Fig 1.
Schematic depicting the three phases of the HTS Assay for PPIP5K.
The schematic describes how the degree of PPIP5K activity is inversely proportional to the magnitude of the HTRF signal. During the kinase reaction 5-InsP7 phosphorylation to 1,5-InsP8 is coupled to ATP conversion to ADP. After 60 minutes the kinase reactions are quenched with EDTA (not shown) and the ADP detection reagents are added. The HTRF signal is measured after another 30 minutes. (A) In the absence of inhibitor there is production of ADP, which competes with the ADP tracer for the ADP antibody, resulting in a low HTRF signal. (B) In the presence of inhibitor, ADP production is decreased thereby allowing ADP tracer to bind to the ADP antibody, resulting in a high HTRF signal. The hypothetical examples shown in (A) and (B) represent two extreme assay outcomes of 100% and 0% phosphorylation respectively.
Table 1.
Steps of the PPIP5K HTS Assay.
Fig 2.
Application of the PKIS library to judge performance of the HTS assay.
(A) Representative technical replicates measured on the same day. (B) Comparison of the mean values of biological replicates (black and white circles) obtained on two different days. R2 = 0.98. (C) Z’ Factor (0.78 ± 0.03) (D) %CV (7.5 ± 1.6) (E) Signal:background ratio (15.8 ± 1.5) (F) These plates included negative controls (DMSO; gray circles, broken line and positive controls (100 μM UNC10225354; gray square, broken line). Each plate also contained one 10-point titration for UNC10225354; data (black circles) depict means and SEMs (n = 5). IC50 = 4.8 ± 0.3 μM. In these experiments, 100% activity is equivalent to consumption of 19.3 ± 1.1% of the ATP.
Fig 3.
HTS of PPIP5K against a kinase-focused library of potential nucleotide antagonists.
(A) Representative technical replicates measured on the same day. (B) Comparison of the mean values of biological replicates (black and white circles) obtained on two different days. R2 = 0.99. (C) Z’ Factor (0.82 ± 0.06) (D) %CV (8.6 ± 1.3) (E) Signal:background ratio (16.4 ± 1.1) (F) These plates included negative controls (0.5% DMSO; gray circle, broken line) and positive controls (20 μM UNC10225354; gray square, broken line). Each plate also contained one 10-point titration for UNC10225354; data (black circles) depict means and SEMs (n = 5). IC50 = 5.2 ± 0.2 μM. In these experiments, 100% activity is equivalent to consumption of 18.9 ± 1.5% of the ATP.
Fig 4.
Structures and dose-response relationships for three inhibitors of PPIP5Ks.
Structures for (A) UNC10112646 (B) UNC10225354 and (C) UNC10225498 (D) Dose-response curves for the inhibition of PPIP5K by UNC10225354 (IC50 = 5.24 ± 0.18 μM), UNC10225498 (IC50 = 2.14 ± 0.07 μM), and UNC10112646 (IC50 = 6.96 ± 0.03 μM). (E) Counterscreen results for the three inhibitors performed in the absence of PPIP5K and 5-InsP7 show that these inhibitors do not interfere with the detection reagents and assay signal. In these experiments, 100% activity is equivalent to consumption of 19.5 ± 0.8% of the ATP.
Fig 5.
Analysis by ITC of the interaction of UNC10225498 and UNC10112646 with PPIP5K The upper panels show the raw data for heat output from the ligand/protein titrations; the lower panels show the least squares fitting of the titration data assuming a single site binding model.
(A) UNC10225498; Kd = 1.37 ± 0.03 μM (B) UNC10112646; Kd = 7.30 ± 0.03 μM. Representative data are shown. Kd values represent means and standard deviations from two experiments
Fig 6.
Analysis of the mechanism of inhibition of PPIP5K by UNC10112646 and UNC10225498.
Assays were performed in HTS format with 2-fold serial dilutions from 100 μM of either UNC10112646 (squares) and UNC10225498 (circles) and varying concentrations of ATP as indicated. Data represent means and standard errors from 3 experiments. The mean Ki values for inhibition of PPIP5K by UNC10225498 and UNC10112646 were 2.0 ± 0.6 μM and 3.6 ± 1.3 μM, respectively. In these experiments, the uninhibited PPIP5K activity is equivalent to consumption of 18.9 ± 0.9% of the ATP.
Fig 7.
The docking poses of UNC10225498 and UNC10112646 with PPIP5K.
(A) UNC10225498 (thick sticks; orange carbons) (B) UNC10112646 (thick sticks; green carbons). The ATP pocket is outlined as a gray transparent surface and ATP itself is depicted by thin magenta sticks. Residues predicted to interact with docked compounds are highlighted (see text for details).
Fig 8.
HPLC analysis of the effects of UNC10225498 and UNC10112646 upon the kinase activities of PPIP5K and IP6K.
Kinase reactions contained either vehicle (0.5% DMSO) or inhibitor in 0.5% DMSO. Reactions were quenched and analyzed by HPLC as described in the methods section. Representative HPLC data are shown for both (A) PPIP5K and (C) IP6K, including no enzyme control (open circles), vehicle control (closed circles), 10 μM UNC10225498 (‘5498’, light gray circles), or UNC10112646 (‘2646’, dark gray circles). Panels B and D show the means and SEM for the percentage of product formed in 3 experiments.