Synthesis and biological evaluation of novel isoxazoloquinone derivatives as potent STAT3-targeting antipsoriasis agents.

Psoriasis is a troublesome scaling skin disease with no high-effective medication available by far. Signal transducer and activator of transcription 3 (STAT3) has recently been revealed as a crucial player in the pathogenesis and progression of psoriasis and emerged as an intriguing antipsoriatic drug target. Naturally occurring lapachol and its quinone analogs had been discovered as effective STAT3 inhibitors, however, their antipsoriatic effects are not well investigated. Previously, we have reported a series of isothiazoloquinone lapachol derivatives. Here, the antipsoriastic potentials of these isothiazoloquinones were investigated and, in addition, 35 novel isoxazoloquinone derivatives were prepared and studied for their anti-psoriasis properties. Among them, the most potent antipsoriatic compound B20 determined by in vitro test on HaCaT cells could directly bind to STAT3, reduce STAT3 level and inhibit STAT3 nuclear translocation. In vivo studies showed that topical application of B20 could effectively alleviate IMQ-induced psoriasis in mice with no obvious side effects. In addition, B20 inhibited the production of interleukin 17 (IL-17A), a STAT3-downstream cytokine essential for the progression of psoriasis, both in vitro and in vivo. Thus, isoxazoloquinone B20 is a potent STAT3-targeting antipsoriatic agent worth of further investigation.

Spinosyn A and Its Derivative Inhibit Colorectal Cancer Cell Growth via the EGFR Pathway.

Spinosyn A (SPA), derived from a soil microorganism, Saccharopolyspora spinosa, and its derivative, LM2I, has potential inhibitory effects on a variety of cancer cells. However, the effects of SPA and LM2I in inhibiting the growth of human colorectal cancer cells and the molecular mechanisms underlying these effects are not fully understood. Cell viability was tested by using a 3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bromide (MTT) assay and a colony formation assay. On the basis of the IC50 values of SPA and LM2I in seven colorectal cancer (CRC) cell lines, sensitive (HT29 and SW480) and insensitive (SW620 and RKO) cell lines were screened. The GSE2509 and GSE10843 data sets were used to identify 69 differentially expressed genes (DEGs) between sensitive and insensitive cell lines. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and protein-protein interactions (PPI) were performed to elucidate the molecular mechanisms of the DEGs. The hub gene of the DEGs was detected by Western blot analysis and verified using the CRISPR/Cas9 system. Our data indicate that SPA and its derivative LM2I have significant antiproliferative activity in seven colorectal cancer cell lines and colorectal xenograft tumors. On the basis of bioinformatics analysis, it was demonstrated that epidermal growth factor receptor (EGFR) was the hub gene of the DEGs and was associated with the inhibitory effects of SPA and LM2I in CRC cell lines. The study also revealed that SPA and LM2I inhibited the EGFR pathway in vitro and in vivo.

An Isoxazoloquinone Derivative Inhibits Tumor Growth by Targeting STAT3 and Triggering Its Ubiquitin-Dependent Degradation.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, with shorter five-year survival than other breast cancer subtypes, and lacks targeted and hormonal treatment strategies. The signal transducer and activator of transcription 3 (STAT3) signaling is up-regulated in various tumors, including TNBC, and plays a vital role in regulating the expression of multiple proliferation- and apoptosis-related genes. RESULTS: By combining the unique structures of the natural compounds STA-21 and Aulosirazole with antitumor activities, we synthesized a class of novel isoxazoloquinone derivatives and showed that one of these compounds, ZSW, binds to the SH2 domain of STAT3, leading to decreased STAT3 expression and activation in TNBC cells. Furthermore, ZSW promotes STAT3 ubiquitination, inhibits the proliferation of TNBC cells in vitro, and attenuates tumor growth with manageable toxicities in vivo. ZSW also decreases the mammosphere formation of breast cancer stem cells (BCSCs) by inhibiting STAT3. CONCLUSIONS: We conclude that the novel isoxazoloquinone ZSW may be developed as a cancer therapeutic because it targets STAT3, thereby inhibiting the stemness of cancer cells.

