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Introduction: Veratrum alkaloids have gained attention due to their toxic effects and potential pharmaceutical applications, particularly in cancer and cardiology. Over 200 alkaloids are found in species of the Veratrum genus. The alkaloid composition and concentrations can greatly vary in plants depending on factors like species, plant part, location, season, weather, or nutrients., Objective: This study aims an analytical approach to analyze and quantify Veratrum alkaloids in different plant parts of Veratrum species. The purpose is to contribute essential alkaloid concentration data for future research on the pharmacological and toxicological aspects of Veratrum alkaloids., Methods: This study focuses on five Veratrum alkaloids (cevadine, jervine, protoveratrine A, veratramine, and veratridine) in three Veratrum species (Veratrum album L., Veratrum californicum Durand, and Veratrum nigrum L.) collected from four German botanical gardens (Dresden, Leipzig, Marburg, and Schellerhau). A liquid-liquid extraction method and a sensitive high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) method operating in multiple reaction monitoring (MRM) mode were applied for the alkaloid determination., Results: Quantification revealed varying alkaloid concentrations among plant parts and Veratrum species in the μg/g to mg/g range. Protoveratrine A exhibited the highest content, while veratramine concentrations were generally lower. Especially in fruit, roots and rootstock of Veratrum album L. alkaloid concentrations were significant high., Conclusion: The developed HPLC-MS/MS method successfully determined Veratrum alkaloid concentrations in plant samples. The study contributes valuable data on Veratrum alkaloid distribution in different species and plant parts, crucial for understanding their potential medicinal and toxicological significance., (© 2024 The Author(s). Phytochemical Analysis published by John Wiley & Sons Ltd.)

The Veratrum alkaloids are a class of highly intricate natural products renowned for their complex structural and stereochemical characteristics, which underlie a diverse array of pharmacological activities ranging from anti-hypertensive properties to antimicrobial effects. These properties have generated substantial interest among both synthetic chemists and biologists. While numerous advancements have been made in the synthesis of jervanine and veratramine subtypes over the past 50 years, the total synthesis of highly oxidized cevanine subtypes has remained relatively scarce. Building on the efficiency of our previously developed strategy for constructing the hexacyclic carbon skeleton of the Veratrum alkaloid family via a stereoselective intramolecular Diels-Alder reaction and radical cyclization, here we show the development of a unified synthetic approach to access highly oxidized Veratrum alkaloids. This includes the total synthesis of (-)-zygadenine, (-)-germine, (-)-protoverine and the alkamine of veramadine A, by capitalizing on a meticulously designed sequence of redox manipulations and a late-stage neighboring-group participation strategy., (© 2024. The Author(s).)

Veratramine and cyclopamine, two of the most representative members of the isosteroidal alkaloids, are valuable molecules in agricultural and medicinal chemistry. While plant extraction of these compounds suffers from uncertain supply, efficient chemical synthesis approaches are in high demand. Here, we present concise, divergent, and scalable syntheses of veratramine and cyclopamine with 11% and 6.2% overall yield, respectively, from inexpensive dehydro-epi-androsterone. Our synthesis readily provides gram quantities of both target natural products by utilizing a biomimetic rearrangement to form the C-nor-D-homo steroid core and a stereoselective reductive coupling/(bis-)cyclization sequence to establish the (E)/F-ring moiety., (© 2024. The Author(s).)

Cyclopamine (1), the teratogenic steroidal alkaloid isolated from corn lily (Veratrum californicum), has recently gained renewed interest due to its anticancer potential, that has been translated into the FDA approval of three Hedgehog (Hh) pathway inhibiting antitumor drugs. A chemical analysis of mother liquors obtained from crystallization of cyclopamine, extracted from roots and rhizomes of V. californicum, resulted in the isolation of two unprecedented cyclopamine analogues, 18-hydroxycyclopamine (2) and 24R-hydroxycyclopamine (3), the first compounds of this class to show modifications on rings D-F. The stereostructures of these new natural compounds have been established based on a detailed MS and 1D/2D NMR investigation. The isolated compounds were evaluated with the dual-luciferase bioassay for their inhibition of the hedgehog pathway in comparison to cyclopamine, providing new insights into the structure-activity relationships for this class of compounds., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Three new steroidal alkaloids, veratrasines A - C (1-3), along with ten known analogues (4-13) were isolated from the roots of Veratrum stenophyllum. Their structures were elucidated by NMR and HRESIMS data and comparison with the reported data in the literatures. A plausible biosynthetic pathway for 1 and 2 were proposed. Compounds 1, 3, and 8 showed moderate cytotoxic activity against MHCC97H and H1299 cell lines., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Chinese Materia Medica and Jiangsu New Medical College record that Radix Veratri root is Liliaceae Veratrum taliense Loses. f. and the root of Veratrum stenophyllum Diels . According to traditional Chinese medicine ( TCM ) example , Radix Veratri is a Liliaceae plant Veratrum taliense. Another literature pointed out that the aliases of Veratrum taliense and Veratrum angustifolia are both Radix Veratri, and their effects are basically the same. The main active ingredient of Veratrum is veratramine, of which veratramine and Jervine are higher in content, reaching 24.60% and 21.28% of the total alkaloids, respectively. Veratrum alkaloids are both toxic and effective ingredients. In addition to its good clinical efficacy, attention should also be paid to its pharmacokinetic characteristics in vivo. It is particularly important to study the pharmacokinetic characteristics of veratramine and Jervine in vivo., Objective: The goal of this study was to develop a simple and effective method for measuring veratramine and Jervine in rat plasma at the same time. This method was used to study the pharmacokinetic characteristics of veratramine and Jervine in the alcohol extract of Radix Veratri in rats, to provide a reasonable basis for the clinical use of Radix Veratri., Methods: Eighteen SD rats were randomly assigned into three groups, half male and half female, and were given 0.04 g/kg, 0.08g/kg, and 0.16 g/kg Radix Veratri alcohol extract, respectively. Blood samples were collected at different time points and were analyzed by LC-MS/MS after protein precipitation. Bullatine was set as the internal standard; the plasma samples were extracted with ethyl acetate. After the sample was processed, acetonitrile-10 mM ammonium acetate, whose pH was adjusted to 8.8 with ammonia water, was taken as the mobile phase. Veratramine quantitative ion pair was 410.1⟶295.1 m / z , Jervine quantitative ion pair was 426.2⟶114.1 m / z , and Bullatine B (IS) quantitative ion pair was 438.2⟶420.1 m / z . In the positive ion mode, the multireaction monitoring (MRM) mode was used to determine the blood concentration of veratramine and Jervine. DAS 3.3.0 was used to calculate the relevant pharmacokinetic parameters., Results: Veratramine had a good linear relationship in the concentration range of 0.0745~18.2 ng/mL, and that of Jervine was 1.11~108 ng/mL. The correlation coefficient r of three consecutive batches of the standard curve was greater than 0.995. Veratramine's lower quantification limit was 0.745 ng/mL, Jervine's was 1.11 ng/mL, and precision and accuracy were both less than 15%. The accuracy of veratramine was between 88.96% and 101.85%, and the accuracy of Jervine was between 92.96% and 104.50%. This method was adopted for the pharmacokinetic study of alcohol extracts of Radix Veratri. The results showed that only C max of veratramine female rats did not show linear kinetic characteristics in the dose range of Radix Veratri alcohol extract from 0.04 g/kg to 0.16 g/kg. For AUC 0- t and C max of veratramine and Jervine, it could not determine whether the Radix Veratri alcohol extract showed linear kinetic characteristics within the dosage range of 0.04 g/kg~0.16 g/kg. Veratramine and Jervine showed obvious gender differences in the absorption and elimination stages. The absorption rate of veratramine and Jervine by male mice was about 10 times higher than that of female mice, and the elimination rate of male mice is about 20 times lower than that of female mice. It was suggested that the clinical application of the steroidal alkaloids veratramine and Jervine in Radix Veratri required rational use of drugs based on gender., Conclusion: An LC-MS/MS analysis method suitable for the pharmacokinetic study of veratramine and Jervine in Radix Veratri in SD rats was established to provide a basis for in vivo pharmacokinetic studies. The pharmacokinetic characteristics of veratramine and Jervine in the alcohol extract of Radix Veratri were significantly different in female and male rats. During the clinical use of Radix Veratri, it should pay close attention to the obvious gender differences that may occur after the medication., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2022 Song Wang et al.)

