Structure-based virtual screening and optimization of modulators targeting Hsp90-Cdc37 interaction.

Identification of novel Hsp90 inhibitors to disrupt Hsp90-Cdc37 protein-protein interaction (PPI) could be an alternative strategy to achieve Hsp90 inhibition. In this paper, a series of small molecules targeting Hsp90-Cdc37 complex are addressed and characterized. The molecules' key characters are determined by utilizing a structure-based virtual screening workflow, derivatives synthesis, and biological evaluation. Structural optimization and structure-activity relationship (SAR) analysis were then carried out on the virtual hit of VS-8 with potent activity, which resulted in the discovery of compound 10 as a more potent regulator of Hsp90-Cdc37 interaction with a promising inhibitory effect (IC50 = 27 µM), a moderate binding capacity (KD = 40 µM) and a preferable antiproliferative activity against several cancer lines including MCF-7, SKBR3 and A549 cell lines (IC50 = 26 µM, 15 µM and 38 µM respectively). All the data suggest that compound 10 exhibits moderate inhibitory effect on Hsp90-Cdc37 and could be regard as a first evidence of a non-natural compound targeting Hsp90-Cdc37 PPI.

Discovery, design and synthesis of 6H-anthra[1,9-cd]isoxazol-6-one scaffold as G9a inhibitor through a combination of shape-based virtual screening and structure-based molecular modification.

Protein lysine methyltransferase G9a is widely considered as an appealing antineoplastic target. Herein we present an integrated workflow combining shape-based virtual screening and structure-based molecular modification for the identification of novel G9a inhibitors. The shape-based similarity screening through ROCS overlay on the basis of the structure of UNC0638 was performed to identify CPUY074001 contained a 6H-anthra[1,9-cd]isoxazol-6-one scaffold as a hit. Analysis of the binding mode of CPUY074001 with G9a and 3D-QSAR results, two series compounds were designed and synthesized. The derivatives were confirmed to be active by in vitro assay and the SAR was explored by docking stimulations. Besides, several analogues showed acceptable anti-proliferative effects against several cancer cell lines. Among them, CPUY074020 displayed potent dual G9a inhibitory activity and anti-proliferative activity. Furthermore, CPUY074020 induced cell apoptosis in a dose-dependent manner and displayed a significant decrease in dimethylation of H3K9. Simultaneously, CPUY074020 showed reasonable in vivo PK properties. Altogether, our workflow supplied a high efficient strategy in the identification of novel G9a inhibitors. Compounds reported here can serve as promising leads for further study.

Wogonin inhibits multiple myeloma-stimulated angiogenesis via c-Myc/VHL/HIF-1α signaling axis.

Angiogenesis is associated with the progression of multiple myeloma (MM). Wogonin is an active mono-flavonoid with remarkable antitumor activity. However, its impact on MM-stimulated angiogenesis remains largely unknown. Here, we demonstrated that wogonin decreased expression and secretion of pro-angiogenic factors in MM cells via c-Myc/HIF-1α signaling axis, reducing MM-stimulated angiogenesis and MM cell proliferation in vivo. Overexpression of c-Myc in MM cells disrupted the balance between VHL SUMOylation and ubiquitination, and thus inhibited proteasome-mediated HIF-1α degradation. Impaired function of VHL ubiquitination complex in c-Myc-overexpressing cells was fully reversed by wogonin treatment via increasing HIF-1α-VHL interaction and promoting HIF-1α degradation. Collectively, our in vitro and in vivo studies reveal for the first time that wogonin represses MM-stimulated angiogenesis and tumor progression via c-Myc/VHL/HIF-1α signaling axis.

Discovery and Modification of in Vivo Active Nrf2 Activators with 1,2,4-Oxadiazole Core: Hits Identification and Structure-Activity Relationship Study.

Induction of phase II antioxidant enzymes by activation of Nrf2/ARE pathway has been recognized as a promising strategy for the regulation of oxidative stress-related diseases. Herein we report our effort on the discovery and optimization of Nrf2 activators with 1,2,4-oxadiazole core. Screening of an in-house collection containing 7500 compounds by ARE-luciferase reporter assay revealed a moderate Nrf2 activator, 1. Aimed at obtaining more derivatives efficiently, molecular similarity search by the combination of 2D fingerprint-based and 3D shape-based search was applied to virtually screening the Chemdiv collection. Three derivatives with the same core were identified to have better inductivity of Nrf2 than 1. The best hit 4 was selected as starting point for structurally optimization, leading to a much more potent derivative 32. It in vitro upregulated gene and protein level of Nrf2 as well as its downstream markers such as NQO1, GCLM, and HO-1. It remarkably suppressed inflammation in the in vivo LPS-challenged mouse model. Our results provide a new chemotype as Nrf2-ARE activators which deserve further optimization with the aim to obtain active anti-inflammatory agents through Nrf2-ARE pathway.