Reducing false positive rate of docking-based virtual screening by active learning.

Machine learning-based scoring functions (MLSFs) have become a very favorable alternative to classical scoring functions because of their potential superior screening performance. However, the information of negative data used to construct MLSFs was rarely reported in the literature, and meanwhile the putative inactive molecules recorded in existing databases usually have obvious bias from active molecules. Here we proposed an easy-to-use method named AMLSF that combines active learning using negative molecular selection strategies with MLSF, which can iteratively improve the quality of inactive sets and thus reduce the false positive rate of virtual screening. We chose energy auxiliary terms learning as the MLSF and validated our method on eight targets in the diverse subset of DUD-E. For each target, we screened the IterBioScreen database by AMLSF and compared the screening results with those of the four control models. The results illustrate that the number of active molecules in the top 1000 molecules identified by AMLSF was significantly higher than those identified by the control models. In addition, the free energy calculation results for the top 10 molecules screened out by the AMLSF, null model and control models based on DUD-E also proved that more active molecules can be identified, and the false positive rate can be reduced by AMLSF.

ZLM-7 Blocks Breast Cancer Progression by Inhibiting MDM2 via Upregulation of 14-3-3 Sigma.

Breast cancer is one of the most prevalent malignancies with poor prognosis. Inhibition of angiogenesis is becoming a valid and evident therapeutic strategy to treat cancer. Recent studies uncovered the antiangiogenic activity of ZLM-7 (a combretastain A-4 derivative), but the regulatory mechanism is unclear. ZLM-7 treatment was applied in estrogen receptor-positive cell MCF-7, triple-negative breast cancer cell MDA-MB-231 and xenograft models. Transfections were conducted to overexpress or knockdown targeted genes. The gene and protein expressions were measured by qPCR and Western blotting assay, respectively. Cell proliferation and apoptosis were evaluated using the CCK8 method, clone formation assay and flow cytometry. We found that ZLM-7 upregulated 14-3-3 sigma expression but downregulated MDM2 expression in breast cancer cells. ZLM-7 delayed cell proliferation, promoted apoptosis and blocked cell-cycle progression in human breast cancer cells in vitro, while those effects were abolished by 14-3-3 sigma knockdown; overexpression of 14-3-3 sigma reproduced the actions of ZLM-7 on the cell cycle, which could be reversed by MDM2 overexpression. In xenograft models, ZLM-7 treatment significantly inhibited tumor growth while the inhibition was attenuated when 14-3-3 sigma was silenced. Collectively, ZLM-7 could inhibit MDM2 via upregulating 14-3-3 sigma expression, thereby blocking the breast cancer progression.

Naturally-occurring spinosyn A and its derivatives function as argininosuccinate synthase activator and tumor inhibitor.

Argininosuccinate synthase (ASS1) is a ubiquitous enzyme in mammals that catalyzes the formation of argininosuccinate from citrulline and aspartate. ASS1 genetic deficiency in patients leads to an autosomal recessive urea cycle disorder citrullinemia, while its somatic silence or down-regulation is very common in various human cancers. Here, we show that ASS1 functions as a tumor suppressor in breast cancer, and the pesticide spinosyn A (SPA) and its derivative LM-2I suppress breast tumor cell proliferation and growth by binding to and activating ASS1. The C13-C14 double bond in SPA and LM-2I while the Cys97 (C97) site in ASS1 are critical for the interaction between ASS1 and SPA or LM-2I. SPA and LM-2I treatment results in significant enhancement of ASS1 enzymatic activity in breast cancer cells, particularly in those cancer cells with low ASS1 expression, leading to reduced pyrimidine synthesis and consequently the inhibition of cancer cell proliferation. Thus, our results establish spinosyn A and its derivative LM-2I as potent ASS1 enzymatic activator and tumor inhibitor, which provides a therapeutic avenue for tumors with low ASS1 expression and for those non-tumor diseases caused by down-regulation of ASS1.

ZLM-7 inhibits the occurrence and angiogenesis of breast cancer through miR-212-3p/Sp1/VEGFA signal axis.

