ABSTRACT
Second harmonic generation (SHG) is an emergent biophysical method that sensitively measures real-time conformational change of biomolecules in the presence of biological ligands and small molecules. This study describes the successful implementation of SHG as a primary screening platform to identify fragment ligands to oncogenic Kirsten rat sarcoma (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 fragment binder to KRasG12D and used 1H 15N transverse relaxation optimized spectroscopy (TROSY) heteronuclear single-quantum coherence (HSQC) NMR to characterize its binding site as a pocket adjacent to the switch 2 region. The unique sensitivity of SHG furthered our study by revealing distinct conformations induced by our hit fragment compared with 4,6-dichloro-2-methyl-3-aminoethyl-indole (DCAI), a Ras ligand previously described to bind the same pocket. This study highlights SHG as a high-throughput screening platform that reveals structural insights in addition to ligand binding.
Subject(s)
Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/chemistry , Amino Acid Substitution , Binding Sites , Humans , Mutation, Missense , Nuclear Magnetic Resonance, Biomolecular , Proto-Oncogene Proteins p21(ras)/geneticsABSTRACT
Second-harmonic generation (SHG) is a biophysical tool that senses ligand-induced conformational changes in biomolecules. The Biodesy Delta™ has been developed as a high-throughput screening platform to monitor conformational changes in proteins and oligonucleotides by SHG to support drug discovery efforts. This work will outline (1) an overview of this technology, (2) detailed protocols for optimizing screening-ready SHG assays on RNA targets, (3) practical considerations for developing robust and informative SHG measurements, and (4) a case study that demonstrates the application of these recommendations on an RNA target. The previously published theophylline aptamer SHG assay [1] was further optimized to maximize the assay window between the positive control (theophylline) and the negative control (caffeine). Optimization of this assay provides practical considerations for building a robust SHG assay on an RNA target, including testing for specific tethering of the conjugate to the surface as well as testing tool compound response stability, reversibility, and concentration-dependence/affinity. A more robust, better-performing theophylline aptamer SHG assay was achieved that would be more appropriate for conducting a screen.
