Discovery of Clinical Candidate (5-(3-(4-Chlorophenoxy)prop-1-yn-1-yl)-3-hydroxypicolinoyl)glycine, an Orally Bioavailable Prolyl Hydroxylase Inhibitor for the Treatment of Anemia.

The design and discovery of a new series of (5-alkynyl-3-hydroxypicolinoyl)glycine inhibitors of prolyl hydroxylase (PHD) are described. These compounds showed potent in vitro inhibitory activity toward PHD2 in a fluorescence polarization-based assay. Remarkably, oral administration of 17, with an IC50 of 64.2 nM toward PHD2, was found to stabilize HIF-α, elevate erythropoietin (EPO), and alleviate anemia in a cisplatin-induced anemia mouse model with an oral dose of 25 mg/kg. Rat and dog studies showed that 17 has good pharmacokinetic properties, with oral bioavailabilities of 55.7 and 54.0%, respectively, and shows excellent safety profiles even at a high dose of 200 mg/kg in these animals. Based on these results, 17 is currently being evaluated in a phase I clinical trial for anemia.

Application of in-vitro screening methods on hypoxia inducible factor prolyl hydroxylase inhibitors.

Anemia resulting from the reduced expression of erythropoietin (EPO) is a common complication of patients with chronic kidney diseases (CKD). Hypoxia inducible transcription factor-α (HIF-α), which adapts cellular hypoxia condition, regulates the expression of many downstream genes including the EPO gene. Hypoxia inducible transcription factor prolyl hydroxylase 2 (HIF-PHD2), as the key regulator of hypoxia response, is function of hydroxylating specify proline residues of HIF-α, which may lead to the degradation of HIF-α and eventually cause disenabling the expression of erythropoietin. Therefore, it is valid to improve anemia by inhibiting HIF-PHD2. In-vitro screening plays a vital role in searching for novel small molecule HIF-PHD2 inhibitors, thus, this review classified in-vitro screening methods which are used to hit novel HIF-PHD2 inhibitors.

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.

Novel naphtho[2,1-d]oxazole-4,5-diones as NQO1 substrates with improved aqueous solubility: Design, synthesis, and in vivo antitumor evaluation.

A new series of ortho-naphthoquinone analogs of ß-lapachone were designed, synthesized and evaluated. The biological results indicated that most of our compounds were efficient substrates for NQO1. The new scaffold with water-soluble side chain resulted in greater solubility under acidic condition compared to ß-lapachone. Thus avoiding the use of hydroxylpropyl ß-cyclodextrin which would finally cause the rapid drug clearance from the blood and dose-limiting toxicity in the form of hemolytic anemia. The most soluble and promising compound in this series was 2-((4-benzylpiperazin-1-yl)methyl)naphtho[2,1-d]oxazole-4,5-dione (3k), which inhibited cancer cell (NQO1-rich A549 cell line) growth at IC50 values of 4.6±1.0µmol·L(-1). Furthermore, compound 3k had in vivo antitumor activity in an A549 tumor xenografts mouse model comparable to the activity obtained with ß-lapachone. The results indicated that these ortho-naphthoquinones could serve as promising leads for further optimization as novel substrates for NQO1.

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.

Wogonin reverses multi-drug resistance of human myelogenous leukemia K562/A02 cells via downregulation of MRP1 expression by inhibiting Nrf2/ARE signaling pathway.

Constitutive NF-E2-related factor 2 (Nrf2) activation has been recently reported to play a pivotal role in enhancing cell survival and resistance to anticancer drugs in many tumors. Previously, much effort has been devoted to the investigation of blocking Nrf2 function in cultured cells and cancer tissues, but few researches have been undertaken to evaluate the precise mechanism of flavonoids-induced sensitivity by inhibiting Nrf2. In this study, we investigated the reversal effect of Wogonin, a flavonoid isolated from the root of Scutellaria baicalensis Georgi, in resistant human myelogenous leukemia. Data indicated that Wogonin had strong reversal potency by inhibiting functional activity and expression of MRP1 at both protein and mRNA in adriamycin (ADR)-induced resistant human myelogenous leukemia K562/A02 cells. Consequently, the inhibition of MRP1 by Wogonin was dependent on Nrf2 through the decreased binding ability of Nrf2 to antioxidant response element (ARE). Further research revealed Wogonin modulated Nrf2 through the reduction of Nrf2mRNA at transcriptional processes rather than RNA degradation, which is regulated by the PI3K/Akt pathway. Moreover, DNA-PKcs was found to be involved in the Wogonin-induced downregulation of Nrf2 mRNA at transcriptional levels. In summary, these results clearly demonstrated the effectiveness of using Wogonin via inhibiting Nrf2 to combat chemoresistance and suggested that Wogonin can be developed into an efficient natural sensitizer for resistant human myelogenous leukemia.

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.

A concise and efficient total synthesis of α-mangostin and ß-mangostin from Garcinia mangostana.

The concise, efficient synthesis of alpha-mangostin is described in eight simple steps with 8.3% overall yield. Highlights include a practical approach to construct the isopentene groups and other diverse groups at C-2 and C-8 of the xanthene skeleton through Claisen rearrangement and Wittig reaction. Meanwhile the first total synthesis of beta-mangostin is presented with a similar approach.

Discovery and design of tricyclic scaffolds as protein kinase CK2 (CK2) inhibitors through a combination of shape-based virtual screening and structure-based molecular modification.