BACKGROUND: Breast cancer (BC) is a common malignant tumor with poor prognosis. Angiogenesis is related to the growth and progression of solid tumors and associated with prognosis. ZLM-7, SP1, VEGFA and miR-212-3p were associated with BC angiogenesis and proliferation, however the detailed mechanism was not clear. This study aimed to reveal the regulatory mechanism of angiogenesis of BC. METHODS: BC cell lines were treated with 10 nM ZLM-7 for 8 h. We detected protein expression level by western blot and RNA expression level by qRT-PCR. Overexpression or inhibition of miR-212-3p is performed using miR-212-3p mimics or miR-212-3p inhibitor, Sp1 overexpression using pcDNA3.1 vector. Angiogenesis was analyzed by co-culturing BC cell lines and HUVEC cells. To evaluate regulatory relationship between miR-212-3p and Sp1, dual luciferase assay was performed. Besides, the direct interaction between Sp1 and VEGFA was analyzed by ChIP. Migration and invasion were analyzed by transwell assay and proliferation was detected by clone formation assay. In mice xenograft model developed using BC cells, we also detected angiogenesis marker CD31 through immunohistochemistry. RESULTS: ZLM-7 up-regulated miR-212-3p and inhibited invasion, migration, proliferation and angiogenesis of BC, while miR-212-3p inhibitor antagonized such effects. Binding sequence was revealed between miR-212-3p and Sp1, and expression of Sp1 was inhibited by miR-212-3p on both protein and mRNA level. Sp1 could interact with VEGFA and promoted its expression. Overexpression of miR-212-3p inhibited migration, invasion, proliferation and angiogenesis of BC cell lines, while Sp1 overexpression showed the opposite effect and could antagonize these effects of miR-212-3p overexpression. ZLM-7 decreased VEGFA expression, which was rescued by co-transfection with miR-212-3p inhibitor. Similar, ZLM-7 could inhibit tumor growth and angiogenesis through the miR-212-3p/Sp1/VEGFA axis in vivo. CONCLUSIONS: ZLM-7 could directly up-regulate miR-212-3p in BC. MiR-212-3p could inhibit VEGFA expression through Sp1, thereby inhibiting angiogenesis and progression of BC.

Synthesis and biological evaluation of novel isothiazoloquinoline quinone analogues.

Natural quinones and their analogues have attracted growing attention because of their novel anticancer activities. A series of novel isothiazoloquinoline quinone analogues were synthesized and evaluated for antitumor activities against four different kind of cancer cells. Among them, isothiazoloquinolinoquinones inhibited cancer cells proliferation effectively with IC50 values in the nanomolar range, and isothiazoloquinolinoquinone 13a induced the cell apoptosis. Further exploration of possible mechanism of action indicates that 13a not only activates ROS production through NQO1-directed redox cycling but also inhibits the phosphorylation of STAT3. These findings indicate that 13a has potential use for the development of new skeleton drug candidate as an efficient substrate of NQO1 and STAT3 inhibitor.

Ginsenoside metabolite 20(S)­protopanaxadiol promotes neural stem cell transition from a state of proliferation to differentiation by inducing autophagy and cell cycle arrest.

20(S)­Protopanaxadiol (PPD) is an active ginseng metabolite and is the final form of protopanaxadiol saponins metabolized by human intestinal microflora. The neuroprotective effects and mechanisms underlying PPD on neural stem cells (NSCs) are not completely understood. The aim of the present study was to assess the effects of PPD on the proliferation and differentiation of neural stem cells. In the present study, following treatment with different concentrations of PPD for 24 h, the percentage of BrdU­positive cells decreased significantly with increasing concentrations of PPD. Moreover, flow cytometric analysis results indicated that PPD treatment increased the proportion of cells in the G0/G1 and G2/M phase and decreased the proportion of cells in the S phase. The activation of autophagy, determined by an increased number of autophagic vacuoles and light chain 3 lipidation, was associated with an increase in the expression of the neuronal marker tubulin­ß3 following PPD treatment. PPD also partially rescued NSCs from the inhibitory effects of the autophagic inhibitor wortmannin, suggesting that the effect of PPD on NSC differentiation was associated with autophagy. Collectively, the results indicated that PPD promoted the transition of NSCs from a state of proliferation to differentiation through the induction of autophagy and cell cycle arrest. Therefore, the present study may provide a basis for the development of regenerative therapies based on ginsenoside, an approved and safe drug.

