Allosteric MEK1/2 Inhibitor Refametinib (BAY 86-9766) in Combination with Sorafenib Exhibits Antitumor Activity in Preclinical Murine and Rat Models of Hepatocellular Carcinoma

OBJECTIVE: The objectives of the study were to evaluate the allosteric mitogen-activated protein kinase kinase (MEK) inhibitor BAY 86-9766 in monotherapy and in combination with sorafenib in orthotopic and subcutaneous hepatocellular carcinoma (HCC) models with different underlying etiologies in two species. DESIGN: Antiproliferative potential of BAY 86-9766 and synergistic effects with sorafenib were studied in several HCC cell lines. Relevant pathway signaling was studied in MH3924a cells. For in vivo testing, the HCC cells were implanted subcutaneously or orthotopically. Survival and mode of action (MoA) were analyzed. RESULTS: BAY 86-9766 exhibited potent antiproliferative activity in HCC cell lines with half-maximal inhibitory concentration values ranging from 33 to 762 nM. BAY 86-9766 was strongly synergistic with sorafenib in suppressing tumor cell proliferation and inhibiting phosphorylation of the extracellular signal-regulated kinase (ERK). BAY 86-9766 prolonged survival in Hep3B xenografts, murine Hepa129 allografts, and MH3924A rat allografts. Additionally, tumor growth, ascites formation, and serum alpha-fetoprotein levels were reduced. Synergistic effects in combination with sorafenib were shown in Huh-7, Hep3B xenografts, and MH3924A allografts. On the signaling pathway level, the combination of BAY 86-9766 and sorafenib led to inhibition of the upregulatory feedback loop toward MEK phosphorylation observed after BAY 86-9766 monotreatment. With regard to the underlying MoA, inhibition of ERK phosphorylation, tumor cell proliferation, and microvessel density was observed in vivo. CONCLUSION: BAY 86-9766 shows potent single-agent antitumor activity and acts synergistically in combination with sorafenib in preclinical HCC models. These results support the ongoing clinical development of BAY 86-9766 and sorafenib in advanced HCC

Development of a high-throughput fluoroimmunoassay for Syk kinase and Syk kinase inhibitors

Syk is a tyrosine kinase which is indispensable in immunoglobulin Fc receptor- and B cell receptor-mediated signal transduction in various immune cells. This pathway is important in the pathophysiology of allergy. In this study we established a quantitative nonradioactive kinase assay to identify inhibitors of Syk. We used recombinant GST-tagged Syk purified from baculovirus-infected insect cells. As a substrate, biotinylated peptide corresponding to the activation loop domain of Syk, whose tyrosine residues are autophosphorylated upon activation, was employed to screen both ATP- and substrate-competitive inhibitors. After the kinase reaction in solution phase, substrate was trapped on a streptavidin-coated plate, followed by detection of the phosphorylated tyrosine with europium-labeled anti-phosphotyrosine antibody. The kinase reaction in solution phase greatly enhanced phosphorylation of substrate compared to that of plate-coated substrate. High signal-to-background ratio and low data scattering were obtained in the optimized high-throughput screening (HTS) format. Further, several kinase inhibitors showed concentration-dependent inhibition of recombinant Syk kinase activity with almost the same efficacy for immunoprecipitated Syk from a human cell line. These data suggest that this assay is useful to screen Syk kinase inhibitors in HTS

Identification of Antibiotic Stress-Inducible Promoters: A Systematic Approach to Novel Pathway-Specific Reporter Assays for Antibacterial Drug Discovery

As present antibiotics therapy becomes increasingly ineffectual, new technologies are required to identify and develop novel classes of antibacterial agents. An attractive alternative to the classical target-based approach is the use of promoter-inducible reporter assays for high-throughput screening. The wide usage of these assays is, however, limited by the small number of specifically responding promoters that are known at present. This work describes a novel approach for identifying genetic regulators that are suitable for the design of pathway-specific assays. The basis for the proposed strategy is a large set of antibiotics-triggered expression profiles (“Reference Compendium”). Pattern recognition algorithms applied to the expression data pinpoint the relevant transcription-factor-binding sites in whole-genome sequences. Using this technique, we constructed a fatty-acid-pathway-specific reporter assay that is based on a novel stress-inducible promoter. In a proof-of-principle experiment, this assay was shown to enable screening for new small-molecule inhibitors of bacterial growth

Virulence and hyphal formation of Candida albicans require the Ste20p-like protein kinase CaCla4p

UI - 97411146NRC publication: Ye

Novel Whole-Cell Antibiotic Biosensors for Compound Discovery▿

Cells containing reporters which are specifically induced via selected promoters are used in pharmaceutical drug discovery and in environmental biology. They are used in screening for novel drug candidates and in the detection of bioactive compounds in environmental samples. In this study, we generated and validated a set of five Bacillus subtilis promoters fused to the firefly luciferase reporter gene suitable for cell-based screening, enabling the as yet most-comprehensive high-throughput diagnosis of antibiotic interference in the major biosynthetic pathways of bacteria: the biosynthesis of DNA by the yorB promoter, of RNA by the yvgS promoter, of proteins by the yheI promoter, of the cell wall by the ypuA promoter, and of fatty acids by the fabHB promoter. The reporter cells mainly represent novel antibiotic biosensors compatible with high-throughput screening. We validated the strains by developing screens with a set of 14,000 pure natural products, representing a source of highly diverse chemical entities, many of them with antibiotic activity (6% with anti-Bacillus subtilis activity of ≤25 μg/ml]). Our screening approach is exemplified by the discovery of classical and novel DNA synthesis and translation inhibitors. For instance, we show that the mechanistically underexplored antibiotic ferrimycin A1 selectively inhibits protein biosynthesis