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The human epidermal growth factor receptor 2 (HER2)-positive breast cancer with overexpression of HER2 accounts for approximately 25% of breast cancers and is more aggressive than other types of breast cancer. Lapatinib has been widely used as a HER2-targeted therapy, however, a number of patients develop lapatinib resistance and still suffer from poor prognosis. Therefore, it is essential to identify novel therapeutic targets that could overcome lapatinib resistance. In this study, we carried out phosphoproteomic analysis of lapatinib sensitive and resistant cell lines (SKBR3 and SKBR3-LR) using stable isotope labeling with amino acids in cell culture (SILAC). We identified 3808 phosphopeptides from 1807 proteins and then analyzed signaling pathways, Gene Ontology, and protein-protein interaction networks. Finally, we identified PAK2 as a therapeutic target from the network analysis and validated that PAK2 knockdown and PAK inhibitor treatment resensitize the lapatinib resistant cells to lapatinib. This results suggest that PAK2 is a potent therapeutic target to overcome acquired lapatinib resistance in HER2-positive breast cancer cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Hypoxia is a general characteristic of most solid malignancies and intimately related to neoplastic diseases and cancer progression. Homeostatic response to hypoxia is primarily mediated by hypoxia inducible factor (HIF)-1α that elicits transcriptional activity through recruitment of the CREB binding protein (CBP)/p300 coactivator. Targeted blockade of HIF-1α binding to CBP/p300 would thus constitute a novel approach for cancer treatment by suppressing tumor angiogenesis and metastasis. Here, we identified inhibitors against the interaction between HIF-1α and p300 by a fluorescence polarization-based assay employing a fluorescently-labeled peptide containing the C-terminal activation domain of HIF-1α. Two small molecule inhibitors, menadione (MD) and ethacrynic acid (EA), were found to decrease expression of luciferase under the control of hypoxia-responsive elements in hypoxic cells as well as to efficiently block the interaction between the full-length HIF-1α and p300. While these compounds did not alter the expression level of HIF-1α, they down-regulated expression of a HIF-1α target vascular endothelial growth factor (VEGF) gene. Considering hypoxia-induced VEGF expression leading to highly aggressive tumor growth, MD and EA may provide new scaffolds for development of tumor therapeutic reagents as well as tools for a better understanding of HIF-1α-mediated hypoxic regulation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Farnesoid X receptor (FXR) serves as a receptor for chenodeoxycholic acid (CDCA) and other bile acids, and it coordinates cholesterol and lipid metabolism. Because targeting the FXR-CDCA interaction might provide a way to regulate lipid homeostasis, we developed an FXR binding assay based on fluorescence polarization. Employing a fluorescently labeled CDCA (CDCA-F), we showed that CDCA-F selectively bound to the ligand binding domain of FXR (FXR-LBD) among nuclear receptors. The assay was then used for screening inhibitors against the FXR-CDCA interaction, thereby discovering four relatively potent inhibitors. The selected inhibitors were further studied for changes in intrinsic tryptophan fluorescence of FXR-LBD to gain structural insights into the interaction. Furthermore, transactivation effects of the inhibitors on the human bile salt excretory pump (BSEP) promoter were examined to reveal their cellular activities in the FXR-mediated pathway. Therefore, we demonstrated that the developed assay would offer an efficient primary screening tool for identifying FXR modulators., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

A highly sensitive nanomechanical cantilever sensor assay based on an electrical measurement has been developed for detecting activated cyclic adenosine monophosphate (cyclic AMP)-dependent protein kinase (PKA). Employing a peptide derived from the heat-stable protein kinase inhibitor (PKI), a magnetic bead system was first selected as a vehicle to immobilize the PKI-(5-24) peptide for capturing PKA catalytic subunit and the activity assay was applied for indirectly assessing the binding. Synergistic interactions of adenosine triphosphate (ATP) and the peptide inhibitor with the kinase were then investigated by a solution phase capillary electrophoretic assay, and by surface plasmon resonance technology which involved immobilization of the peptide inhibitor. After systemically evaluated by a homogeneous direct binding assay, the ATP-dependent recognition of the catalytic subunit of PKA by PKI-(5-24) was successfully transferred on to the nanomechanical cantilevers at protein concentrations of 6.6 pM-66 nM, exhibiting much higher sensitivity and wider dynamic range than the conventional activity assay. Thus, direct assessment of activated kinases using the cantilever sensor system functionalized with specific peptide inhibitors holds great promise in analytical applications and clinical medicine.

A series of alkyl tetrahydrofuran-2-carboxylates (1-4) bearing a new set of three pharmacophoric groups were tested as protein kinase C (PKC) ligands. The compounds were synthesized from commercially available glycidyl 4-methoxyphenyl ether. The correlation between their binding affinities for PKC-alpha and a conformational fit to phorbol ester indicates they mimic a pharmacophore model comprising the C20-OH, C3-C=O and C9-OH rather than that including the C13-C=O moiety.