Biological screening of heterocyclic derivatives: in-vitro evaluation of anticancer activity and adme profiling

Abstract

Malignancy is categorised among the most ruthless diseases that embodies a significant threat to human health worldwide. Epidermal growth factor receptors (EGFRs) are associated with cancer initiation, growth and development. Despite the development of several EGFR inhibitors, most of the human cancers eventually develop resistance towards these drugs. Therefore, the synthesis and development of much better and efficacious EGFR tyrosine kinase inhibitors is an unmet clinical need. Nitrogen-oxygen and sulphur-containing heterocyclic compounds represent the vital structural components of many of the anticancer drugs available on the market today. Almost two-thirds contained heterocyclic moieties within their frameworks of the novel molecular anticancer agents approved from 2010 to 2015 by FDA (U.S. Food and Drug Administration). The prevalence of heterocyclic moieties in anticancer drug design is partly credited to their nature, accompanied by their involvement in various cellular processes and exciting mechanisms that interact with them. To understand the accurate binding of ligands with an active site of the target and to predict the stability of the protein-ligand complex and change in interaction within the active site, molecule dynamics and docking studies were performed initially. A molecular dynamics-based sampling of epidermal growth factor receptor tyrosine kinase (EGFR-TK) was conducted to identify energetically more stable protein, then used for molecular docking. Fourteen molecules displaying the best fit were picked from the library of compounds and studied under in-silico and in-vitro experimental conditions. Further, physicochemical properties of all the compounds were calculated, suggesting that all the compounds show suitable drug-like properties based on solubility and caco-2 cell permeability data. Further, none of the compounds was anticipated to inhibit the hERG channel, which indicates the cardiac safety of the compounds. All the compounds were then screened for their ability to produce cytotoxicity in four different cell lines overexpressing EGFR (A549, HCT-116, HEPG2, MCF‐7) and one EGFR negative cancer cell line (SW620)at three concentrations (10, 1 and 0.1 µM). None of the compounds showed activity against SW620, which suggested that the compounds show cytotoxicity through inhibition of EGFR. Eight test compounds (6a, 6b, 6c, 6e, 6g, 6k, 6m, and 6n) that showed promise in this 3-concentration screen were subjected to multiple dose-response curves to identify the IC50 values. Four test compounds (6a, 6b, 6e and 6m) showed promising IC50 (ranging from 8 to 13µM) values against EGFR positive cancer cell lines (A549, HEPG2, and MCF-7), which were then selected for further in-silico study, additional biological activity assessment, and in-vitro ADME profiling. A molecular dynamics was performed to understand the correlation of non-covalent binding energies with biological activity. Compounds 6a, 6b, and 6m are the active compounds. Further, the EGFR binding activity was confirmed with EGFRWT and EGFRT790M kinase activity assay using the Luminescence-based method. These compounds (6a, 6b, and 6m) also showed activity against both EGFRWT and mutant EGFRT790M, exhibiting IC50 values of <10 and <50 micromolar, respectively. The drug-like properties of the selected four compounds (6a, 6b, 6e and 6m) were evaluated by in-vitro ADME studies, which revealed that all four compounds possess moderate aqueous solubility in the range of 40 to 70 µg/ml at physiological pH – 7.4 and 30 to 100 µg/ml at pH 4.0. Further, 6a, 6b, and 6m showed balanced lipophilicity with LogD values ranging from 1 to 3 and demonstrated good correlation in Caco-2 cell permeability with Papp – around 6 x 10-6 cm/s in comparison with 6e, which was found to be highly lipophilic (Log D >5) and showed high permeability (Papp 17 x 10-6 cm/s). Lastly, all these compounds were moderately stable in liver microsomes at alkaline pH with a half-life between 30 to 60 min, while at highly acidic pH (2.0), the compounds were stable up to 15-20 min. Overall, in-vitro ADME results of these molecules showed good drug-like properties, which are well correlated with "in-silico" ADME data, making them ideal for developing oral drug delivery formulation. Overall, this thesis has been divided into six chapters. The details of the contents of each chapter are provided in subsequent sections.