Biochemical assay for compounds that inhibit AlF4- anion-dependent RGS4 binding to Galpha-o
Name: Biochemical validation assay for compounds that inhibit AlF4- anion-dependent RGS4 binding to Galpha-o ..more
BioActive Compounds: 18
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 validation assay for compounds that inhibit AlF4- anion-dependent RGS4 binding to Galpha-o
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, Galphao, FCPIA, inhibitors, antagonist, allosteric, AlexaFluor, flow cytometry, biotin, 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. Blazer, L.L., et al., Use of flow cytometric methods to quantify protein-protein interactions. Curr Protoc Cytom, 2010. Chapter 13: p. Unit 13 11 1-15.
A flow cytometry protein interaction assay (FCPIA)  was employed to monitor compound-mediated inhibition of RGS4 binding to Galphao in an aluminum fluoride-dependent manner. In this assay, RGS4 (52-205)-MBP fusion protein is biotinylated with NHS-biotin and coupled to avidin-coated microspheres. An AlexaFluor 532-labeled rat Galphao is activated with GDP, aluminum, magnesium, and fluoride (AMF), irreversibly inducing the nucleotide hydrolysis transition state for which RGS has the highest affinity. RGS4-labeled microspheres are then incubated for 30 minutes at room temperature with activated Galphao to reach equilibrium. Binding is measured on a 96-well plate using a Luminex 200 flow cytometer to detect microsphere bead-associated fluorescence. Compounds were tested in a concentration-response format ranging from 100 uM to 30nM. Activity was based on a reduction in median fluorescent intensity (MFI) associated with the microsphere beads in each well. Concentration-response curves for each compound, performed in duplicates, were used to calculate IC50 values constrained to 100% inhibition in every case. Compounds with IC50 values > 100 uM were considered inactive.
Protocol for FPCIA confirmatory assay:
Procedures for performing the FCPIA experiments are as previously described , with the exception of the following
1. Compounds were prepared at 2x concentration (200 uM) in assay buffer (50mM Hepes, 100mM NaCl, 1%BSA, 0.1% Luberol, pH7.4) from 100mM DMSO stocks, or less if 100mM concentration could not be reached in DMSO.
2. Compounds were diluted by half-log molar concentration increments in 50 ul of assay buffer down the assay plate.
3. Two controls were setup on every plate: Control one comprised assay buffer plus 0.2% DMSO (the highest concentration of DMSO present) in order to measure total binding on each plate. Control two measured non-specific binding of Galphao to microspheres by adding a vast excess (0.5 uM final) of unbiotinylated RGS4-MBP to antagonize all specific binding of Galphao to RGS4-labeled microspheres.
4. 20 ul of RGS4-coupled microspheres, approximately 75 microspheres/ul, were added to each well and allowed to incubate with the compound for 15 minutes at room temperature.
5. 30 ul of 3.3x (66nM) of AMF-activated Galphao-AF532 was added to each well and allowed to incubate at room temperature for 30 minutes.
6. The compound plate was then read to measure MFI associated with the microsphere beads in each well.
7. Data analysis: Values from control 2 were used to subtract signals due to non-specific binding from all of the experimental values on each plate.
8. MFI at each concentration of compound was normalized using control 1 on each plate by plotting as percent of DMSO control-Galphao binding
9. The above procedure was repeated twice for each compound tested.
10. Replicates from each of the two experiments for each compound were compiled. Percent DMSO control-Galphao binding for each compound concentration was fit to a variable slope IC50 curve constrained to 100% inhibition.
11. Outcome assignment: IC50 values > 100 uM (IC50 denoted 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.
12. Score assignment: An active test compound is assigned a score between 0 and 100. If Outcome=2, it is assigned as 100. If Outcome=1, it is assigned as 0.
List of Reagents
1.NHS Biotin (SigmaB2643-100mg)
2.AlexaFluor 532 carboxylic acid (Invitrogen A20001)
4.Hexahistidine Galphao labeled with AlexaFluor 532
5.Avidin coated microspheres (XMap Reagents L100-LXXX-01)
6.96-well PCR plate (ISC Bioexpress, cat. no. T-3082-1)
7.HEPES (Sigma, Cat#H4034)
8.BSA (Fisher BP1600-100)
9.Luberol (MP Biomedicals 195299)
10.GDP (Simga G7127-500mg)
11.DMSO (Fisher BP231-100)
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.
Categorized Comment - additional comments and annotations
* Activity Concentration.
Data Table (Concise)