Second harmonic generation (SHG) is a biophysical method that sensitively measures real-time conformational change of biomolecules attached to membranes. SHG has recently been applied to detect protein conformational changes in the presence of biological ligands and small molecules. I will describe implementation of SHG as a primary screening platform to identify novel fragment ligands to oncogenic KRas. KRas is the most frequently mutated driver of pancreatic, colon, and lung cancers; however, there are few well-characterized small molecule ligands due to a lack of deep binding pockets. Using SHG, we identified a novel fragment binder to KRasG12D and then used 1H 15N HSQC NMR to characterize its binding site as a pocket adjacent to the Switch II region. The unique sensitivity of SHG furthered our study by revealing distinct conformations induced by our hit fragment compared to DCAI, a Ras ligand previously described to bind the same pocket, identified by fragment based NMR screening. This study highlights SHG as a high-throughput screening platform that reveals structural insights in addition to ligand binding.

Learning Objectives: 

1. Ras proteins, once considered “undruggable”, are now being subjected to a variety of new chemical and biophysical techniques to identify potential drugs that bins to shallow pockets in the protein
2. The biophysical phenomenon referred to as second harmonic generation (SHG) can be used as a tool for discovering molecules that bind to proteins of interest