Bioassay-Guided Fractionation and Biological Activity of Cardenolides from Streptocaulon juventas.

The discovery that Na/K-ATPase acts as a signal transducer led us to investigate the structural diversity of cardiotonic steroids and study their ligand effects. By applying Na/K-ATPase activity assay-guided fractionation, we isolated a total of 20 cardiotonic steroids from Streptocaulon juventas, including an undescribed juventasoside B (10: ) and 19 known cardiotonic steroids. Their structures have been elucidated. Using our platform of purified Na/K-ATPase and an LLC-PK1 cell model, we found that 10: , at a concentration that induces less than 10% Na/K-ATPase inhibition, can stimulate the Na/K-ATPase/Src receptor complex and selectively activate downstream pathways, ultimately altering prostate cancer cell growth. By assessing the ligand effect of the isolated cardiotonic steroids, we found that the regulation of cell viability by the isolated cardiotonic steroids was not associated with their inhibitory potencies against Na/K-ATPase activity but reflected their ligand-binding affinity to the Na/K-ATPase receptor. Based on this discovery, we identified a unique active cardiotonic steroid, digitoxigenin (1: ), and verified that it can protect LLC-PK1 cells from hypoxic injury, implicating its potential use in ischemia/reperfusion injury and inducing collagen synthesis in primary human dermal fibroblast cells, and implicating that compound 2: is the molecular basis of the wound healing activity of S. juventas.

Discovery of Coixol Derivatives as Potent Anti-inflammatory Agents.

Coixol, a derivative of 2-benzoxazolinone extracted from coix (Coix lachryma-jobi L. var. ma-yuen Stapf), has demonstrated promising anti-inflammatory activity and low cytotoxicity. In this study, 26 coixol derivatives were designed and synthesized by hybridization with cinnamic acid to identify new anti-inflammatory agents. The anti-inflammatory activities of the derivatives were screened using LPS-induced overexpression of nitric oxide (NO) in RAW264.7 macrophages. On the basis of the screening results, compounds containing furan (9c) or nitrofuran (9j) moieties displayed more pronounced activity than coixol and celecoxib. Mechanistic investigations revealed that 9c and 9j suppressed the expression of induced nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1ß, which was associated with the inhibition of the nuclear factor (NF)-κB signaling pathway. In vivo studies confirmed the anti-inflammatory activity of 9c and 9j in a xylene-induced mice auricles edema model. The preliminary in vitro and in vivo research findings suggest that 9c and 9j have the potential to be developed as anti-inflammatory agents.

Design and synthesis of novel indole and indazole-piperazine pyrimidine derivatives with anti-inflammatory and neuroprotective activities for ischemic stroke treatment.

Microglia-mediated neuroinflammation plays an important role in ischemic stroke (IS). In this work, a series of novel indole and indazole-piperazine pyrimidine derivatives with anti-neuroinflammatory and neuroprotective activities were designed and synthesized for treatment of IS. Among these compounds, 5j displayed the most attractive cytoprotective effect against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced damage in BV2 cells. Meanwhile, it significantly ameliorated the release of inflammatory mediators, including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), IL-6, nitric oxide (NO) and prostaglandin E2 (PGE2), from lipopolysaccharide (LPS)-induced BV2 cells. Moreover, 5j can decrease the release of TNF-α and IL-1ß form LPS-induced mouse brain neuroinflammation model. As a potent inhibitor against both cyclooxygenase-2 (COX-2, IC50 = 92.54 nM) and 5-lipoxygenase (5-LOX, IC50 = 41.86 nM), 5j inhibited the M1 phenotype polarization of microglia and promoted the M2 phenotype polarization of microglia. Additionally, 5j exhibited remarkable neuroprotection in middle cerebral artery occlusion (MCAO) rats by reducing their infarct volumes and neurological deficit scores. In conclusion, 5j has the potential for the treatment of stroke as an anti-inflammatory and neuroprotective agent.

Neuroprotective Activities of Constituents from Phyllosticta capitalensis, an Endophyte Fungus of Loropetalum chinense var. rubrum.

One new dioxolanone derivative, guignardianone G (1) and twelve known compounds (2-13) were isolated from the 95 % ethanol extract of the plant endophytic fungus Phyllosticta capitalensis cultured in rice medium. Among these known compounds, isoaltenuene (3), brassicasterol (7), 5,6-epoxyergosterol (8), citreoanthrasteroid A (9), demethylincisterol A (10), and chaxine C (11) were reported from Phyllosticta sp. for the first time. The structure of 1 was elucidated by 1D- and 2D-NMR experiments and HR-ESI-MS data analysis, and its absolute configuration was established through the comprehensive use of the methods of modified Mosher methods, calculations of ECD spectra and optical rotation values. The neuroprotective activity of compounds (1-9, 11-13) were evaluated on PC12 cells damage induced by glutamate, and compounds 9 and 12 showed potential neuroprotective activities with half effective concentration (EC50 ) of 24.2 and 33.9 µM, respectively.

