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. Overall design: 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.