Development and Validation of 2D Difference Intensity Analysis for Chemical Library Screening by Protein-Detected NMR Spectroscopy. Chembiochem 2018 Mar 02;19(5):448-458
Date
12/15/2017Pubmed ID
29239081Pubmed Central ID
PMC5844354DOI
10.1002/cbic.201700386Scopus ID
2-s2.0-85041064927 (requires institutional sign-in at Scopus site) 12 CitationsAbstract
An academic chemical screening approach was developed by using 2D protein-detected NMR, and a 352-chemical fragment library was screened against three different protein targets. The approach was optimized against two protein targets with known ligands: CXCL12 and BRD4. Principal component analysis reliably identified compounds that induced nonspecific NMR crosspeak broadening but did not unambiguously identify ligands with specific affinity (hits). For improved hit detection, a novel scoring metric-difference intensity analysis (DIA)-was devised that sums all positive and negative intensities from 2D difference spectra. Applying DIA quickly discriminated potential ligands from compounds inducing nonspecific NMR crosspeak broadening and other nonspecific effects. Subsequent NMR titrations validated chemotypes important for binding to CXCL12 and BRD4. A novel target, mitochondrial fission protein Fis1, was screened, and six hits were identified by using DIA. Screening these diverse protein targets identified quinones and catechols that induced nonspecific NMR crosspeak broadening, hampering NMR analyses, but are currently not computationally identified as pan-assay interference compounds. The results established a streamlined screening workflow that can easily be scaled and adapted as part of a larger screening pipeline to identify fragment hits and assess relative binding affinities in the range of 0.3-1.6 mm. DIA could prove useful in library screening and other applications in which NMR chemical shift perturbations are measured.
Author List
Egner JM, Jensen DR, Olp MD, Kennedy NW, Volkman BF, Peterson FC, Smith BC, Hill RBAuthors
Francis C. Peterson PhD Professor in the Biochemistry department at Medical College of WisconsinBrian C. Smith PhD Associate Professor in the Biochemistry department at Medical College of Wisconsin
Brian F. Volkman PhD Professor in the Biochemistry department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
Cell Cycle ProteinsChemokine CXCL12
Drug Discovery
Humans
Ligands
Membrane Proteins
Mitochondrial Proteins
Models, Molecular
Nuclear Magnetic Resonance, Biomolecular
Nuclear Proteins
Principal Component Analysis
Protein Binding
Recombinant Proteins
Small Molecule Libraries
Transcription Factors
