qHTS Assay for Identifying the Cell-Membrane Permeable IMPase Inhibitors: Potentiation with Lithium
Lithium has been widely used for the treatment of bipolar disorder. But lithium has a narrow therapeutic index and it can cause side effects such as thirst, weight gain, tremor, polyuria and memory problems. Although the mechanism for lithium action in treatment of bipolar disorder is still not fully understood, inhibition of inositol monophosphatase (IMPase) and the subsequent depletion of the more ..
BioActive Compounds: 727
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production centers Network [MLPCN]
MLPCN Grant: X01 MH082413-01
Assay Submitter (PI): Wei Zheng
NCGC Assay Overview:
Lithium has been widely used for the treatment of bipolar disorder. But lithium has a narrow therapeutic index and it can cause side effects such as thirst, weight gain, tremor, polyuria and memory problems. Although the mechanism for lithium action in treatment of bipolar disorder is still not fully understood, inhibition of inositol monophosphatase (IMPase) and the subsequent depletion of the inositol pool in living cells have been implicated as the primary therapeutic benefit of lithium therapy. Therefore, a selective IMPase inhibitor with reduced side effects might be a good drug candidate to replace lithium for the treatment of bipolar disorder. Several IMPase inhibitors have been discovered previously. However, they are not suitable for therapeutic use because of their poor cell membrane penetrability or oral bioavailability; the development of new cell-permeable IMPase inhibitors is needed.
IMPase catalyzes the hydrolysis of inositol-1-phosphate (IP1) to release free inositol. Binding of an agonist to a Gq coupled GPCR results in the activation of PLC, which hydrolyzes the PIP2 to form inositol-1,4,5-trisphosphate (IP3) and diacyl glycerol (DAG). While DAG activates protein kinase C (PKC), IP3 triggers the elevation of intracellular free Ca2+. The IP3 is very rapidly inactivated by hydrolysis to IP2, IP1 and then inositol by a series of enzymes including IMPase. Free inositols are used for synthesis of PI and thus start another inositol cycle.
To identify new cell-permeable IMPase inhibitors, we developed a cell-based IMPase assay in which the cytosolic IP1 is measured by a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. In this assay, an IP1 specific antibody is used to detect the labeled IP1 tracer. When the cell lysate contains the elevated IP1 due to the inhibition of IMPase, the TR-FRET between the labeled antibody and IP1 tracer is disrupted resulting in a reduction in the total signal. Since the TR-FRET is used, this cell-based assay is a homogenous assay without cell wash and medium change that is suitable for assay miniaturization and HTS. In this particular assay, a small amount of lithium is added to potentiate the response of weak activators.
NCGC Assay Protocol Summary:
A Chinese hamster ovary (CHO) cell line stably expressing the M1 receptor (CHO-M1) was obtained from ATCC and maintained in F-12 Kaighn's media (Invitrogen, Carlsbad, CA) supplemented with 10 % FBS, 100 units/ml penicillin, 100 ug/ml streptomycin and 250 ug/ml geneticin at 37C, 5% CO2 in a humidified atmosphere. Before the assay, aliquots of cells were frozen and stored at -135C. The assay was performed in 1536-well format. Data are reported for both the ratio of the two emission wavelengths, and also for the component 'donor' channel, Em2=620. Data were normalized to the controls for basal activity (DMSO only) and 100% inhibition (18.4 mM LiCl). AC50 values were determined from concentration-response data modeled with the standard Hill equation.
(1) Frozen CHO-M1 cells were thaw, washed once with fresh media and resuspended in F12K media containing 10% FBS, 100 units/ml penicillin and 100 ug/ml streptomycin. Cells were plated at 4ul/well (2000 cells) in white, tissue culture treated 1536-well plates, and then cultured at 37C, 5 % CO2 for 16 to 30 hours.
(2) Add 23 nl/well of compound in DMSO solution. The final titration for each compound was between 0.6 nM and 46 uM.
(3) Add 1 ul of stimulation reagent (250 uM carbachol and 500 uM LiCl in stimulation buffer containing10mM HEPES, 1mM CaCl2, 0.5 mM MgCl2, 4.2 mM KCl, 146 mM NaCl, 5.5 mM glucose, 50mM LiCl, pH 7.4)
(4) Incubation at 37C, 5 % CO2 for 30 min.
(5) Add 1 ul/well of d2-conjugated IP1 (d2-IP1) and 1 ul/well of Eu+ cryptate conjugated anti-IP1 antibody (Eu+-Ab) prepared in the cell lysis buffer, both from the IP-One kit (Cisbio, MA).
(6) Incubate at room temperature for 10 minutes.
(7) Detect the assay plate (Ex = 320, Em1 =665 and Em2 620) in a ViewLux plate reader.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)