Fluorogen/soluble FAP binding competition assay
Assay Provider: Jonathan Jarvik, Carnegie Mellon University Screening Center/ PI: UNMCMD/ Larry Sklar ..more
BioActive Compounds: 2
Depositor Specified Assays
University of New Mexico Assay Overview:
Assay Support: R03 DA031668-01
Project Title: HTS for Non-Canonical Ligands for Beta 2 Adrenergic Receptor Internalization
Assay Provider: Jonathan Jarvik, Carnegie Mellon University Screening Center/ PI: UNMCMD/ Larry Sklar
Lead Biologist: Yang Wu
Chemistry Center/ PI: Vanderbilt Specialty Chemistry Center/Craig Lindsley Chemistry Center Lead: Shaun Stauffer
Assay Implementation: Greg Fisher, CMU
Assay Background and Significance:
The Carnegie Mellon University Technology Center for Networks and Pathway (CMU TCNP) has developed a novel fluorogen activating peptide (FAP) technology. Fluorogens are small molecules that are not fluorescent, but acquire fluorescence when rotational motion about certain bonds within the molecule is constrained. Within the NIH Roadmap-funded CMU TCNP, single chain antibodies (scFvs) that bind specific fluorogens and activate their fluorescence were identified and improved by successive rounds of mutagenesis and selection [Fischer et al, 2010]. These proteins are called FAPs, or fluorogen activating proteins.
The strong (Kd < 10nM) and specific binding between a particular FAP and fluorogen can, in principle, be blocked or reversed by a specific competitive or noncompetitive interaction with a different molecule. The screen that was undertaken in the original MH093192 (HTS for Non-canonical Ligands for Beta 2 Adrenergic Receptor Internalization) project was designed to reveal new effectors of beta2-AR function, but its design was such as to also reveal inhibitors of the FAP-fluorogen interaction. In fact, further analysis using the first 20 compounds provided for validation by VUSCC indicates that some of them interfere with fluorogen activation rather than receptor stimulation. This opens the door for the design of new tool sets for FAP based technology.
FAP technology is an innovative technology that was first published on in 2008. It is not surprising to discover amongst the hits from the MLPCN screen targeting beta2-AR modulators, a new class of molecules that interfere with FAP-fluorogen binding. There are no such molecules reported in the literature to date, and the proposed probes will be the first in this class. These compounds will help in advancing science by providing the opportunity to efficiently separate false positives from target-specific compounds in the hit list of high-throughput screening, to monitor receptor trafficking and receptor location in real time, and with the potential to extend the application to animal models.
Competition binding assays will be performed by adding the compounds to mixtures of soluble AM2.2 (FAP-tag) and TO1-2p (fluorogen), and the fluorescent signal measured by fluorimetry.
* purified AM2-2 was diluted 50/200uL in cPBS and TO1-2p was added (10X Kd concentration)
* 10mM of each of the two compounds was made up in DMSO
* antagonists were diluted 1/10 into ddH20 and then diluted 1/20 first well and 1/10 thereafter
* plates were scanned in the SafireII Tecan plate reader (541nm emission maximum)
Raw fluorescence readings for the entire concentration range of a test compound were fitted by Prism(R) software (GraphPad Software, Inc., San Diego, CA) using nonlinear least-squares regression in a sigmoidal dose response model with variable slope, also known as the four parameter logistic equation. Curve fit statistics were used to determine the following parameters of the model: EC50, microM - concentration of added test compound competitor that inhibited fluorescent ligand binding by 50 percent; LOGEC50 - the logarithm of EC50; TOP - the response value at the top plateau; BOTTOM - the response value at the bottom plateau; HILLSLOPE - the slope factor, or the Hill coefficient; STD_LOGEC50, STD_TOP, STD_BOTTOM, STD_HILLSLOPE - standard errors of LOGEC50, TOP, BOTTOM, and HILLSLOPE ; EC50_95CI_LOW, EC50_95CI_HIGH - the low and high boundaries of the 95% confidence interval of the EC50 estimate, RSQR - the correlation coefficient (r squared) indicative of goodness-of-fit.
Compounds with EC50 less than 30 microM were active.
Keywords: UNMCMD, CMU
** Test Concentration.
Data Table (Concise)