The X-linked deubiquitinase, USP9X, is implicated in multiple cancers by targeting various substrates. Increased expression of USP9X is observed in non-small-cell lung cancer (NSCLC) and is correlated with poor prognosis. However, the molecular mechanism for USP9X regulation of tumor cell survival and tumorigenesis in NSCLC is less defined.Methods: In this study, chemical labeling, quantitative proteomic screening was applied to analyze A549 cells with or without USP9X RNA interference. Functional in vitro and in vivo experiments were performed to confirm the oncogenic effects of USP9X in NSCLC and to investigate the underlying mechanisms.Results: The resulting data suggested that dual specificity protein kinase TTK is a potential substrate of USP9X. Further experimental evidences confirmed that USP9X stabilized TTK via direct interaction and efficient deubiquitination of TTK on K48 ubiquitin chain. Moreover, knockdown of USP9X or TTK inhibited cell proliferation, migration and tumorigenesis, and the immunohistochemical analysis of clinical NSCLC samples showed that the protein expression levels of USP9X and TTK were significantly elevated and positively correlated in tumor tissues.Conclusions: In summary, our data demonstrated that the USP9X-TTK axis may play a critical role in NSCLC, and could be considered as a potential therapeutic target.

Activation of the stimulator of interferon gene (STING) has emerged as an exciting immuno-oncology therapeutic strategy; however, the first-generation STING agonists, cyclic dinucleotide (CDN) analogues, have suffered from many disadvantages and failed in clinical trials. Therefore, non-CDN small-molecule STING agonists are urgently needed. In view of the unique structure of the high potency of dimeric amidobenzimidazole STING agonist 5, a structural elaboration was conducted by modifying several structural hotspots of this scaffold. Triazole 40 was identified as a new potent STING activator, possessing EC50 values of 0.24 and 39.51 μM for h- and m-STING, respectively. This compound has a slightly better pharmacokinetic profile and is >20-fold more aqueously soluble than 5. It activated the STING signaling dramatically by directly binding and stabilizing all h-STING isoforms and m-STING. In vivo, intermittent administration of 40 was found to have significant antitumor efficacy with good tolerance in two mouse tumor models.

The activation of cyclic GMP–AMP synthase (cGAS) by double-stranded DNA is implicated in the pathogenesis of many hyperinflammatory and autoimmune diseases, and the cGAS-targeting small molecule has emerged as a novel therapeutic strategy for treating these diseases. However, the currently reported cGAS inhibitors are far beyond maturity, barely demonstrating in vivo efficacy. Inspired by the structural novelty of compound 5 (G140), we conducted a structural optimization on both its side chain and the central tricyclic core, leading to several subseries of compounds, including those unexpectedly cyclized complex ones. Compound 25 bearing an N-glycylglycinoyl side chain was identified as the most potent one with cellular IC50 values of 1.38 and 11.4 μM for h- and m-cGAS, respectively. Mechanistic studies confirmed its direct targeting of cGAS. Further, compound 25 showed superior in vivo anti-inflammatory effects in the lipopolysaccharide-induced mouse model. The encouraging result of compound 25 provides solid evidence for further pursuit of cGAS-targeting inhibitors as a new anti-inflammatory treatment.

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The coronavirus disease 2019 (COVID-19) pandemic caused by the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has cast a notorious damage to the public health and global economy. The Stimulator of Interferon Genes (STING) is a crucial element of the host antiviral pathway and plays a pivotal but complex role in the infection and development of COVID-19. Herein, we discussed the antagonistic mechanism of viral proteins to the STING pathway as well as its activation induced by host cells. Specifically, we highlighted that the persistent activation of STING by SARS-CoV-2 led to abnormal inflammation, and STING inhibitors could reduce the excessive inflammation. In addition, we also emphasized that STING agonists possessed antiviral potency against diverse coronavirus and showed adjuvant efficacy in SARS-CoV-2 vaccines by inducing IFN responses.