Second harmonic generation detection of Ras conformational changes and discovery of a small molecule binder

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 KRas^G12D 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.

Keywords: KRAS; cancer; second harmonic generation; small G protein; small molecule inhibitors.

Fig. 1.

SHG schematic. (A) When a labeled protein tethered to a membrane-coated surface is pulsed with an infrared laser, the SHG probe converts a portion of the incident light into blue light, the SHG signal. The intensity of the SH signal is highly dependent on the orientation of the dye probe relative to the surface normal (z axis) and is sensitive to relative changes in the time- and space-averaged orientation of the dye probe that reports on conformational changes. (B) A conformational change that alters the orientation of the label in relation to the z axis increases or decreases the signal intensity. In general, when the SHG dye shifts away from the z axis (s₁ → s₂), the signal decreases, and when it shifts toward the z axis, it increases (s₂ → s₁).

Fig. 2.

KRas^G12D G-domain SHG assay development. (A) KRas^G12D structure illustrating the solvent-accessible lysine residues labeled by SHG probe conjugation (green). The dominant labeling sites are indicated in bold. (B) GST-Raf-RBD beads were incubated in the presence of unconjugated or SH-active KRas^G12D loaded with GDP or ‘GTP’ as indicated. The reaction input and Ras:Raf-RBD complex was analyzed with anti-Ras antibody. (C) His-tagged GDP KRas^G12D-SHG1 was tethered to an Ni-NTA bilayer, and the SHG signal intensity was monitored in the presence of increasing SOS_cat (micromolar). (D) His-tagged GDP KRas^G12D-SHG1 was tethered to an Ni-NTA bilayer, and the SHG signal was monitored in the presence of 1 µM buffer-matched monoclonal antibody. (E) GDP or ‘GTP’ KRas^G12D-SHG1 was monitored in the presence of 1 µM buffer-matched monoclonal antibody (mean ± SD [error bars]; n = 3). All SHG measurements were recorded before and 2 min after ligand addition.

Fig. 3.

Primary SHG-based screen reveals the KRas^G12D fragment ligand. (A) The percentage SHG signal change (ΔSHG [%]) was monitored in the presence or absence of 50 µM mepazine in a 384-well Biodesy Delta plate 2 min postinjection. (B) ‘GTP’ KRas^G12D-SHG1 SHG signal change in the presence of (Left) 500 or (Right) 250 µM fragment concentration across the library. The dotted lines indicate ±10% threshold. (C) Fragment 18 concentration response curve at T = 4 min postinjection (mean ± SD [error bars]; n = 3). (D) The chemical structure of fragment 18, 4-(cyclopent-2-en-1-yl)phenol. (E) The SHG signals of (Left) ‘GTP’ KRas^G12D-SHG1 and (Right) GDP KRas^G12D-SHG1 were monitored in the presence of 0 to 500 µM fragment 18 for 10 min, with measurements taken at 2-min intervals postinjection.

Fig. 4.

The 2D ¹H ¹⁵N TROSY HSQC NMR validates fragment (cmpd) 18 binding to KRas^G12D. (A) The ¹H ¹⁵N TROSY HSQC NMR spectra of 50 µM GDP KRas^G12D in the presence of increasing ligand concentration. The KRas^G12D ribbon model shows the predicted binding interface in yellow. (Left) TROSY HSQC GDP KRas^G12D spectra in the presence of 0 to 5 mM fragment 18. (Right) TROSY HSQC GDP KRas^G12D spectra in the presence of 0 to 10 mM DCAI. KRas^G12D ribbon model shows the predicted DCAI binding interface in yellow and red. (B) GDP KRas^G12D-SHG1 10-min SHG time course in the presence of 0 to 500 µM DCAI. (C) Fragment 18 docking to KRas^G12D in DCAI pocket. The docking pose of fragment 18 (yellow carbon atoms) was predicted with SILCS FragMaps and overlaid on the crystal binding mode of DCAI (white carbon atoms; PDB ID code 4DST). The apolar (green) and hydrogen bonding donor (blue) FragMaps are shown in the KRas^G12D small molecule binding pocket (surface in white) overlapping corresponding functional groups from both compounds. (D) Predicted binding pose of fragment 18 and its interactions with surrounding KRas residues.