miR-584-5p / Ykt6 - mediated autophagy - lysosome - exosome pathway as a critical route affecting the toxic effects of lead on HK-2 cells.

Lead is a widespread environmental pollutant with serious adverse effects on human health, but the mechanism underlying its toxicity remains elusive. This study aimed to investigate the role of miR-584-5p / Ykt6 axis in the toxic effect of lead on HK-2 cells and the related mechanism. Our data suggested that lead exposure caused significant cytotoxicity, DNA and chromosome damage to HK-2 cells. Mechanistically, lead exposure down-regulated miR-584-5p and up-regulated Ykt6 expression, consequently, autophagosomal number and autophagic flux increased, lysosomal number and activity decreased, exosomal secretion increased. Interestingly, when miR-584-5p level was enhanced with mimic, autophagosomal number and autophagic flux decreased, lysosomal number and activity increased, ultimately, exosomal secretion was down-regulated, which resulted in significant aggravated toxic effects of lead. Further, directly blocking exosomal secretion with inhibitor GW4869 also resulted in exacerbated toxic effects of lead. Herein, we conclude that miR-584-5p / Ykt6 - mediated autophagy - lysosome - exosome pathway may be a critical route affecting the toxic effects of lead on HK-2 cells. We provide a novel insight into the mechanism underlying the toxicity of lead on human cells.

Lysosome-related exosome secretion mediated by miR-26b / Rab31 pathway was associated with the proliferation and migration of MCF-7 cells treated with BPA.

Bisphenol A (BPA), one of the typical environmental endocrine disruptors (EEDs), can promote the proliferation and migration of cancer cells, but the mechanism of which remains largely unclear. Exosome secretion plays an important role in the stress response of cells to environmental stimuli. This study was designed to explore whether exosome secretion was involved in the toxic effect of BPA on the proliferation and migration of MCF-7 cells, and the related mechanism. Our data shows that the IC50 value of MCF-7 exposure to BPA was about 65.82 µM. The exposure of MCF-7 to 10 µM BPA resulted in a decreased miR-26b expression and the activation of miR-26b/Rab-31 pathway, consequently, the number and activity of lysosomes decreased, the secretion of exosomes increased, cell proliferation and migration were enhanced obviously. Interestingly, miR-26b mimic up-regulated the number and activity of lysosomes via miR-26b/miR-31 pathway, exosome secretion was down-regulated, cell proliferation and migration decreased. Further, when GW4869 was used to directly inhibit the exosome secretion of MCF-7 treated with BPA, their proliferation and migration were down-regulated. Herein, we concluded that the stimulating effect of BPA on the proliferation and migration of MCF-7 cells was associated with the lysosome - related exosome secretion via miR-26b / Rab31 pathway.

Circulating exosome level of indigenous fish may be a novel biomarker for the integrated ecotoxicity effect of water environment.

The deficiency of effective biomarker for the toxic effects of water pollutants greatly limits the application of biological monitoring. This study aimed to investigate the possibility of circulating exosomes of indigenous fish acting as biomarker for the ecotoxicity effect of water environment. The Helong Reservoir in Guangzhou, China, was chosen as the investigating field, of which the water quality belongs to Class V (2013) (GB 3838-2002, China). The clean drinking water source of the upper reaches of the Liuxihe Reservoir was selected as the control. Indigenous fishes including Oreochromis niloticus (Nile tilapia), Labeo rohita (Rohu), Carassius auratus (Crucian carp) were sampled during the period from July 2020 to April 2021. Circulating exosomes of fish samples were isolated by using ultracentrifugation, characterized with transmission electron microscopy (TEM) and quantified by using bicinchoninic acid (BCA) assay. Oxidative stress, DNA and chromosome damage in liver, kidney, brain, gill and blood of fish samples were measured. The results showed that there were significant differences in superoxide dismutase (SOD) activity, glutathione (GSH) and malondialdehyde (MDA) contents, DNA and chromosome damage in fish samples between the Helong Reservoir and the control. Interestingly, there were also significant differences in circulating exosome levels of fish samples between them. Our data suggested that circulating exosome level of indigenous fish may be a novel biomarker for the ecotoxicity effects of water environment.

Discovery of Novel Indoleamine 2,3-Dioxygenase 1 (IDO1) and Histone Deacetylase 1 (HDAC1) Dual Inhibitors Derived from the Natural Product Saprorthoquinone.

