Lightly stroking the lips or gently poking some skin regions can evoke mechanical itch in healthy human subjects. Sensitization of mechanical itch and persistent spontaneous itch are intractable symptoms in chronic itch patients. However, the underlying neural circuits are not well defined. We identified a subpopulation of excitatory interneurons expressing Urocortin 3::Cre (Ucn3 + ) in the dorsal spinal cord as a central node in the pathway that transmits acute mechanical itch and mechanical itch sensitization as well as persistent spontaneous itch under chronic itch conditions. This population receives peripheral inputs from Toll-like receptor 5-positive (TLR5 + ) Ab low-threshold mechanoreceptors and is directly innervated by inhibitory interneurons expressing neuropeptide Y::Cre (NPY + ) in the dorsal spinal cord. Reduced synaptic inhibition and increased intrinsic excitability of Ucn3 + neurons lead to chronic itch sensitization. Our study sheds new light on the neural basis of chronic itch and unveils novel avenues for developing mechanism-specific therapeutic advancements.

A novel series of tertiary amines as retinoid-related orphan receptor gamma-t (RORγt) inverse agonists was discovered through agonist/inverse agonist conversion. The level of RORγt inhibition can be enhanced by modulating the conformational disruption of H12 in RORγt LBD. Linker exploration and rational design led to the discovery of more potent indole-based RORγt inverse agonists.KEYWORDS: RORγt, agonists, inverse agonists, Th17 cell differentiation, cocrystal structure, structure-based design R etinoid-related orphan receptor gamma-t (RORγt) is a member of the nuclear receptor superfamily. RORγt is a key regulator of the development and functions of T-helper 17 (Th17) cells which are implicated in the pathology of a variety of human inflammatory and autoimmune disorders. 1,2 The RORγt inhibitors have potential utility in controlling the activity of Th17 cells and can be developed as therapeutic agents for treatment of Th17-related autoimmune diseases. A few small molecule inhibitors of RORγt have been reported in the literature. 3−10 In this paper, we report the discovery of tertiary amines and indoles as potent RORγt inverse agonists using structure-and knowledge-based compound design.A high-throughput screen (HTS) of the GSK in-house compound collection using a RORγ fluorescence resonance energy transfer (FRET) assay 11 resulted in identification of thiazole amide 1 as a RORγt inverse agonist with IC 50 of 1.0 μM. The binding of 1 to the RORγt ligand binding domain (LBD) was confirmed with a thermal shift of 7.1°C in a thermal shift assay. 11 SAR exploration on the left-hand side (LHS) of 1 led to the identification of tertiary amine 2 as a potent RORγt agonist with a EC 50 of 0.02 μM in dual FRET assay (Scheme 1). 12 Dual FRET assay, using the same technology as the FRET assay but without adding a surrogate agonist, only relies on the basal level of RORγt activity and is able to measure both agonists and inverse agonists. Peptide profiling study using dual FRET assay showed that coactivator peptide (e.g., steroid receptor coactivator 1 (SRC1)) was recruited upon binding of 2 to RORγt LBD whereas corepressor peptide (e.g., nuclear receptor corepressor 2 (NCOR2)) was not. 12 Given the structure similarity of 1 and 2, we assume that they adopt a similar binding mode within RORγt LBD despite their difference as agonist and inverse agonist. To understand the binding mode of the chemical series, an in-silico docking study for 2 based on a reported RORγt crystal structure 13 was conducted.A RORγt LBD crystal structure (PDB accession code: 3KYT) was selected and processed for the docking study. A total of 40 poses with the best scores were obtained and visually inspected after docking with Surflex-Dock v2.3 14−16 in Sybyl 8.1, 17 among which the top 10 poses were found to be representative and thus further ranked using MM/GBSA 18−20 affinity scores based on the VSGB2.0 solvent model. 21,22 As a

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Glycyrrhizin is an important bioactive compound that is used clinically to treat chronic hepatitis and is also used as a sweetener world-wide. However, the key UDP-dependent glucuronosyltransferases (UGATs) involved in the biosynthesis of glycyrrhizin remain unknown. To discover unknown UGATs, we fully annotated potential UGATs from Glycyrrhiza uralensis using deep transcriptome sequencing. The catalytic functions of candidate UGATs were determined by an in vitro enzyme assay. Systematically screening 434 potential UGATs, we unexpectedly found one unique GuUGAT that was able to catalyse the glucuronosylation of glycyrrhetinic acid to directly yield glycyrrhizin via continuous two-step glucuronosylation. Expression analysis further confirmed the key role of GuUGAT in the biosynthesis of glycyrrhizin. Site-directed mutagenesis revealed that Gln-352 may be important for the initial step of glucuronosylation, and His-22, Trp-370, Glu-375 and Gln-392 may be important residues for the second step of glucuronosylation. Notably, the ability of GuUGAT to catalyse a continuous two-step glucuronosylation reaction was determined to be unprecedented among known glycosyltransferases of bioactive plant natural products. Our findings increase the understanding of traditional glycosyltransferases and pave the way for the complete biosynthesis of glycyrrhizin.

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