Protein kinase CK2 (CK2), a ubiquitous serine/threonine protein kinase for hundreds of endogenous substrates, serves as an attractive anticancer target. One of its most potent inhibitors, CX-4945, has entered a phase I clinical trial. Herein we present an integrated workflow combining shape-based virtual screening for the identification of novel CK2 inhibitors. A shape-based model derived from CX-4945 was built, and the subsequent virtual screening led to the identification of several novel scaffolds with high shape similarity to that of CX-4945. Among them two tricyclic scaffolds named [1,2,4]triazolo[4,3-c]quinazolin and [1,2,4]triazolo[4,3-a]quinoxalin attracted us the most. Combining strictly chemical similarity analysis, a second-round shape-based screening was performed based on the two tricyclic scaffolds, leading to 28 derivatives. These compounds not only targeted CK2 with potent and dose-dependent activities but also showed acceptable antiproliferative effects against a series of cancer cell lines. Our workflow supplies a high efficient strategy in the identification of novel CK2 inhibitors. Compounds reported here can serve as ideal leads for further modifications.

Wogonin induced G1 cell cycle arrest by regulating Wnt/ß-catenin signaling pathway and inactivating CDK8 in human colorectal cancer carcinoma cells.

Wogonin, a naturally occurring mono-flavonoid, has been reported to have tumor therapeutic potential and good selectivity both in vitro and in vivo. Herein, we investigated the anti-proliferation effects and associated mechanisms of wogonin in human colorectal cancer in vitro. The flow-cytometric analysis showed that wogonin induced a G1 phase cell cycle arrest in HCT116 cells in a concentration- and time-dependent manner. Meanwhile, the cell cycle-related proteins, such as cyclin A, E, D1, and CDK2, 4 were down-regulated in wogonin-induced G1 cell cycle arrest. Furthermore, we showed that the anti-proliferation and G1 arrest effect of wogonin on HCT116 cells was associated with deregulation of Wnt/ß-catenin signaling pathway. Wogonin-treated cells showed decreased intracellular levels of Wnt proteins, and activated degradation complex to phosphorylated and targeted ß-catenin for proteasomal degradation. Wogonin inhibited ß-catenin-mediated transcription by interfering in the transcriptional activity of TCF/Lef, and repressing the kinase activity of CDK8 which has been considered as an oncogene involving in the development of colorectal cancers. Moreover, CDK8 siRNA-transfected HCT116 cells showed similar results to wogonin treated cells. Thus, our data suggested that wogonin induced anti-proliferation and G1 arrest via Wnt/ß-catenin signaling pathway and it can be developed as a therapeutic agent against human colorectal cancer.

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.

GSK3ß/ß-catenin signaling is correlated with the differentiation of glioma cells induced by wogonin.

Malignant gliomas are the most common and most aggressive primary brain tumor, and for which differentiation therapy has emerged as a promising candidate strategy. In this study, we used in vitro and in vivo assays to examine the differentiation effects of wogonin, a major active constituent of Scutellaria baicalensis, on glioma C6 and U251 cells. We found that wogonin can suppress cell proliferation and induce G0/G1 arrest under a concentration-dependent manner. Wogonin also triggered significant reduction in the G1 cell-cycle regulatory proteins cyclin D1, cyclin-dependent kinase 2 and 4 along with overexpression of cell-cycle inhibitory proteins p27. Immunofluorescence and western blot analysis indicated that wogonin increased the expression of lineage-specific differentiation marker glial fibrillary acidic protein (GFAP). In mechanisms, we verified that wogonin significantly diminished the phosphorylated level of protein kinase B (AKT), and maintenance of low ß-catenin expression level was dependent on glycogen synthase kinase 3ß (GSK3ß) activation at Ser9. Blocking GSK3ß/ß-catenin pathway was required for wogonin-induced proliferation inhibition and terminal differentiation by using canonical activator lithium chloride (LiCl) and inhibitor dickkopf-1 (Dkk1). Moreover, intravenous administration of wogonin delayed the growth of C6 glioma in the intracranial tumor model. These findings provide the evidence and mechanistic support for wogonin-based differentiation therapies for malignant glioblastoma. Furthermore, inhibition of GSK3ß/ß-catenin pathway may be a key and requisite factor in glioma differentiation.

Identification, design and bio-evaluation of novel Hsp90 inhibitors by ligand-based virtual screening.

Heat shock protein 90 (Hsp90), whose inhibitors have shown promising activity in clinical trials, is an attractive anticancer target. In this work, we first explored the significant pharmacophore features needed for Hsp90 inhibitors by generating a 3D-QSAR pharmacophore model. It was then used to virtually screen the SPECS databases, identifying 17 hits. Compound S1 and S13 exhibited the most potent inhibitory activity against Hsp90, with IC50 value 1.61±0.28 µM and 2.83±0.67 µM, respectively. Binding patterns analysis of the two compounds with Hsp90 revealed reasonable interaction modes. Further evaluation showed that the compounds exhibited good anti-proliferative effects against a series of cancer cell lines with high expression level of Hsp90. Meanwhile, S13 induced cell apoptosis in a dose-dependent manner in different cell lines. Based on the consideration of binding affinities, physicochemical properties and toxicities, 24 derivatives of S13 were designed, leading to the more promising compound S40, which deserves further optimization.

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.