Synthesis and anti-OXPHOS, antitumor activities of DLC modified spinosyn derivatives.

A series of DLC (delocalized lipophilic cation) modified spinosyn derivatives were synthesized and evaluated for antitumor efficacies both in vitro and in vivo. Cancer cell based antiproliferative assays indicated that the more lipophilic derivatives had stronger inhibitory effects on the tested cancer cell lines. Compound 7b and 8b exhibited strong anti-OXPHOS and apoptosis inducing ability. Notable antitumor efficacies of 7b (5 mg/kg) and 8b (2.5 mg/kg) were observed in the in vivo tumor xenograft experiments, however, lethal toxicities were observed on higher dosages. Our findings indicated that DLC modification is a viable strategy to enhance the anti-OXPHOS and antitumor efficacies of spinosyn derivatives.

Synthesis and antiproliferative activities of novel quartenary ammonium spinosyn derivatives.

In order to enhance the mitochondria-targeting ability of spinosad. A series of quartenary ammonium spinosyn derivatives was designed and synthesized. Some of the derivatives displayed greatly enhanced antiproliferative ability towards tested human cancer cell lines. The structure activity relationship study indicated that lipophilicity has a great influence on the antiproliferative effects of these derivatives. The most active compound 11d exhibited remarkably enhanced OXPHS inhibition and apoptosis inducing ability than spinosyn A.

7-O-aminoalkyl-2,3-dehydrosilibinins: Synthesis and in vitro Anti-cancer Efficacy.

BACKGROUND: The heptaprotective flavonolignan silibinin and dehydrosilibinin have exhibited moderate antiproliferative activities toward many cancer cell lines. Considering of the nontoxic profile of these natural products, chemical modification to enhance the anticancer potentials is promising. METHOD: A series of 7-O-aminoalkyl-2,3-dehydrosilibinin derivatives were synthesized and evaluated for their antiproliferative activities against several cancer cell lines. RESULTS: A number of the synthesized dehydrosilibinin derivatives exhibited greatly enhanced potency with 50% growth inhibition at low micromolar concentrations. Structure activity study indicated that the distance between N and 7-O on the side chain has a limited influence on the antiproliferative activity, while the presence of a morpholino group decreases the antiproliferative activities dramatically. Flow cytometry based assays on human colon cancer HCT116 cells revealed that 6a and 6c, two of the most potent derivatives, effectively arrested the cell cycle in the G2 phase and stimulated cell apoptosis. CONCLUSION: Our findings suggest that attaching an appropriate tertiary amino alkyl side chain through 7-Oalkylation on 2,3-dehydrosilibinin, would be a viable strategy for the development of silibinin derivatives as anticancer agents.

The anti-angiogenic effect and novel mechanisms of action of Combretastatin A-4.

Combretastatin A-4 (CA4) is the lead compound of a relatively new class of vascular disrupting agents that target existing tumor blood vessels. Recent studies showed the CA4 might inhibit angiogenesis. However, the underlying molecular mechanisms by which CA4 exerts its anti-angiogenic effects are not fully understood. In this study, we revealed that CA4 inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration and capillary-like tube formation of human umbilical vascular endothelial cells (HUVECs). In in vivo assay, CA4 suppressed neovascularization in chicken chorioallantoic membrane (CAM) model and decreased the microvessel density in tumor tissues of a breast cancer MCF-7 xenograft mouse model. In addition, CA4 decreased the expression level and secretion of VEGF both in MCF-7 cells and HUVECs under hypoxia, as well as the activation of VEGFR-2 and its downstream signaling mediators following VEGF stimulation in HUVECs. Moreover, VEGF and VEGFR-2 expression in tumor tissues of the mouse xenograft model were down-regulated following CA4 treatment. Taken together, results from the current work provide clear evidence that CA4 functions in endothelial cell system to inhibit angiogenesis, at least in part, by attenuating VEGF/VEGFR-2 signaling pathway.