Naphtho-γ-pyrone Dimers from an Endozoic Aspergillus niger and the Effects of Coisolated Monomers in Combination with Cisplatin on a Cisplatin-Resistant A549 Cell Line.

Chemotherapy resistance is one of the main causes of lung cancer treatment failure, and a combination regimen may be an effective way to overcome this. Here we report 5 new (1-3, 7, and 9) and 15 known polyketides, isolated from an endozoic Aspergillus niger. The structures of the new compounds were determined by the interpretation of IR, HRESIMS, NMR, and ECD spectra. The ESI-MS/MS fragmentation of the isolated naphtho-γ-pyrone isomers in positive mode is discussed. The effects of isolated compounds in combination with cisplatin (DDP) on a DDP-resistant A549 cell line (A459/DDP) are investigated. The most active compound, 12, could reduce the ratio of GSH/GSSG, promote the generation of intracellular ROS, and cooperate with DDP to down-regulated levels of Nrf2, Akt, HO-1, and NQO1, suggesting that inhibition of Nrf2 and Akt pathways might be involved in the combined effect of 12 and DDP in A549/DDP cells.

Synthesis and activity of miconazole derivatives as dual BChE/IDO1 inhibitors for the treatment of Alzheimer's disease.

Background: Alzheimer's disease is a multifactorial neurological disorder seen in elderly people. Loss of cholinergic transmission and unbalanced tryptophan metabolism kynurenine pathway have been demonstrated in neuropsychiatric diseases. Methods & results: Among the two series of synthesized compounds, compounds 5c and 5h were identified as effective dual BChE/IDO1 inhibitors, with well-balanced micromolar activity. Compounds 5c and 5h exhibited promising ability to ameliorate behavioral impairment by Morris water maze. The safety of miconazole analogs was also validated by PC12 and SH-SY5Y cell lines. Conclusion: These results highlight the ability of 5c and 5h to treat Alzheimer's disease.

Discovery of potent glycogen synthase kinase 3/cholinesterase inhibitors with neuroprotection as potential therapeutic agent for Alzheimer's disease.

In the present work, a novel series of pyridinethiazole bearing benzylpiperidine hybrids were designed and synthesized as dual-target inhibitors of GSK-3ß/AChE. Among them, GD29 was the most promising candidate, with an IC50 value of 0.3 µM for hAChE and an IC50 value of 0.003 µM for hGSK-3ß, respectively. The compounds exhibited good drug-like properties with optimal inhibitory enzyme activities. Moreover, GD29 showed anti-inflammatory properties at micromolar concentrations and displayed interesting neuroprotective profiles in an in vitro model of oxidative stress-induced neuronal death. Notably, the compounds also exhibited good permeability across the blood-brain-barrier (BBB) both in vitro. Central cholinomimetic activity was confirmed using a scopolamine-induced cognition impairment model in Institute of Cancer Research (ICR) mice upon oral administration. The current work identified optimized compounds and explored the therapeutic potential of glycogen synthase kinase 3/cholinesterase inhibition for the treatment of AD.

A previously undescribed phenylethanoid glycoside from Callicarpa kwangtungensis Chun acts as an agonist of the Na/K-ATPase signal transduction pathway.

The new concept that Na/K-ATPase acts as a receptor prompted us to look for new ligands from Callicarpa kwangtungensis Chun. Using column chromatography, an undescribed phenethyl alcohol glycoside, callicarpanoside A, and an undescribed benzyl alcohol glycoside, callicarpanoside B, along with twelve known polyphenols were isolated from Callicarpa kwangtungensis Chun. All the isolated compounds were evaluated for their Na/K-ATPase (NKA) inhibitory activities. Using our NKA technology platform-based screening assay protocols, callicarpanoside B was identified as an undescribed Na/K-ATPase agonist. In particular, the newly identified benzyl alcohol glycoside was found to bind NKA and activate the receptor NKA/Src complex, resulting in the activation of protein kinase cascades. These cascades included extracellular signal-regulated kinases and protein kinase C epsilon, as well as NKA α1 endocytosis at nanomolar concentrations. Unlike the class of cardiotonic steroids, callicarpanoside B showed less inhibition of NKA activity and caused less cellular toxicity. Moreover, callicarpanoside B was found to bind NKA at a different site other than the cardiotonic steroids binding site. Thus, we have identified an undescribed NKA α1 agonist that may be used to enhance the physiological processes of NKA α1 signaling.

