Identification of small molecule inhibitors of human COQ7.

Given that the associated clinical manifestations of ubiquinone (UQ, or coenzyme Q) deficiency diseases are highly heterogeneous and complicated, effective new research tools for UQ homeostasis studies are awaited. We set out to develop human COQ7 inhibitors that interfere with UQ synthesis. Systematic structure-activity relationship development starting from a screening hit compound led to the identification of highly potent COQ7 inhibitors that did not disturb physiological cell growth of human normal culture cells. These new COQ7 inhibitors may serve as useful tools for studying the balance between UQ supplementation pathways: de novo UQ synthesis and extracellular UQ uptake.

A fluorescent-based high-throughput screening assay for small molecules that inhibit the interaction of MdmX with p53.

A fluorescent-based high-throughput screening (HTS) assay for small molecules that inhibit the interaction of MdmX with p53 was developed and applied to identify new inhibitors. The assay evaluated the MdmX-p53 interaction by detecting the quenching of the fluorescence of green fluorescent protein (GFP) fused to the MdmX protein, after its interaction with a p53 peptide labeled with a fluorescence quencher. In this report, the developed HTS assay was applied to about 40 000 compounds, and 255 hit compounds that abrogated the GFP quenching were selected. Next, the obtained hits were reevaluated by other assays. First, their effects on the diffusion time of a fluorescently-labeled p53 peptide after incubation with the MdmX protein were tested by measuring the diffusion time using fluorescence correlation spectroscopy, and six stable hit compounds with IC(50) values less than 5 µM were selected. Next, we further confirmed their inhibition of the MdmX-p53 interaction by surface plasmon resonance. To indicate the efficacy of the hit compound as a candidate anticancer drug, we showed that the hit compound triggered apoptosis after p53 and p21 accumulation in cultured MV4;11 leukemia cells. Thus, the new HTS assay is effective for obtaining novel MdmX-p53 interaction inhibitors that are valuable as candidate compounds for cancer treatment.

Development of a novel fluorescent probe for fluorescence correlation spectroscopic detection of kinase inhibitors.

We have developed a fluorescently labeled probe for high-throughput screening of kinase inhibitors using fluorescence correlation spectroscopy. With this probe, we have successfully evaluated the inhibitory activities of known inhibitors of a model kinase, ASK1. Because the probe contains a general kinase inhibitor, staurosporine, we believe that this homogeneous, high-throughput, and simple method can be applied to the inhibitor screening of other kinases as well.

Photo-cross-linked small-molecule microarrays as chemical genomic tools for dissecting protein-ligand interactions.

We have developed a unique photo-cross-linking approach for immobilizing a variety of small molecules in a functional-group-independent manner. Our approach depends on the reactivity of the carbene species generated from trifluoromethylaryldiazirine upon UV irradiation. It was demonstrated in model experiments that the photogenerated carbenes were able to react with every small molecule tested, and they produced multiple conjugates in most cases. It was also found in on-array immobilization experiments that various small molecules were immobilized, and the immobilized small molecules retained their ability to interact with their binding proteins. With this approach, photo-cross-linked microarrays of about 2000 natural products and drugs were constructed. This photo-cross-linked microarray format was found to be useful not merely for ligand screening but also to study the structure-activity relationship, that is, the relationship between the structural motif (or pharmacophore) found in small molecules and its binding affinity toward a protein, by taking advantage of the nonselective nature of the photo-cross-linking process.