Activity assay of epidermal growth factor receptor tyrosine kinase inhibitors in triple-negative breast cancer cells using peptide-conjugated magnetic beads.

Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer with limited treatment options. Epidermal growth factor receptor I (EGFR) has emerged as a promising target in TNBC. Limited success of the EGFR kinase inhibiting small molecules in clinical trials may be attributed in part to inaccuracy in identifying EGFR signatures in patient tumors. In light of the absence of a simple correlation between EGFR expression and its degree of activation, a simple and reliable tool that can quantify EGFR kinase activity in tumor samples may be of therapeutic value in predicting patient-specific EGFR targeted therapies. This study reports the development of an assay that can quantitatively profile EGFR kinase activities and inhibitor sensitivities in TNBC cell lysates by using peptide reporters covalently tethered to magnetic beads in a controlled orientation. The use of magnetic beads provides rapid sample handling and easy product isolation. The potential of this approach was demonstrated by screening a set of five clinically relevant EGFR tyrosine kinase inhibitors. Formatted for microwell plates, this magnetic bead-based kinase assay may be used as a complementary approach for direct high-throughput screening of small molecule inhibitors.

A pairwise chemical genetic screen identifies new inhibitors of glucose transport.

Oxidative phosphorylation (OXPHOS) and glycolysis are the two main pathways that control energy metabolism of a cell. The Warburg effect, in which glycolysis remains active even under aerobic conditions, is considered a key driver for cancer cell proliferation, malignancy, metastasis, and therapeutic resistance. To target aerobic glycolysis, we exploited the complementary roles of OXPHOS and glycolysis in ATP synthesis as the basis for a chemical genetic screen, enabling rapid identification of novel small-molecule inhibitors of facilitative glucose transport. Blocking mitochondrial electron transport with antimycin A or leucascandrolide A had little effect on highly glycolytic A549 lung carcinoma cells, but adding known glycolytic inhibitors 2-deoxy-D-glucose, iodoacetate or cytochalasin B, rapidly depleted intracellular ATP, displaying chemical synthetic lethality. Based on this principle, we exposed antimycin A-treated A549 cells to a newly synthesized 955 member diverse scaffold small-molecule library, screening for compounds that rapidly depleted ATP levels. Two compounds potently suppressed ATP synthesis, induced G1 cell-cycle arrest and inhibited lactate production. Pathway analysis revealed that these novel probes inhibited GLUT family of facilitative transmembrane transporters but, unlike cytochalasin B, had no effect on the actin cytoskeleton. Our work illustrated the utility of a pairwise chemical genetic screen for discovery of novel chemical probes, which would be useful not only to study the system-level organization of energy metabolism but could also facilitate development of drugs targeting upregulation of aerobic glycolysis in cancer.

A solid-phase Bcr-Abl kinase assay in 96-well hydrogel plates.

Regulated phosphorylation by protein tyrosine kinases (PTKs), such as c-Abl, is critical to cellular homeostasis. In turn, once deregulated as in the chronic myeloid leukemia (CML) fusion protein Bcr-Abl, PTKs can promote cancer onset and progression. The dramatic success of the Bcr-Abl inhibitor imatinib as therapy for CML has inspired interest in other PTKs as targets for cancer drug discovery. Here we report a novel PTK activity and inhibition screening method using hydrogel-immobilized peptide substrates. Using acrylate crosslinkers, we tether peptides via terminal cysteines to thiol-presenting hydrogels in 96-well plates. These surfaces display low background and high reproducibility, allowing semiquantitative detection of peptide phosphorylation by recombinant c-Abl or by Bcr-Abl activity in cell extracts using traditional anti-phosphotyrosine immunodetection and chemifluorescence. The capabilities of this assay are demonstrated by performing model screens for inhibition with several commercially available PTK inhibitors and a collection of pyridopyrimidine Src/Abl dual inhibitors. This assay provides a practical method to measure the activity of a single kinase present in a whole cell lysate with high sensitivity and specificity as a valuable means for efficient small molecule screening.