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Updated: Apr 15, 2026
Identification of Kinase-substrate Pairs Using High Throughput Screening
Published on: August 29, 2015
Combined rational design and a high throughput screening platform for identifying chemical inhibitors of a
Chris R Evelyn1, Jacek Biesiada2, Xin Duan1
1From the Division of Experimental Hematology and Cancer Biology.
Abstract:
The Ras family small GTPases regulate multiple cellular processes, including cell growth, survival, movement, and gene expression, and are intimately involved in cancer pathogenesis. Activation of these small GTPases is catalyzed by a special class of enzymes, termed guanine nucleotide exchange factors (GEFs). Herein, we developed a small molecule screening platform for identifying lead hits targeting a Ras GEF enzyme, SOS1. We employed an ensemble structure-based virtual screening approach in combination with a multiple tier high throughput experimental screen utilizing two complementary fluorescent guanine nucleotide exchange assays to identify small molecule inhibitors of GEF catalytic activity toward Ras. From a library of 350,000 compounds, we selected a set of 418 candidate compounds predicted to disrupt the GEF-Ras interaction, of which dual wavelength GDP dissociation and GTP-loading experimental screening identified two chemically distinct small molecule inhibitors. Subsequent biochemical validations indicate that they are capable of dose-dependently inhibiting GEF catalytic activity, binding to SOS1 with micromolar affinity, and disrupting GEF-Ras interaction. Mutagenesis studies in conjunction with structure-activity relationship studies mapped both compounds to different sites in the catalytic pocket, and both inhibited Ras signaling in cells. The unique screening platform established here for targeting Ras GEF enzymes could be broadly useful for identifying lead inhibitors for a variety of small GTPase-activating GEF reactions.
Insights
Researchers developed a novel screening platform to identify small molecule inhibitors of SOS1, a key enzyme regulating Ras proteins. This platform successfully identified two distinct inhibitors that block Ras signaling, offering potential new cancer therapies.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Ras GTPases are crucial regulators of cellular functions like growth and survival, and their dysregulation is implicated in cancer.
- Guanine nucleotide exchange factors (GEFs) activate Ras GTPases by catalyzing nucleotide exchange.
- SOS1 is a key GEF enzyme that activates Ras, making it a significant target for cancer therapy.
Purpose of the Study:
- To develop and validate a small molecule screening platform for identifying inhibitors of the Ras GEF enzyme, SOS1.
- To discover novel small molecules that inhibit SOS1's catalytic activity and disrupt the SOS1-Ras interaction.
- To explore the potential of targeting Ras GEF enzymes for cancer treatment.
Main Methods:
- Ensemble structure-based virtual screening of 350,000 compounds.
- High-throughput screening using dual wavelength fluorescent assays for GDP dissociation and GTP-loading.
- Biochemical validation, mutagenesis, and structure-activity relationship (SAR) studies.
Main Results:
- Identified two chemically distinct small molecule inhibitors of SOS1 from the screening campaign.
- Validated inhibitors dose-dependently inhibit SOS1 catalytic activity and bind with micromolar affinity.
- Demonstrated that inhibitors disrupt the SOS1-Ras interaction and inhibit Ras signaling in cellular assays.
Conclusions:
- The developed screening platform is effective for identifying inhibitors of Ras GEF enzymes like SOS1.
- The identified small molecules represent promising lead compounds for targeting Ras-driven cancers.
- This approach can be broadly applied to discover inhibitors for other small GTPase-activating GEF reactions.
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