Fluorescence polarization-based biochemical high throughput dose response screening assay to identify inhibitors of ADP-ribosylation factor GTPase activating protein 1 (ARFGAP1)
Name: Fluorescence polarization-based biochemical high throughput dose response screening assay to identify inhibitors of ADP-ribosylation factor GTPase activating protein 1 (ARFGAP1). ..more
BioActive Compounds: 3
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: University of North Carolina at Chapel Hill
Assay Provider: Qisheng Zhang, University of North Carolina at Chapel Hill
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1R21NS073041 (Fast Track)
Grant Proposal PI: Qisheng Zhang, University of North Carolina at Chapel Hill
External Assay ID: ARFGAP1_INH_FP_1536_3XIC50 DRUN
Name: Fluorescence polarization-based biochemical high throughput dose response screening assay to identify inhibitors of ADP-ribosylation factor GTPase activating protein 1 (ARFGAP1).
The low molecular weight ADP-ribosylation factors (Arfs) regulate actin remodeling, vesicle trafficking, membrane lipid composition, and phospholipid metabolism (1). Arfs are members of the Ras family of GTP-binding proteins, switching between the GTP- and GDP-bound forms (2). Arf GTP binding and GTP hydrolysis is regulated by ARFGAPs (ARF GTPase-activating proteins) which associate with the Golgi apparatus and possess a conserved zinc-finger GAP catalytic domain. ARFGAPs such as ARFGAP1 and ASAP1 are found in cell structures involved in vesicle production and trafficking, adhesion, migration, and development (3, 4). ARFGAP1 promotes hydrolysis of ARF1-bound GTP and is required for dissociation of coat proteins from Golgi-derived membranes and vesicles. ARFGAP1 is stimulated by phosphoinosides and inhibited by phosphatidylcholine (4). Dysfunctional regulation of ARFGAPs has been implicated in various diseases, including cancer, alzheimer disease, and autism (3). However, the catalytic mechanism and specific disease-associated roles of ARFGAPs are unclear (2), but recent studies suggest a role for Ca2+ in stimulating ARFGAP-mediated GTP hydrolysis (2). As a result the identification of modulators of ARFGAPs would provide useful tools to elucidate ARFGAP biology (4,5).
1. Donaldson, J.G. and C.L. Jackson, ARF family G proteins and their regulators: roles in membrane transport, development and disease. Nat Rev Mol Cell Biol, 2011. 12(6): p. 362-75.
2. Ismail, S.A., I.R. Vetter, B. Sot, and A. Wittinghofer, The structure of an Arf-ArfGAP complex reveals a Ca2+ regulatory mechanism. Cell, 2010. 141(5): p. 812-21.
3. Sabe, H., Y. Onodera, Y. Mazaki, and S. Hashimoto, ArfGAP family proteins in cell adhesion, migration and tumor invasion. Curr Opin Cell Biol, 2006. 18(5): p. 558-64.
4. Hashimoto, S., A. Hashimoto, A. Yamada, Y. Onodera, and H. Sabe, Assays and properties of the ArfGAPs, AMAP1 and AMAP2, in Arf6 function. Methods Enzymol, 2005. 404: p. 216-31.
5. Sun, W., J.L. Vanhooke, J. Sondek, and Q. Zhang, High-Throughput Fluorescence Polarization Assay for the Enzymatic Activity of GTPase-Activating Protein of ADP-Ribosylation Factor (ARFGAP). J Biomol Screen, 2011.
biochemical, protein, end-point, endpoint, ADP-ribosylation factor GTPase activating protein 1, rat, ARF, GAP, ARFGAP, Arfgap1, GTPase activating protein for ADP-ribosylation factor, FP, fluorescence, fluorescence polarization, inhibitor, inhibit, primary screen, HTS, DRUN, dose response, high throughput screen, AD, cancer, metastatic, neurodegenerative, 1536, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to determine dose response curves for compounds that confirmed ARFGAP activity in a set of previous experiments entitled, "Fluorescence polarization-based biochemical primary high throughput screening assay to identify inhibitors of ADP-ribosylation factor GTPase activating protein 1 (ARFGAP1)" (AID 651572) and "Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of ADP-ribosylation factor GTPase activating protein 1 (ARFGAP1)" (AID 651590). This biochemical assay employs fluorescence polarization to monitor the ability of ARFGAPs to reduce the capacity of ARF1 to hydrolyze guanosine triphosphate. Specifically, this assay monitors the capacity of the GAP domain of rat Arfgap1(residues 1-136) to enhance the intrinsic GTPase activity of human ARF1 lacking its first 17 residues. Although using the GAP domain instead of the full-length Arfgap1 might potentially miss allosteric inhibitors, the His6-[1-136]Arfgap1 has better solubility than the full-length protein and can be produced in bacteria in large quantities. Polarization is a measure of the change in molecular movement of a labeled species and is defined as the ratio of the difference between the vertical and horizontal components of emitted light over their sum. Because polarization is a dimensionless value, it is independent of the emitted light or concentration of fluorophore.