ZLM-7 exhibits anti-angiogenic effects via impaired endothelial cell function and blockade of VEGF/VEGFR-2 signaling.

Inhibition of angiogenesis is a promising therapeutic strategy against cancer. In this study, we reported that ZLM-7, a combretastain A-4 (CA-4) derivative, exhibited anti-angiogenic activity in vitro and in vivo. In vitro, ZLM-7 induced microtubule cytoskeletal disassembly. It decreased VEGF-induced proliferation, migration, invasion and tube formation in endothelial cells, which are critical steps in angiogenesis. In vivo, ZLM-7 significantly inhibited neovascularization in a chicken chorioallantoic membrane (CAM) model and reduced the microvessel density in tumor tissues of MCF-7 xenograft mouse model. ZLM-7 also displayed comparable antiangiogenic and anti-tumor activities associated with the lead compound CA-4, but exhibited lower toxicity compared with CA-4. The anti-angiogenic effect of ZLM-7 was exerted via blockade of VEGF/VEGFR-2 signaling. ZLM-7 treatment suppressed the expression and secretion of VEGF in endothelial cells and MCF-7 cells under hypoxia. Further, ZLM-7 suppressed the VEGF-induced phosphorylation of VEGFR-2 and its downstream signaling mediators including activated AKT, MEK and ERK in endothelial cells. Overall, these results demonstrate that ZLM-7 exhibits anti-angiogenic activities by impairing endothelial cell function and blocking VEGF/VEGFR-2 signaling, suggesting that ZLM-7 might be a potential angiogenesis inhibitor.

Facile photochemical transformation of alkyl aryl selenides to the corresponding carbonyl compounds by molecular oxygen: use of selenides as masked carbonyl groups.

Alkyl aryl selenides with and without functional groups on the alkyl group were transformed efficiently into the corresponding carbonyl compounds, particularly primary alkyl aryl selenides in good yields, by a simple photolysis in the presence of air or oxygen. This transformation can be conducted without protection of functional groups. The yield of carbonyl compounds was much affected by the solvent viscosity, reaction temperature, concentration of dissolved oxygen in the solvents, wavelength of light, and structure of the aryl substituents. The present study indicates that aryl selenides can be considered as a masked carbonyl group that can be easily converted to a carbonyl group by very mild reaction conditions even in the presence of various unprotected functional groups. Therefore, this functional group transformation can be used as an important tool in organic synthesis due to its simplicity and mild reaction condition.

Factors controlling photochemical cleavage of the energetically unfavorable Ph-Se bond of alkyl phenyl selenides.

Primary photochemical paths of alkyl phenyl selenides (1) were investigated, and an origin of large deviations in the chemical yields of products obtained by carbon radical reactions induced by photolysis of phenyl selenides was clarified. KrF excimer laser photolyses of n-pentyl phenyl selenide (1a) yielded 1-pentene (2a), n-pentane (3a), n-decane (4a), dipentyl selenide (5a), benzene (6), dipentyl diselenide (7a), and diphenyl diselenide (7) as major photoproducts, with compounds 2a, 3a, 4a, 5a, and 7 formed by pentyl-Se bond cleavage, and 5a, 6, and 7a by Ph-Se bond cleavage. The selectivity of the photoproducts revealed the occurrence of an unexpected amount of Ph-Se bond cleavage (35% in n-hexane at 248 nm) during photolysis. Solvent viscosity, wavelength of light, and the structure of alkyl substituents were the major factors that controlled Ph-Se bond cleavage. The ratio of Ph-Se bond cleavage decreased with increasing solvent viscosity and laser wavelength. The effect of alkyl substituents on the ratio of bond cleavages, Ph-Se/total C-Se, was investigated for five alkyl phenyl selenides; the ratio decreased in the order pentyl > 2-methylallyl > allyl > 1-ethylpropyl > tert-butyl groups. The contribution of Ph-Se bond cleavage is most probably the origin of the large deviations in the yields of radical reactions induced by photolyses of 1, which can be minimized by selecting appropriate solvents and wavelength of light.