Fig 1.
Promega ADP-Glo Max assay workflow.
IP6K1 was incubated with ATP and IP6 to generate ADP and PP-IP5. The excess ATP in the reaction was removed with the ADP-Glo reagent leaving the ADP concentration unchanged. The Kinase Detection reagent converts ADP to ATP, which can then react with luciferase to generate a bioluminescent signal that is directly proportional to IP6K1 activity.
Fig 2.
Optimization parameters of the IP6K1 high throughput assay.
Various assay parameters were investigated and optimized. (A) Buffer pH was explored using buffers with 6 different pH values between 5.7 and 7.2 units, 1mM ATP, 500μM IP6, and 120nM IP6K1 for 30 minutes at 37°C. (B) Three IP6K1 concentrations were tested at three time points with 300μM ATP and 250μM IP6 to determine conditions that gave good signal and displayed linearity. (C) The percentage of DMSO tolerated by the assay was measured with 0.3125–20% DMSO, 1mM ATP, 500μM IP6, and 120nM IP6K1 for 30 minutes at 37°C. Concentrations higher than 5% were detrimental to assay performance. (D) ATP concentrations of 62.μM, 250μM, and 2000μM were tested with 400μM IP6 and 60nM IP6K1 for 30 minutes at 37°C. (E) IP6 concentrations of 6.25–200μM were tested with 1mM ATP and 60nM IP6K1 for 30 minutes at 37°C. The signal/background ratio is defined as the luminescent signal of the reaction divided by the signal generated from the same reaction in the absence of IP6. ADP concentrations are calculated by correlating the luminescence produced by a known standard of ADP to that of the experimental condition. Percent activity is defined as 100*(μexperimental–μnegative)/(μpositive–μnegative) where positive and negative controls are the experiment with and without IP6 present. All data points are replicated in quadruplicate and represented as the mean ± SEM. Non-significance was determined via one-way ANOVA testing where p > 0.05.
Fig 3.
Michaelis-Menten kinetic parameters were determined for IP6K1 at various concentrations of ATP. The Km and Vmax parameters for ATP were determined to be 382±44μM and 1116±41nmol/min/mg respectively. The experiment was conducted with 400μM IP6 with variable amounts of ATP and 15ng IP6K1 for 15 minutes. All data points (n = 4–6) are represented as the mean ± SEM and fit to the Michaelis-Menten equation for analysis.
Fig 4.
Dose response inhibition of IP6K1 by TNP in the presence of different concentrations of ATP and IP6.
TNP was confirmed to compete with ATP by binding to IP6K1. (A) IC50 values increased from 12±1.1μM to 39±1.1μM when ATP concentrations increased from 62.5μM to 2000μM indicating competitive inhibition. The reaction was run with 400μM IP6 and 60nM IP6K1 for 30 minutes at 37°C. (B) IC50 values decreased slightly from 24±1.5μM to 9.5±1.5μM as IP6 concentrations were increased from 50μM to 400μM. Reactions were carried out with 300μM ATP and 60nM IP6K1 for 15 minutes. Percent activity is defined as 100*(μexperimental-μnegative)/(μpositive–μnegative) where positive is the experimental result with no inhibitor present and negative is the signal produced by the reaction with no IP6 present. All data points are replicated in quadruplicate and represented as the mean ± SEM.
Fig 5.
Assay validation and LOPAC screening results.
The LOPAC was screened to assess the degree to which Promega’s ADP-Glo Max assay can be used to measure IP6K1 activity in a high-throughput manner. (A) Signal to background ratio represents the DMSO control divided by 100μM Myricetin inhibition control. Signal/background = 6.00±0.47. (B) Z’ factor = 0.62±0.05. (C) CV = 8.50±1.57%. (D) Plot of Z score for LOPAC compounds. Compounds below Z of -2.0 are considered hits. Myricetin is highlighted in red and 6-Hydroxy-DL-Dopa in blue. Data presented here are representative of a single LOPAC screen where each compound is screened at one 10μM concentration.
Fig 6.
Chemical structure of inhibitors found in LOPAC screen.
(A) Myricetin and (B) 6-Hydroxy-DL-Dopa.
Fig 7.
LOPAC hit validation and counter-screening.
(A) Dose response assay for inhibition of IP6K1 by 6-Hydroxy-DL-Dopa and Myricetin. Reactions were run with 1mM ATP, 100μM IP6, 60nM IP6K1, for 30 minutes at 37°C. Myricetin and 6-Hydroxy-DL-Dopa were able to inhibit IP6K1 with an IC50 of 4.96±1.06μM and 1.84±1.03μM respectively. Percent activity is defined as 100*(μexperimental-μnegative)/(μpositive–μnegative) where positive is the experimental result with no inhibitor present and negative is the signal produced at the highest concentration of inhibitor. (B) IP6K1 inhibitors Myricetin, TNP, and 6-Hydroxy-DL-Dopa were tested at 10μM against 25μM ADP and 975μM ATP. Percent signal is defined as 100*(μexperimental–μnegative)/(μpositive–μnegative) where the positive is the 5% DMSO control and the negative contains no IP6. All data points are replicated in quadruplicate and represented as the mean ± SEM. Significance was determined via one-way ANOVA testing where p < 0.05.
Fig 8.
PAGE separation of IP6 and 5PP-IP5 shows Myricetin and 6-Hydroxy-DL-Dopa inhibit IP6K1 catalytic activity.
A representative result of numerous gel separations is shown here. When incubated with Myricetin or 6-Hydroxy-DL-Dopa 5PP-IP5 production by IP6K1 is dramatically reduced. Each reaction is carried out with 10μM inhibitor, 1mM ATP, and 250μM IP6 with 60nM IP6K1 for 2 hours at 37°C.
Fig 9.
LOPAC hits inhibit IP6K2 and IP6K3.
Dose response assay for inhibition of (A) IP6K2 and (B) IP6K3 by Myricetin and 6 -Hydroxy-DL-Dopa. Myricetin and 6-Hydroxy-DL-Dopa were able to inhibit IP6K2 with an IC50 of 23.41±1.10μM and 1.66±1.06μM respectively and inhibit IP6K3 with an IC50 of 4.93±1.14μM and 11.10±1.13μM respectively. Reactions were run with (A) 1mM ATP, 100μM IP6, 7.5nM IP6K2, for 30 minutes at 37°C or (B) 1mM ATP, 100μM IP6, 120nM IP6K3, for 120 minutes at 37°C. Percent activity is defined as 100*(μexperimental-μnegative)/(μpositive–μnegative) where positive is the experimental result with no inhibitor present and negative is the signal produced at the highest concentration of inhibitor.