Structure-Based Discovery of Novel EGFR Tyrosine Kinase Inhibitor
There are approximately 518 kinases encoded in the human genome. As the increasing knowledge of the structures and functions of the tyrosine kinases families, it is clear that the kinases play an important role in initiation and progression of various cancers. Epidermal growth factor receptor (EGFR) tyrosine kinase is one of the best studied tyrosine kinase subfamilies and studies of various tumors have shown that many tumors either overexpress EGFR or one of its common ligands, epidermal growth factor (EGF) and transforming growth factor-α (TGFα), and some tumors overexpress both the receptor and at least one of the ligands. EGFR is a transmembrane glycoprotein with an extracellular ligand binding domain and an intracellular tyrosine kinase domain (TKD). When the ligand binds to the EGFR, the receptor dimerization induces the activation of the TKD. The TKD catalyzes the transfer of the γ-phosphate of bound ATP to the tyrosine residues at intracellular autophosphorylation sites in the C-terminal region. The autophosphorylation of the tyrosine residues leads to the recruitment of various adaptor proteins such as SHC, growth factor receptor-bound protein 2 (GRB2) and activating different intracellular signaling cascades which effects gene transcriptions, which in turn effects cell survival, proliferation, angiogenesis, metastasis, and apoptosis. Therefore, inhibition of the EGFR TKD should lead to inhibition of the downstream signaling cascade and eventually cause cancer cell death. Currently, there are three generations of EGFR tyrosine kinase inhibitors (TKIs). First and second generation EGFR TKIs both have quinazoline based scaffold, and the third generation EGFR TKIs have pyrimidine based scaffold. All of them mimic adenosine triphosphate (ATP) and bind to the ATP binding pocket of the EGFR TKD. However, the efficacy of these EGFR tyrosine kinase inhibitors (TKIs) is limited greatly due to the mutation found in EGFR TKD (T790M, L858R, L718Q, L844V, C797S mutations). My goal is to develop a novel inhibitor that binds to substrate binding pocket of the mutant EGFR TKD with higher affinity and selectivity. The ligands binding to the substrate binding pocket were identified by docking a library of substrate peptides using an integrated computational protein-protein molecular docking approach. The four substrate peptides with the best docking score were used to determine the pharmacophore. Pharmacophore is molecular structures and chemical functionalities that are necessary for a compound to be recognized by a biological macromolecule and evoke biological responses. Using ligand-based pharmacophores, we have identified potential EGFR TKD targeting compounds from a large database of 10 million compounds. Top 25 compounds from each pharmacophoric query were docked in to both the ATP and substrate binding pockets of EGFR TKD using SYBYL-X suite (Tripos, Inc). SYBYL-X suite is a comprehensive software designed for molecular modeling and drug discovery. The lead compound ZL354 preferentially docked well into the EGFR TKD pockets in various mutations compared to the wild type EGFR TKD. ZL354 was able to inhibit cell viability in various EGFR expressing cancer cells (Breast, Lung and Prostate) and suppress angiogenesis as demonstrated in HUVEC cells. We believe the lead compound ZL354 will provide structural clues towards the identification of novel EGFR kinase inhibitors to combat various cancers including breast and lung cancers.