The class Frizzled of G protein-coupled receptors (GPCRs), consisting of ten Frizzled (FZD 1-10 ) subtypes and Smoothened (SMO), remains one of the most enigmatic GPCR families. While SMO relies on cholesterol binding to the 7TM core of the receptor to activate downstream signaling, underlying details of receptor activation remain obscure for FZDs. Here, we aimed to investigate the activation mechanisms of class F receptors utilizing a computational biology approach and mutational analysis of receptor function in combination with ligand binding and downstream signaling assays in living cells. Our results indicate that FZDs differ substantially from SMO in receptor activation-associated conformational changes. SMO manifests a preference for a straight TM6 in both ligand binding and functional readouts. Similar to the majority of GPCRs, FZDs present with a kinked TM6 upon activation owing to the presence of residue P 6.43 . Functional comparison of FZD and FZD P 6.43 F mutants in different assay formats monitoring ligand binding, G protein activation, DVL2 recruitment and TOPflash activity, however, underlines further the functional diversity among FZDs and not only between FZDs and SMO.

Progress toward a convergent approach for the enantioselective synthesis of the Veratrum alkaloid jervine is presented. The two requisite fragments were stereoselectively and efficiently fashioned from economical and readily available reagents. Key reactions include (a) a highly diastereoselective Ireland-Claisen rearrangement to establish the necessary cis- relationship between the amine and methyl group on the tetrahydrofuran E-ring; (b) a diastereoselective selenoetherification reaction that enabled the assembly of the D/E oxaspiro[4.5]decene in the needed configuration; and (c) an enzymatic desymmetrization of an abundant achiral diol en route to a key four-carbon building block as a practical alternative to a protected Roche ester reduction.

An efficient synthetic strategy for three natural seco -type cholestane alkaloids isolated from the Veratrum plants, based on commercially available naturally occurring and abundant (-)-diosgenin ( 1 ), as exemplified in the concise asymmetric synthesis of (-)-verazine ( 4 ), (-)-veramiline ( 5 ) (proposed structure), and its 22-epimer, (-)-oblonginine ( 6 ), is presented. This work highlights the application of a cascade ring-switching process of (-)-diosgenin to achieve the E-ring opening and construction of chiral six-membered lactone challenges in seco -type cholestane alkaloid synthesis. This approach enables the synthesis of related natural and nature-like novel cholestane alkaloids, opening up opportunities for more extensive exploration of cholestane alkaloid biology.

Veratrum plant contains a family of compounds called steroidal alkaloids which have been previously reported to cause DNA damage and blood pressure decrease in vivo. In this study, the antihypertensive effects and DNA damage in brain cells of 12 steroidal alkaloids separated from Veratrum plant were all evaluated to develop a relationship among chemical structure, antihypertensive activity and neurotoxicity by utilization of chemical principal component analysis (PCA) and hierarchical cluster analysis (HCA). Twelve steroidal alkaloids markedly reduced high blood pressure of hypertensive mice and also similarly induced varying degrees of DNA single-strand breaks in mouse cerebellum and cerebral cortex after oral administration. On the basis of the PCA and HCA results, it was suggested that the 3-carboxylic esters and benzene group play a core role in the DNA damage of brain cells, while more hydroxy groups in the A-ring and B-ring structure of jervine-type alkaloid led to stronger antihypertensive activity. The primary structure, activity and neurotoxicity relationship were discussed briefly., (© 2020 Wiley-VHCA AG, Zurich, Switzerland.)

