Ward W H, Cook P N, Slater A M, Davies D H, Holdgate G A, Green L R
ZENECA Pharmaceuticals, Macclesfield, Cheshire, U.K.
Biochem Pharmacol. 1994 Aug 17;48(4):659-66. doi: 10.1016/0006-2952(94)90042-6.
Inhibition of tyrosine kinases is a possible approach for the treatment of cancer. We have investigated the catalytic mechanism of the epidermal growth factor receptor tyrosine kinase (EGF-RTK) in order to obtain information for use in structure-based searching for inhibitors. Initial rate studies imply that EGF-RTK forms a ternary complex together with ATP and peptide substrate. Investigation of pH and temperature dependence suggests that the kinase reaction requires the ionised form of a carboxylate (pK = 6.3) and the protonated form of another group (pK = 9.1). These characteristics are consistent with a mechanism where the carboxylate of Asp813(pK = 6.3) facilitates deprotonation of the tyrosyl hydroxyl of the peptide substrate, activating it as a nucleophile to attack the gamma-phosphorus of ATP which interacts with a protonated enzyme side-chain (pK = 9.1), possibly the guanidinium group of Arg817. This proposed catalytic mechanism was used to define a query when searching for inhibitors in a database of predicted three-dimensional structures. The procedure involved searching for compounds that mimic the ATP gamma-phosphate, tyrosyl hydroxyl and the tyrosyl aromatic ring, all of which seem to interact strongly with the enzyme during catalysis. This search allowed identification of inhibitors of EGF-RTK which were used to define queries for two-dimensional searching of a larger database, leading to the discovery of 4-(3-chloroanilino)quinazoline (CAQ) which is a potent inhibitor (Ki = 16 nM) of the enzyme. The compound is believed to be the first representative from a new structural class of anilinoquinazoline tyrosine kinase inhibitors. It follows competitive kinetics with respect to ATP and noncompetitive kinetics when the peptide is varied, implying that it functions as an analogue of ATP. CAQ is a novel and potent lead in the search for tyrosine kinase inhibitors as potential agents for the treatment of cancer.
抑制酪氨酸激酶是治疗癌症的一种可能方法。我们研究了表皮生长因子受体酪氨酸激酶（EGF-RTK）的催化机制，以便获取用于基于结构搜索抑制剂的信息。初始速率研究表明，EGF-RTK与ATP和肽底物形成三元复合物。对pH和温度依赖性的研究表明，激酶反应需要一种羧酸盐的离子化形式（pK = 6.3）和另一个基团的质子化形式（pK = 9.1）。这些特征与一种机制相符，即Asp813的羧酸盐（pK = 6.3）促进肽底物酪氨酸羟基的去质子化，将其激活为亲核试剂以攻击与质子化酶侧链（pK = 9.1）相互作用的ATP的γ-磷，可能是Arg817的胍基。在预测的三维结构数据库中搜索抑制剂时，这种提出的催化机制被用于定义一个查询。该过程涉及搜索模拟ATPγ-磷酸、酪氨酸羟基和酪氨酸芳环的化合物，所有这些在催化过程中似乎都与酶强烈相互作用。这种搜索使得能够鉴定出EGF-RTK的抑制剂，这些抑制剂被用于定义对更大数据库进行二维搜索的查询，从而发现了4-(3-氯苯胺基)喹唑啉（CAQ），它是该酶的一种强效抑制剂（Ki = 16 nM）。该化合物被认为是苯胺基喹唑啉酪氨酸激酶抑制剂新结构类别的首个代表。它对ATP遵循竞争性动力学，而当肽变化时遵循非竞争性动力学，这意味着它作为ATP的类似物起作用。CAQ是寻找作为潜在癌症治疗药物的酪氨酸激酶抑制剂的一种新型强效先导化合物。
