Validation of the Japanese version of Algoplus® for the assessment of acute postoperative pain in older patients with cancer.

AIM: This study aimed to validate a Japanese version of the Algoplus® tool by assessing postoperative pain in older Japanese patients with cancer and examining the scale's psychometric properties. METHODS: After translating Algoplus® into Japanese, we conducted a cross-sectional study of patients aged 65 years or older who underwent surgery to remove malignant tumors. Two registered nurse-certified investigators used the Numerical Rating Scale, the Japanese version of Algoplus®, and the Japanese version of the Abbey Pain Scale before and after analgesic use on postoperative days 3 and 5 to evaluate response to pharmacologic therapy. Validity was tested by a correlation analysis between the Japanese version of Algoplus®, two pain scales, and nine hypotheses related to demographic variables and surgical invasions. The Kuder-Richardson-20 test and Cohen's Kappa coefficient were used for internal consistency and inter-rater reliability, respectively. RESULTS: The total score of the Japanese version of Algoplus® showed strong to moderate correlations with the two pain scales. Demographic characteristics were not associated with the total score of the Japanese version, but significant correlations with operative time and postoperative analgesia administration existed. The scale demonstrated good internal consistency (Kuder-Richardson-20 α: .70) and inter-rater reliability (Kappa coefficient .72). The total score of the Japanese version decreased significantly after analgesic use in both postoperative assessments. CONCLUSIONS: The Japanese version of Algoplus® is a reliable and valid instrument for nurses to easily assess acute postoperative pain in older Japanese patients with cancer and shows good responsiveness for detecting the change in pain status.

A fluorescence correlation spectroscopy-based assay for fragment screening of slowly inhibiting protein-peptide interaction inhibitors.

A fluorescence correlation spectroscopy (FCS)-based competitive binding assay to screen fragment-size compounds that weakly and slowly inhibit protein-peptide interactions was established. The interactions were detected by the increased diffusion time of a fluorescently labeled peptide probe after binding to its interacting protein. We analyzed the interactions between the c-Cbl TKB domain and phosphopeptides derived from ZAP-70, APS, and EGFR with the FCS assay and obtained 6 hit fragments that bound to the c-Cbl interaction sites. The binding amounts of the fragments were measured by direct binding measurements using surface plasmon resonance, and 5 fragments were found to bind selectively. The effect of 2 of the 5 fragments on the interaction with c-Cbl and the peptide exhibited strong time dependency. Furthermore, the inhibition by the selected 5 fragments on the protein-peptide interaction was confirmed by their effect on pull-down assays of c-Cbl with the biotin-conjugated interaction peptides. These results indicate the advantage of our FCS-based assay to study the time-dependent binding of compounds to their target protein.

Contribution of structural biology to clinically validated target proteins.

We identified six groups of diseases expected to cause serious future health issues on the basis of a WHO report. Approved drugs for these diseases were associated with 409 target proteins; however, the percentage of selected proteins with full-length structural information deposited in the Protein Data Bank (PDB) was only 9.8%. The reason for the low percentage may be as a result of a disproportionate number of intractable proteins with multiple transmembrane regions and variable, or undefined glycosylation patterns, which impede protein preparation and crystallization, in such druggable proteins. We stress the importance of structural analysis of proteins, especially approved-drug target proteins, and the development of new methods to enable structural analyses of presently intractable proteins. In addition, we present an overview of large structural biology projects.

Two-colored fluorescence correlation spectroscopy screening for LC3-P62 interaction inhibitors.

The fluorescence correlation spectroscopy (FCS)-based competitive binding assay to screen for protein-protein interaction inhibitors is a highly sensitive method as compared with the fluorescent polarization assay used conventionally. However, the FCS assay identifies many false-positive compounds, which requires specifically designed orthogonal screenings. A two-colored application of the FCS-based screening was newly developed, and inhibitors of a protein-protein interaction, involving selective autophagy, were selected. We focused on the interaction of LC3 with the adaptor protein p62, because the interaction is crucial to degrade the specific target proteins recruited by p62. First, about 10,000 compounds were subjected to the FCS-based competitive assay using a TAMRA-labeled p62-derived probe, and 29 hit compounds were selected. Next, the obtained hits were evaluated by the second FCS assay, using an Alexa647-labeled p62-derived probe to remove the false-positive compounds, and six hit compounds inhibited the interaction. Finally, we tested all 29 compounds by surface plasmon resonance-based competitive binding assay to evaluate their inhibition of the LC3-p62 interaction and selected two inhibitors with IC50 values less than 2 µM. The two-colored FCS-based screening was shown to be effective to screen for protein-protein interaction inhibitors.

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.

A novel Pim-1 kinase inhibitor targeting residues that bind the substrate peptide.

A new screening method using fluorescent correlation spectroscopy was developed to select kinase inhibitors that competitively inhibit the binding of a fluorescently labeled substrate peptide. Using the method, among approximately 700 candidate compounds selected by virtual screening, we identified a novel Pim-1 kinase inhibitor targeting its peptide binding residues. X-ray crystal analysis of the complex structure of Pim-1 with the inhibitor indicated that the inhibitor actually binds to the ATP-binding site and also forms direct interactions with residues (Asp128 and Glu171) that bind the substrate peptide. These interactions, which cause small side-chain movements, seem to affect the binding ability of the fluorescently labeled substrate. The compound inhibited Pim-1 kinase in vitro, with an IC(50) value of 150 nM. Treatment of cultured leukemia cells with the compound reduced the amount of p21 and increased the amount of p27, due to Pim-1 inhibition, and then triggered apoptosis after cell-cycle arrest at the G(1)/S phase. This screening method may be widely applicable for the identification of various new Pim-1 kinase inhibitors targeting the residues that bind the substrate peptide.