In this assay, purified Arfgap1 protein (residues 1-136) is incubated with ARF (lacking its first 17 residues, previously loaded with GTP). After GTP hydrolysis the mixture is then incubated with of a fluorescent version GDP (GDP Alexa 633 Tracer), initially antibody-bound and having high fluorescence polarization (mP). Free GDP derived from ARF-mediated GTP hydrolysis displaces the fluorescent tracer from the antibody, leading to decreased mP. As designed, compounds that act as Arfgap1 inhibitors will reduce GTP hydrolysis, thereby preventing displacement of the tracer, resulting in no decrease in mP. Compounds are tested in triplicate using a dilution series starting at a nominal maximum concentration of 165 uM.
Prior to the start of the assay, 2 uL of a solution containing 5 uM Arfgap1 in assay buffer (25 mM HEPES, 150 mM NaCl, 1mM MgCl2, 1 mM DTT, pH 7.5) were dispensed into wells 3-48 and 2 uL assay buffer alone were added to wells 1-3 of a 1536 well-plate. Next, 67.41 nL of test compound in DMSO or DMSO alone (1.26% final concentration) were added to the appropriate wells and incubated for 20 minutes at 25 C.The assay was started by the addition of 2 uL of 10 uM ARF1-GTP to all wells. Plates were centrifuged and after 120 minutes of incubation at 25 C, 4 uL of detection buffer (1X Stop and Detection Buffer, 8 nM GDP Alexa 633 Tracer, and 3.8 ug/mg GDP antibody in assay buffer, were added to all wells. Plates were centrifiged and after 60 minutes incubation at 25 C, fluorescence polarization was read on an EnVision microplate reader (PerkinElmer, Turku, Finland) using a Cy5 FP filter set and a Cy5 dichroic mirror (excitation = 620 nm, emission = 688 nm). Fluorescence polarization was read for 50 seconds for each polarization plane (parallel and perpendicular).
Prior to further calculations, the following formula was used to calculate fluorescence polarization (FP):
FP = ( Raw2 - Raw1 ) / ( Raw1 + Raw2 )
Raw1 is defined as the P channel.
Raw2 is defined as the S channel.
The percent inhibition for each compound was calculated as follows:
%_Inhibition = 100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )
Low_Control is defined as wells containing Arfgap1, ARF1-GTP and DMSO.
Test_Compound is defined as wells containing Arfgap1 and ARF1-GTP in the presence of test compound.
High_Control is defined as wells containing DMSO, ARF1-GTP but, no Arfgap1 protein.
For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Accelrys Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. In cases where the highest concentration tested (i.e. 165 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 165 uM.
PubChem Activity Outcome and Score:
Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.
Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score greater than zero.
Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for active compounds is 100-97, and for inactive compounds 95-0.
List of Reagents:
Arfgap1 protein (GAP domain) (supplied by Assay Provider)
ARF1-GTP (supplied by Assay Provider)
Assay buffer containing 25 mM HEPES, 150 mM NaCl, 1 mM MgCl2, 1 mM DTT at pH 7.5
Detection buffer containing 1X Stop and Detection Buffer (20 mM HEPES, 40 mM EDTA, 0.02% Brij-35), 4 nM GDP Alexa 633 Tracer (BellBrook Labs, Madison, WI), and 3.8 ug/mg GDP antibody (BellBrook Labs, Madison, WI) in assay buffer.
Ethylenediaminetetraacetic acid (Fisher BP2482-1)
Sodium chloride (Fisher, part S640-500)
Dithiothreitol (Acros, part 16568-0250)
HEPES (Life Technologies, part 15630080)
Magnesium chloride (Fisher, part M35-500)
Brij-35 (Astoria-Pacifica, part 90-0710-04)
1536-well plates (Corning, part 7561)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR. The MLSMR was unable to provide all compounds selected for this assay.
Assay: Dictionary: Version: 0.1
Assay: CurveFit : Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]
Assay: CurveFit : Mask: Excluded Points
* Activity Concentration. ** Test Concentration.
Data Table (Concise)