Smoothened (SMO) is a GPCR that mediates hedgehog signaling. Hedgehog binds the transmembrane protein Patched, which in turn regulates SMO activation. Overactive SMO signaling is oncogenic and is therefore a clinically established drug target. Here we establish a nanoluciferase bioluminescence resonance energy transfer (NanoBRET)-based ligand binding assay for SMO providing a sensitive and high throughput-compatible addition to the toolbox of GPCR pharmacologists. In the NanoBRET-based binding assay, SMO is N terminally tagged with nanoluciferase (Nluc) and binding of BODIPY-cyclopamine is assessed by quantifying resonance energy transfer between receptor and ligand. The assay allowed kinetic analysis of ligand-receptor binding in living HEK293 cells, competition binding experiments using commercially available SMO ligands (SANT-1, cyclopamine-KAAD, SAG1.3 and purmorphamine), and pharmacological dissection of two BODIPY-cyclopamine binding sites. This high throughput-compatible assay is superior to commonly used SMO ligand binding assays in the separation of specific from non-specific ligand binding and, provides a suitable complement to chemical biology strategies for the discovery of novel SMO-targeting drugs. SIGNIFICANCE STATEMENT: We established a NanoBRET-based binding assay for SMO with superior sensitivity compared to fluorescence-based assays. This assay allows distinction of two separate binding sites for BODIPY-cyclopamine on the SMO transmembrane core in live cells in real time. The assay is a valuable complement for drug discovery efforts and will support a better understanding of Class F GPCR pharmacology., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

In this study, we investigated whether jervine (J) could prevent gastrointestinal (GI) side effects of abdominopelvic radiotherapy (RT) in Wistar-Albino female rats. Rats were divided into five groups: control (C), J only (J), J administered at 5 mg/kg/days for 7 days, RT only (RT), J before RT (J + RT), J administered for seven days before RT, J both before and after RT (J + RT + J), and J administered for 7 days before RT and after RT for 3 days. The weights of rats were measured on the 1st, 7th, and 10th days of the study. Rats were sacrificed to obtain tissues from the liver and intestine, which was followed by taking blood samples intracardially. In addition, the tissues were stained with pyruvate dehydrogenase (PDH) immunohistochemically. In our study, J supplementation markedly reduced weight loss, and histopathological, immunohistochemical, biochemical results suggest that J had a protective effect on GI toxicity following RT.

Jervine is a natural teratogenic compound isolated from Veratrum californicum. In this study, for the first time, we revealed a novel activity of jervine in sensitizing the anti-proliferation effect of doxorubicin (DOX). We demonstrated that the synergistic mechanism was related to the intracellular accumulation of DOX via modulating ABCB1 transportation. Jervine did not affect the expression of ABCB1 in mRNA nor protein levels. However, jervine increased the ATPase activity of ABCB1 and possibly served as a substrate of ABCB1. The molecular docking results indicated that jervine was bound to a closed ABCB1 conformation and blocked drug entrance to the central binding site at the transmembrane domain. The present study identifies jervine acts as a substrate of ABCB1, and has potential to be developed as a novel and potent chemotherapy sensitizer used for patients developing multidrug resistance., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The prominent stromal compartment surrounds pancreatic ductal adenocarcinoma and protects the tumor cells from chemo- or radiotherapy. We hypothesized that our nano formulation carrying cyclopamine (CPA, stroma modulator) and paclitaxel (PTX, antitumor agent) could increase the permeation of PTX through the stromal compartment and improve the intratumoral delivery of PTX. In the present study a sensitive, reliable UPLC-MS/MS method was developed and validated to quantify PTX and CPA simultaneously in mouse whole blood, pancreas, liver and spleen samples. Docetaxel was used as the internal standard. The method demonstrated a linear range of 0.5-2000 ng/mL for whole blood and tissue homogenates for both PTX and CPA. The accuracy and precision of the assay were all within ±15%. Matrix effects for both analytes were within 15%. Recoveries from whole blood, liver, spleen and pancreas homogenates were 92.7-105.2% for PTX and 72.8-99.7% for CPA. The stability was within ±15% in all test biomatrices. The validated method met the acceptance criteria according to US Food and Drug Administration regulatory guidelines. The method was successfully applied to support a pharmacokinetic and biodistribution study for PTX and CPA in mice biomatrices., (© 2019 John Wiley & Sons, Ltd.)

The oncoprotein Smoothened (SMO), a G-protein-coupled receptor (GPCR) of the Frizzled-class (class-F), transduces the Hedgehog signal from the tumour suppressor Patched-1 (PTCH1) to the glioma-associated-oncogene (GLI) transcription factors, which activates the Hedgehog signalling pathway 1,2 . It has remained unknown how PTCH1 modulates SMO, how SMO is stimulated to form a complex with heterotrimeric G proteins and whether G-protein coupling contributes to the activation of GLI proteins 3 . Here we show that 24,25-epoxycholesterol, which we identify as an endogenous ligand of PTCH1, can stimulate Hedgehog signalling in cells and can trigger G-protein signalling via human SMO in vitro. We present a cryo-electron microscopy structure of human SMO bound to 24(S),25-epoxycholesterol and coupled to a heterotrimeric G i protein. The structure reveals a ligand-binding site for 24(S),25-epoxycholesterol in the 7-transmembrane region, as well as a G i -coupled activation mechanism of human SMO. Notably, the G i protein presents a different arrangement from that of class-A GPCR-G i complexes. Our work provides molecular insights into Hedgehog signal transduction and the activation of a class-F GPCR.

