Summary of HTS Screening Project for Inhibitors of fluorogen-FAP tag interactions
Project Title: Small molecule inhibitors of fluorogen-FAP tag interactions: case study of non-fluorescent analogs of thiazole orange ..more
University of New Mexico Assay Overview:
Assay Support: 1 R03 DA031668-01
Project Title: Small molecule inhibitors of fluorogen-FAP tag interactions: case study of non-fluorescent analogs of thiazole orange
Assay Provider: Jonathan Jarvik, Carnegie Mellon University
Screening Center/ PI: UNMCMD/ Larry Sklar
Lead Biologist: Yang Wu
Assay Implementation: Yang Wu, Phillip Tapia, Terry Foutz, Stephanie Chavez, Dominique Perez, Annette Evangelisti, Anna Waller, Cristian Bologa, Mark Carter
Chemistry: Vanderbilt Specialized Chemistry Center for Accelerated Probe Development
Vanderbilt Specialized Chemistry Center PI: Craig Lindsley, Ph.D.
Vanderbilt Chemistry Lead: Shaun Stauffer, Ph.D.
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.
1. Spin down Am2.2-beta2AR cells, discard supernatant, and resuspend in fresh RPMI-1640 full medium. Final cell density will be 5x10^6 cells/mL.
2. Add 5 microL serum free RPMI to Columns 1-10, 12-22 and 24 of the assay plate by Nanoquot.
3. Add 5 microL of freshly prepared 20 microM ISO in RPMI full media to Column 11 and 23 of all the plates as PCntrls by Microflow.
4. Add 100 nanoL of library compounds ranging from 1.5 microM to 10 milliM to assay plates by FX or NX.
5. Add 3 microL of cells to Columns 1 - 11, and 13-23 of the assay plates by Microflow
6. Shake the plates and put them in 37oC incubator for 90mins then transfer the plate to 4oC fridge for 30mins.
7. Add 3 microL 650 nanoM TO1-2p to assay plates by Microflow or Nanoquot to assay plates, allow binding between TO1-2p and compounds for 30mins and read by high-throughput flow cytometers.
Categorized Comment - additional comments and annotations
From MLP Probe Report:
Probe count: 1
MLP Probe ML# for probe 1: ML342
PubChem Substance ID (SID) for probe 1: 136348701
PubChem Compound ID (CID) for probe 1: 2953239
Probe type for probe 1: Inhibitor
IC50/EC50 (nM) for probe 1: 2.24
Target for probe 1: FAPtag (gi: 163932450)
Disease relevance for probe 1: Reasearch Tool
Anti-target for probe 1: MG13
Fold selectivity for probe 1: >800
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK148495/ (ID: 3040604)
Grant number for probe 1: DA031668-01
NCBI Book chapter title for probe 1: Inhibitors of FAP-fluorogen interaction as a multiplex assay tool compound for receptor internalization assays