A high-throughput platform to identify selective allosteric inhibitors of the PLC-y isozymes

摘要 两种PLC-Gamma同工酶(PLC-Gamma1、-Gamma2)是多种受体酪氨酸激酶和免疫受体(包括B和T细胞受体)信号传递和放大的中心。当这种调控失调时，PLC-γ同工酶会导致炎症和免疫性疾病，如各种白血病和淋巴瘤。例如，PLC-Gamma1是成人T细胞白血病/淋巴瘤患者中最常见的(~40%)突变蛋白；突变蛋白总是具有结构性活性。同样，突变的、构成活性形式的PLC-Gamma2在B细胞白血病的治疗中出现，并导致难治性疾病。尽管PLC-Gamma同工酶与血液病癌症之间存在明显的因果关系，但目前还没有专门针对PLC-Gamma同工酶的化学探针或突破性疗法。这一缺陷主要是由于我们对PLC-Gamma同工酶是如何调控的了解不够充分，以及没有足够的工具以适应高通量筛选的方式监测这种调控。 我们最近在原子分辨率下确定了全长PLC-伽马同工酶的第一个结构，并用它来建立其调控的变构模型。此外，我们还创造了XY-69，这是第一个荧光的、膜结合的PIP2类似物，它可靠地捕捉到PLC-伽马同工酶对细胞外刺激或突变的变构激活。在初步研究中，我们表明XY-69可用于在高通量筛选中鉴定正构和变构抑制剂。因此，这项提案的总体目标是确定PLC-伽马同工酶的选择性抑制剂，用作药物开发的化学探针和先导。 我们将追求两个具体目标。在目标1中，我们将使用一种新的高通量筛选来鉴定选择性抑制PLC-伽马同工酶的类药物化合物。在目标2中，将利用一系列强调T细胞生物学和趋化性的生化、生物物理和细胞分析，对这些化合物进行分析并优先进行进一步优化。最终，这些抑制剂将是必不可少的、高需求的探针，用于剖析由PLC-伽马同工酶控制的细胞过程。此外，这些抑制剂将成为开发治疗由PLC-伽马同工酶驱动的各种血液病癌症的药物的有希望的线索。

ABSTRACT The two PLC-gamma isozymes (PLC-gamma1, -gamma2) are central to the relay and amplification of signals originating from numerous receptor tyrosine kinases and immune receptors, including the B and T cell receptors. When this control is dysregulated, the PLC-gamma isozymes contribute to inflammatory and immunological diseases as exemplified by various leukemias and lymphomas. For example, PLC-gamma1 is the most frequently (~40%) mutated protein in patients with adult T cell leukemia/lymphoma; the mutated proteins are invariably constitutively active. Similarly, mutated, constitutively active forms of PLC-gamma2 arise in response to treatment of B cell leukemias and lead to refractory disease. Despite the obvious causality between the PLC-gamma isozymes and hematologic cancers, there are no chemical probes or breakthrough therapeutics that specifically target the PLC-gamma isozymes. This deficiency arises mainly from our incomplete understanding of how the PLC-gamma isozymes are regulated as well as inadequate tools to monitor this regulation in a manner amenable to high-throughput screens. We recently determined the first structure of a full-length PLC-gamma isozyme at atomic resolution and used it to develop an allosteric model of its regulation. In addition, we created XY-69, the first fluorescent, membrane-bound analog of PIP2 that reliably captures the allosteric activation of PLC-gamma isozymes in response to either extracellular stimuli or mutation. In preliminary studies, we show that XY-69 can be used to identify both orthosteric and allosteric inhibitors in high-throughput screens. Consequently, the overall objective of this proposal is to identify selective inhibitors of the PLC-gamma isozymes to be used as chemical probes and leads for drug development. Two Specific Aims will be pursued. In Aim 1, we will use a novel high-throughput screen to identify drug-like compounds that selectively inhibit the PLC-gamma isozymes. In Aim 2, these compounds will be profiled and prioritized for further optimization using a battery of biochemical, biophysical and cellular assays that emphasize T cell biology and chemotaxis. Ultimately, these inhibitors will be essential, high-demand probes used to dissect cellular processes controlled by the PLC-gamma isozymes. Moreover, these inhibitors will be promising leads for the development of drugs to treat various hematologic cancers driven by the PLC-gamma isozymes.