| High throughput screening (HTS) is an important tool in current drug discovery. A complete HTS platform is consisted of a set of compatible equipments which is capable of precision liquid handling and highly sensitive signal acquisition, a chemical compound library which includes compounds with variety chemical scaffolds and biological activities, and a collection of HTS-compatible methods to measure biological activities. Natural product library is a good source for the drug discovery because of its abaundant and diversity. The chemical library used in this study includes10,000flash fractions and50,000HPLC fractions extracted from respectively500and171different herbs widely used in Traditional Chinese Medicine (TCM). This study combined liquid handling, dispenser, molecular device M5and384pin tool to esdablish a384format HTS platform. During the construction of the HTS system, eight assays to measure the cellular kinase activity of different tyrosine kinase, two assays to measure different GLP1R agonist activity, and10assays to masure cytotoxcicity were established. A throughput of50,000wells/day was routinely achieved in our subsequent screens using this HTS platform.Leukemia and lymphoma are hematologic malignancies, which may have different mechanisms and pathology from solid tumors. Specific drug discovery for leukemia treatment is crucial for personal cancer treatment. Because BaF3is rat pro-B cells, which expand rapidly it was used to as a cellular model to screen for inhibitors against leukemia. After the screening, one active fraction was found with high potency against the proliferation of BaF3. This fraction has little inhibitory effect on the growth of solid tumor cells, such as A549, SNU16and SNU638. After further extraction and purification procedures, the structure of active component is identified as panaxydol. Using the flow cytometry analysis, panaxydol was found to induce the apoptosis of BaF3/, but not of A549cells. Cytotoxic activity of panaxydol is also confirmed in Jurkat and Raji cells which are originally isolated from patients with leukemia and lymphoma.Glucagon-like peptide1receptor (GLP1R) is a validated target for type2diabetes treatment. In vivo, GLP1R can be activated by binding with GLP1peptide and subsequently increase glucose-dependent insulin gene expression and insulin secretion in L cells. Due to the activity DPP-IV, the endogenous GLP1peptide is cleaved and lost its activity, so a longer half-life GLP1R agonist is in great interest as a candidate in treating type2diabetes. A HTS to screen for GLP1R agonists from a natural product compound library derived from Traditional Chinese Medicine (TCM) was conducted. One fraction extracted from dry Ophisaurus harti was found to have specific GLP1agonist activity. Similar to Exenatide, a clinical-proven GLP1R agonist in treating type2diabetes, active fraction also showed sensitivity to trypsin digestion and resistant to DPP-Ⅳ cleavage, revealing its peptidic nature and DPP-Ⅳ resistance. Interestingly, Exenatide was originally identified from Heloderma Suspectum, a lizard species that belong to the same suborder of Anguimorpha as the host animals of active fraction. The active ingredient was later identified as O. harti GLP1through transcriptome analysis. The O. harti GLP1peptide was chemically synthesized based on its sequence, and was demonstrated to preserve its GLP1R agonist activity. However, chemically synthesized GLP1peptide is not DPP-Ⅳ resistant, and we suspect this was due to lack of special secondary structure, or post-translational modification which only presented in biologically generated GLP1. |
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