The discovery of IDO1 and HDAC1 dual inhibitors may provide a novel strategy for cancer treatment by taking advantages of both immunotherapeutic and epigenetic drugs. In this paper, saprorthoquinone (1) and 13 of its analogues from Salvia prionitis Hance were investigated for their SAR against IDO1, the results demonstrated the ortho-quinone was a key pharmacophore. Then a series of IDO1 and HDAC dual inhibitors connected by appropriate linkers were designed, synthesized, and evaluated from the hit compound saprorthoquinone (1). Among them, compound 33d showed balanced activity against both IDO1 (IC50 = 0.73 µM) and HDAC1 (IC50 = 0.46 µM). Importantly, the structure of 33d suggested that an ortho-quinone pharmacophore and a N-(2-aminophenyl) amide pharmacophore were necessary for the IDO inhibition and HDAC inhibition respectively. Meanwhile, these two pharmacophore groups should be combined by a pentane linker. Moreover, the binding modes of 33d to the enzyme active site showed that the hydrogen bond with Leu234 of IDO1 appeared to confer increased potency to this class of inhibitors, which may explain the higher activity of 33d. This study provides a new strategy for future IDO1/HDAC dual inhibitors with synergistic antitumor activity started from lead compound 33d.

A Concise Approach to N-Substituted Rhodanines through a Base-Assisted One-Pot Coupling and Cyclization Process.

An efficient approach to obtain functionalized rhodanines was developed through a base-assisted one-pot coupling and continuous cyclization of a primary amine, carbon disulfide, and methyl (2-chloroacetyl)carbamate. This conversion tolerates a broad range of functional groups and can be used to scale the preparation of N-substituted rhodanines in excellent yields.

Sulfoxide-Based Enantioselective Nazarov Cyclization: Divergent Syntheses of (+)-Isopaucifloral†F, (+)-Quadrangularin†A, and (+)-Pallidol.

The synthesis of enantiomerically pure 3-aryl substituted indanones is developed using an enantioselective sulfoxide-based Knoevenagel condensation/Nazarov cyclization procedure. After the reductive desulfonation of the methyl para-tolyl sulfoxide-containing chiral auxiliary under mild conditions, selected enantiomerically pure indanone is used for the divergent total syntheses of three resveratrol natural products (+)-isopaucifloral F, (+)-quadrangularin A, and (+)-pallidol.

From Hit to Lead: Discovery of First-In-Class Furanone Glycoside D228 Derived from Chimonanthus salicifolius for the Treatment of Inflammatory Bowel Disease.

TNFα and related inflammatory factor antibody drugs have been orchestrated for the treatment of inflammatory bowel disease (IBD). However, antibody drugs elicited inevitable disadvantages and small molecule drugs are in an urgent need. Herein, we described the discovery, design, synthesis, and SAR studies from furanone glycoside compound Phoenicein (hit) isolated from Chimonanthus salicifolius to D228 (lead). Remarkably, D228 exhibited good inhibitory activity on B and T lymphocyte and excellent anti-IBD efficacy in vivo. Mechanistically, D228 alleviated the inflammation response by downregulating the MyD88/TRAF6/p38 signaling. Importantly, the relationship of D228, Phoenicein, and their aglycone 7a was deduced: D228 could be considered as a prodrug and metabolized to intermediate Phoenicein. In turn, Phoenicein released their shared active aglycone 7a. Additionally, D228 demonstrated good and balanced profiles of safety and efficacy both in vitro and in vivo. These results suggested that D228 could be used as an ideal lead and potentially utilized for IBD chemotherapy.

MCF-7 cell - derived exosomes were involved in protecting source cells from the damage caused by tributyltin chloride via transport function.

