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Application: Study enzyme kinetics and screen small molecular inhibitors of KRAS Isoform B for drug discovery and High Throughput Screening (HTS) applications.
Background: High levels of wild-type KRAS cause a slowing of cell replication and growth, and increased apoptosis. This can be induced by cellular stress, certain types of radiation, chemical signals, and other prompts. Wild-type KRAS may also protect against mutant KRAS over-activation by dimerizing with mutant KRAS protein. The study of differences in the behavior of the wild-type and mutated forms of KRAS is especially important since KRAS mutations are responsible for more than 30% of human cancers. Compounds that affect the nucleotide exchange (GDP to GTP) reaction provide a novel approach to the inhibition of tumor cell growth in KRAS-driven tumors. In 2021, the Food and Drug Administration (FDA) granted approval for use of AMG510 (Sotorasib), a potent KRAS G12C inhibitor for non-squamous non-small cell lung cancer. BODIPY FL-GDP is a mixed isomer in which the fluorophore has been attached to the 2’ or 3’ position of the ribose ring via a linker. It is a green-fluorescent dye with similar excitation and emission to fluorescein or Alexa Fluor™ 488, characterized by a high extinction coefficient and high quantum yield and is relatively insensitive to pH changes. The dye has an excited-state lifetime of 5 nanoseconds or longer. It is a commonly used dye to fluorescently label GTPases and determine their GTP/GDP status.
Contraindications: The final concentration of DMSO in the assay should not exceed 1%.Compounds that are fluorescent may interfere with the results, depending on their spectral excitation and emission properties.It is recommended that the compound alone is tested to determine any potential interference of the compound on the assay results.
Description: The KRAS Isoform B Nucleotide Exchange Assay Kit is a fluorogenic homogeneous assay designed for the screening and profiling of KRAS Isoform B antagonists/inhibitors by using BODIPY®-GDP to monitor the KRAS Isoform B GDP or GTP binding status. The KRAS Isoform B Nucleotide Exchange Assay Kit comes in a convenient format, with enough purified recombinant KRAS Isoform B labeled with BODIPY®-GDP, GTP, assay buffer and additives for 100 enzyme reactions. Two different protocols can be used with this kit, allowing greater experimental flexibility. It can used to either titrate the inhibitor at a fixed GTP concentration, or to titrate GTP in the presence of a fixed inhibitor concentration.Figure 1: Schematic representation of the mechanism of the KRAS Isoform B Nucleotide Exchange Assay Kit. KRAS is activated upon binding GTP and undergoes a conformational change. KRAS then returns to a GDP-bound inactive state following the hydrolysis of GTP to GDP. In this assay, KRAS is pre-loaded with fluorescent BODIPY-GDP and therefore is inactive. Adding GTP in the presence of EDTA displaces BODIPY-GDP because the affinity of KRAS for GTP is greater than its affinity for GDP. The fluorescence intensity decreases as the BODIPY-GDP is displaced, since free BODIPY-GDP has low fluorescence versus when protein-bound. Several KRAS inhibitors lock KRAS in the (inactive) GDP-bound conformation and prevent GDP/GTP exchange. In this scenario the fluorescence intensity remains high and corelates with drug concentration as more BODIPY-GDP stays bound to KRAS.Interested in having us perform the screening and profiling of KRAS inhibitors? Visit our KRAS Screening and Profiling Services.
Storage Stability: This assay kit will perform optimally for up to 6 months from date of receipt when the materials are stored as directed.
Target: KRAS
Uniprot: P01116
Warnings: Avoid freeze/thaw cycles.
Biosafety Level: Not applicable (BSL-1)
References: Ostrem J.M., et al., 2013. Nature 503: 548-551.Patricelli M.P., et al., 2016. Cancer Discover. 6: 316-329.
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