Discovering modulators of exonucleases PLD3 and PLD4 for immunoregulation

摘要 PLD 3和PLD 4以前被认为是磷脂酶，最近被发现是内体磷脂酶。 通过消化核酸传感器的配体来调节先天免疫应答的核酸外切酶。这些 酶可以抑制RNA和DNA先天免疫传感器，如TLR 7 -9，TLR 13和未知的STING 传感器.缺乏PLD 3或PLD 4的细胞对TLR 9的某些配体具有增强的应答。此外，Pld 4- /-小鼠过度产生细胞因子并上调驻留细胞表面的主要组织相容性复合物II类 腹腔巨噬细胞考虑到强大的免疫调节作用， 核酸配体在B细胞、巨噬细胞和树突细胞，特别是浆细胞样树突细胞上的表达， 产生大量的I型干扰素（IFN）。最近发现的免疫调节作用， 酶有助于解释它们的多态性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、肾纤维化和其它炎性疾病。除了 作为这些疾病的治疗剂，PLD 3和PLD 4的调节剂可以用作佐剂、药物稳定剂、或药物的载体。 寡核苷酸疗法以及免疫抑制剂和刺激剂。 核酸传感器激动剂与靶向TLR和STING途径的几种化合物一起受到高度追捧 正在进行临床试验，作为癌症免疫反应和其他疫苗的佐剂。 咪喹莫特证明了靶向TLR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。PLD 3和PLD 4调节剂呈现独特的 靶向核酸传感器的策略有两个原因。(1)而不是针对特定的传感器，PLD 3和 PLD 4调节剂将影响几种免疫传感器的核酸配体的可用性。(2)由于 由于细胞类型表达的差异，PLD 4调节剂将允许选择性靶向免疫细胞， 这与PLD 3调制器的较宽效应相反。为了实现任何有希望的治疗潜力，PLD 3和 PLD 4通路及其在疾病中的作用必须明确。为此，我们寻求识别小分子 选择性和非选择性激活或抑制PLD 3和PLD 4的探针。 我们已经开发了一种高通量荧光酶活性测定法来鉴定PLD 3的调节剂， PLD 4功能。多样性文库的中试筛选（N = 1056）已经鉴定了非选择性和选择性激活剂 以及PLD 3和PLD 4的抑制剂。我们寻求通过筛选~ 100 K多样性来探索更广阔的化学空间 图书馆有了这些数据，结构活性关系将被确定，并通过药物研究进行探索。 化学.将采用评估效价、特异性和毒性的细胞测定来审查命中。最后，绑定 将评价动力学、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.