Discovery of a Highly Potent Oxysterol Receptor GPR183 Antagonist Bearing the Benzo[d]thiazole Structural Motif for the Treatment of Inflammatory Bowel Disease (IBD).

Accumulating evidence has demonstrated a critical pathological role of oxysterol receptor GPR183 in various inflammatory and autoimmune diseases, including inflammatory bowel disease (IBD). However, the currently reported GPR183 antagonists are very limited and not qualified for in vivo studies due to their inferior druglike properties. Herein, we conducted a structural elaboration focusing on improving its PK and safety profile based on a reference antagonist NIBR189. Of note, compound 33, bearing an aminobenzothiazole motif, exhibited reduced hERG inhibition, improved PK properties, and robust antagonistic activity (IC50 = 0.82 nM) with high selectivity against GPR183. Moreover, compound 33 displayed strong in vitro antimigration and anti-inflammatory activity in monocytes. Oral administration of compound 33 effectively improved the pathological symptoms of DSS-induced experimental colitis. All of these findings demonstrate that compound 33 is a novel and promising GPR183 antagonist suitable for further investigation to treat IBD.

Discovery of Pyridinone Derivatives as Potent, Selective, and Orally Bioavailable Adenosine A2A Receptor Antagonists for 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 (t1/2 = 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.

HERC3 directly targets RPL23A for ubiquitination degradation and further regulates Colorectal Cancer proliferation and the cell cycle.

Aims: Colorectal cancer (CRC) has high mortality and morbidity rates; however, the mechanism of CRC cells uncontrolled proliferation is unclarified, E3 ligases are widely reported to have crucial functions in cancers. HERC3 was once recognized as an important role in CRC, however its effects on CRC cell proliferation and cell cycle are blank. Methods: Correlation between HERC3 and clinical characteristics was analyzed. Coimmunoprecipitation, mass spectrometry analysis and GST-pull down were performed to identify interacting-proteins of HERC3. Expression pattern of RPL23A and its correlation between HERC3 was researched via qRT-PCR, western blot and immunohistochemistry. In vivo and vitro gain-and loss-of-function assays and rescue experiments concentrating HERC3-RPL23A axis in terms of cell proliferation and cell cycle were conducted. The ubiquitination regulatory mechanism between HERC3 and RPL23A were identified via vivo ubiquitylation assays, cycloheximide analysis and mass spectrometry analysis. GSEA aided to research the potential functional mechanism of RPL23A and validated by western blot and in vivo ubiquitylation assays. Results: HERC3 expression decreased gradually from colorectal tissues in healthy individuals to adjacent-tumors normal tissue in CRC patients, and to tumor tissues and HERC3 could inhibit CRC cell proliferation and arrest cells in the G0-G1 phase. RPL23A which was recognized as one potential target of HERC3 was identified to be overexpressed in CRC and could serve as a prognostic biomarker in CRC. RPL23A could also independently regulate the cell cycle and cell proliferation and attenuate the influence of HERC3 on CRC. In addition, HERC3 directly interacted with RPL23A and served as an E3 ligase to ubiquitination degrade RPL23A via K48-dependant manner through the HECT domain. Furthermore, HERC3 could regulate the ubiquitination of p21 and further modulate protein expression of c-Myc and p21 via regulating RPL23A. Conclusion: HERC3 controlled CRC proliferation, the cell cycle and regulated the c-Myc/p21 axis via directly targeting RPL23A for ubiquitination degradation.

Epilepsy-Associated UBE3A Deficiency Downregulates Retinoic Acid Signalling Pathway.

Ubiquitin-protein ligase E3A (UBE3A) has dual functions as a E3 ubiquitin-protein ligase and coactivator of nuclear hormone receptors. Mutations or deletions of the maternally inherited UBE3A gene cause Angelman syndrome. Here, we performed transcriptome profiling in the hippocampus of Ube3a m+/p+ and Ube3a m-/p+ mice, and determined that the expression of the retinoic acid (RA) signalling pathway was downregulated in Ube3a-deficient mice compared to WT mice. Furthermore, we demonstrated that UBE3A directly interacts with RARα and may function as a coactivator of the nuclear receptor RARα to participate in the regulation of gene expression. Loss of UBE3A expression caused the downregulation of the expression of RA-related genes, including Erbb4, Dpysl3, Calb1, Pten, and Arhgap5 in Ube3a m-/p+ mice brain tissues. This work revealed a new role for UBE3A in regulating retinoic acid (RA) signalling downstream genes and hopefully to shed light on the potential drug target of AS.

BAP1 regulates AMPK-mTOR signalling pathway through deubiquitinating and stabilizing tumour-suppressor LKB1.

Liver kinase B1 (LKB1), a tumour suppressor, participates in many cellular processes, including cell survival, growth, apoptosis, transformation, and metabolism. Upon performing yeast two-hybrid screening, co-immunoprecipitation, and GST pull-down, we identified that BRCA1-associated protein 1 (BAP1), a deubiquitinase, interacts with LKB1. Immunoblotting was performed to examine the effect of BAP1 on the activation of 5' AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR), downstream of LKB1. The relationship between BAP1 deficiency and cancer cell proliferation was examined using cell survival assay and soft agar assay. qRT-PCR and oil red O staining were performed to evaluate lipid synthesis. Our findings reveal that BAP1 deubiquitinates LKB1, inhibits its degradation, and stabilises it, thereby affecting AMPK activation and downstream mTOR activity. BAP1 deficiency may enhance cellular proliferation as well as lipid synthesis.

Ubiquitination of IGF2BP3 by E3 ligase MKRN2 regulates the proliferation and migration of human neuroblastoma SHSY5Y cells.

Neuroblastoma (NB) is a paediatric tumour that shows great biomolecule and clinical heterogeneity, and patients with NB often develop various neurological complications. Currently, the disease is mainly treated by surgery and still lacks specific therapeutic drugs; therefore, targets are urgently needed. Makorin ring finger protein 2 (MKRN2) is an E3 ligase whose effects on neuroblastoma have not been illustrated. shRNAs for MKRN2 have been designed, and MKRN2-knockdown human neuroblastoma SHSY5Y cells were established. MKRN2 knockdown promotes the proliferation and migration of SHSY5Y cells. Because MKRN2 is an E3 ligase, we performed a series of experiments, and Insulin-like growth factor-2 mRNA-binding protein 3 (IGF2BP3) was identified as a new substrate for MKRN2. IGF2BP3 is an RNA-binding protein that regulates the stability of many mRNAs, including CD44 and PDPN, and our study demonstrated that MKRN2 regulates the expression of CD44 and PDPN in an IGF2BP3-dependent manner. These results suggest that MKRN2 might be a potential therapeutic target for neuroblastoma.