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. 2013;8(1):e54127.
doi: 10.1371/journal.pone.0054127. Epub 2013 Jan 10.

A homogeneous, high-throughput assay for phosphatidylinositol 5-phosphate 4-kinase with a novel, rapid substrate preparation

Mindy I Davis  1 , Atsuo T Sasaki, Min Shen, Brooke M Emerling, Natasha Thorne, Sam Michael, Rajan Pragani, Matthew Boxer, Kazutaka Sumita, Koh Takeuchi, Douglas S Auld, Zhuyin Li, Lewis C Cantley, Anton Simeonov
Affiliations

A homogeneous, high-throughput assay for phosphatidylinositol 5-phosphate 4-kinase with a novel, rapid substrate preparation

Mindy I Davis et al. PLoS One. 2013.

Abstract

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)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 IC(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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Schematic representation of the PI5P4K reaction using PI5P as the substrate.
The additional carrier substrate DPPS is not shown.
Figure 2
Figure 2. Schematic representation of the 2∶1 DPPS:PI5P lipid preparation protocol.
Figure 3
Figure 3. Lipid dependence, overnight stability and control compound.
(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-32P-ATP and PI5P and measures the radiolabeled enzymatic product, PI(4,5)P2 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)P2 product with two palmitate groups, and the faint spots below represent the product with just one palmitate group.
Figure 4
Figure 4. Performance of the PI5P4Kα assay in 1536-well assay plates.
(A) Z’ factor, (B) signal/background, and (C) % column variance as a function of assay plate. An average Z’  = 0.77±0.04, S/B  = 12.6±1.04 and % CV  = 9.32±2.1 was achieved for the six plates (reported as average ± standard deviation). (D) IC50 data for the control compound AG-82 on the six assay plates depicted with six symbols. MSR of the IC50 = 1.29. Each plate contained 2 16-pt titrations, which were averaged, and the standard deviation is depicted as error bars on the plot (N = 2).
Figure 5
Figure 5. Confirmation of inhibitors.
(A) i, The IC50 inhibition curves of the tyrphostin analogues identified from the Lopac library are shown. Numbers refer to the tyrphostin analog and IC50s were determined to be as follows: tyrphostin I-OMe-AG-538 (2 µM), tyrphostin 51 (5 µM), tyrphostin AG 112 (13 µM), tyrphostin AG 538 (14 µM), tyrphostin AG 808 (18 µM), tyrphostin 47 (20 µM), tyrphostin AG 537 (32 µM), tyrphostin 23 (45 µM), tyrphostin AG 555 (50 µM), tyrphostin AG 698 (50 µM), tyrphostin AG 490 (89 µM), tyrphostin AG 494 (89 µM), tyrphostin AG 1478 (100 µM). ii, Structures of the four most potent tyrphostin analogs. (B) The IC50 curves with standard deviation error bars (N = 2) of tyrphostin I-OMe-AG-538 in the PI5P4Kα assay (squares) and the counterscreen (open circles).
Figure 6
Figure 6. ATP competition with tyrphostin I-OMe-AG-538.
The IC50 of tyrphostin I-OMe-AG-538 for PI5P4Kα is plotted against the [ATP]/Km. The Km of ATP is 5 µM, and seven concentrations were evaluated.
See this image and copyright information in PMC

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