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. 2010 Jul;76(1):1-9.
doi: 10.1111/j.1747-0285.2010.00986.x. Epub 2010 May 4.

Targeting the dimerization of epidermal growth factor receptors with small-molecule inhibitors

Robert Y C Yang  1 , Katherine S Yang, Linda J Pike, Garland R Marshall
Affiliations

Targeting the dimerization of epidermal growth factor receptors with small-molecule inhibitors

Robert Y C Yang et al. Chem Biol Drug Des. 2010 Jul.

Abstract

The epidermal growth factor (EGF) receptor is a receptor tyrosine kinase involved in the control of cell proliferation, and its overexpression is strongly associated with a variety of aggressive cancers. For example, 70-80% of metaplastic (cancer cells of mixed type) breast carcinomas overexpress EGF receptors. In addition, the EGF receptor is a highly significant contributor to common brain tumors (glioblastoma multiforme), both in initiation and progression (Huang P.H., Xu A.M., White F.M. (2009) Oncogenic EGFR signaling networks in glioma. Sci Signal;2:re6.). Brain metastases, an unmet medical need, are also common in metastatic cancer associated with overexpression of EGF receptors. Formation of EGF receptor homodimers is essential for kinase activation and was the basis for exploring direct inhibition of EGF receptor activation by blocking dimerization with small molecules. While inhibitors of protein/protein interactions are often considered difficult therapeutic targets, NSC56452, initially identified by virtual screening, was shown experimentally to inhibit EGF receptor kinase activation in a dose-dependent manner. This compound blocked EGF-stimulated dimer formation as measured by chemical cross-linking and luciferase fragment complementation. The compound was further shown to inhibit the growth of HeLa cells. This first-generation lead compound represents the first drug-like, small-molecule inhibitor of EGF receptor activation that is not directed against the intracellular kinase domain.

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Figures

Figure 1
Figure 1
The target site at the EGF receptor dimerization interface. A) Crystal structures of the extracellular domain of the EGF receptor homodimer (PDB:1MOX) and the dimerization arm (box). B) Critical residues Y246 and Y251 pack into adjacent pockets at the dimer interface.
Figure 2
Figure 2
Evaluation of the vHTS protocol against four test cases shown in an enrichment curve analysis. In each case, multiple known ligands were mixed with ~2000 random compounds to form the screening library. The black diagonal line represents the random distribution of active molecules.
Figure 3
Figure 3
Inhibition of EGF receptor autophosphorylation by NSC56452. Cells were pre-incubated with NSC56452 or 1% DMSO for the control. Inhibition of EGF receptor autophosphorylation of the controls (lane 1 and 2) and increasing doses of NSC56452. Residual kinase activity was estimated by densitometry and plotted to obtain IC50 values. Structure and molecular weights of NSC56452 is also shown.
Figure 4
Figure 4
Specificity of the inhibitors for the EGF receptor. Cells expressing either the insulin receptor or the PDGF receptor were pre-incubated with 1% DMSO (controls) or 100 μM of each of the 20 candidate compounds. A) Insulin receptor kinase activity was assessed by measuring the phosphorylation of IRS-1 in response to 3 nM insulin for 1 minute. The data shown are representative of three separate experiments. B) PDGF receptor kinase activity was assessed by measuring autophosphorylation of the PDGF receptor in response to 2 nM PDGF for 3 minutes. The data shown are representative of two separate experiments.
Figure 5
Figure 5
Testing for inhibition of EGF receptor dimerization by two methods. A) Chemical cross-linking assay. Cells were pre-incubated with 1% DMSO (lane 1 and 2) or 100 μM of different inhibitors (lane 3-6) prior to stimulation with 25 nM EGF (lane 2-6) for 5 minutes. All cells were then treated with 3 mM BS3. NSC11241 (lane 3) and NSC56452 (lane 5) significantly inhibit dimer formation, while Lane 4 and 6 show compounds that did not inhibit dimer formation. B) Luciferase fragment complementation. Cells stably expressing ΔC-EGFR-NLuc and ΔC-EGFR-CLuc were pre-treated with DMSO, the indicated concentrations of NSC56452 or 1 μg/ml cetuximab for 20 min in the presence of 0.6 mg/ml D-luciferin prior to the addition of 3 nM EGF. All assays were performed in quadruplicate. Data represent the change in photon flux between cells treated with or without EGF.
Figure 6
Figure 6
Effect of the A286W mutation on the sensitivity to NSC56452 inhibition. Mixed CHO cell culture expressing either wild type or A286W-EGF receptors were pre-incubated with 1%DMSO (control) or NSC56452 at the indicated doses for 20 min at room temperature before stimulation with 3 nM EGF for 1 minute. Data shown are representative of two separate experiments.
Figure 7
Figure 7
Inhibition of HeLa cell proliferation. Cells were grown in the absence or presence of erlotinib, NSC56452, or a combination of the two inhibitors at the indicated doses. Cell proliferation was measured by the cellTiter 96 Aqueous One Solution Cell Proliferation Assay after 48 hr incubation with the inhibitors. All experiments were performed in triplicate. All cultures contained 1% DMSO.
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