Discovery of Triazolyl Derivatives of Cucurbitacin B Targeting IGF2BP1 against Non-Small Cell Lung Cancer.

Cucurbitacin B (CuB) is a potent but toxic anticancer natural product. Herein, we designed and synthesized 2-OH- and 16-OH-modified CuB derivatives to improve their antitumor efficacy and reduce toxicity. Among them, derivative A11 had the most potent antiproliferative activity against A549 lung cancer cells (IC50 = 0.009 µM) and was approximately 10-fold more potent than CuB, while the cytotoxicity of A11 toward normal L02 cells was about 10-fold less potent, indicating a much wider therapeutic window than CuB. Derivative A11 directly binds to the insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) protein with a KD value of 2.88 nM, which is about 23-fold more potent than CuB, leading to the decreased expression of downstream apoptosis- and cell cycle-related proteins. More importantly, A11 exhibited much more potent anticancer efficacy in an A549 xenograft mouse model with a TGI rate of 80% and a superior in vivo safety profile than that of CuB.

Photochemically controlled activation of STING by CAIX-targeting photocaged agonists to suppress tumor cell growth.

Controllable activation of the innate immune adapter protein - stimulator of interferon genes (STING) pathway is a critical challenge for the clinical development of STING agonists due to the potential "on-target off-tumor" toxicity caused by systematic activation of STING. Herein, we designed and synthesized a photo-caged STING agonist 2 with a tumor cell-targeting carbonic anhydrase inhibitor warhead, which could be readily uncaged by blue light to release the active STING agonist leading to remarkable activation of STING signaling. Furthermore, compound 2 was found to preferentially target tumor cells, stimulate the STING signaling in zebrafish embryo upon photo-uncaging and to induce proliferation of macrophages and upregulation of the mRNA expression of STING as well as its downstream NF-kB and cytokines, thus leading to significant suppression of tumor cell growth in a photo-dependent manner with reduced systemic toxicity. This photo-caged agonist not only provides a powerful tool to precisely trigger STING signalling, but also represents a novel controllable STING activation strategy for safer cancer immunotherapy.

Agrobacterium fabrum gene atu1420 regulates the pathogenicity by affecting the degradation of growth- and virulence-associated phenols.

Agrobacterium fabrum is a phytopathogen that causes the crown gall disease. Some plant-derived molecules, e.g. phenols, directly affect A. fabrum-plant interactions. Here, we characterize a phenolic catabolism-related gene, atu1420, that affects the pathogenicity of A. fabrum. Atu1420 is predicted to be an O-demethylase with high structural homology to Sphingomonas paucimobilis LigM. The HPLC-UV analysis showed that atu1420 affected the degradation of acetosyringone (AS). The deletion of atu1420 gene significantly enhanced the AS-induced virulence (vir) gene expression. atu1420 was shown to relieve the inhibitory effect of vanillic acid on the AS-induced vir gene expression and the growth of A. fabrum. The expression of atu1420 and the degradation of AS in A. fabrum C58 was up-regulated by the addition of indole acetic acid (IAA). The inhibitory effect of IAA on the AS-induced vir gene expression was partially relieved by the deletion of atu1420 gene, indicating that accelerating the degradation of AS is one of the ways that IAA inhibits vir genes induction. Furthermore, atu1420 mutant produced more pronounced tumors on kalanchoe leaves than the wild-type strain. These findings reveal the role of atu1420 in A. fabrum-host interactions and will broaden our understanding of the regulatory network of the interactions.

Involvement of the STING signaling in COVID-19.

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.

Synthesis and anti-tumor activity of nitrogen-containing derivatives of the natural product diphyllin.

