Methods for Identifying Ligand Binding Sites in Drug Discovery

By Daniel Beck and Karson Putt
Edited by Dennis X. Hu, Nicholas Morano, Jen Huen
Reviewed by Pablo Martín-Gago, Ravi Kurumbail

Understanding how a small molecule ligand binds to its target is valuable in drug discovery, because it enables more efficient optimization through structure-based design, better mechanistic understanding of molecular pharmacology, and greater confidence in the therapeutic hypothesis from both safety and efficacy perspectives. Recently, Drug Hunter highlighted methods for target identification when the target is unknown. In this Drug Hunter Minireview, we catalog common methods used in industry to determine or predict a ligand’s binding site, while highlighting advantages, disadvantages, and case studies, including:

  • X-ray crystallography
  • cryogenic electron microscopy (cryo-EM)
  • competition assays
  • nuclear magnetic resonance (NMR)
  • covalent labeling 
  • photoaffinity labeling
  • mutational analysis and site-directed mutagenesis
  • hydrogen/deuterium exchange (HDX) and chemical or proteolytic stability profiling
  • native mass spectrometry
  • computational approaches

Our list begins with techniques providing the highest confidence prediction with the greatest precision and resolution (e.g., X-ray crystallography), and continues toward the other end of the spectrum with economical alternatives (Figure 1). The techniques span a range of classes: biophysical; biochemical; chemical biology; and virtual. For simplicity, proteins are assumed to be the biological target throughout the review, although this may not always be the case.

Figure 1. Overview of the methods covered in this mini-review, with the relative position on the arrow indicating the level of confidence/precision/resolution for the binding site determination or prediction.

X-Ray Crystallography

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