fragment-based screening by nuclear magnetic resonance

Stages

1. 1.
   NMR fragment-based screening for MITF binders(broad_screen)

   This stage identifies initial ligands for the MITF DNA binding domain on a target class described as unprecedented for direct targeting.

   Selection: fragment binding to the MITF DNA binding domain

2. 2.
   Structure-based optimization of weak MITF kink-pocket fragments(secondary_characterization)

   This stage improves very weak initial fragment binders into more potent ligands using structural and biophysical guidance.

   Selection: improved affinity from weak initial fragment binders

3. 3.
   Mechanistic characterization of ligand-induced MITF conformational perturbation(functional_characterization)

   This stage examines how ligand binding affects the dynamic conformational exchange within the asymmetric MITF homodimer.

   Selection: evidence that ligand binding perturbs MITF conformational exchange

Steps

1. 1.
   Screen fragments against the MITF DNA binding domain by NMRscreening method

   Identify initial ligands for the MITF DNA binding domain.

   The campaign first needs direct binders before any optimization can occur.

2. 2.
   Optimize weak MITF kink-pocket fragments by structure-based design using X-ray crystallography and biophysics

   Increase affinity of initial MITF-binding fragments.

   Optimization follows initial hit discovery because the starting fragments bind very weakly.

3. 3.
   Use NMR and molecular dynamics to assess conformational exchange and its perturbation by ligand binding

   Characterize the dynamic mechanism associated with ligand binding to MITF.

   Mechanistic analysis is performed after ligands are available so their effects on MITF conformational exchange can be examined.