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

描述(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)信号转导和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，验证ZAP-70作为治疗靶点也是困难的。我们最近开发了一个ZAP-70的模型化学遗传抑制系统，在细胞系和小鼠中，使用一个庞大的抑制物来特异性地靶向ZAP-70的一个突变等位基因。到目前为止的工作支持ZAP-70功能在NAVE和记忆T细胞功能中至关重要的概念，但有趣的是，在T调节细胞中不是。然而，我们还没有将我们的研究扩展到临床前疾病模型，还需要进一步的验证。此外，开发ZAP-70抑制剂需要一种新的方法，因为野生型激酶催化位点似乎不服从抑制剂的开发。在TCR刺激后，ZAP-70的串联SH2结构域与位于CD3和Zeta链细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结合。一旦与ITAM结合，ZAP-70就被Src激酶Lck的磷酸化和自身的反式自磷酸化激活。我们实验室最近对全长ZAP-70分子的突变和结构研究表明，在与ITAM结合之前，ZAP-70处于一种自抑制构象，这是几次分子内相互作用的结果。此外，我们的新见解表明，ZAP-70在与TCR结合的过程中可能发生构象变化。基于最近的这些研究，我们现在有机会开发一种新的、可能更具特异性的ZAP-70功能变构抑制剂。因此，我们建议：1)鉴定ZAP-70的特异性变构抑制剂的先导化合物，并比较我们开发的模型催化位点抑制剂在阻断信号通路方面的有效性；2)利用我们建立的小鼠对ZAP-70类似物敏感的突变体，我们将确定ZAP-70抑制在关节炎、系统性红斑狼疮和实验性自身免疫性脑脊髓炎中的临床前效用。

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.