Single cell RNA seq analysis of LPS-stimulated PBMCs pre-treated with JRD-SIK1/2i-3

The Salt-Inducible Kinases (SIK) 1-3 are key regulators of pro- versus anti-inflammatory cytokine responses during innate immune activation. The lack of highly SIK-family or SIK isoform-selective inhibitors suitable for repeat, oral dosing has limited the study of which SIK isoforms need to be targeted to suppress inflammation in vivo. To overcome this challenge, we devised a structure-based design strategy for developing potent SIK inhibitors that are highly selective against other kinases by engaging two differentiating features of the SIK catalytic site. This effort resulted in SIK1/2-selective probes that inhibit key intracellular proximal signaling events including reducing phosphorylation of the SIK substrate cAMP response element binding protein (CREB) regulated transcription coactivator 3 (CRTC3) 3 as detected with a newly generated phospho-Ser329-specific antibody. These inhibitors also suppress production of pro-inflammatory cytokines while inducing anti-inflammatory interleukin-10 in activated human and murine myeloid cells as well as in mice following a lipopolysaccharide challenge. To define the functional effects of SIK1/2 inhibitors in primary human immune cells, we profiled the transcriptional responses elicited by SIK1/2-selective probe JRD-SIK1/2i-3 in peripheral blood mononuclear cells (PBMCs) isolated from healthy donors responding to TLR4 activation at single cell resolution. single-cell RNAseq of human PBMCs from two donors pre-treated with 0, 1 uM, or 10 uM JRD-SIK1/2i-3 for 1 hr, followed by 5 ng/mL LPS stimulation for 1, 4, or 24 hours.

盐诱导激酶（Salt-Inducible Kinases, SIK）1~3型是天然免疫激活过程中调控促炎与抗炎细胞因子应答的关键调控因子。目前缺乏适用于重复给药、口服使用的高选择性SIK家族或SIK亚型抑制剂，这极大限制了我们对体内抑制炎症所需靶向的SIK亚型的研究。为解决这一研究瓶颈，我们开发了基于结构的设计策略，通过靶向SIK催化结构域的两个差异化特征，研制出对其他激酶具有高度选择性的强效SIK抑制剂。本研究获得的SIK1/2选择性探针可抑制关键的细胞内近端信号转导事件，包括通过新生成的磷酸化Ser329特异性抗体检测证实的、SIK底物cAMP应答元件结合蛋白（cAMP response element binding protein, CREB）调控的转录共激活因子3（CRTC3）的磷酸化水平降低。此类抑制剂在活化的人源及鼠源髓系细胞中，可抑制促炎细胞因子的产生并诱导抗炎因子白细胞介素-10（interleukin-10, IL-10）的表达；在脂多糖（lipopolysaccharide, LPS）攻毒的小鼠体内同样可观察到这一效应。为明确SIK1/2抑制剂在原代人源免疫细胞中的功能效应，我们以单细胞分辨率分析了健康供者来源的外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）经Toll样受体4（TLR4）激活后，由SIK1/2选择性探针JRD-SIK1/2i-3诱导的转录应答。本研究对两名供者的外周血单个核细胞开展了单细胞RNA测序实验：细胞先经0、1 μM或10 μM的JRD-SIK1/2i-3预处理1小时，随后用5 ng/mL的脂多糖（LPS）分别刺激1、4或24小时。