Although layered double hydroxides (LDH) have been listed as promising nanomaterials in human healthcare, very little has been achieved on osteoblast inflammatory signaling. Thus, osteoblasts were challenged with two LDHs (Mg 2 Al-Cl and Zn 2 Al-Cl, at 0.002 mg/mL) up to 24 h, establishing an acute inflammatory mechanism, as well as identifying whether Sonic hedgehog (Shh) signaling has an influence. Functional experiments were performed by previously treating (2 h) semiconfluent osteoblast cultures with cyclopamine molecule (cyc), a widely used Shh inhibitor. Considering inflammasome complex, the asc1 gene was significantly up-expressed in response to Zn 2 Al-Cl - LDHs, as well as the nrlp3 gene. By treating the osteoblast with cyc, the asc1 gene presented an even higher profile. Our results found a down-modulation of major pro-inflammatory cytokines-related genes, when tnfα and il1ß were significantly down-modulated in response to LDHs. Conversely, anti-inflammatory cytokines were up-modulated considering the same experimental procedures. Except the il6, the other il13, il10, and tgfß genes were up modulated. Additionally, Shh signaling seems to modulate this repertory as both the il13 and il10 genes were significantly up-modulated when the Shh signaling was inhibited. Altogether, our results reveal for the first time the exigency of Shh-dependent anti-inflammatory signals in LDH-induced osteoblast responses., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In this study, a sensitive hydrophilic interaction liquid chromatography (HILIC) method was developed to determine pseudojervine (PJV), veratrosine (VTS), jervine (JV), veratramine (VTM), veramarine (VA) and veratroylzygadenine (VTG) in rat plasma. Separations were carried out using LC-MS/MS with a Chrom Matrix HP amide column (5 m, 10 cm × 3.0 mm i.d.). The mobile phases were (A) 0.01 mm formic acid and (B) acetonitrile. Good linearity was found for all analytes (R 2  > 0.995) in the concentration range from 5 to 1000 μg/L with LLOQ at 5 μg/L for VTM and VTS; and from 1 to 1000 μg/L with LLOQ at 1 μg/L for PJV, JV, VA and VTG. Accuracy of the assay varied from 90.5 to 108.1%. The extraction recovery and matrix effect of six analytes ranged from 72.2 to 95.5% and from 79.2 to 98.4%. According to the stability test, six analytes in rat plasma were stable during the analysis process. On the basis of validation of the assay, the pharmacokinetics of the six steroid alkaloids were investigated after oral administration of Lilu extracts to rats., (© 2018 John Wiley & Sons, Ltd.)

The Hedgehog (Hh) signaling pathway plays a critical role in controlling patterning, growth and cell migration during embryonic development. Aberrant activation of Hh signaling has been linked to tumorigenesis in various cancers, such as basal cell carcinoma (BCC) and medulloblastoma. As a key member of the Hh pathway, the Smoothened (Smo) receptor, a member of the G protein-coupled receptor (GPCR) family, has emerged as an attractive therapeutic target for the treatment and prevention of human cancers. The recent determination of several crystal structures of Smo in complex with different antagonists offers the possibility to perform structure-based virtual screening for discovering potent Smo antagonists with distinct chemical scaffolds. In this study, based on the two Smo crystal complexes with the best capacity to distinguish the known Smo antagonists from decoys, the molecular docking-based virtual screening was conducted to identify promising Smo antagonists from ChemDiv library. A total of 21 structurally novel and diverse compounds were selected for experimental testing, and six of them exhibited significant inhibitory activity against the Hh pathway activation (IC 50  < 10 μM) in a GRE (Gli-responsive element) reporter gene assay. Specifically, the most potent compound (compound 20: 47 nM) showed comparable Hh signaling inhibition to vismodegib (46 nM). Compound 20 was further confirmed to be a potent Smo antagonist in a fluorescence based competitive binding assay. Optimization using substructure searching method led to the discovery of 12 analogues of compound 20 with decent Hh pathway inhibition activity, including four compounds with IC 50 lower than 1 μM. The important residues uncovered by binding free energy calculation (MM/GBSA) and binding free energy decomposition were highlighted and discussed. These findings suggest that the novel scaffold afforded by compound 20 can be used as a good starting point for further modification/optimization and the clarified interaction patterns may also guide us to find more potent Smo antagonists., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

The seven-transmembrane-spanning protein Smoothened is the central transducer in Hedgehog signaling, a pathway fundamental in development and in cancer. Smoothened is activated by cholesterol binding to its extracellular cysteine-rich domain (CRD). How this interaction leads to changes in the transmembrane domain and Smoothened activation is unknown. Here, we report crystal structures of sterol-activated Smoothened. The CRD undergoes a dramatic reorientation, allosterically causing the transmembrane domain to adopt a conformation similar to active G-protein-coupled receptors. We show that Smoothened contains a unique inhibitory π-cation lock, which is broken on activation and is disrupted in constitutively active oncogenic mutants. Smoothened activation opens a hydrophobic tunnel, suggesting a pathway for cholesterol movement from the inner membrane leaflet to the CRD. All Smoothened antagonists bind the transmembrane domain and block tunnel opening, but cyclopamine also binds the CRD, inducing the active transmembrane conformation. Together, these results define the mechanisms of Smoothened activation and inhibition., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Purpose: The aim of this study was to determine whether co-administration of hedgehog (Hh) pathway inhibitor cyclopamine (CYP) and microtubule stabilizer docetaxel (DTX) as polymer-drug conjugates, methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylenecarbonate-graft-dodecanol-graft-cyclopamine) (P-CYP) and methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol-graft-docetaxel) (P-DTX) could synergistically inhibit orthotopic pancreatic tumor growth in NSG mice., Methods: P-DTX and P-CYP were synthesized from mPEG-b-PCC through carbodiimide coupling reaction and characterized by 1 H-NMR. The micelles were prepared by film hydration and particle size was measured by dynamic light scattering (DLS). Cytotoxicity, apoptosis and cell cycle analysis of P-DTX and P-CYP were evaluated in MIA PaCa-2 cells. In vivo efficacy of P-DTX and P-CYP were evaluated in NSG mice bearing MIA PaCa-2 cells derived orthotopic pancreatic tumor., Results: P-CYP and P-DTX self-assembled into micelles of <90 nm and their combination therapy efficiently inhibited the proliferation of MIA PaCa-2 cells, induced apoptosis and cell cycle arrest at M-phase more efficiently than P-CYP and P-DTX monotherapies. Furthermore, the combination therapy of P-CYP and P-DTX significantly reduced Hh component expression compared to P-CYP alone as determined by Western blot analysis. Lastly, the combination therapy induced greater inhibition of orthotopic pancreatic tumor growth in NSG mice compared to their monotherapies., Conclusion: Combination of polymer conjugated anticancer drug (P-DTX) with polymer conjugated Hh inhibitor (P-CYP) enhanced pancreatic cancer cell killing, apoptosis as well as in vivo tumor growth inhibition with no obvious toxicities.

