HTRF-based method for determination of HSP90-HOP inhibition activity and its application / 药学学报

HSP90 is widely expressed in cells with the main function in assisting the maturation of other proteins that are called clients. Many clients play critical roles in the occurrence and development of cancer. Inhibition of HSP90 can lead to degradation of the oncogenic proteins, and result in potent anti-cancer effects. HSP90-HOP interaction is critical for the chaperone function of HSP90, thereby disruption of the HSP90-HOP interaction is a novel strategy in the inhibition of HSP90. Based on the technology of homogeneous time-resolved fluorescence (HTRF), we developed a new assay for the identification of new inhibitors of HSP90-HOP interaction. This method was evaluated in the study of the HSP90-HOP inhibition activity of the pentapeptide MEEVD from HSP90 C-terminal and its derivatives. This study can provide a basis for the screening and discovery of novel HSP90-HOP disruptors.

Activity of anti-epidermal growth factor receptor in Calla Chinensis and UPLC/Q-TOF-MS analysis on its effective part / 中草药

Objective: Using homogeneous time-resolved fluorescence (HTRF) technology to screen the Calla Chinensis extracts with anti-epidermal growth factor receptor activity and to analyze the active ingredients in Calla Chinensis by ultra-performance liquid chromatography/quadrupole-time of flight mass-spectrometry (UPLC/Q-TOF-MS). Methods: After percolation with petroleum ether, ethanol extract, ethyl acetate extraction, and boiling with water, four fractions were obtained. HTRF method was applied to detecting the inhibition of ethyl acetate fraction on EGFR, and the inhibitory rate was calculated. The chromatographic separation was performed on Acquity UPLC BEH C18 column with a gradient elufion of 0.1% formic acid water-acetonitrile. The mass spectrometer equipped with electrospay ionization source was used as defector, data were collected under the positive ion modes, and screened at 200-400 nm. Results: The ethyl acetate fraction of Calla Chinensis showed the strong inhibitory activity on EGFR. Fourteen compounds were analyzed and identified, among which tannins were the main active components, the 11 tannin compounds were punicalin, vanillin, di-HHDP-glucose, ellagitannin, myricetin 3-O-rhamnoside, protocatechuic acid, gallotannin-glucose, ellagic acid-hexose, 3, 5-dicaffeoylquinic acid, monogalloyl-glucose, and gallotannin, and the three others were unknown. Conclusion: The ethyl acetate fraction of Calla Chinensis has the significant EGFR inhibitory activity, and the IC50 value is 5.528 μg/mL. Tannins, including Chinese tannin, gallogen, and ellagitannin as main constituents, are identified through the information of positive ion and relative molecular mass determined by Q-TOF-MS. The results indicate that Calla Chinensis contains tannin ingredients to inhibit EGFR, in the hope to provide a theoretical basis of the application in anticancer and to lay the foundation for the further tracking separation of the active ingredients.

Tyrosine kinase inhibitory activity of dehydroabietylamine derivatives tested by homogeneous time-resolved fluorescence based high throughput screening model / 中国天然药物

Protein tyrosine kinases (PTKs) are attractive targets in searching for therapeutic agents against many diseases. In this study, a series of dehydroabietylamine derivatives were first determined to show PTK inhibitory activity using a high-throughput screening (HTS) method based on homogeneous time-resolved fluorescence (HTRF) technology. The structure-activity relationships of the dehydroabietylamine derivatives were established, and it was found that the compounds with a nitrogen-containing side chain had better inhibitory activity. Further studies showed that the compounds substituted with halogen in the phenyl ring resulted in higher inhibitory activity on the epidermal growth factor receptor (EGFR), and can be a guide to modify the structure of dehydroabietylamine derivatives. Dehydroabietylamine derivatives might be a new class of multi-targeted and effective PTK inhibitors with structure modifications.

Establishment and application of high throughput screening model for kinase insert domain receptor / 中国药学杂志

OBJECTIVE: To establish a KDR high throughput screening model with homogeneous time resolved fluorescence(HTRF) detection technology in order to screen small molecular KDR inhibitors from the compound library. METHODS: A 20 μL assay system in 384-well low-volume white microplate was developed with HTRF based fluorescence detection system to determine the enzyme activity. The optimization steps consisted of the following experiments: enzyme concentration and incubation time optimiztion, ATP and substrate Km determination, quality control experiments including signal noise ratio inspection, the inhibitor SU5416 IC50 validation, and Z' value calculation. After a stable assay system was accomplished, a HTS campaign was started with 10560 samples irom the compound library and the IC50 of some compounds were determined. RESULTS: The optimized KDR activity assay conditions were as follows: the kinase concentration was 0.10 ng · μL-1; ATP Km was 0.75μmol · L-1; substrate Km was 94.90 nmol · L-1; biotin/ SA ratio was 2:1; Z' value was 0.85 and the IC50 of SU5416 inhibitor was 1.03 μmol · L-1. Three compounds with the ID of S2-14, S2-16, and S2-38 showed IC50 on KDR of 1.01 × 10-4, 6.04 × 10-5, 7.23 × 10-6 mol · L-1, respectively. CONCLUSION: A KDR high throughput screening model has been successfully established with HTRF methodology and a focused HTS campaign has been accomplished with several leads. This assay system is reliable and the results are stable. The established assay system is suitable for further application in screening natural anti-angiogenesis products.

Establishment of high-throughput screening system for BACE1 inhibitors in vitro / 国际药学研究杂志

Objective: To establish the high-throughput screening system for β-secretase (β-site amyloid precursor protein cleaving enzyme 1, BACE1) inhibitors in vitro. Methods: Using the homogeneous time-resolved fluorescence (HTRF) technique to establish a high-throughput screening system for BACE1 inhibitors according to the extent that test compounds inhibited BACE1 activity by optimizing the incubation time, enzyme concentration and the detector. Results: A high-throughput screening system for BACE1 inhibitors based on HTRF methods was established. The incubation time was 6 h, the BACE1 concentration is 670 U/L. According to the detector of VICTOR3, the signal-to-noise (S/N) ratio reached 649.6, while the signal-to-background (S/B) ratio and Z′ factor were 44.6 and 0.91, respectively. The coefficient of variation (CV) was much lower than 10%. Using this method, the inhibitors with IC50<106mol/L could be screened out. Conclusion: The high-throughput screening system based on HTRF Could assure a high sensitivity, specificity and stability, which could be used to high-through put screening for the BACE1 inhibitors.