An allosteric inhibitor of ZAP-70 as a novel therapeutic for autoimmune disease

描述（由申请人提供）：ZAP-70是一种细胞质蛋白酪氨酸激酶，在T细胞抗原受体（TCR）信号传导和T细胞生物学的大多数方面发挥关键作用。大型制药和生物技术行业已经花费了相当大的努力来开发ZAP-70催化抑制剂，但没有成功。因此，也难以验证ZAP-70作为治疗靶标。我们最近在细胞系和小鼠中开发了ZAP-70的模型化学遗传抑制剂系统，其中大体积抑制剂用于特异性靶向ZAP-70的突变等位基因。迄今为止的工作支持ZAP-70功能在naove和记忆T细胞功能中至关重要的观点，但有趣的是在T调节细胞中不重要。然而，我们还没有将我们的研究扩展到临床前疾病模型，需要进一步验证。此外，需要开发ZAP-70抑制剂的新方法，因为野生型激酶催化位点似乎不适合抑制剂开发。 在TCR刺激后，ZAP-70的串联SH 2结构域与位于CD 3和ζ链的胞质结构域的保守序列基序（称为ITAM）内的双酪氨酸磷酸化残基缔合。一旦与ITAMS结合，ZAP-70通过Src激酶Lck的磷酸化和其自身的反式自磷酸化而被激活。我们实验室最近对全长ZAP-70分子的诱变和结构研究表明，在与ITAM结合之前，ZAP-70处于自抑制构象，这是由几种分子内相互作用引起的。此外，我们的新见解表明ZAP-70的构象变化可能发生在其与TCR结合期间。基于这些最近的研究，我们现在有机会采用新方法开发一种新型且可能更特异的ZAP-70功能变构抑制剂。 因此，我们建议：1）鉴定作为ZAP-70的特异性变构抑制剂的先导化合物，并通过我们开发的模型催化位点抑制剂比较其阻断信号传导途径的功效;以及，2）使用我们已经建立的小鼠中的ZAP-70的类似物敏感突变体，我们将确定ZAP-70抑制在关节炎中的临床前效用，系统性红斑狼疮和实验性自身免疫性脑脊髓炎。

DESCRIPTION (provided by applicant): ZAP-70 is a cytoplasmic protein tyrosine kinase that plays a critical function in T cell antigen receptor (TCR) signaling and in most aspects of T cell biology. Considerable effort has been expended by the big pharma and biotech industries to develop ZAP-70 catalytic inhibitors, without success. Thus, it has also been difficult to validate ZAP-70 as a therapeutic target. We have recently developed a model chemical genetic inhibitor system for ZAP-70 in cell lines and in mice in which a bulky inhibitor is used to specifically target a mutant allele of ZAP-70. Work to date supports the notion that ZAP-70 function is critically important in naove and memory T cell function, but interestingly not in T regulatory cells. However, we have not yet extended our studies to preclinical disease models and further validation is needed. In addition, a new approach towards developing a ZAP-70 inhibitor is needed since the wild-type kinase catalytic site does not seem amenable to inhibitor development. Following TCR stimulation, the tandem SH2 domains of ZAP-70 associate with dual tyrosine phosphorylated residues located within conserved sequence motifs (referred to as ITAMs) of the cytoplasmic domains of the CD3 and zeta chains. Once associated with the ITAMS, ZAP-70 is activated by its phosphorylation by the Src kinase Lck and by its own trans- autophosphorylation. Recent mutagenesis and structural studies from our labs on the full-length ZAP-70 molecule suggest that, prior to association with the ITAMs, ZAP-70 is in an autoinhibited conformation that results from several intramolecular interactions. Moreover, our new insights suggest a conformational change in ZAP-70 is likely to occur during its binding to the TCR. Based on these recent studies, we now have the opportunity for a new approach towards developing a novel and potentially more specific allosteric inhibitor of ZAP-70 function. Thus, we propose to: 1) identify a lead compound that is a specific allosteric inhibitor of ZAP-70 and compare its efficacy on blocking signaling pathways by the model catalytic site inhibitor that we have developed; and, 2) using the analog-sensitive mutant of ZAP-70 in mice that we have established, we will determine the preclinical utility of ZAP-70 inhibition in arthritis, systemic lupus erythematosus and experimental autoimmune encephalomyelitis.