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Our nucleotide exchange assay detects inhibitors against any step in the reaction mechanism, whereas the nucleotide release assay specifically identifies inhibitors against the first phase of the exchange reaction, nucleotide release. Õâ¶Î»°ÖУ¬nucleotide exchange assay ºÍnucleotide release assay ÊÇʲôÒâ˼¡£ DCAI identified by Genentech, was found to bind in the hydrophobic pocket located between the ¦Á2 helix (of the switch II region) and the core ¦Â-sheet of the KRAS protein and thereby interrupt KRAS/SOS association.Titration studies with DCAI indicated that it blocked both nucleotide exchange and release reactions, with IC50 values of 342 ¡À 22 and 155¡À 36¦ÌM, respectively. It was also noted that a secondary electronegative binding cleft is formed in the protein upon fragment binding. It is proposed that DCAI blocks the first phase of the exchange reaction by inhibiting nucleotide release from KRAS.Õâ¶Î»°ÖÐsecondary electronegative binding cleft ÊÇʲôÒâ˼£¿ Çó½âÊÍ£¬Ð»Ð»£¡ |
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