Synthesis and biological evaluation of novel oxazolo[5,4-d]pyrimidines as potent VEGFR-2 inhibitors.

Tumor angiogenesis is mediated by vascular endothelial growth factor receptor (VEGFR) and other protein kinases. Inhibition of these kinases presents an attractive approach for developing anticancer therapeutics. In this work, a series of 2,5,7-trisubstituted oxazolo[5,4-d]pyrimidines were synthesized, and their inhibitory activities were investigated against VEGFR-2 and human umbilical vein endothelial cells (HUVEC) in vitro. Compound 9n exhibited the most potent inhibitory activity with IC50 values of 0.33 and 0.29 µM for VEGFR-2 kinase and HUVEC, respectively. A further kinase selectivity assay revealed that these compounds exhibit good VEGFR and moderate EGFR inhibitory activities. Docking analysis suggested a common mode of interaction at the ATP-binding site of VEGFR-2.

Wogonin suppresses human alveolar adenocarcinoma cell A549 migration in inflammatory microenvironment by modulating the IL-6/STAT3 signaling pathway.

Increasing evidence from various clinical and experimental studies has demonstrated that the inflammatory microenvironment facilitates tumor metastasis. Clinically, it will be a promising choice to suppress tumor metastasis by targeting inflammatory microenvironment. Our previous studies have demonstrated that wogonin (a bioflavonoid isolated from the traditional Chinese medicine of Huang-Qin) possesses the anti-metastatic and anti-inflammatory activity, but we have little idea about its efficacy on inflammatory-induced tumor metastasis and the mechanism underlying it. In this study, we focused on epithelial mesenchymal transition (EMT), the first step of tumor metastasis, to evaluate the effects of wogonin on tumor metastasis in inflammatory microenvironment. We found that wogonin inhibited THP-1 conditioned-medium- (CM-) and IL-6-induced EMT by inactivating STAT3 signal. And in wogonin-treated A549 cells which pretreated with THP-1 CM or IL-6, the expression level of E-cadherin, an EMT negative biomarker, increased while that of N-cadherin, Vimentin, and EMT-related transcription factors including Snail and Twist decreased. Moreover, wogonin inhibited IL-6-induced phosphorylation of STAT3, prevented p-STAT3 dimer translocation into the nucleus, and suppressed the DNA-binding activity of p-STAT3. Interestingly, similar results were obtained in the tumor xenografts mice, including downregulation of p-STAT3, N-cadherin, and Vimentin while up-regulation of E-cadherin. Wogonin also inhibit the metastasis of A549 cells in vivo. Taken all data together, we concluded that wogonin suppresses tumor cells migration in inflammatory microenvironment by inactivating STAT3 signal.

Identification and optimization of novel Hsp90 inhibitors with tetrahydropyrido[4,3-d]pyrimidines core through shape-based screening.

Rapid Overlay of Chemical Structures (ROCS), which can rapidly identify potentially active compounds by shape comparison, is recognized as a powerful virtual screening tool. By ROCS, a class of novel Hsp90 inhibitors was identified. The calculated binding mode of the most potent hit 36 guided us to design and synthesize a series of analogs (57a-57h). Over 100-fold improvement was achieved in the target-based assay. The most potent compound 57h inhibited Hsp90 with IC50 0.10 ± 0.01 µM. It also showed much improved cell potency and ligand efficiency. Our study showed that ROCS is efficient in the identification of novel cores of Hsp90 inhibitors. 57h can be ideal leads for further optimization.

Wogonin reverses hypoxia resistance of human colon cancer HCT116 cells via downregulation of HIF-1α and glycolysis, by inhibiting PI3K/Akt signaling pathway.

