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.