The unique tumour microenvironment (TM) of pancreatic ductal adenocarcinoma (PDA) including highly desmoplastic ECM and low tumour perfusion supports a considerable barrier for effective delivery of nanomedicines. Effectively modulating PDA microenvironment to enhance tumour drug delivery represents a pinpoint in the field of PDA treatment. In this study, it was the first time that biomimetic nanoparticles, which were designed in the form of erythrocyte membrane-camouflaged PLGA nanoparticles (MNP), were utilized for PDA microenvironment modulation. Cyclopamine (CYC), an inhibitor of Hedgehog pathway that contributed a lot to desmoplastic ECM of PDA, was selected as the model drug and successfully encapsulated into MNP. Advantages of CYC-loaded MNP (CMNP) included favourable biocompatibility, long circulation time, and powerful TM modulation effect. CMNP could effectively deliver CYC to the tumour site, disrupt tumour ECM, increase functional vessels, and improve tumour perfusion significantly. The combination treatment with CMNP and PTX-loaded MNP (PMNP) successfully improved PTX delivery to tumour, resulting in remarkable tumour growth inhibition in vivo. Therefore, biomimetic nanoparticles provide a new strategy for modulating PDA TM and will have great potential to improve the therapeutic effects of nanomedicines for PDA patients.

Isosteroidal alkaloids are a category of promising bioactive compounds which mostly exist in plants of genus Veratrum and Fritillaria. The pharmacological activities of isosteroidal alkaloids include antihypertensive, antitussive, anti-inflammatory, antithrombosis, among others. Recently, some studies show that this kind of alkaloids exhibited significant antitumor activity. To the best of our knowledge, there is no review focusing on their antitumor activity and mechanism of their antitumor activity. To fill the gap, in this review, we summarized antitumor effects of the isosteroidal alkaloids from genus Veratrum and Fritillaria on different tumors and the mechanisms of their antitumor activity. In conclusion, this kind of alkaloids has extensive antitumor activity, and there are several main mechanisms of their antitumor activity, including the Hedgehog signaling pathway, caspase-3 dependent apoptosis, cell cycle, and autophagy., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Cell-based assays enable monitoring of small-molecule bioactivity in a target-agnostic manner and help uncover new biological mechanisms. Subsequent identification and validation of the small-molecule targets, typically employing proteomics techniques, is very challenging and limited, in particular if the targets are membrane proteins. Herein, we demonstrate that the combination of cell-based bioactive-compound discovery with cheminformatic target prediction may provide an efficient approach to accelerate the process and render target identification and validation more efficient. Using a cell-based assay, we identified the pyrazolo-imidazole smoothib as a new inhibitor of hedgehog (Hh) signaling and an antagonist of the protein smoothened (SMO) with a novel chemotype. Smoothib targets the heptahelical bundle of SMO, prevents its ciliary localization, reduces the expression of Hh target genes, and suppresses the growth of Ptch +/- medulloblastoma cells., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Three new steroidal alkaloids 1-3, together with four known compounds 4-7, were isolated from the ethanol extract of Veratrum grandiflorum Loes. Their structures were elucidated by NMR (1D and 2D NMR) and MS spectroscopic data. The inhibition activities on Hedgehog (Hh) pathway were evaluated using a cell-based bioassay system (Shh-LIGHT 2 cells). The results showed that compounds 1-3 and 5 displayed inhibitory activities obviously with the IC50 values of 0.63-3.11μM. Among them, compound 5 showed the most prominent inhibition activity (IC50=0.63±0.02μM). Thus, these active alkaloids may be potent natural compounds as Hh pathway inhibitors for the treatment of various cancers., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Human respiratory syncytial virus (hRSV) is a leading cause of acute lower respiratory tract infection in infants, elderly and immunocompromised individuals. To date, no specific antiviral drug is available to treat or prevent this disease. Here, we report that the Smoothened receptor (Smo) antagonist cyclopamine acts as a potent and selective inhibitor of in vitro and in vivo hRSV replication. Cyclopamine inhibits hRSV through a novel, Smo-independent mechanism. It specifically impairs the function of the hRSV RNA-dependent RNA polymerase complex notably by reducing expression levels of the viral anti-termination factor M2-1. The relevance of these findings is corroborated by the demonstration that a single R151K mutation in M2-1 is sufficient to confer virus resistance to cyclopamine in vitro and that cyclopamine is able to reduce virus titers in a mouse model of hRSV infection. The results of our study open a novel avenue for the development of future therapies against hRSV infection.

Covering: 1950s to 2015During the 1950s, sheep ranchers in the western United States experienced episodic outbreaks of cyclopic lambs. In this highlight I describe how these mysterious incidents were traced to the grazing of Veratrum californicum wildflowers by pregnant ewes, leading to the discovery of cyclopamine () as a plant-derived teratogen. The precise mechanism of cyclopamine action remained enigmatic for 30 years, until this steroid alkaloid was found to be the first specific inhibitor of Hedgehog (Hh) signalling and a direct antagonist of the transmembrane receptor Smoothened (SMO). In addition to being a valuable probe of Hh pathway function, cyclopamine has been used to demonstrate the therapeutic potential of Hh pathway inhibitors. I discuss the development of SMO antagonists as anticancer therapies and emerging challenges.

