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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.

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.

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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 Ube3am+/p+ and Ube3am–/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 Ube3am–/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.