Category: Assay Development and Screening

1322-C - Identifying allosteric modulators of KRas using second harmonic generation

Tuesday, February 6, 2018
2:00 PM - 3:00 PM

The Ras protein family consists of small GTPases that are critical signaling transducers regulating cellular processes including proliferation, differentiation, and survival. As monomeric GTPases, Ras proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Oncogenic mutations in one of the Ras isoforms, H-, N-, and K-Ras, impair GTP hydrolysis, thus de-regulating Ras signaling.  Ras mutations are found in one-third of human cancers, and tumors bearing these mutations are notoriously difficult to treat. Conventional approaches to inhibit Ras with small molecules have been largely unsuccessful leading many to characterize the Ras family as an “undruggable” target. However, there is renewed enthusiasm towards Ras drug discovery in response to a recent surge of reports revealing structural complexities to Ras proteins that provide opportunity for innovative drug discovery efforts.      


In light of findings suggesting KRas contains transient and dynamic binding pockets, many of which are undetectable with traditional structural techniques, we used the Biodesy Delta second harmonic generation (SHG) platform to screen for conformational modulators of KRas. SHG is an exquisitly sensitive biophysical technique that detects real-time changes in protein conformation.  When a labeled protein tethered to a membrane-coated surface is pulsed with infrared light, the SHG probe converts  a portion of the incident infrared into blue light, termed the 'second harmoic signal.' The intensity of the second harmonic signal is highly dependent on the orientation of the dye probe in relation to the surface, and is extremely sensitive to relative shifts and alterations, indicative of conformational change.

We implemented the Biodesy SHG platform to identify Ras modulators from a chemically diverse ~2700 fragment library. Using ±3σ criteria, our hit rate was 1.4%; however, we selected a larger collection of compounds for secondary screening at lower concentrations against both GDP KRas and 'GTP' KRas. 160 hit fragments were then characterized in dose response SHG assays, and ~50 were for selected for orthoganol validation by SPR.  The top 20% of detectable binders are currently under evaluation in a series of HSQC NMR experiments to characterize the binding site(s) of each compound of interest. Concurrently, we are running classical biochemical assays in the presence of these compounds to determine how KRas conformational modulators affect its function. Moving forward we will use our structural and functional insights to guide medicinal chemistry on these fragments towards the development of lead targeted KRas therapies. 


 


 


 


 


 


 

Elizabeth Donohue Vo

Scientist
Biodesy
San Francisco, California

Elizabeth Donohue Vo, Ph.D. is a scientist at Biodesy, Inc. Dr. Vo previously worked as a postdoctoral fellow in Dr. Frank McCormick's laboratory at UCSF where she developed and carried out multiple small molecule screens against oncogenic KRas using innovative strategies. Prior to joining UCSF, Dr. Vo earned her Ph.D. in biochemistry and molecular biology at The University of British Columbia under Dr. Michel Roberge where she identified and characterized a novel autophagy inhibitor and its therapeutic potential in cancer.