Biochemical assay for compounds that inhibit RGS4 stimulation of Galpha1 nucleotide hydrolysis under receptor-independent steady-state conditions
Name: Biochemical assay for compounds that inhibit RGS4 stimulation of Galpha1 nucleotide hydrolysis under receptor-independent steady-state conditions. ..more
BioActive Compounds: 28
Depositor Specified Assays
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
MLPCN Screening Center: Johns Hopkins Ion Channel Center
MLPCN Screening Center PI: Min Li, Ph.D.
Screening Center: Owen McManus, Ph.D., Meng Wu Ph.D.
Grant Proposal Number: 1 R03 MH087441-01A1
Grant Proposal PI: Richard Neubig, M.D., Ph.D. (Department of Pharmacology, University of Michigan)
Assay Execution: Jian P. Mei, Andrew Storaska (Department of Pharmacology, University of Michigan)
Name: Biochemical assay for compounds that inhibit RGS4 stimulation of Galpha1 nucleotide hydrolysis under receptor-independent steady-state conditions.
Regulators of G-protein signaling (RGS)-proteins regulate G-protein coupled receptor (GPCR) signal transduction by enhancing the intrinsic rate of guanine nucleotide hydrolysis on the Galpha subunit [1, 2]. The significant influence over GPCR signaling has garnered interest in targeting RGS proteins therapeutically [3-5]. In particular, RGS4 is expressed in the SA-node and widely across the CNS, but is limited in peripheral tissues [6, 7]. RGS4 has potential involvement in the pathology of pain where RGS4 mRNA is upregulated in response to nerve injury models of neuropathic pain . Additionally, RGS4 may be involved in negative regulation of insulin release from pancreatic beta cells, indicated by enhanced glucose tolerance and insulin release in pancreatic beta-cell-specific RGS4 k/o mice . Antagonism of RGS4 could allow tissue-specific potentiation of Gi and Gq signaling pathways, while maintaining the natural receptor signaling patterns. Furthermore, an RGS4 inhibitor in combination with a GCPR agonist could provide a synergistic effect, allowing a lower dose of drug to be used, and thereby reducing side effects.
Key words: RGS4, Galphai1, GAP, steady state, kinetic, malachite green, colorimetric, inhibitor, allosteric, antagonist, absorbance, Molecular Libraries Probe Production Centers Network, MLPCN, Michigan, Pharmacology
1. Berman, D.M., T. Kozasa, and A.G. Gilman, The GTPase-activating protein RGS4 stabilizes the transition state for nucleotide hydrolysis. J Biol Chem., 1996. 271(44): p. 27209-12.
2. Berman, D.M., T.M. Wilkie, and A.G. Gilman, GAIP and RGS4 are GTPase-activating proteins for the Gi subfamily of G protein alpha subunits. Cell, 1996. 86(3): p. 445-52.
3. Kimple, A.J., et al., Regulators of G-protein signaling and their Galpha substrates: promises and challenges in their use as drug discovery targets. Pharmacol Rev, 2011. 63(3): p. 728-49.
4. Neubig, R.R. and D.P. Siderovski, Regulators of G-protein signalling as new central nervous system drug targets. Nat Rev Drug Discov, 2002. 1(3): p. 187-97.
5. Zhong, H. and R.R. Neubig, Regulator of G protein signaling proteins: novel multifunctional drug targets. J Pharmacol Exp Ther, 2001. 297(3): p. 837-45.
6. Cifelli, C., et al., RGS4 regulates parasympathetic signaling and heart rate control in the sinoatrial node. Circ Res, 2008. 103(5): p. 527-35.
7. Gold, S.J., et al., Regulators of G-protein signaling (RGS) proteins: region-specific expression of nine subtypes in rat brain. J Neurosci, 1997. 17(20): p. 8024-37.
8. Garnier, M., et al., Up-regulation of regulator of G protein signaling 4 expression in a model of neuropathic pain and insensitivity to morphine. J Pharmacol Exp Ther, 2003. 304(3): p. 1299-306.
9. Ruiz de Azua, I., et al., RGS4 is a negative regulator of insulin release from pancreatic beta-cells in vitro and in vivo. Proc Natl Acad Sci U S A, 2010. 107(17): p. 7999-8004.
10. Zielinski, T., et al., Two Galpha(i1) rate-modifying mutations act in concert to allow receptor-independent, steady-state measurements of RGS protein activity. J Biomol Screen, 2009. 14(10): p. 1195-206.
