In HEK-293 cells expressing AT1a receptors, 250 nM PIK93 given 10 min before angiotensin II did not detectably alter the reported InsP3 or calcium responses. This model-specific negative finding limits generalization from biochemical PI4KIIIβ inhibition to all phosphoinositide-dependent cellular responses. [3]
In COS-7 cells, 250 nM PIK93 given for 10 min before the assay reduced fluorescent ceramide accumulation in the Golgi. This transport phenotype and the negative angiotensin II signaling result concern different models and endpoints; neither establishes exclusive target attribution. [4,3]
Structural binding of PIK-93 to PI3K p110γ
Biochemical assay · Recombinant human p110γ; inhibitor-bound X-ray crystal structure [2]
The PIK-93 cocrystal places the inhibitor in the p110γ binding site, with reported contacts to Val882 and Asp964. This establishes PI3K binding as a limitation on interpreting PIK-93 as a PI4KIIIβ-specific probe.
Literature-Reported Treatment Conditions
- Method
- Inhibitor soaking of recombinant p110γ crystals and X-ray diffraction.
- Reported limits
- Inhibitor soaking concentration and detailed refinement statistics require the unacquired supplement; no quantitative binding affinity adopted.
Methods and source locations
Compound reported in the paper: PIK-93; Compound label in the paper: PIK-93
Research compound-level evidence; no validation of A10731 material, purity, formulation or lot. No salt conversion or stereochemical extrapolation.
Results P21–P22; Figure 2D (PIK-93 x-ray); Experimental Procedures P50; Figure 1A identity
PIK-93 contacts in the p110γ cocrystal: X-ray crystallography; qualitative contacts reported in Results P22, not inferred affinity. — Results P21–P22; Figure 2D (PIK-93 x-ray); Experimental Procedures P50; Figure 1A identity
PI4KA/PI4KB inhibition across two assay formats
Biochemical assay · Affinity-purified FLAG-tagged human PI4KA and PI4KB [1]
PIK93 inhibited PI4KB more potently than PI4KA in both assay formats. The radiometric IC50 values were higher than the ADP-Glo values; these are assay-dependent endpoints, not treatment doses.
Literature-Reported Treatment Conditions
- Treatment concentration
- PIK93 concentration-response series; individual series concentrations not tabulated.
- Exposure duration
- 10 min inhibitor preincubation. Radiometric reaction: 30 min at room temperature. ADP-Glo shared methods allow reactions up to 60 min; exact Figure 6 reaction duration is not separately specified.
- Method
- Purified-enzyme comparison; ADP-Glo inhibitor assays use 100 μM ATP and 800 μM phosphatidylinositol. Radiometric conditions are described separately in P14.
- Reported limits
- PIK93 stock solvent/final vehicle fraction is not specified in the acquired main text. No IC50 confidence intervals are given in the adopted results.
Methods and source locations
Compound reported in the paper: PIK-93; Compound label in the paper: PIK93
Research compound-level evidence; no validation of A10731 material, purity, formulation or lot. No salt conversion or stereochemical extrapolation.
Results P22, inhibitor profiling of PI4KA and PI4KB; Figure 6 and caption; Materials P6; assay methods P11–P14
Inhibition of purified human PI4KB activity (ADP-Glo): ADP-Glo detects reaction ADP; radiometric assay quantifies labeled PtdIns4P. Blank-subtracted signal normalized to inhibitor-free enzyme control; four-parameter nonlinear regression. Means ± SD from triplicates in three independent experiments. — Results P22, inhibitor profiling of PI4KA and PI4KB; Figure 6 and caption; Materials P6; assay methods P11–P14
Inhibition of purified human PI4KA activity (ADP-Glo): ADP-Glo detects reaction ADP; radiometric assay quantifies labeled PtdIns4P. Blank-subtracted signal normalized to inhibitor-free enzyme control; four-parameter nonlinear regression. Means ± SD from triplicates in three independent experiments. — Results P22, inhibitor profiling of PI4KA and PI4KB; Figure 6 and caption; Materials P6; assay methods P11–P14
Inhibition of purified human PI4KB activity (radiometric): ADP-Glo detects reaction ADP; radiometric assay quantifies labeled PtdIns4P. Blank-subtracted signal normalized to inhibitor-free enzyme control; four-parameter nonlinear regression. Means ± SD from quadruplicates in two independent experiments. — Results P22, inhibitor profiling of PI4KA and PI4KB; Figure 6 and caption; Materials P6; assay methods P11–P14
Inhibition of purified human PI4KA activity (radiometric): ADP-Glo detects reaction ADP; radiometric assay quantifies labeled PtdIns4P. Blank-subtracted signal normalized to inhibitor-free enzyme control; four-parameter nonlinear regression. Means ± SD from quadruplicates in two independent experiments. — Results P22, inhibitor profiling of PI4KA and PI4KB; Figure 6 and caption; Materials P6; assay methods P11–P14
Limited effect on angiotensin II–evoked signaling
Cell-based study · HEK-293 cells stably expressing AT1a angiotensin receptors [3]
At 250 nM, PIK93 did not detectably alter the reported InsP3 or cytoplasmic calcium responses to angiotensin II, unlike the comparator inhibitor responses. This negative result is specific to the tested concentration and model; it is not a statistical equivalence result.
