NMR Based Screening Assay For Novel Chemical Probes Targeting The FRB Domain of mTOR
The mammalian target of rapamycin (mTOR) is a protein that is intricately involved in signaling pathways controlling cell growth (1). Rapamycin is a natural product that binds and inhibits mTOR function by interacting with its FKBP-Rapamycin-Binding (FRB) domain (2) and the proline cis-trans isomerase FKBP12. Several attempts have been made to design molecules that separate the FKBP12 binding more ..
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Probe Production Network (MLPCN)
Grant Number: R03 MH084221
Assay Provider: Dr. Maurizio Pellecchia, Sanford-Burnham Medical Research Institute
The mammalian target of rapamycin (mTOR) is a protein that is intricately involved in signaling pathways controlling cell growth (1). Rapamycin is a natural product that binds and inhibits mTOR function by interacting with its FKBP-Rapamycin-Binding (FRB) domain (2) and the proline cis-trans isomerase FKBP12. Several attempts have been made to design molecules that separate the FKBP12 binding activity from the mTOR inhibitory activity of Rapamycin, but to date no such viable compounds has been reported that are efficient against mTOR. However, we found that Rapamycin binds the FRB domain and inhibits the kinase activity of mTOR even in the absence of FKBP12 albeit at much higher concentrations (low micromolar versus the low nanomolar inhibition observed in presence of FKBP12). Hence, we propose an unbiased NMR-fragment-based approach to develop novel high affinity chemical probes that interfere with the function of mTOR by targeting its FRB domain. These ligands could result very useful in deciphering the complex regulation of mTOR in the cell and in validating the FRB domain as a possible target for the development of novel therapeutic compounds. The chemical structures, SAR data and range of biochemical activities of the resulting compounds will provide a framework onto which to develop potentially novel anti-cancer therapies.
1. Schmelzle, T, and Hall, M. N. (2000) TOR, a Central controller of Cell Growth, Cel. 103, 253-262.
2. Banaszynski, L. A., Liu, C. W., and Wandless, T. J. (2005) Characterization of the FKBP-rapamycin-FRB ternary complex, J. Am. Chem. Soc. 127, 4715-21
1. Protein expression and purification
Expression Plasmid The region encoding for the rapamycin associated protein FRAP2 / FK506 binding protein 12-rampamycin associated protein 1 (Gene ID 2475 FRAP1) (2013-2113) was synthesized by Celtek Bioscience (Nashville, TN 37210, USA) and subcloned into pET15a using the NdeI and BamHI cloning sites. The resulting protein contain the frb domain of FRAP1 (2013-2113) with extra 21 amino acid residues (MGSSHHHHHHSSGLVPRGSHM ) at the N-terminus.
Purification from inclusion bodies and refolding procedure. The recombinant protein was expressed as insoluble inclusion bodies in the Escherichia coli strain BL21(DE3) at 37 0C in minimal M9 media (either unlabeled or with 15NH4Cl as sole source of nitrogen for the preparation of 15N-labeled FRB for hit confirmation). Pure soluble recombinant protein was obtained by using the following protocol:
A. Preparation of inclusion bodies The bacteria pellet was lysed by sonication in the lysis buffer (100mM Tris-HCl, pH 8.0, 300mM NaCl, 0.1% NaN3 and 0.1mM PMSF ) and washed three times using the same buffer. The recombinant protein was dissolved in 6M guanidine-HCl.
B. Refolding. Protein refolding was done by adding solublized inclusion bodies drop-wise into a refolding buffer under rapid stirring conditions. The refolding buffers include 100mM Tris-HCl, pH 8.0, 50mM L-Arginine 300mM NaCl, 5mM EDTA, 0.1% NaN3 and 0.1mM PMSF.
C. Purification The refolded protein was centrifuged 20min at 19000rpm to remove the precipitant, and then purified using Ni2+ affinity chromatography.
The final NMR samples were buffer exchange to 100mM phosphate buffer (pH 7.0) containing 90%/10% (H2O/2H2O) or 99.5% 2H2O.
2. Compound Library Screened by NMR
We have assembled a scaffold library composed of ~ 4,000 compounds. The compounds have been selected based on their anticipated use as building blocks or scaffolds components of further optimized molecules. The scaffold library has been acquired from three different sources and the chemical structures of the library have been deposited into PubChem. In line with the general NIH Molecular Libraries Screening Centers Network (MLSCN) library, we have also included a collection of 602 Natural Products (MicroSource) that could be screened by NMR
3. Screening description
Primary ligand binding was monitored by comparing the aliphatic region of 1D 1H NMR spectra of a 10 uM FRB solution (100 mM sodium phosphate buffer at pH 7.5 containing 100% D2O; T= 300 K) in the presence and in absence of compounds tested at a final concentration of 100 uM. Compounds were initially tested at mixtures of 20, and then individual compounds for those mixtures that caused significant perturbations in the spectrum were further deconvoluted.
Secondary, hit confirmation was accomplished by 2D [15N,1H] NMR HSQC spectra with 15N labeled FRB domain measured at 100 uM concentration in presence and absence of test compounds (at 500 uM).
Several hits were identified initially; however, further 2D NMR analysis revealed that these compounds simply denatured the protein. Hence, no viable hits were identified from the screen in the current form.
A. The score is an estimate that reflects the binding affinity of the compound for the target and the accuracy of the method used to measure the dissociation constants. Compounds with an estimated Kd > 4000 uM are assigned a Kd of 4001 uM. Compounds for which we do not have a complete titration curve are defined as inactive and scored on a scale from 0 to 40 according to the following scale:
Kd > 4000 uM Assigned Score 0
1000 < Kd < 4000 uM Assigned Score 10
500 < Kd < 1000 uM Assigned Score 20
100 < Kd < 500 uM Assigned Score 30
Kd < 100 uM Assigned Score 40
Finally, compounds that reduce protein solubility as detected by an overall reduction of target NMR signal intensities upon addition of a 1:10 protein-ligand ratio are considered potential aggregators and are assigned Kd of 501. This phenomenon is accounted in the Score as -1.
Data Table (Concise)