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Applications: Study enzyme kinetics and screen small molecular inhibitors of KRAS(G12V) for drug discovery and High Throughput (HTS) applications.
Background: It is well established that RAS mutations are responsible for more than 30% of human cancers. Although new treatments have been recently developed to mitigate the tumor-promoting effects of RAS mutations, one mutant, KRAS(G12V), has been resistant to most known inhibitors. For example, small molecule AMG510 (Amgen) can block the signaling pathway mediated by several KRAS(G12) mutants by locking the KRAS conformation in the GDP-bound state. However, KRAS(G12V) has been insensitive to this inhibitor. New compounds that affect the nucleotide exchange (GDP to GTP) reaction in KRAS mutant G12V are expected to inhibit tumor cell growth in KRAS(G12V)-driven tumors. MRTX1133, which selectively inhibits KRAS G12D mutant but not KRAS wild-type, has shown activity on KRAS(G12V) in vitro.
Description: The KRAS(G12V) Nucleotide Exchange Assay is a homogeneous assay designed for the screening and profiling of KRAS(G12V) antagonists/inhibitors by using BODIPY®-GDP to monitor the GDP or GTP binding status. The kit can be used with two different protocols for greater flexibility, either titrating the inhibitor at a fixed GTP concentration or titrating the GTP at a fixed inhibitor concentration.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.Assay Principle Illustration: KRAS is activated upon binding GTP, when it 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 KRAS affinity for GTP is greater than its affinity for GDP. The fluorescence intensity decreases as the BODIPY®-GDP is displaced. Several KRAS inhibitors lock KRAS in the (inactive) GDP-bound conformation and prevent GDP/GTP exchange. In this scenario the fluorescence intensity increases with drug concentration as more BODIPY®-GDP remains bound to KRAS.
Storage Stability: This assay kit will perform optimally for up to 6 months from date of receipt when the materials are stored as directed.
Supplied As: The KRAS(G12V) Nucleotide Exchange Assay Kit comes in a convenient 384-well format, with enough purified recombinant KRAS(G12V) labeled with BODIPY®-GDP, GTP, assay buffer and additives for 400 enzyme reactions.
Uniprot: P01116
Warnings: Avoid freeze/thaw cycles.
Biosafety Level: Not applicable (BSL-1)
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