Tributyltin chloride (TBTC) is a ubiquitous environmental pollutant with various adverse effects on human health. Exosomes are cell - derived signaling and substance transport vesicles. This investigation aimed to explore whether exosomes could impact the toxic effects caused by TBTC via their transport function. Cytotoxicity, DNA and chromosome damage caused by TBTC on MCF-7 cells were analyzed with CCK-8, flow cytometry, comet assay and micronucleus tests, respectively. Exosomal characterization and quantitative analysis were performed with ultracentrifugation, transmission electron microscope (TEM) and bicinchoninic acid (BCA) methods. TBTC content in exosomes was detected with Liquid Chromatography-Mass Spectrometry (LC-MS). The impacts of exosomal secretion on the toxic effects of TBTC were analyzed. Our data indicated that TBTC caused significant cytotoxicity, DNA and chromosome damage effects on MCF-7 cells, and a significantly increased exosomal secretion. Importantly, TBTC could be transported out of MCF-7 cells by exosomes. Further, when exosomal secretion was blocked with GW4869, the toxic effects of TBTC were significantly exacerbated. We concluded that TBTC promoted exosomal secretion, which in turn transported TBTC out of the source cells to alleviate its toxic effects. This investigation provided a novel insight into the role and mechanism of exosomal release under TBTC stress.

Baicalin maintains late-stage functional cardiomyocytes in embryoid bodies derived from murine embryonic stem cells.

BACKGROUND/AIMS: Low efficiency of cardiomyocyte (CM) differentiation from embryonic stem (ES) cells limits their therapeutic use. The objective of this study was to investigate the effect of baicalin, a natural flavonoid compound, on the in vitro cardiac differentiation of murine ES cells. METHODS: The induction of ES cells into cardiac-like cells was performed by embryoid body (EB)-based differentiation method. The electrophysiological properties of the ES cell-derived CMs (ES-CMs) were measured by patch-clamp. The biomarkers of ES-CMs were determined by quantitative RT-PCR and immunofluorescence. RESULTS: Continuous baicalin treatment decreased the size of EBs, and increased the proportion of α-actinin-positive CMs and transcript level of cardiac specific markers in beating EBs by inducing cell death of non-CMs. Baicalin increased the percentage of working ES-CMs which had typical responses to ß-adrenergic and muscarinic stimulations. CONCLUSION: Baicalin maintains the late-stage functional CMs in EBs derived from murine ES cells. This study describes a new insight into the various biological effects of baicalin on cardiac differentiation of pluripotent stem cells.

Discovery of novel tubulin CBSI (R)-9k from the indanone scaffold for the treatment of colorectal cancer.

In view of the serious adverse reactions and clinical toxicity of first line therapy 5-fluorouracil and lack of small molecule therapeutics in colorectal cancer chemotherapy, a series of natural scaffold-based 3-arylindanone derivatives (9a-q) were designed, synthesized and evaluated as tubulin polymerization inhibitors targeting the colchicine site. The most potent colchicine binding site inhibitor (CBSI), (R)-9k, exhibited 14-38 times more dominant anti-proliferative activity against three colon cancer cell lines than 5-fluorouracil. Particularly, (R)-9k showed higher selectivity against human normal cells compared with 5-fluorouracil and colchicine, and displayed negligible cardiotoxicity through hERG assessment. Furthermore, the binding of (R)-9k to the colchicine site was strongly supported by EBI competition assay and (R)-9k inhibited more tubulin polymerization than colchicine. Besides, the mechanism of action and binding modes of (R)-9k were verified by molecular dynamics simulations and docking. Therefore, (R)-9k could be regarded as a promising CBSI for colorectal cancer therapy.

Discovery of First-in-Class TAK1-MKK3 Protein-Protein Interaction (PPI) Inhibitor (R)-STU104 for the Treatment of Ulcerative Colitis through Modulating TNF-α Production.

Tumor necrosis factor α (TNF-α) has been demonstrated to be a therapeutic target for autoimmune diseases. However, this biological therapy exhibits some inevitable disadvantages, such as risk of infection. Thus, small-molecule alternatives by targeting TNF-α production signaling pathway are still in demand. Herein, we describe the design, synthesis, and structure-activity relationships of 3-aryindanone compounds regarding their modulation of TNF-α production. Among them, (R)-STU104 exhibited the most potent inhibitory activity on TNF-α production, which suppressed the TAK1/MKK3/p38/MnK1/MK2/elF4E signal pathways through binding with MKK3 and disrupting the TAK1 phosphorylating MKK3. As a result, (R)-STU104 demonstrated remarkable dose-effect relationships on both acute and chronic mouse UC models. In addition to its good pharmacokinetic (PK) and safety profile, (R)-STU104 showed better anti-UC efficacy in vivo at 10 mg/kg/d than mesalazine at the dose of 50 mg/kg/d. These results suggested that TAK1-MKK3 interaction inhibitors could be potentially utilized for the treatment of UC.