Steroidal alkaloids are a class of secondary metabolites isolated from plants, amphibians, and marine invertebrates. Evidence accumulated in the recent two decades demonstrates that steroidal alkaloids have a wide range of bioactivities including anticancer, antimicrobial, anti-inflammatory, antinociceptive, etc., suggesting their great potential for application. It is therefore necessary to comprehensively summarize the bioactivities, especially anticancer activities and mechanisms of steroidal alkaloids. Here we systematically highlight the anticancer profiles both in vitro and in vivo of steroidal alkaloids such as dendrogenin, solanidine, solasodine, tomatidine, cyclopamine, and their derivatives. Furthermore, other bioactivities of steroidal alkaloids are also discussed. The integrated molecular mechanisms in this review can increase our understanding on the utilization of steroidal alkaloids and contribute to the development of new drug candidates. Although the therapeutic potentials of steroidal alkaloids look promising in the preclinical and clinical studies, further pharmacokinetic and clinical studies are mandated to define their efficacy and safety in cancer and other diseases., (© 2015 Wiley Periodicals, Inc.)

The natural steroidal alkaloid cyclopamine has been identified as the first inhibitor of the Hedgehog (Hh) signaling pathway, which is implicated in embryonic development and tumorigenesis, as well as is hyperactivated in cancer stem cells (CSCs). The list of Hh-dependent tumors is steadily growing, and it has been estimated that about 25% of all cancer deaths show signs of aberrant Hh pathway activation. Notably, cyclopamine has been found to exert anticancer activity against several types of human cancer and to inhibit CSCs proliferation, thus highlighting the druggability of the Hh pathway and paving new opportunities in anticancer drug discovery. The aim of the present work is to review the main synthetic strategies to cyclopamine and its derivatives, with particular emphasis on the challenging chemical modifications aimed at improving the biological activity of the molecule.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers. Cyclopamine (CPA), a potent inhibitor for sonic hedgehog pathway (SHH), shows great promises in PDAC treatment, including the disruption of tumor-associated stroma, and enhancement of radiation therapy. However, CPA is insoluble in water and therefore requires a nanometric delivery platform to achieve satisfactory performance. We herein encapsulated CPA in a core-cross-linked polymeric micelle system (M-CPA). M-CPA was combined with Cs-137 radiation and evaluated in vitro in PDAC cell lines and a human pancreatic stellate cell line. The results showed that M-CPA had higher cytotoxicity than CPA, abolished Gli-1 expression (a key component of SHH), and enhanced the radiation therapy of Cs-137. M-CPA radiosensitization correlated with its ability to disrupt the repair of radiation-induced DNA damage. These findings indicate that the combination therapy of M-CPA and radiation is an effective strategy to simultaneously treat pancreatic tumors and tumor-associated stroma.

Cancer stem cells (CSCs) have the ability to transform into bulk cancer cells, to promote tumor growth and establish tumor metastasis. To effectively inhibit tumor growth and prevent metastasis, treatments with conventional chemotherapy drugs should be combined with CSC targeted drugs. In this study, we describe the synthesis and characterization of a new amphiphilic polymer, hyaluronic acid-cystamine-polylactic-co-glycolic acid (HA-SS-PLGA), composed of a hydrophobic PLGA head and a hydrophilic HA segment linked by a bioreducible disulfide bond. With a double emulsion method, a nano delivery system was constructed to deliver doxorubicin (DOX) and cyclopamine (CYC, a primary inhibitor of the hedgehog signaling pathway of CSCs) to both a CD44-overexpressing breast CSC subpopulation and bulk breast cancer cells and allow an on-demand release. The resulting drug-loaded NPs exhibited a redox-responsive drug release profile. Dual drug-loaded particles potently diminished the number and size of tumorspheres and HA showed a targeting effect towards breast CSCs. In vivo combination therapy further demonstrated a remarkable synergistic anti-tumor effect and prolonged survival compared to mono-therapy using the orthotopic mammary fat pad tumor growth model. The co-delivery of drug and the CSC specific inhibitor towards targeted cancer chemotherapeutics provides an insight into anticancer strategy with facile control and high efficacy.

Background: Intercellular communication by the hedgehog cell signaling pathway is necessary for tooth development throughout the vertebrates, but it remains unclear which specific developmental signals control cell behavior at different stages of odontogenesis. To address this issue, we have manipulated hedgehog activity during zebrafish tooth development and visualized the results using confocal microscopy., Results: We first established that reporter lines for dlx2b, fli1, NF-κB, and prdm1a are markers for specific subsets of tooth germ tissues. We then blocked hedgehog signaling with cyclopamine and observed a reduction or elimination of the cranial neural crest derived dental papilla, which normally contains the cells that later give rise to dentin-producing odontoblasts. Upon further investigation, we observed that the dental papilla begins to form and then regresses in the absence of hedgehog signaling, through a mechanism unrelated to cell proliferation or apoptosis. We also found evidence of an isometric reduction in tooth size that correlates with the time of earliest hedgehog inhibition., Conclusions: We hypothesize that these results reveal a previously uncharacterized function of hedgehog signaling during tooth morphogenesis, regulating the number of cells in the dental papilla and thereby controlling tooth size., (© 2015 Wiley Periodicals, Inc.)

The Hedgehog pathway is critical for animal development and has been implicated in multiple human malignancies. Despite great interest in targeting the pathway pharmacologically, many of the principles underlying the signal transduction cascade remain poorly understood. Hedgehog ligands are recognized by a unique receptor system that features the transporter-like protein Patched and the G protein-coupled receptor (GPCR)-like Smoothened (SMO). The biochemical interaction between these transmembrane proteins is the subject of intensive efforts. Recent structural and functional studies have provided great insight into the small-molecule regulation of SMO through identification of two distinct ligand-binding sites. In this Perspective, we review these recent findings and relate them to potential mechanisms for the endogenous regulation of SMO.

