Discovering modulators of exonucleases PLD3 and PLD4 for immunoregulation

摘要 以前被认为是磷脂酶的PLD3和PLD4最近被发现是内涵体 通过消化核酸传感器的配体来调节先天免疫反应的核酸外切酶。这些 酶可以抑制TLR7-9、TLR13等先天免疫感受器和未知的刺痛 传感器。PLD3或PLD4缺失的细胞对TLR9的某些配体的反应增强。此外，Pld4- /-小鼠过度产生细胞因子并上调居民表面主要组织相容性复合体II类 腹膜巨噬细胞。考虑到强大的免疫调节作用，这些反应并不令人惊讶 B细胞、巨噬细胞和树突状细胞，特别是浆细胞样树突状细胞上的核酸配体 产生大量I型干扰素(干扰素)。最近发现的这些细胞的免疫调节作用 酶有助于解释它们的多态与类风湿性关节炎等疾病之间的相关性， 系统性硬化症、阿尔茨海默病、肾脏纤维化和其他炎症性疾病。除了……之外 对于这些疾病的治疗，PLD3和PLD4的调节子可以作为佐剂，稳定剂 寡核苷酸疗法、免疫抑制药和刺激剂。 核酸感受器激动剂与几种靶向TLRs和刺痛通路的化合物一起受到追捧 正在进行临床试验，作为对癌症和其他疫苗的免疫反应的佐剂。 咪喹莫特证明了靶向TLRs的可行性，并被批准用于人类治疗 光化性角化病、生殖器疣和非黑色素瘤皮肤癌。PLD3和PLD4调制器提供了独特的 以核酸传感器为目标的战略有两个原因。(1)PLD3和PLD3不是针对特定传感器 PLD4调节剂会影响几种免疫传感器的核酸配体的可用性。(2)到期 细胞类型表达的差异，PLD4调节剂将允许选择性靶向免疫细胞，因为 与PLD3调制器的边界效应相反。为了实现任何有希望的治疗潜力，PLD3和 必须明确PLD4通路及其在疾病中的作用。为此，我们试图识别小分子 选择性和非选择性地激活或抑制PLD3和PLD4的探针。 我们已经建立了一种高通量的荧光酶活性分析来鉴定PLD3和PLD3的调节因子 PLD4函数。多样性文库(N=1056)的初步筛选已鉴定出非选择性和选择性激活剂 以及PLD3和PLD4的抑制剂。我们试图通过筛选~100K的多样性来探索更广阔的化学空间 图书馆。利用这些数据，将通过药物来识别和探索结构活性关系 化学反应。用于评估效力、特异性和毒性的细胞分析将被用于审查HITS。最后，捆绑 将评估动力学、蛋白质异构体活性和体外化学稳定性，以指导探针的优化 用于小鼠模型的分子。

ABSTRACT Previously thought to be phospholipases, PLD3 and PLD4 have recently been revealed to be endosomal exonucleases that regulate the innate immune response by digesting the ligands of nucleic acid sensors. These enzymes can suppress RNA and DNA innate immune sensors like TLR7-9, TLR13 and an unknown STING sensor. Cells deficient in PLD3 or PLD4 have enhanced responses to certain ligands of TLR9. Additionally, Pld4– /– mice overproduce cytokines and upregulate major histocompatibility complex class II on the surface of resident peritoneal macrophages. These responses are unsurprising considering the powerful immunomodulatory effects of nucleic acid ligands on B cell, macrophages, and dendritic cells, particularly plasmacytoid dendritic cells, which produce significant quantities of type I interferon (IFN). The recently discovered immunoregulatory role of these enzymes helps explain the correlation between their polymorphisms and diseases like rheumatoid arthritis, systemic sclerosis, Alzheimer’s disease, Kidney Fibrosis, and other inflammatory diseases. In addition to therapeutics for these diseases, modulators of PLD3 and PLD4 may serve as adjuvants, stabilizers of oligonucleotide therapies, and immune suppressants and stimulators. Nucleic acid sensors agonists are highly sought with several compounds that target TLRs and STING pathways undergoing undergoing clinical trials as adjuvants for immune responses to cancer and in other vaccines. Imiquimod demonstrates the feasibility of targeting TLRs and is approved for use in humans for the treatment of actinic keratosis, genital warts and non-melanoma skin cancers. PLD3 and PLD4 modulators present a unique strategy of targeting nucleic acid sensors for two reasons. (1) Rather than target specific sensors, PLD3 and PLD4 modulators would affect the availability of the nucleic acid ligands of several immune sensors. (2) Due differences in cell type expression, PLD4 modulators would allow for selective targeting of immune cells as opposed to the boarder effects of PLD3 modulators. To realize any promising therapeutic potential, PLD3 and PLD4 pathways, and their role in disease, must be defined. To this end, we seek to identify small molecule probes that selectively and nonselectively activate or inhibitor PLD3 and PLD4. We have developed a high-throughput fluorescence enzymatic activity assay to identify modulators of PLD3 and PLD4 function. A pilot screen of a diversity library (N = 1056) has identified nonselective and selective activators and inhibitors of PLD3 and PLD4. We seek to explore a broader chemical space by screening a ~100K diversity library. With these data, structural activity relationships will be identified and explored through medicinal chemistry. Cell assays to assess potency, specificity, and toxicity will be employed to vet hits. Finally, binding kinetics, protein isoform activity, and in vitro chemical stability will be evaluated to guide optimization of probe molecules for mouse models.