Saturation Transfer Difference NMR for Fragment Screening

Darren W. Begley1, Spencer O. Moen1, Phillip G. Pierce1, Edward R. Zartler2

1 Emerald Bio, Bainbridge Island, Washington, 2 Quantum Tessera Consulting, Collegeville, Pennsylvania
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch130118
Online Posting Date:  December, 2013
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Fragment screening by saturation transfer difference nuclear magnetic resonance (STD‐NMR) is a robust method for identifying small molecule binders and is well suited to a broad set of biological targets. STD‐NMR is exquisitely sensitive for detecting weakly binding compounds (a common characteristic of fragments), which is a crucial step in finding promising compounds for a fragment‐based drug discovery campaign. This protocol describes the development of a library suitable for STD‐NMR fragment screening, as well as preparation of protein samples, optimization of experimental conditions, and procedures for data collection and analysis. Curr. Protoc. Chem. Biol. 5:251‐268 © 2013 by John Wiley & Sons, Inc.

Keywords: saturation transfer difference; STD; NMR; fragment screening; fragment‐based hit generation

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Direct Identification of Protein Binding Fragments Using One‐Dimensional Proton STD‐NMR Spectroscopy
  • Support Protocol 1: Select NMR Parameters for STD‐NMR Experiments
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Direct Identification of Protein Binding Fragments Using One‐Dimensional Proton STD‐NMR Spectroscopy

  • Fragment library molecules, available from a variety of vendors (e.g., Sigma‐Aldrich, TCI, ChemBridge, ChemDiv, Life Chemicals, Maybridge, Alfa Aesar, Acros)
  • Deuterated dimethyl sulfoxide (d 6‐DMSO, >99% D, Aldrich, cat. no. 151882)
  • 1.0 mM 4,4‐dimethyl‐4‐silapentane‐1‐sulfonic acid (DSS, Aldrich, cat. no. 178837) stock in deuterium oxide (D 2O > 99% D)
  • NMR QC buffer (see recipe)
  • 50 µM purified target protein in NMR QC buffer
  • 0.5‐ to 200‐µl pipettors and pipet tips
  • 1.7‐ml microcentrifuge tubes
  • NMR spectrometer (≥ 500 MHz field strength) and processing software

Support Protocol 1: Select NMR Parameters for STD‐NMR Experiments

  • NMR sample in NMR tube with cap
  • 500‐MHz NMR spectrometer with acquisition and data processing software
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Literature Cited

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