04 November 2019

Second harmonic generation (SHG) vs KRAS

Practical Fragments is currently running a poll on fragment-finding methods used by readers – please vote on the right-hand side. One biophysical method that perhaps we should have included is second harmonic generation (SHG). A recent paper in Proc. Nat. Acad. Sci. USA by Josh Salafsky, Frank McCormick, and collaborators at Biodesy, University of California San Francisco, and elsewhere describes the technique and its application to find fragments that bind to the oncogenic protein KRAS.

In SHG, two photons of the same energy are absorbed by a material which then emits a single photon with twice the energy. In the commercial instrument developed by Biodesy, a powerful 800 nm laser irradiates a dye, and the 400 nm photon it emits is detected. The intensity of the signal is exquisitely sensitive to the precise orientation of the dye. If a protein is labeled with an SHG-active dye and then immobilized on a glass surface, even subtle changes in conformation will be detected.

The researchers chose the G12D mutant form of KRAS, which is one of the most common variants and is associated with particularly aggressive tumors. They labeled the protein with a lysine-reactive SHG dye under conditions in which each protein would, on average, have one covalently-bound dye molecule (though some would have none and others would have more than one). Proteolysis and mass-spectrometry analysis revealed that the dye molecule labeled three different lysine residues, which the researchers viewed as a feature since a ligand causing a conformational change to any of the lysine residues would generate a signal. The researchers also demonstrated that the dye modification did not interfere with the ability of KRAS to bind to the RAS-binding domain of RAF.

Labeled KRAS was then immobilized and tested against several proteins known to bind it, including antibodies and the nucleotide exchange factor SOS. These produced SHG signals, presumably by causing conformational changes to KRAS, while non-binders such as tubulin did not.

Having established that the assay could detect binders, the researchers screened 2710 fragments at 250 and 500 µM, and obtained a whopping 490 hits. These were then triaged by screening at lower concentrations and performing dose-titrations, and 60 were then characterized by SPR.

Fragment 18, 4-(cyclopent-2-en-1-yl)phenol, showed binding by both SHG and SPR, and was further studied by 2-dimensional NMR (1H-15N HSQC). This technique allowed measurement of the weak 3.3 mM dissociation constant. More importantly, it allowed the researchers to establish the binding location as being near the so-called “switch 2” region where SOS normally binds. This is the same region where a previous NMR screen had identified the slightly more potent fragment DCAI. The current paper confirmed that finding, though the researchers found evidence that DCAI may bind to other sites too. Docking studies using SILCS suggested that fragment 18 likely binds in a similar orientation as DCAI. Not surprising given the low affinity, the new fragment did not show functional activity in a biochemical screen.

SHG is an interesting approach, and the ability to rapidly assess protein conformational changes distinguishes it from other biophysical techniques. Site-specific labeling would produce more informative data on which regions of a protein move. However, I wonder if SHG is perhaps too sensitive, as evidenced by the large number of hits. Indeed, the researchers demonstrated that the promiscuous lipophilic amine mepazine also generated a strong SHG signal with KRAS. It would be interesting to do a head-to-head comparison with other similarly rapid techniques such as DSF or MST. Have you tried using SHG, and if so, how did it perform for you?


Josh Salafsky said...

Thanks for taking the time to review our paper, Dan. Typical hit rates for a fragment screen are 10-15% as reported in our publication in MIE (https://www.sciencedirect.com/science/article/pii/S0076687918303781). We published an earlier fragment screen in which we identified a single aggregation inhibiting compound in vitro and in cells against synuclein: http://www.jbc.org/content/290/46/27582.short. As SHG's throughput is thousands/day, following up on the hits at lower doses and running DRC's is pretty fast.

Elizabeth Donohue Vo said...

Thank you for this publication review. I want to clarify a couple points regarding hit rate for this study. This was largely an exploratory study as it was the first automated screen of its kind. Therefore, we purposely set a loose hit-calling threshold to allow ample follow-up since we were unsure of the platform sensitivity compared to other fragment-based drug discovery methods. Secondly, we obtained an academic fragment library rather than a rigorously curated proprietary library, and this likely contributed to a noisier hit:noise distribution. As Josh mentions, we were quickly able to funnel our hits through successive SHG experiments to achieve a manageable number of compounds for follow-up. Thank you for your interest and for highlighting this study.