Literature-Reported Treatment Conditions
- Treatment concentration
- 250 nM PIK93; angiotensin II stimulus 10^-7 M.
- Exposure duration
- PIK93 added 10 min before angiotensin II; Figure 1A follows responses through 600 s.
- Method
- Cellular signaling time courses; stimulated control without PIK93. Comparators: 10 μM wortmannin or 10 μM PAO with 1 mM β-mercaptoethanol.
- Reported limits
- PIK93 vehicle and final vehicle fraction not specified in the acquired material. No statistical equivalence test reported for this negative comparison.
Methods and source locations
Compound reported in the paper: PIK-93; Compound label in the paper: PIK93
Research compound-level evidence; no validation of A10731 material, purity, formulation or lot. No salt conversion or stereochemical extrapolation.
Results: Pharmacological Manipulations of Type III PI 4-Kinase Activities Affect Agonist-regulated PtdIns4P Pools; Figure 1A/B and caption; Materials; calcium/inositol-phosphate methods
Angiotensin II–stimulated InsP3 time course: Myo-[3H]inositol labeling and HPLC separation of soluble inositol phosphates. Figure 1A: means ± range of duplicate determinations; two additional experiments at the 10-min time point had similar results. — Results: Pharmacological Manipulations of Type III PI 4-Kinase Activities Affect Agonist-regulated PtdIns4P Pools; Figure 1A/B and caption; Materials; calcium/inositol-phosphate methods
Angiotensin II–stimulated cytoplasmic calcium time course: Fura-2/AM spectrofluorometry. Figure 1B is representative of three similar observations; no numeric effect size or equivalence interval adopted. — Results: Pharmacological Manipulations of Type III PI 4-Kinase Activities Affect Agonist-regulated PtdIns4P Pools; Figure 1A/B and caption; Materials; calcium/inositol-phosphate methods
Reduced ceramide analogue accumulation in the Golgi
Cell-based study · COS-7 cells; fluorescent C5-BODIPY-ceramide transport assay [4]
PIK93 reduced fluorescent ceramide accumulation in the Golgi compared with untreated control; wortmannin produced a similar transport phenotype. This observed response does not establish exclusive PI4KIIIβ target attribution.
Literature-Reported Treatment Conditions
- Treatment concentration
- 250 nM PIK93; 10 μM wortmannin comparator.
- Exposure duration
- 10 min inhibitor pretreatment; uptake time course through 25 min at 37°C in Figure 5C.
- Method
- Fluorescent ceramide transport measured by confocal imaging after analogue loading and warming.
- Reported limits
- PIK93 vehicle fraction is not specified in the acquired manuscript. Exact effect size and uncertainty are not tabulated for Figure 5C.
Methods and source locations
Compound reported in the paper: PIK-93; Compound label in the paper: PIK93
Compound-level research evidence; no A10731 lot, purity or formulation validation.
JBC Papers in Press manuscript: Materials p. 2; confocal/uptake-ratio methods p. 3; Results p. 5; Figure 5B/C p. 17 and caption p. 11
Golgi-to-cytoplasmic fluorescence uptake-ratio time course: Confocal imaging and linescan analysis. Figure 5C reports three independent experiments with 6–8 cells per time point; error bars omitted in the original. No exact curve-derived percentage adopted. — JBC Papers in Press manuscript: Materials p. 2; confocal/uptake-ratio methods p. 3; Results p. 5; Figure 5B/C p. 17 and caption p. 11
