靶點(diǎn) EGFRL858R EGFRL858R/T790M

IC50 2 nM 8 nM

體外研究 WZ4002也抑制其他EGFR基因型，如作用于E746_A750和E746_A750/T790M時(shí)IC50分別為2和6 nM。WZ4002作用于野生型EGFR時(shí)IC50為32 nM。WZ4002作用于非小細(xì)胞肺癌（NSCLC）細(xì)胞時(shí)抑制EGFR, AKT和ERK1/2的磷酸化作用。WZ4002作用于表達(dá)不同EGFRT790M突變等位基因的NIH-3T3細(xì)胞時(shí)抑制EGFR的磷酸化作用。測(cè)定激酶的解離常數(shù)，WZ4002作用下的實(shí)驗(yàn)組比DMSO作用下的對(duì)照組高95%。WZ4002由于在C2苯胺取代時(shí)獲得一個(gè)正甲氧基團(tuán)，因而作用于EGFR比WZ3146效果更高。與喹唑啉類抑制劑相比，WZ4002作用于野生型EGFR磷酸化作用的效果要低100倍。WZ4002抑制重組L858R/T790M蛋白EGFR激酶活性的效果比抑制野生型EGFR高很多，而HKI-272和Gefitinib的抑制效果剛好與WZ4002相反。[1]此外,抗Src TKI的H1975細(xì)胞和HCC827細(xì)胞的EGFR的磷酸化完全被第三代EGFR TKI, WZ4002抑制。

體內(nèi)研究 處理含有T790M突變鼠模型2周，用于研究WZ4002的功效，發(fā)現(xiàn)與對(duì)照組相比WZ4002處理導(dǎo)致明顯的腫瘤退化。[1]用低劑量WZ4002,和高劑量WZ4002處理，導(dǎo)致平均攝取分別下降26%和36%。

The structure of EGFR contains the extracellular domains and transmembrane domains. Among them, the intracellular domain is mainly divided into different parts including juxtamembrane domain, tyrosine kinase domain, and regulatory region domain. The tyrosine kinase domain of EGFR consists of the N-lobe and C-lobe flanking the activation loop and active site cleft. Threonine 790 is the gatekeeper residue of EGFR, which locates at the entrance to a hydrophobic pocket in the back of the ATP binding cleft and thus determines inhibitor specificity in protein kinases. Substitution of threonine 790 with methionine (T790M) causes resistance by steric interference with binding of tyrosine kinase inhibitors. ?

The crystal structure reveals the covalent binding of WZ4002 to the ATP-binding cleft of EGFR T790M mutant. The covalent bond is formed between Cys797 and the acrylamide group of WZ4002. Non-covalent interactions also enhance the affinity between WZ4002 and kinase domain. The anilinopyrimidine core of WZ4002 forms a bidentate hydrogen bonding interaction with Met793, and the chlorine substituent on the pyrimidine ring contacts Met790, the mutant gatekeeper residue. Besides, the aniline ring also contacts the α-carbon of Gly796 via a hydrophobic interaction, with its methoxy substituent extending toward Leu792 and Pro794 in the hinge region. Furthermore, the ‘linker’ phenyl ring lies roughly perpendicular to the pyrimidine core, and this orientation juxtaposes the acrylamide with the thiol of Cys797 for covalent bond formation. The interactions determine the potency and relative selectivity of WZ4002 for EGFR T790M.?

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