PROTACs suppression of GSK-3ß, a crucial kinase in neurodegenerative diseases.

Glycogen synthase kinase 3ß (GSK-3ß) is involved in a variety of diseases such as neurodegenerative diseases, bipolar disorder, and diabetes. In this study, a series of heterobifunctional small molecule proteolysis targeting chimera (PROTAC) were designed and synthesized based on E3 ubiquitin ligase cereblon (CRBN). Most of PROTACs displayed good inhibitory activity, with the IC50 values at the double-digits nanomolar levels and moderate protein degradation ability against GSK-3ß. Western-blot data showed compound PG21 can effectively degrade GSK-3ß in a dose-dependent manner, which can induce 44.2% protein degradation at 2.8 µM. Further pharmacological experiments revealed that the ability of PG21 to degrade GSK-3ß is mediated by the ubiquitin-proteasome system (UPS). In addition, PG21 protects against glutamate-induced cell death in HT-22 cells. As the first PROTAC example to degrade GSK-3ß protein, the present study has provided potential candidates for further investigation in the biological function of GSK-3ß protein and its association with diseases.

Degradation of proteins by PROTACs and other strategies.

Blocking the biological functions of scaffold proteins and aggregated proteins is a challenging goal. PROTAC proteolysis-targeting chimaera (PROTAC) technology may be the solution, considering its ability to selectively degrade target proteins. Recent progress in the PROTAC strategy include identification of the structure of the first ternary eutectic complex, extra-terminal domain-4-PROTAC-Von-Hippel-Lindau (BRD4-PROTAC-VHL), and PROTAC ARV-110 has entered clinical trials for the treatment of prostate cancer in 2019. These discoveries strongly proved the value of the PROTAC strategy. In this perspective, we summarized recent meaningful research of PROTAC, including the types of degradation proteins, preliminary biological data in vitro and in vivo, and new E3 ubiquitin ligases. Importantly, the molecular design, optimization strategy and clinical application of candidate molecules are highlighted in detail. Future perspectives for development of advanced PROTAC in medical fields have also been discussed systematically.

Targeted degradation of anaplastic lymphoma kinase by gold nanoparticle-based multi-headed proteolysis targeting chimeras.

Anaplastic lymphoma kinase (ALK) is a major target in treating non-small-cell lung cancer, and several ALK inhibitors have been developed to antagonize its kinase activity. However, patients treated with inhibitors ultimately develop drug resistance. Therefore, therapies with new mechanisms of action are needed. Proteolysis targeting chimeras (PROTACs) are molecules that comprise a ligand for binding a protein of interest (POI), a connecting linker and a ligand for recruiting E3 ligase, and cause degradation of the target POI. Here, the first multi-headed PROTAC, as a proof of concept, is developed as a gold nanoparticle (GNP)-based drug delivery system for delivering PROTACs to target ALK. Pegylated GNPs loaded with both ceritinib and pomalidomide molecules, termed Cer/Pom-PEG@GNPs, showed good stability in several media. The GNP conjugates potently decreased the levels of ALK fusion proteins in a dose- and time-dependent manner, and specifically inhibited the proliferation of NCI-H2228 cells. In comparison with small molecule PROTACs, the new multi-headed PROTAC promoted the formation of coacervates of POIs/multi-headed PROTAC/E3 ubiquitin ligases, and POI and E3 ubiquitin ligase interacted through multidirectional ligands and a flexible linker, thereby avoiding the need for complicated structure optimization of PROTACs. In conclusion, Cer/Pom-PEG@GNPs can degrade intracellular ALK fusion proteins with minor off-target toxicity and can be applied in patients resistant to ALK inhibitors. As a nano-based drug carrier, Cer/Pom-PEG@GNPs have the potential to enable prolonged circulation and specifically distribute drugs to tumor regions in vivo; thus, further investigation is warranted.

Anti-angiogenic and anticancer effects of baicalein derivatives based on transgenic zebrafish model.

