Benzoimidazoquinazoline and benzimidazotriazine had excellent
permeability and oral absorptivity. Moreover, the novel molecules
exhibited promising binding to plasma protein and can penetrate BBB,
criteria essential in drug-like molecules. The physiochemical properties
of the compounds determined in silico along with their
anti-cancer effects in vivo in EAC animals give the compounds potential
for use in treatment of breast cancer and warrant further clinical
study.
CONCLUSION
Modulation of the activity of VEGF/VEGFR and mi-RNA-122 is a promising
for the management of cancer. Two heterocyclic compounds,
benzoimidazoquinazoline and benzimidazotriazine, exhibited anticancer
properties through reducing the angiogenesis effect and enhancing the
expression of miRNA-122. The efficacy of the two molecules at inhibition
of VEGFR was validated in vitro , in vivo , and in
silico using molecular modeling. The designed compounds anchored inside
the hinge region of VEGFR forming a hydrogen bonding with key amino
acids; cysteine mimics the adenine ring of ATP. Both compounds induced
up-regulation of liver specific miRNA-122. The small molecules perturbed
angiogenesis of EAC in mice by inhibition of both CD34 and VEGF/VEGFR
signaling pathway. These data indicate that both molecules can be used
as therapeutics for the treatment of breast cancer, with a better
toxicity profile.