Hypoxia induced drug resistance is a major obstacle in the development of effective cancer therapy. In the present study, the reversal abilities of wogonin on the hypoxia resistance and the underlying mechanisms were discovered. MTT assay revealed that hypoxia increased maximal 1.71-, 2.08-, and 2.15-fold of IC50 toward paclitaxel, ADM, and DDP in human colon cancer cell lines HCT116, respectively. Furthermore, wogonin showed strong reversal potency in HCT116 cells in hypoxia and the RF reached 2.05. hypoxia-inducible factor-1α (HIF-1α) can activate the expression of target genes involved in glycolysis. Wogonin decreased the expression of glycolysis-related proteins (HKII, PDHK1, LDHA), glucose uptake, and lactate generation in a dose-dependent manner. Further, Western blot experiments exhibited that wogonin could down regulate HIF-1α expression and glycolysis through inhibiting PI3K/Akt signaling pathway, which might be the mechanism of reversal resistance of wogonin. Also, wogonin could inhibit the growth of transplantable tumors and the expression of HIF-1α, glycolysis-related proteins and PI3K/Akt in vivo. In summary, wogonin could be a good candidate for the development of new multidrug resistance (MDR) reversal agent and its reversal mechanism probably is due to the suppression of HIF-1α expression via inhibiting PI3K/Akt signaling pathway.

Enhanced 5-fluorouracil cytotoxicity in high COX-2 expressing hepatocellular carcinoma cells by wogonin via the PI3K/Akt pathway.

Combination therapies may increase the antitumor effects and reduce the adverse effects for the treatment of hepatocellular carcinoma. In this study, we determined the effects of 5-fluorouracil alone or in combination with wogonin in vitro and in vivo, and we investigated the possible mechanisms. The combination of these 2 drugs led to a decrease in survival and a significant synergistic inhibitory effect on high COX-2 expression in SMMC-7721 hepatocellular carcinoma (HCC) cells. Furthermore, the results show that this combination inhibits COX-2 expression and increases sensitivity to chemotherapeutic agents partly through regulating the PI3K/Akt signaling pathway. Moreover, the combination treatment caused a significant growth inhibition of human tumor xenografts in vivo. In conclusion, wogonin may increase the cytotoxicity of some antineoplastic agents and it can be used in combination with these agents as a novel therapeutic regimen for HCC treatment.

Synthesis and antibacterial evaluation of a novel series of 10-hydroxyl ketolide derivatives.

A novel series of 10-hydroxyl ketolide derivatives were synthesized, during which a distinctive intermediate, 3-O-descladinosyl-3-oxo-11-deoxy-10,11-epoxy-6-O-methylerythromycin A, was obtained from 6-O-methylerythromycin A. The structure and stereochemistry of this novel structure were confirmed via NMR and X-ray crystallography. Moreover, antibacterial evaluations were established in order to assess our modifications and acquire a deep understanding of the ketolides' structure-activity relationship (SAR).

[Recent advances in chemistry and biology of gamboge].

Gamboge, the resin of Garcinia hanburyi has had a long history of use as the traditional dye as well as a complementary and alternative medicine. The antitumor activities of gamboge have been well demonstrated by inhibiting the growth and progression of cancer cells both in vitro and in vivo. In order to further clarify the mode of action of gamboge, there are three key questions needed to be answered, including what's in gamboge? How do the chemical components from gamboge work on cancer cells? How do biological systems work on the chemical components from gamboge after administration? In this review, we summarize the explorations of the answers toward these questions according to the recent progress in both of chemistry and biology research of gamboge. In addition, the implication in the future research and discovery of the caged G. xanthones as anticancer agents is also discussed.

Novel IKKß inhibitors discovery based on the co-crystal structure by using binding-conformation-based and ligand-based method.

IκB kinase ß (IKKß), an attractive anti-inflammation and anti-cancer target, plays a crucial role in the activation of NF-κB signalling pathway. To identify novel IKKß inhibitors, we combined structure-based and ligand-based methods based on the co-crystal structure of IKKß. According to the chemical similarity, 162 reported IKKß inhibitors were divided into five classes. For each class, a 3D pharmacophore model was established based on the binding conformations of the compounds. The validated models were further used in virtual screening. Twelve drugable compounds were retained for biological test, resulting in two novel inhibitors with IC50 values lower than 10 µM. Compared to other models, our method considers the crystal structure of IKKß for the first time.

Pharmacophore-based drug design and biological evaluation of novel ABCB1 inhibitors.

Overexpression of ABCB1 is one of major barriers for multidrug resistance in chemotherapy and limits drug oral bioavailability. Inhibition of ABCB1 would sensitize multidrug resistance in clinical cancer chemotherapy. With this aim, a 3D pharmacophore model was created based on known ABCB1 inhibitors with correlation coefficient of 0.94, comprising three hydrophobic features and one hydrogen bond acceptor. It was further validated and used to search our in-house 3D database for potential ABCB1 inhibitors. The inhibitory activities of the best hits were evaluated by several biological assays, such as rhodamine 123 accumulation assay, chemosensitization assay, multidrug resistance 1-Madin-Darby canine kidney cells/Madin-Darby canine kidney cells permeability assay. Finally, compounds YZ-3 and YZ-16 were identified as potential leads to be developed in the designing of novel potent ABCB1 inhibitors.