The natural product diphyllin has demonstrated great potential in the treatment of various human cancers, especially pancreatic cancer. However, its relative weak potency, low aqueous solubility, and poor metabolic stability limits its development ability. In this study, we designed and synthesized two series of novel nitrogen-containing diphyllin derivatives with the aim to improve both antitumor efficacy and drug-like properties. Among them, the amino derivative 15 showed an IC50 value of 3 nM against pancreatic cancer CFPAC-1 cells and is about 69-fold more potent than diphyllin. In addition, compound 15 possesses improved aqueous solubility and metabolic stability in liver microsomes. This compound not only significantly induced cell cycle arrest at G0/G1 phase with down-regulation of CDK4 and cyclinD1 in a dose-dependent manner, but also blocked the later stage of autophagy in CFPAC-1 cells. In pancreatic cancer xenograft model, treatment of 15 with 10 mg/kg exhibited much more potent efficacy in suppressing the growth of transplanted PANC02 tumors than diphyllin without obvious safety concern.

Structure-Activity relationship study of benzothiophene oxobutanoic acid analogues leading to novel stimulator of interferon gene (STING) agonists.

Pharmacological activation of stimulator of interferon genes (STING) by agonists has emerged as a new modality of cancer immunotherapy. However, current available STING agonists remain in early developmental stage or failed in clinic trials due to limited efficacy in humans. In this report, we performed a structure-activity relationship study based on the benzothiophene oxobutanoic acid scaffold of MSA-2, a well-documented STING agonist by Merck, leading to a series of N-substituted acyloxyamino derivatives with potent STING activating effect. Among them, compounds 57 and 60 displayed the most potent activity specifically targeting both h- and m-STING. Particularly, 57 displayed more potent and rapid activation of the STING signaling pathway than ADU-S100 in THP1-Dual cells. In vivo anti-tumor efficacy of 57 by intratumoral or oral administration was also demonstrated in several mouse tumor models. Intriguingly, treatment with 57 eradicated all the CT26 tumor without further recurrence in all treated mice, which could also reject the same tumor re-inoculation, indicating an induction of immune memory by 57. Taken together, acyloxyamino derivative 57 represents a new chemotype of STING agonist with well-demonstrated in vivo anti-tumor activity, which is deserved for further investigation.

Small molecules targeting cGAS-STING pathway for autoimmune disease.

Autoimmune diseases represent a class of over 80 illnesses with high incidence and prevalence and share a common pathogenesis of immune system disorders and self-attack. Over the past decade, extensive studies have demonstrated that imbalance of cGAS-STING mediated innate immune signaling is closely involved in autoimmune diseases. Over-activation of cGAS-STING pathway by mutations of STING or several exonucleases can cause accumulation of interferon and systemic inflammation. Therefore, suppression of the upregulated cGAS-STING pathway holds great potential in the treatment of human inflammatory and autoimmune diseases. Inhibitors targeting cGAS, STING and the downstream factors have been developed and pharmacologically evaluated recently. Herein, we summarize the recent advance on development of small molecular inhibitors targeting the key effectors in cGAS-STING axis as promising treatment for autoimmune diseases.

Synthesis and pharmacological validation of fluorescent diarylsulfonylurea analogues as NLRP3 inhibitors and imaging probes.

The NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) is a key cytosolic pattern recognition receptor that senses diverse pathogen- and host-originated threat signals. Aberrant activation of NLRP3 inflammasomes is closely associated with the pathogenesis of various complex inflammatory diseases. Nevertheless, the detailed regulation mechanism of NLRP3 inflammasome and its pathogenic roles in the inflammation progression remain to be fully elucidated. Fluorescent imaging with small molecule probe can provide valuable visualization information on the expression, occupancy and bio-distribution of target protein. Herein, we reported a series of diarylsulfonylurea NLRP3 fluorescent inhibitors bearing an amino benzodiazole fluorophore. Compared to the previously reported NLRP3 fluorescent probes, these inhibitors are more structurally concise and membrane permeable due to no additionally appended fluorophore via a linker. Among this series, compound 13a exhibited the most potent cellular NLRP3 inhibitory effect with an IC50 value of 49 nM, and significantly suppressed LPS/Nigericin-induced secretion of active caspase-1 and mature IL-1ß in a dose-dependent manner to block the activation of NLRP3 inflammasome. Meanwhile, this new probe exhibited promising fluorescent properties for specifically detecting and imaging the LPS-induced or constitutively expressed NLRP3 proteins in RAW264.7 cells. Collectively, probe 13a is a potent NLRP3 fluorescent inhibitor with cellular NLRP3 imaging ability, which is useful for NLRP3 inhibitor screening and related mechanism study.