11. Chang, L., et al., High-throughput screen for small molecules that modulate the ATPase activity of the molecular chaperone DnaK. Anal Biochem, 2008. 372(2): p. 167-76.
A steady-state GTPase activating protein (SS-GAP) assay was used to monitor compound-mediated inhibition of RGS4 activity. The assay was run in the absence of any receptor, utilizing a recombinant Galphai1 (R178M, A326S) mutant previously described . RGS4 stimulation of basal G-protein nucleotide hydrolysis was measured using malachite green dye, which increases absorbance at 630nm in the presence of inorganic phosphate released from GTP. Compounds were tested in a concentration-response format ranging from 100 uM to 100nM. Activity was based on a reduction in absorbance at 630nm after addition of malachite green dye. On a 384-well plate in triplicates, concentration-response curves for each compound were used to calculate IC50 values constrained to 100% inhibition in every case. Compounds with IC50 values > 100 uM (tagged as 110 uM) were considered inactive.
The procedure for malachite green based detection was adapted from Chang, et al., 2008 .
Protocol for malachite green SS-GAP confirmatory assay:
1. Compounds were prepared at 4x (400 uM) in assay buffer (50mM Hepes, 100mM NaCl, 5 ug/ml BSA, 0.05% Luberol, 5mM EDTA, 10mM MgCl2, pH 7.4)
2. Compounds were serially diluted in 20 ul of assay buffer in half-log molar increments, and 2 ul of each compound concentration was transferred to the assay plate well.
3. Three controls were run on every plate: Control one contained 0.4% DMSO (the highest concentration of DMSO present), RGS4, GTP, and G-protein in order to measure maximum RGS4 stimulation of phosphate release. Control 2 contained G-protein, DMSO (as above), and GTP in order to measure basal G-protein phosphate release. Control 3 contained GTP and DMSO (as above) in order to measure spontaneous GTP hydrolysis during the assay. All concentrations were identical to test wells.
4. 2 ul of 4x (0.8 uM) RGS4-MBP was added to the appropriate wells and allowed to incubate at room temperature with the compounds for 15 minutes.
5. 2 ul of 4x (24 uM) G-protein was added to the appropriate wells.
6. The reaction was initiated with the addition of 2 ul of 4x (1.2mM) GTP, by rows, up the plate at 30 second intervals.
7. After incubating the reaction at room temperature for 2 hours, the reaction was quenched with 10 ul malachite green dye.
8. The plate was put on a plate shaker at 300rpm for 20 seconds and then centrifuged for 30 seconds at 500 rpm.
9. Immediately following, 2 ul 32% sodium citrate was added to each well and the plate was shook and centrifuged as before.
10. Color change was allowed to stabilize by incubating the plate at room temperature for 30 minutes.
11. The plate was read on Victor2 Wallac plate reader measuring absorbance at 630nm.
12. The above procedure was repeated twice for each compound tested and the replicates from each experiment were compiled.
13. Data analysis: Basal G-protein activity (control 2) was subtracted off all values to remove basal signal and then values were normalized to control 1on each plate
14. Data was plotted as Percent DMSO control GTP hydrolysis versus compound concentration and fit to a variable slope IC50 curve, constrained to 100% inhibition.
15. Outcome assignment: IC50 values > 100 uM (tagged as 110 uM) were classified as inactive (value score=1), or compounds with IC50 < 100 uM are considered active and given a value score=2.
List of Reagents:
1. HEPES (Sigma, Cat#H4034)
2. Luberol (MP Biomedicals 195299)
3. BSA (Fisher BP1600-100)
4. Malachite Green Oxalate Salt (MP Biomedicals 152642)
5. Polyvinyl Alcohol (Sigma 363162-500G)
6. Ammonium heptomolybdate (Sigma 431346-50g)
8. Galphai1 (R178M, A326S)
9. GTP (Sigma G5884-100mg)
10. DMSO (Fisher BP231-100)
11. 384-well plate (Corning 3680 clear flat bottom)
12. Sodium Chloride
13. Magnesium Chloride
Possible artifacts of this assay can include, but are not limited to: non-intended chemicals or dust, compounds that non-specifically modulate the cell host or the targeted activity, and compounds that quench or emit light or fluorescence. All test compound concentrations reported are nominal.
* Activity Concentration.
Data Table (Concise)