Singh J, Dobrusin E M, Fry D W, Haske T, Whitty A, McNamara D J
Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, Michigan 48106-1047, USA. juswinder_singh@biogen. com
J Med Chem. 1997 Mar 28;40(7):1130-5. doi: 10.1021/jm960380s.
We report the use of structure-based drug design to create a selective erbB-1 (a.k.a. epidermal growth factor receptor) and erbB-2 (a.k.a. neu/her2 growth factor receptor) tyrosine kinase inhibitor. Using the X-ray crystal structure of the ternary complex of the cAMP-dependent Ser/Thr kinase together with a sequence alignment of the catalytic domains of a representative set of Ser/Thr and Tyr protein kinases, we have examined the nucleotide binding site for potential positions to attach an irreversible inhibitor. This information, combined with homology modeling of the erbB-1 and erbB-2 tyrosine kinase catalytic domains, has led to the identification of Cys797 of erbB1 and Cys805 of erbB2, which are structurally equivalent to Glu127 in the cAMP dependant Ser/Thr kinase as potential target residues. The X-ray structure of the cAMP Ser/Thr kinase shows Glu127 to be involved in a hydrogen-bonding interaction with the 2'-OH of the ribose portion of ATP. Using molecular modeling, it was predicted that the Cys side chains in erbB-1 and erbB-2 performed an analogous role, and it was postulated that the replacement of the 2'-OH of adenosine with a thiol might allow for a covalent bond to form. Since only erbB-1 and erbB-2 have a Cys at this position, the inhibitor should be selective. This model was subsequently tested experimentally by chemical synthesis of 2'-thioadenosine and assayed against the full length erbB-1 receptor and the catalytic domains of erbB-2, insulin receptor, beta-PDGF receptor, and the FGF receptor. Our results show that thioadenosine covalently inactivates erbB-1 with a second-order rate constant of k(max)/K(S) = 2000 +/- 500 M(-1) s(-1). Inactivation is fully reversed by 1 mM dithiothreitol, suggesting that inactivation involves the modification of a cysteine residue at the active site, presumably Cys797. The rate of inactivation saturates with increasing thioadenosine concentrations, suggesting that inactivation occurs through initial formation of a noncovalent complex with K(D) = 1.0 +/- 0.3 microM, followed by the slow formation of a disulfide bond with a rate constant of k(max) = (2.3 +/- 0.2) x 10(-3) s(-1). This approach may have application in the design of selective irreversible inhibitors against other members of the kinase family.
我们报告了基于结构的药物设计方法，用于创建一种选择性的erbB-1（又称表皮生长因子受体）和erbB-2（又称neu/her2生长因子受体）酪氨酸激酶抑制剂。利用环磷酸腺苷（cAMP）依赖性丝氨酸/苏氨酸激酶的三元复合物的X射线晶体结构，以及一组代表性的丝氨酸/苏氨酸和酪氨酸蛋白激酶催化结构域的序列比对，我们研究了核苷酸结合位点中可能连接不可逆抑制剂的位置。这些信息，结合erbB-1和erbB-2酪氨酸激酶催化结构域的同源建模，已确定erbB1的Cys797和erbB2的Cys805，它们在结构上等同于cAMP依赖性丝氨酸/苏氨酸激酶中的Glu127，作为潜在的靶标残基。cAMP丝氨酸/苏氨酸激酶的X射线结构显示，Glu127参与与ATP核糖部分2'-OH的氢键相互作用。通过分子建模预测，erbB-1和erbB-2中的半胱氨酸侧链发挥类似作用，并推测用硫醇取代腺苷的2'-OH可能允许形成共价键。由于只有erbB-1和erbB-2在该位置有一个半胱氨酸，该抑制剂应该具有选择性。随后通过化学合成2'-硫代腺苷并针对全长erbB-1受体以及erbB-2、胰岛素受体、β-血小板衍生生长因子受体和FGF受体的催化结构域进行检测，对该模型进行了实验验证。我们的结果表明，硫代腺苷以二级速率常数k(max)/K(S)=2000±500 M⁻¹ s⁻¹共价使erbB-1失活。1 mM二硫苏糖醇可完全逆转失活，这表明失活涉及活性位点处半胱氨酸残基的修饰，推测为Cys797。失活速率随着硫代腺苷浓度的增加而饱和，这表明失活是通过首先形成K(D)=1.0±0.3 μM的非共价复合物，然后以k(max)=(2.3±0.2)×10⁻³ s⁻¹的速率常数缓慢形成二硫键而发生的。这种方法可能在设计针对激酶家族其他成员的选择性不可逆抑制剂方面具有应用价值。