Angiogenesis leads to tumor neovascularization by promoting tumor growth and metastatic spread, therefore, angiogenesis is considered as an attractive target for potential small molecule anticancer drug discovery. Herein, we report the structural modification and biological evaluation of baicalein derivatives, among which compound 42 had potent in vivo anti-angiogenic activity and wide security treatment window in transgenic zebrafish model. Further, 42 exhibited the most potent inhibitory activity on HUVEC proliferation, migration and tube formation in vitro. Moreover, 42 significantly inhibited growth of human lung cancer A549 cells and weak influence on human normal fibroblast L929 cells. The present research demonstrated that the significant anti-angiogenic and anticancer effects, which provided the supportive evidence for 42 could be used as a potential compound of cancer therapy.

Small molecule KDM4s inhibitors as anti-cancer agents.

Histone demethylation is a vital process in epigenetic regulation of gene expression. A number of histone demethylases are present to control the methylated states of histone. Among these enzymes, KDM4s are one subfamily of JmjC KDMs and play important roles in both normal and cancer cells. The discovery of KDM4s inhibitors is a potential therapeutic strategy against different diseases including cancer. Here, we summarize the development of KDM4s inhibitors and some related pharmaceutical information to provide an update of recent progress in KDM4s inhibitors.

Dual GSK-3ß/AChE Inhibitors as a New Strategy for Multitargeting Anti-Alzheimer's Disease Drug Discovery.

Designing multitarget-directed ligands (MTDLs) is considered to be a promising approach to address complex and multifactorial maladies such as Alzheimer's disease (AD). The concurrent inhibition of the two crucial AD targets, glycogen synthase kinase-3ß (GSK-3ß) and human acetylcholinesterase (hAChE), might represent a breakthrough in the quest for clinical efficacy. Thus, a novel family of GSK-3ß/AChE dual-target inhibitors was designed and synthesized. Among these hybrids, 2f showed the most promising profile as a nanomolar inhibitor on both hAChE (IC50 = 6.5 nM) and hGSK-3ß kinase activity (IC50 = 66 nM). It also showed good inhibitory effect on ß-amyloid self-aggregation (inhibitory rate = 46%) at 20 µM. Western blot analysis revealed that compound 2f inhibited hyperphosphorylation of tau protein in mouse neuroblastoma N2a-Tau cells. In vivo studies confirmed that 2f significantly ameliorated the cognitive disorders in scopolamine-treated ICR mice and less hepatotoxicity than tacrine. This study provides new leads for assessment of GSK-3ß and AChE pathway dual inhibition as a promising strategy for AD treatment.

Therapeutic Agents in Alzheimer's Disease Through a Multi-targetdirected Ligands Strategy: Recent Progress Based on Tacrine Core.

Alzheimer's Disease (AD) is one of the most common forms of dementia in elderly people. To date, efficacious therapeutic agent for the treatment of AD is still very limited, so it has long been a challenging and attractive task to discover new anti-AD drugs. Considering the multifactorial nature of AD, recently, the concept of Multi-Target-Directed Ligands (MTDLs) has emerged as a new strategy for designing therapeutic agents on AD. MTDLs are believed to exert their effects through simultaneously affecting multiple targets which contribute to etiology of AD. Therefore, MTDLs are considered to be more efficacious than mono-target agents. Tacrine is the first drug approved by Food and Drug Administration (FDA). Although the clinical use of tacrine is restricted because of its hepatotoxicity, the high Ligand Efficiency (LE) of this compound makes it an ideal component for designing MTDLs. This article provides an update review of the advances on the development of MTDLs based on tacrine. Case studies are carefully selected to show the detailed strategy on medicinal modification of Tacrine-Based MTDLs. Finally, several concerns and opinions on designing new MTDLs are discussed as well.

Gamma secretase inhibitors: a patent review (2013 - 2015).

INTRODUCTION: Gamma secretase (GS) is an intricate and multi-subunits complex, and it can cut various transmembrane proteins. Now it is a therapeutic target for a number of diseases. However, due to some side effects, the clinical development of GSI is not successful. Therefore, searching for effective GSIs has become a key point in drug discovery. Areas covered: This review discusses the structure and function of GS and various types of GSIs. And this article seeks to give an overview of the patents or applications published from 2013 to 2015 in which novel chemical classes are claimed to inhibit the GS. Expert opinion: Firstly, further understanding the structure and function of GS to elucidate the disease mechanism and develop AD therapies is urgent. Secondly, if the bioequivalence, pharmacokinetics and selectivity can be improved greatly, some failed clinical inhibitors still can become the promising compounds for clinical trials. Thirdly, some weaknesses are exposed during the development of GSI, especially the insufficient potency, low brain penetration and poor selectivity. Finally, to find potent and selective GSI is the major direction in future. Moreover, to find new indications and dosing regimens in a trial of GSIs also can be seen as new ways.