The relapse of cancer after first line therapy with anticancer agents is a common occurrence. This recurrence is believed to be due to the presence of a subpopulation of cells called cancer stem cells in the tumor. Therefore, a combination therapy which is susceptible to both types of cells is desirable. Delivery of this combinatorial approach in a nanoparticulate system will provide even a better therapeutic outcome in tumor targeting. The objective of this study was to develop and characterize nanoparticulate system containing two anticancer agents (cyclopamine and paclitaxel) having different susceptibilities toward cancer cells. Both drugs were entrapped in glyceryl monooleate (GMO)-chitosan solid lipid as well as poly(glycolic-lactic) acid (PLGA) nanoparticles. The cytotoxicity studies were performed on DU145, DU145 TXR, and Wi26 A4 cells. The particle size of drug-loaded GMO-chitosan nanoparticles was 278.4 ± 16.4 nm with a positive zeta potential. However, the PLGA particles were 234.5 ± 6.8 nm in size with a negative zeta potential. Thermal analyses of both nanoparticles revealed that the drugs were present in noncrystalline state in the matrix. A sustained in vitro release was observed for both the drugs in these nanoparticles. PLGA blank particles showed no cytotoxicity in all the cell lines tested, whereas GMO-chitosan blank particles showed substantial cytotoxicity. The types of polymer used for the preparation of nanoparticles played a major role and affected the in vitro release, cytotoxicity, and uptake of nanoparticles in the all the cell lines tested.

The chick early B-cell factor 1 (cEbf1) is a member of EBF family of helix loop helix transcription factors. Recently, we have proved that cEbf1 expression in feather is regulated by Shh. It is therefore possible that the somitic expression of cEbf1 is controlled by Shh signals from the notochord. To assess this hypothesis, the expression profile of cEbf1 was first detailed in somites of chick embryos (from HH8 to HH28). cEbf1 expression was mainly localised in the medial sclerotome and later around the vertebral cartilage anlagen of body and pedicles. Tissue manipulations (notochord ablation) and Shh gain and loss of function experiments were then performed to analyse whether the notochord and/or Shh regulate cEbf1 expression. Results from these experiments confirmed our hypothesis that the medial somitic expression of cEbf1 is regulated by Shh from the notochord. In conclusion, cEbf1 gene is considered as a medial sclerotome marker, downstream to and regulated by the notochord derived Shh, which may be functionally involved in somitogenesis., (Copyright © 2014 Elsevier B.V. All rights reserved.)

This survey on steroidal alkaloids of the Veratrum and Solanum family isolated between 1974 and 2014 includes 187 compounds and 197 references. New developments in the chemistry and biology of this family of natural products with a special focus on the medicinal relevance of the jervanine alkaloid cyclopamine are discussed.

Recent data suggest that inhibition of the Hedgehog pathway could be a therapeutic target for glioblastoma. Alkaloid cyclopamine inhibits Hedgehog signaling, depleting stem-like cancer cells derived from glioblastoma. However, this compound is toxic for somatic stem cells, preventing its use for clinical applications. In this study, we tested a derivatization product of cyclopamine in the form of cyclopamine glucuronide prodrug (CGP-2). This compound was used in vitro and in vivo toward glioblastoma-initiating cells (GIC). Results obtained in vitro indicate that CGP-2 is active only in the presence of β-glucuronidase, an enzyme detected in high levels in necrotic areas of glioblastomas. CGP-2 decreased proliferation and inhibited the self-renewal of all GIC lines tested. Hedgehog pathway blockade by 10 μmol/L of CGP-2 induced a 99% inhibition of clonogenicity on GICs, similar to cyclopamine treatment. Combination of CGP-2 with radiation decreased clonogenic survival in all GIC lines compared with CGP-2 alone. In a subcutaneous glioblastoma xenograft model, a two-week CGP-2 treatment prevented tumor growth with 75% inhibition at 8 weeks, and this inhibition was still significant after 14 weeks. Unlike cyclopamine, CGP-2 had no detectable toxic effects in intestinal crypts. Our study suggests that inhibition of the Hedgehog pathway with CGP-2 is more effective than conventional temozolomide adjuvant, with much lower concentrations, and seems to be an effective therapeutic strategy for targeting GICs., (©2014 American Association for Cancer Research.)

In the late 1960s, the steroidal alkaloid cyclopamine was isolated from the plant Veratrum californicum and identified as the teratogen responsible for craniofacial birth defects including cyclops in the offspring of sheep grazing on mountain ranges in the western United States. Cyclopamine was found to inhibit the hedgehog (Hh) signaling pathway, which plays a critical role in embryonic development. More recently, aberrant Hh signaling has been implicated in several types of cancer. Thus, inhibitors of the Hh signaling pathway, including cyclopamine derivatives, have been targeted as potential treatments for certain cancers and other diseases associated with the Hh signaling pathway. A brief history of cyclopamine and cyclopamine derivatives investigated for the treatment of cancer is presented.

Background: Fibrochondrocytes are involved in entheses repair, but their response to mechanical strain (MS) is ill known., Objective: To determine if parathyroid hormone-related protein (PTHrP) expression in fibrochondrocytes from fibrocartilaginous entheses is modulated by MS, and to further observe the regulatory effects of human (h) PTHrP on fibrochondrocyte differentiation., Methods: Fibrochondrocytes from fibrocartilaginous entheses of Guizhou miniature pig׳s Achilles tendon were submitted or not to MS (4%, 8% or 12% cyclic tensile strain; 1 Hz). Fibrochondrocytes were also exposed to: cyclopamine (Indian hedgehog (Ihh) inhibitor) (10 μM), hPTHrP (10 nM) or cyclopamine/hPTHrP (cyclopamine 10 uM+hPTHrP 10 nM). Types I, II and X collagen and PTHrP expressions were measured by real-time RT-PCR and Western blot., Result: Under 4% strain load for 12 h, types I and II collagen mRNA expressions were increased (+324% and +659%, P<0.001), while type X collagen was decreased (-89%, P<0.001). At 12%, types I and II collagen mRNA expressions were decreased (-62% and -62%, P<0.001), while type X collagen was increased (+375%, P<0.05). Under 4% strain load, PTHrP mRNA expression was increased in relation with strain duration (from 3 to 12 h: +168%, P<0.001), while at 12%, PTHrP expression decreased with time (from 3 to 12 h: -81%, P<0.001). Using cyclopamine for 24 h, PTHrP mRNA expression was significantly decreased (-88%, P<0.05), types I and II collagen were decreased (-90% and -82%, P<0.001), and type X collagen was increased (+261%, P<0.001)., Conclusions: Dynamic MS modulate PTHrP expressions. Thus, PTHrP might play an important role in fibrochondrocyte differentiation, indirectly revealing a role in entheses׳ formation and repair., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Interference of the Hedgehog (Hh) signaling pathway by cyclopamine leads to abnormal embryonic development. We monitor this dynamical drug effect in zebrafish embryos with highly precise microenvironment control using an integrated microfluidic device. This chip-based platform, which is programmable and automated, greatly facilitates the accuracy and reproducibility of the in vivo assays.