Synthesis of aryldifluoromethyl aryl ethers via nickel-catalyzed suzuki cross-coupling between aryloxydifluoromethyl bromides and boronic acids.

As a unique organofluorine fragment, gem-difluoromethylated motifs have received widespread attention. Here, a convenient and efficient synthesis of aryldifluoromethyl aryl ethers (ArCF2OAr') was established via Nickel-catalyzed aryloxydifluoromethylation with arylboronic acids. This approach features easily accessible starting materials, good tolerance of functionalities, and mild reaction conditions. Diverse late-stage difluoromethylation of many pharmaceuticals and natural products were readily realized. Notably, a new difluoromethylated PD-1/PD-L1 immune checkpoint inhibitor was conveniently synthesized and showed both improved metabolic stability and enhanced antitumor efficacy. Preliminary mechanistic studies suggested the involvement of a Ni(I/III) catalytic cycle.

Synthesis and Pharmacological Evaluation of Tetrahydro-γ-carboline Derivatives as Potent Anti-inflammatory Agents Targeting Cyclic GMP-AMP Synthase.

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.

Structure-Activity Relationship Study of Amidobenzimidazole Analogues Leading to Potent and Systemically Administrable Stimulator of Interferon Gene (STING) Agonists.

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.

Suppression of asparagine synthetase enhances the antitumor potency of ART and artemalogue SOMCL-14-221 in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality. Artemisinin (ART) and SOMCL-14-221 (221), a spirobicyclic analogue of ART, have been reported to inhibit the proliferation of A549 cells with unclear underlying mechanism. In the present study, we validated that both ART and 221 inhibited the proliferation and migration of NSCLC cells and the growth of A549 xenograft tumors without appreciable toxicity. The proteomic data revealed proteins upregulated in ART and 221 groups were involved in "response to endoplasmic reticulum stress" and "amino acid metabolism". Asparagine synthetase (ASNS) was identified as a key node protein in these processes. Interestingly, knockdown of ASNS improved the antitumor potency of ART and 221 in vitro and in vivo, and treatments with ART and 221 disordered the amino acid metabolism of A549 cells. Moreover, ART and 221 activated ER stress, and inhibition of ER stress abolished the anti-proliferative effects of ART and 221. In conclusion, this study demonstrates that ART and 221 suppress tumor growth by triggering ER stress, and the inhibition of ASNS enhances the antitumor activity of ART and 221, which provides new strategy for drug combination therapy.

A novel USP9X substrate TTK contributes to tumorigenesis in non-small-cell lung cancer.

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.

Deep Phosphoproteomic Measurements Pinpointing Drug Induced Protective Mechanisms in Neuronal Cells.

Alzheimer's disease (AD) is a progressive and irreversible neurological disorder that impairs the living quality of old population and even life spans. New compounds have shown potential inneuroprotective effects in AD, such as GFKP-19, a 2-pyrrolidone derivative which has been proved to enhance the memory of dysmnesia mouse. The molecular mechanisms remain to be established for these drug candidates. Large-scale phosphoproteomic approach has been evolved rapidly in the last several years, which holds the potential to provide a useful toolkit to understand cellular signaling underlying drug effects. To establish and test such a method, we accurately analyzed the deep quantitative phosphoproteome of the neuro-2a cells treated with and without GFKP-19 using triple SILAC labeling. A total of 14,761 Class I phosphosites were quantified between controls, damaged, and protected conditions using the high resolution mass spectrometry, with a decent inter-mass spectrometer reproducibility for even subtle regulatory events. Our data suggests that GFKP-19 can reverse Aß25-35 induced phosphorylation change in neuro-2a cells, and might protect the neuron system in two ways: firstly, it may decrease oxidative damage and inflammation induced by NO via down regulating the phosphorylation of nitric oxide synthase NOS1 at S847; Secondly, it may decrease tau protein phosphorylation through down-regulating the phosphorylation level of MAPK14 at T180. All mass spectrometry data are available via ProteomeXchange with identifier PXD005312.