Inflammatory bowel disease (IBD) is a multifactorial autoimmune disease, representing a major clinical challenge. Herein, a strategy of dual-targeting approach employing retinoic acid receptor-related orphan receptor γ-t (RORγt) and dihydroorotate dehydrogenase (DHODH) was proposed for the treatment of IBD. Dual RORγt/DHODH inhibitors are expected not only to reduce RORγt-driven Th17 cell differentiation but also to mitigate the expansion and activation of T cells, which may enhance anti-inflammatory effects. Starting from 2-aminobenzothiazole hit 1, a series of 2-aminotetrahydrobenzothiazoles were discovered as potent dual RORγt/DHODH inhibitors. Compound 14d stands out with IC 50 values of 0.110 μM for RORγt and of 0.297 μM for DHODH. With acceptable mouse pharmacokinetic profiles, 14d exhibited remarkable in vivo anti-inflammatory activity and dose-dependently alleviated the severity of dextran sulfate sodium (DSS)-induced acute colitis in mice. Taken together, the present study provides a novel framework for the development of therapeutic agents for the treatment of IBD.

In recent years, the adenosine A2A receptor (A2AR) has shown exciting progress in the development of immunotherapies for the treatment of cancer. Herein, a 2-amino-7,9-dihydro-8H-purin-8-one compound (1) was identified as an A2AR antagonist hit through in-house library screening. Extensive structure–activity relationship (SAR) studies led to the discovery of 2-aminopteridin-7­(8H)-one derivatives, which showed high potencies on A2AR in the cAMP assay. Compound 57 stood out with an IC50 value of 8.3 ± 0.4 nM against A2AR at the 5′-N-ethylcarboxamidoadenosine (NECA) level of 40 nM. The antagonistic effect of 57 was sustained even at a higher NECA concentration of 1 μM, which mimicked the adenosine level in the tumor microenvironment (TME). Importantly, 57 enhanced T cell activation in both the IL-2 production assay and the cancer-cell-killing model, thus demonstrating its potential as a lead for developing novel A2AR antagonists in cancer immunotherapy.

Recent studies and clinical evidence have strongly supported the development of adenosine A2A receptor (A2AR) antagonists as novel approaches for cancer immunotherapy. By screening our in-house compound library, a pyridinone hit compound (1) with weak A2AR antagonistic activity was identified. Further structure–activity relationship studies revealed a series of pyridinone derivatives with strong potency. Compound 38 stood out with a potent A2AR antagonistic activity (IC50 = 29.0 nM), good mouse liver microsomal metabolic stability (t 1/2 = 86.1 min), and excellent oral bioavailability (F = 86.1%). Of note, 38 effectively enhanced the activation and killing ability of T cells in vitro by down-regulation of immunosuppressive molecules (LAG-3 and TIM-3) and up-regulation of effector molecules (GZMB, IFNG, and IL-2). Moreover, 38 exhibited excellent in vivo antitumor activity with a tumor growth inhibition (TGI) of 56.0% in the MC38 tumor model via oral administration, demonstrating its potential as a novel A2AR antagonist candidate for cancer immunotherapy.

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BACKGROUND AND PURPOSE Human dihydroorotate dehydrogenase (DHODH) represents a promising therapeutic target for chronic inflammatory and autoimmune diseases. The aim of this study was to discover novel DHODH inhibitor and evaluate the potential of DHODH inhibition in treating multiple sclerosis (MS), a popular chronic inflammatory disease of the central nervous system. EXPERIMENTAL APPROACH Biochemical and Biophysical methods, including enzymatic kinetic analysis, thermofluor assay, isothermal titration calorimetry and X-ray crystallography were used to assess DHODH inhibition. The immunomodulatory activity was assessed by using concanavalin a-triggered T-cell assay and mixed lymphocyte reaction assay. MOG-induced experimental allergic encephalomyelitis (EAE) was used to assess the in vivo therapeutic effects. Myelin destruction and blood-brain barrier (BBB) was evaluated via in vivo imaging KEY RESULTS Piperine was identified as a natural inhibitor of human DHODH with an IC50 value of 0.88 ± 0.04 μM. In addition, we resolved the co-complex crystal structure of DHODH and piperine at 1.98Å resolution and found that Tyr356 residue of DHODH is critical for piperine binding. Moreover, piperine can markedly suppress T cell overactivation via a DHODH dependent-manner. Finally, we found that piperine exhibits strong preventive and therapeutic effect in the MOG-induced EAE by restricting inflammatory cells infiltration into the CNS and by preventing myelin destruction and blood-brain barrier (BBB) disruption. CONCLUSION AND IMPLICATIONS Taken together, these findings highlight DHODH as a therapeutic target for autoimmune disease of the nervous system, and demonstrate a novel pharmacological role for piperine in the treatment of MS. EXPERIMENTAL APPROACH Biochemical and Biophysical methods, including enzymatic kinetic analysis, thermofluor assay, isothermal titration calorimetry and X-ray crystallography were used to assess DHODH inhibition. The immunomodulatory activity was assessed by using concanavalin a-triggered T-cell assay and mixed lymphocyte reaction