Drug resistance associates with activation of Nrf2 in MCF-7/DOX cells, and wogonin reverses it by down-regulating Nrf2-mediated cellular defense response.

Acquired resistance to doxorubicin (DOX) is a serious therapeutic problem in breast cancer patients. In this study, we investigated whether nuclear factor erythroid 2-related factor 2 (Nrf2) was associated with drug resistant in DOX resistant MCF-7 (MCF-7/DOX) cells, and if wogonin, a flavonoid isolated from the root of Scutellaria baicalensis Georgi, could reverse drug resistance in MCF-7/DOX cells. We found that the endogenous expression of Nrf2 as well as its target proteins heme oxygenase-1 (HO-1), NADP(H):quinone oxidoreductase (NQO) in MCF-7/DOX cells was higher than that in MCF-7 cells. Tert-butylhydroquinone treatment increased the expression Nrf2, HO-1, and NQO-1, and enhanced resistance of MCF-7 cells to DOX. Similarly, intracellular Nrf2 protein level was significantly decreased in MCF-7/DOX cells and DOX resistance was partially reversed by Nrf2 siRNA. Wogonin downregulated the Nrf2-dependent response and partly reversed DOX resistance in MCF-7/DOX cells. These results suggested that activation of Nrf2 was associated with drug resistance in MCF-7/DOX cells. Wogonin reversed drug resistance and its reversal mechanism might be due to the suppression of Nrf2 signaling pathway, indicating the feasibility of using Nrf2 inhibitors to increase efficacy of chemotherapeutic drugs.

The role of Nrf2 and apoptotic signaling pathways in oroxylin A-mediated responses in HCT-116 colorectal adenocarcinoma cells and xenograft tumors.

Oroxylin A is a flavonoid found in the roots of Scutellaria baicalensis Georgi, a herbal medicine commonly used as an antipyretic, analgesic, antitumor, and anti-inflammatory agent. It has recently been investigated for its anticancer activities in hepatoma, gastric, and breast tumors. Here, we investigated the antitumor effects of oroxylin A in human colon carcinoma HCT-116 cells in vitro and in vivo. We characterized the proapoptotic effect of oroxylin A using diamidino-phenyl-indole (DAPI) and annexin V/PI staining. We then found that both caspase-3 and caspase-9 were activated, the expression of Bcl-2 protein decreased, and the expression of Bax protein increased after treatment with oroxylin A. In addition, oroxylin A increased nuclear transcription factor erythroid-related factor 2 (Nrf2) expression and induced Nrf2 translocation into the nucleus. Furthermore, we found that oroxylin A treatment elevated intracellular reactive oxygen species levels and increased the protein expression level of two of the Nrf2 target genes heme oxygenase-1 and NADP(H):quinone oxidoreductase-1 in HCT-116 cells. Finally, our study demonstrated that oral administration of oroxylin A significantly decreased tumor volume and weight in immunodeficient mice that were inoculated with HCT-116 cells. The in-vivo chemopreventive efficacy of oroxylin A against HCT-116 human colon cancer was accompanied by its proapoptotic and Nrf2-inducing activities, which correlates with the in-vitro study. This is the first demonstration of oroxylin A-dependent chemoprevention in colon cancer and may offer a potential mechanism for its anticancer action in vivo.

Wogonin potentiates the antitumor effects of low dose 5-fluorouracil against gastric cancer through induction of apoptosis by down-regulation of NF-kappaB and regulation of its metabolism.

Traditional Chinese medicines have been recognized as a new source of anticancer drugs or chemotherapy adjuvant to enhance the efficacy of chemotherapy and to ameliorate the side effects. Wogonin (WOG) has a potential for therapeutic use in the treatment of antitumor and chemoprophylaxis. 5-Fluorouracil (5-FU) is a key systemic chemotherapy drug and widely use in the treatment of solid tumors. In this study, we found that combination of WOG and 5-FU inhibited the viability of MGC-803 cells in a concentration-dependent manner and exhibited a synergistic anticancer effect (CI<1) when 5-FU was used at relatively low concentrations. The pro-apoptotic activity of two-drug combination was much stronger than single. Furthermore, WOG could decrease the mRNA levels of dihydropyrimidine dehydrogenase (DPD), the metabolic enzymes of 5-FU. WOG could inhibit the NF-kappaB nuclear translocation and I-kappaB phosphorylation. Moreover, combined treatment caused significantly growth inhibition of human tumor xenografts. In addition, WOG markedly enhanced the antitumor activity of low dose 5-FU (i.p. 10mg/kg/day), however there is no toxicity and influence on diet consumption in experimental animals. Taken together, our data's showed that WOG increased 5-FU retention for a prolonged catabolism by modulating 5-FU metabolic enzymes and sensitized the MGC-803 cells to 5-FU induced apoptosis by inhibiting the NF-kappaB nuclear translocation. The anti-gastric cancer effect of two-drug combination was much stronger than that of WOG or 5-FU alone. These results may be relevant to design new clinical therapeutic strategies against gastric cancer in future.

