Ligand Screening and Discovery Using Cocktail Soaking and Automated Microcrystal Electron Diffraction.

Area of Science:

• Structural Biology
• Biophysics
• Drug Discovery

Background:

• Single-crystal X-ray diffraction (SCXRD) with cocktail soaking enables high-throughput screening of ligands against protein targets.
• Protein microcrystals that are too small for SCXRD pose a challenge for ligand screening and structure determination.

Purpose of the Study:

• To develop an integrated workflow combining cocktail soaking with automated microcrystal electron diffraction (AMED).
• To enable rapid ligand screening, structure determination, and binding analysis directly from protein microcrystals.

Main Methods:

• Integration of cocktail soaking with automated microcrystal electron diffraction (AMED).
• Application of the workflow to known ligands of thermolysin and proteinase K.

Main Results:

• Successful validation of the AMED workflow with known ligands.
• Identification of novel binding interactions for proteinase K ligands.
• Rapid structure solution for multiple protein-ligand complexes, including those with weak binding affinities.
• Estimated relative binding affinities align with previous studies and microscale thermophoresis measurements.

Conclusions:

• The developed workflow significantly improves the hit rate for ligand screening using small protein microcrystals.
• AMED provides a rapid and effective method for structure determination and binding analysis of protein-ligand complexes.
• This approach facilitates the discovery of novel binding interactions and characterization of weak-affinity ligands.