Discovery of Pyridone-Substituted Triazolopyrimidine Dual A2A/A1 AR Antagonists for the Treatment of Ischemic Stroke.

Ischemic stroke is a complex systemic disease characterized by high morbidity, disability, and mortality. The activation of the presynaptic adenosine A2A and A1 receptors modifies a variety of brain insults from excitotoxicity to stroke. Therefore, the discovery of dual A2A/A1 adenosine receptor (AR)-targeting therapeutic compounds could be a strategy for the treatment of ischemic stroke. Inspired by two clinical phase III drugs, ASP-5854 (dual A2A/A1 AR antagonist) and preladenant (selective A2A AR antagonist), and using the hybrid medicinal strategy, we characterized novel pyridone-substituted triazolopyrimidine scaffolds as dual A2A/A1 AR antagonists. Among them, compound 1a exerted excellent A2A/A1 AR binding affinity (K i = 5.58/24.2 nM), an antagonistic effect (IC50 = 5.72/25.9 nM), and good metabolic stability in human liver microsomes, rat liver microsomes, and dog liver microsomes. Importantly, compound 1a demonstrated a dose-effect relationship in the oxygen-glucose deprivation/reperfusion (OGD/R)-treated HT22 cell model. These findings support the development of dual A2A/A1 AR antagonists as a potential treatment for ischemic stroke.

Indigenous Fish-Based Assessment of Genotoxic Potentials of the Helong Reservoir in Guangzhou, China.

The present study was conducted to assess the genotoxic potential of water from the Helong Reservoir, which was designated as a strategic drinking water source by the Guangdong Provincial Government of China in October 2016. Four kinds of common indigenous fish samples (Labeo rohita, Cirrhinus molitorella, red tilapia, and Oreochromis niloticus) were collected at 6 sampling sites during the period from July to November 2020. Fish from the clean drinking water source of the upper reaches of the Liuxihe Reservoir in Guangzhou were collected as the control. Both the alkaline single cell gel electrophoresis assay and the micronucleus test were used to detect DNA damage and the micronucleus rate in erythrocytes of fish samples, respectively. The results indicated that there was a significant increase in comet tail length, Olive tail moment, and micronucleus rates of all fish samples compared with those of the control (p < 0.05). The order of sensitivity to DNA damage and micronucleus formation was Labeo rohita > Cirrhinus molitorella > red tilapia > Oreochromis niloticus. The results of the 2 kinds of experiments were in perfect agreement with each other. We conclude that there are obvious genotoxic effects from the water in the Helong Reservoir. As a strategic drinking water source, the safety of the Reservoir water quality should be considered. The local government should put the restoration of the Helong Reservoir water quality on the agenda as soon as possible. Environ Toxicol Chem 2021;40:1919-1927. © 2021 SETAC.

A pre-screening strategy to assess resected tumor margins by imaging cytoplasmic viscosity and hypoxia.

To assure complete tumor removal, frozen section analysis is the most common procedure for intraoperative pathological assessment of resected tumor margins. However, during one operation, multiple biopsies may be sent for examination, but only few of them are made into cryosections because of the complex preparation protocols and time-consuming pathological analysis, which potentially increases the risk of overlooking tumor involvement. Here, we propose a fluorescence-based pre-screening strategy that allows high-throughput, convenient, and fast gross assessment of resected tumor margins. A dual-activatable cationic fluorescent molecular rotor was developed to specifically illuminate live tumor cells' cytoplasm by emitting two different fluorescence signals in response to elevations in hypoxia-induced nitroreductase (a biochemical marker) and cytoplasmic viscosity (a biophysical marker), two characteristics of cancer cells. The ability of the fluorescent molecular rotor in detecting tumor cells was evaluated in mouse and human specimens of multiple tissues by comparing with hematoxylin and eosin staining. Importantly, the fluorescent molecular rotor achieved 100 % specificity in discriminating lung and liver cancers from normal tissue, allowing pre-screening of the tumor-free surgical margins and promoting clinical decision. Altogether, this type of fluorescent molecular rotor and the proposed strategy may serve as a new option to facilitate intraoperative assessment of resected tumor margins.

Novel octapeptide-DTX prodrugs targeting MMP-7 as effective agents for the treatment of colorectal cancer with lower systemic toxicity.

Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer death around the world. The current treatments of CRC exhibited high occurrence rate of side effects. Docetaxel (DTX), an important drug widely used in cancer chemotherapy, showed serious toxicity in CRC. Reducing toxicity of DTX could be a feasible and promising way to achieve the new indication of DTX for CRC. In this study, a series of MMP-7 activated octapeptide-DTX/4FDT prodrugs (6a-10a and 6b-10b) were designed and synthesized based on the features of MMP-7 which is highly expressed in CRC and could specially recognize octapeptides with specific sequences. Among them, 9a and 9b, both possessing an octapeptide Gly-Pro-Gln-Gly-Ile-Ala-Met-Gln moiety, were the most potent prodrugs. Compounds 9a and 9b were also tested their release rate in HCT116 cell culture fluids and tumor homogenate along with in vivo anti-CRC activity and systemic toxicity. Since 9a showed better anti-CRC activity and lower systemic toxicity than 9b in CRC tumor bearing mice, it was further evaluated for its acute toxicity, pharmacokinetics and tissue distribution in comparison with its parent drug DTX. These results revealed that 9a possessed good systemic stability, rapid release rate in CRC and reduced systemic toxicity, while retaining similar anti-CRC activity to its parent drug DTX. Thus, 9a, an MMP-7 polypeptide prodrug of DTX, has been identified as a promising candidate for the treatment of CRC.

Novel Mercapto Propionamide Derivatives with Potent New Delhi Metallo-ß-Lactamase-1 Inhibitory Activity and Low Toxicity.

The emergence and worldwide prevalence of New Delhi metallo-ß-lactamase 1 (NDM-1) expressing Gram-negative bacteria with resistance against most ß-lactam antibiotics pose a serious threat to human health. However, no NDM-1 inhibitors are clinically approved at present. Herein, based on the lead compound captopril, a series of compounds were designed, synthesized, and evaluated for NDM-1 inhibitory activities. All designed compounds showed single digit micromolar or submicromolar NDM-1 inhibitory activities, which were much more potent than that of captopril. Among them, compounds 14a and 14m exhibited excellent NDM-1 inhibitory activities, with IC50 values of 0.10 and 0.12 µM, respectively. Further studies demonstrated that compound 14m displayed low cytotoxicity, good water solubility, high metabolic stability, and low acute toxicity in mice. Importantly, compound 14m exhibited potent synergistic antimicrobial activities with Meropenem (MEM) for the treatment of clinically isolated NDM-1-expressing strains.

Zinc-Mediated Decarboxylative Alkylation of Gem-difluoroalkenes.

An efficient and mild zinc-mediated decarboxylative alkylation of gem-difluoroalkenes with N-hydroxyphthalimide (NHP) esters, to give monofluoroalkenes in moderate to excellent yields with high Z-selectivity is reported. The reaction tolerates a broad range of functional groups and can be easily scaled up, which thus may pave the way for its further applications in medicinal chemistry and materials science.

Discovery of novel sesquistilbene indanone analogues as potent anti-inflammatory agents.

To develop novel anti-inflammatory agents with improved pharmaceutical profiles, twenty-eight novel sesquistilbene indanone analogues were synthesized and evaluated for anti-inflammatory activity using RAW264.7 cells. Among these compounds, compound 11k was found to be one of the most potent analogues in inhibiting NO production in LPS-stimulated RAW264.7 cells. Furthermore, it could also significantly suppress LPS-induced iNOS and COX-2 expression and NO production through TLR4/JNK/NF-κB signaling pathway in a concentration dependent manner.

Differential metabolism of 3FDT and docetaxel in RLMs, rats, and HLMs.

3FDT, an analog of docetaxel with a blocked metabolism at its 3'-N-tert-butyloxyl group with three fluorine atoms, exhibits more potent cytotoxicity than docetaxel both with human cancer cell line SK-OV-3 in vitro and with human non-small cell lung cancer A549 xenografts in vivo. To further develop pharmacodynamically and pharmacokinetically favorable fluorinated docetaxel analogs as anticancer agents, we chose 3FDT as the model compound to identify the metabolites of 3FDT in RLMs, rats, and HLMs and the cytochrome P450 enzymes responsible for the metabolism of 3FDT. Our findings indicated that the major metabolic site switched from the C3' appendage for docetaxel to the taxane ring for 3FDT, and the main metabolizing P450 enzymes switched from CYP3A to CYP3A4 and CYP2E1.