14 research outputs found

    Multimodal action of KRP203 on phosphoinositide kinases in vitro and in cells

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    Increased phosphoinositide signaling is commonly associated with cancers. While "one-drug one-target" has been a major drug discovery strategy for cancer therapy, a "one-drug multi-targets" approach for phosphoinositide enzymes has the potential to offer a new therapeutic approach. In this study, we sought a new way to target phosphoinositides metabolism. Using a high-throughput phosphatidylinositol 5-phosphate 4-kinase-alpha (PI5P4Kα) assay, we have identified that the immunosuppressor KRP203/Mocravimod induces a significant perturbation in phosphoinositide metabolism in U87MG glioblastoma cells. Despite high sequence similarity of PI5P4K and PI4K isozymes, in vitro kinase assays showed that KRP203 activates some (e.g., PI5P4Kα, PI4KIIβ) while inhibiting other phosphoinositide kinases (e.g., PI5P4Kβ, γ, PI4KIIα, class I PI3K-p110α, δ, γ). Furthermore, KRP203 enhances PI3P5K/PIKFYVE's substrate selectivity for phosphatidylinositol (PI) while preserving its selectivity for PI(3)P. At cellular levels, 3 h of KRP203 treatment induces a prominent increase of PI(3)P and moderate increase of PI(5)P, PI(3, 5)P₂, and PI(3, 4, 5)P₃ levels in U87MG cells. Collectively, the finding of multimodal activity of KRP203 towards multi-phosphoinositide kinases may open a novel basis to modulate cellular processes, potentially leading to more effective treatments for diseases associated with phosphoinositide signaling pathways

    A Homogeneous, High-Throughput Assay for Phosphatidylinositol 5-Phosphate 4-Kinase with a Novel, Rapid Substrate Preparation

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    Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phosphoinositide kinases often use lipid substrates prepared from lyophilized lipid preparations by sonication, which result in variability in the liposome size from preparation to preparation. Herein, we report a homogeneous 1536-well luciferase-coupled bioluminescence assay for PI5P4Kα. The substrate preparation is novel and allows the rapid production of a DMSO-containing substrate solution without the need for lengthy liposome preparation protocols, thus enabling the scale-up of this traditionally difficult type of assay. The Z’-factor value was greater than 0.7 for the PI5P4Kα assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4Kα by assessing the degree of phospho transfer of γ-32^{32}P-ATP to PI5P; its inhibitory activity against PI5P4Kα was confirmed in the present miniaturized assay. From a pilot screen of a library of bioactive compounds, another tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538), was identified as an ATP-competitive inhibitor of PI5P4Kα with an IC50_{50} of 1 µM, affirming the suitability of the assay for inhibitor discovery campaigns. This homogeneous assay may apply to other lipid kinases and should help in the identification of leads for this class of enzymes by enabling high-throughput screening efforts

    <i>De Novo</i> Synthesis of l-Colitose and l-Rhodinose Building Blocks

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    A divergent, practical, and efficient <i>de novo</i> synthesis of fully functionalized l-colitose (3,6-dideoxy-l-galactose), 2-<i>epi</i>-colitose (3,6-dideoxy-l-talose), and l-rhodinose (2,3,6-trideoxy-l-galactose) building blocks has been achieved using inexpensive, commercially available (<i>S</i>)-ethyl lactate as the starting material. The routes center around a diastereoselective Cram-chelated allylation that provides a common homoallylic alcohol intermediate. Oxidation of this common intermediate finally resulted in the synthesis of the three monosaccharide building blocks

    Multimodal action of KRP203 on phosphoinositide kinases in vitro and in cells

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    Increased phosphoinositide signaling is commonly associated with cancers. While "one-drug one-target" has been a major drug discovery strategy for cancer therapy, a "one-drug multi-targets" approach for phosphoinositide enzymes has the potential to offer a new therapeutic approach. In this study, we sought a new way to target phosphoinositides metabolism. Using a high-throughput phosphatidylinositol 5-phosphate 4-kinase-alpha (PI5P4Kα) assay, we have identified that the immunosuppressor KRP203/Mocravimod induces a significant perturbation in phosphoinositide metabolism in U87MG glioblastoma cells. Despite high sequence similarity of PI5P4K and PI4K isozymes, in vitro kinase assays showed that KRP203 activates some (e.g., PI5P4Kα, PI4KIIβ) while inhibiting other phosphoinositide kinases (e.g., PI5P4Kβ, γ, PI4KIIα, class I PI3K-p110α, δ, γ). Furthermore, KRP203 enhances PI3P5K/PIKFYVE's substrate selectivity for phosphatidylinositol (PI) while preserving its selectivity for PI(3)P. At cellular levels, 3h of KRP203 treatment induces a prominent increase of PI(3)P and moderate increase of PI(5)P, PI(3, 5)P₂, and PI(3, 4, 5)P₃ levels in U87MG cells. Collectively, the finding of multimodal activity of KRP203 towards multi-phosphoinositide kinases may open a novel basis to modulate cellular processes, potentially leading to more effective treatments for diseases associated with phosphoinositide signaling pathways

    Lipid dependence, overnight stability and control compound.

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    <p>(A) The overnight (16 hour) stability of the assay reagents at 4°C when the enzyme and lipid were premixed, stored separately or made up fresh as compared to a no enzyme and 5 µM ADP (representing 0% and 100% conversion, respectively). The error bars represent the standard deviation (N = 2). (B) The PI5P lipid dependence of the PI5P4Kα enzyme reaction. The error bars represent the standard deviation (N = 2) and are not discernable on the plot. (C) and (D) Tyrphostin AG-82 (AG82) was identified as a weak inhibitor of PI5P4Kα (decreases the enzyme activity by 75%) by a radiometric assay that uses γ-<sup>32</sup>P-ATP and PI5P and measures the radiolabeled enzymatic product, PI(4,5)P<sub>2</sub> after the separation by thin layer chromatography. Five additional compounds were tested and found not to significantly inhibit PI5P4Kα (AG17 =  tyrphostin AG-17, AG18 =  tyrphostin AG-18, MP = mycophenolate, PVB = purvalanol B and SU6668). All compounds were tested at 100 µM, except for PVB, which was tested at 10 µM due to solubility limitations at higher concentrations. The raw image and the extracted data are shown in (C) and (D), respectively. The commercial PI5P substrate predominantly contains two palmitate groups with a very small amount of deacylated lipid lyso-PI5P that contains only one palmitate group. The intense top spots in (C) represent the PI(4,5)P<sub>2</sub> product with two palmitate groups, and the faint spots below represent the product with just one palmitate group.</p
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