Flavonoid baicalein suppresses adhesion, migration and invasion of MDA-MB-231 human breast cancer cells.

Baicalein is a widely used Chinese herbal medicine that has been used historically in anti-inflammatory and anti-cancer therapy. However, the molecular mechanism of its anti-cancer activity remains poorly understood and warrants further investigations. The purpose of this study is to verify the activity of baicalein to inhibit the invasion of MDA-MB-231 human breast cancer cells. The results indicated that baicalein suppressed MDA-MB-231 cell adhesion to fibronectin-coated substrate, wound healing migration and invasion through the Matrigel in a concentration-dependent manner. Western blot and gelatin zymography analysis showed that baicalein significantly inhibited the expression and secretion of matrix metalloproteinases 2/9 (MMP-2/9) in MDA-MB-231 cells. Additionally, treatment of MDA-MB-231 cells with baicalein down-regulated the expression of MMP-2/9 involved mitogen-activated protein kinases (MAPK) signaling pathway. Taken together, baicalein had potential to suppress the adhesion, migration and invasion of MDA-MB-231 cancer cells in vitro and it could serve as a promising drug for the treatment of cancer metastasis.

Inhibition of glioblastoma growth and angiogenesis by gambogic acid: an in vitro and in vivo study.

Gambogic acid (GA) is the major active ingredient of gamboge, a brownish to orange resin exuded from Garcinia hanburryi tree in Southeast Asia. The present study aims to demonstrate that gambogic acid (GA) has potent anticancer activity for glioblastoma by in vitro and in vivo study. Rat brain microvascular endothelial cells (rBMEC) were used as an in vitro model of the blood-brain barrier (BBB). To reveal an involvement of the intrinsic mitochondrial pathway of apoptosis, the mitochondrial membrane potential and the western blot evaluation of Bax, Bcl-2, Caspase-3, caspase-9 and cytochrome c released from mitochondria were performed. Angiogenesis was detected by CD31 immunochemical study. The results showed that the uptake of GA by rBMEC was time-dependent, which indicated that it could pass BBB and represent a possible new target in glioma therapy. GA could cause apoptosis of rat C6 glioma cells in vitro in a concentration-dependent manner by triggering the intrinsic mitochondrial pathway of apoptosis. In vivo study also revealed that i.v. injection of GA once a day for two weeks could significantly reduce tumor volumes by antiangiogenesis and apoptotic induction of glioma cells. Collectively, the current data indicated that GA may be of potential use in treatment of glioblastoma by apoptotic induction and antiangiogenic effects.

Gambogic acid inhibits angiogenesis through suppressing vascular endothelial growth factor-induced tyrosine phosphorylation of KDR/Flk-1.

Previous studies revealed that gambogic acid (GA), the major active ingredient of gamboge, a brownish to orange resin exuded from Garcinia hanburryi tree in Southeast Asia, possessed significant anticancer activity both in vitro and in vivo. In this study, we explored the high antiangiogenic activities of GA for the first time. GA inhibits the VEGF-stimulated proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) as well as microvessel sprouting from rat aortic rings in vitro. Moreover, GA inhibits vessel growth in matrigel plugs and CAM in vivo and transplanted tumor in mice. The results also indicated that GA decreases VEGF production of cultured tumor cells and inhibits VEGF-induced tyrosine phosphorylation of KDR/Flk-1. This inhibition of receptor phosphorylation is correlated with a significant decrease in VEGF-triggered phosphorylated forms of ERK, AKT and p38. Taken together, these findings strongly suggest that GA might be a structurally novel angiogenesis inhibitor.

Pharmacophore identification of KSP inhibitors.

A three-dimensional pharmacophore model was developed based on 25 currently available KSP (kinesin spindle protein) inhibitors in Catalyst software package. The best pharmacophore hypothesis (Hypo1), consisting of four chemical features (one hydrogen-bond acceptor, one hydrogen-bond donor, one aromatic ring, and one hydrophobic group), has a correlation coefficient of 0.965. The results of our study provide a valuable tool in designing new leads with desired biological activity by virtual screening.