Current treatments for prostate cancer are still not satisfactory, often resulting in tumor regrowth and metastasis. One of the main reasons for the ineffective anti-prostate cancer treatments is the failure to deplete cancer stem-like cells (CSCs) - a subset of cancer cells with enhanced tumorigenic capacity. Thus, combination of agents against both CSCs and bulk tumor cells may offer better therapeutic benefits. Several molecules with anti-cancer stem/progenitor cell activities have been under preclinical evaluations. However, their low solubility and nonspecific toxicity limit their clinical translation. Herein, we designed a combination macromolecular therapy containing two drug conjugates: HPMA copolymer-cyclopamine conjugate (P-CYP) preferentially toxic to cancer stem/progenitor cells, and HPMA copolymer-docetaxel conjugate (P-DTX) effective in debulking the tumor mass. Both conjugates were synthesized using RAFT (reversible addition-fragmentation chain transfer) polymerization resulting in narrow molecular weight distribution. The killing effects of the two conjugates against bulk tumor cells and CSCs were evaluated in vitro and in vivo. In PC-3 or RC-92a/hTERT prostate cancer cells, P-CYP preferentially kills and impairs the function of CD133+ prostate cancer stem/progenitor cells; P-DTX was able to kill bulk tumor cells instead of CSCs. In a PC-3 xenograft mice model, combination of P-DTX and P-CYP showed the most effective and persistent tumor growth inhibitory effect. In addition, residual tumors contained less CD133+ cancer cells following combination or P-CYP treatments, indicating selective killing of cancer cells with stem/progenitor cell properties., (© 2013 Elsevier B.V. All rights reserved.)

Organocatalyzed enantioselective allylation of 2-iodocyclohexenone followed by methylation and oxy-Cope rearrangement delivered enantiomerically enriched 2-methyl 3-allyl cyclohexanone, which engaged in acid-catalyzed Robinson annulation to give the bicyclic enone. Subsequent elaboration of the pendant allyl group into an α-diazo β-keto ester set the stage for Rh-mediated cyclization to deliver the tricyclic A-B-C core of the Veratrum alkaloids.

Four steroidal alkaloids extracted from the roots and rhizomes of Veratrum californicum were separated by high performance liquid chromatography (HPLC) and identified using high resolution electrospray ionization time of flight tandem mass spectrometry (ESI-TOF-MS/MS) as veratrosine, cycloposine, veratramine, and cyclopamine. This paper compares ethanol and benzene as extraction solvents, HPLC solvent conditions leading to good separation of steroidal alkaloids, and MS/MS fragmentation patterns for the four steroidal alkaloids which have been released to the public database MassBank.jp. The reported Soxhlet extraction method nearly triples the recovery of steroidal alkaloids from V. californicum.

A practical approach to the synthesis of the A, B and C-ring subunit of cyclopamine has been developed. This synthetic tactic highlights the utility of mandelate acetal-mediated resolution of the fused ring ketone (±)-4 and IBX-mediated oxidation cascades from 12 to 9. The availability of advanced intermediates from enantiomerically pure (+)-4 and 2 could provide efficient access to biologically active and structurally diverse C-nor-D-homo-steroidal alkaloids such as cyclopamine., (Copyright © 2013 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Ovarian cancer is the most lethal gynecological malignancy, characterized by a high rate of chemoresistance. Current treatment strategies for ovarian cancer focus on novel drug combinations of cytotoxic agents and molecular targeted agents or novel drug delivery strategies that often involve intraperitoneal (IP) injection. Poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-b-PCL) micelles were loaded with paclitaxel (cytotoxic agent), cyclopamine (hedgehog inhibitor), and gossypol (Bcl-2 inhibitor). After physicochemical studies focusing on combination drug solubilization, 3-drug PEG-b-PCL micelles were evaluated in vitro in 2-D and 3-D cell culture and in vivo in xenograft models of ovarian cancer, tracking bioluminescence signals from ES-2 and SKOV3 human ovarian cancer cell lines after IP injection. 3-Drug PEG-b-PCL micelles were not significantly more potent in 2-D cell culture in comparison to paclitaxel; however, they disaggregated ES-2 tumor spheroids, whereas single drugs or 2-drug combinations only slowed growth of ES-2 tumor spheroids or had no noticeable effects. In ES-2 and SKOV3 xenograft models, 3-drug PEG-b-PCL micelles had significantly less tumor burden than paclitaxel based on bioluminescence imaging, 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) PET imaging, and overall survival. (18)F-FLT-PET images clearly showed that 3-drug PEG-b-PCL micelles dramatically reduce tumor volumes over paclitaxel and vehicle controls. In summary, PEG-b-PCL micelles enable the IP combination drug delivery of paclitaxel, cyclopamine and gossypol, resulting in tumor growth inhibition and prolonged survival over paclitaxel alone. These results validate a novel treatment strategy for ovarian cancer based on drug combinations of cytotoxic agents and molecular targeted agents, delivered concurrently by a nanoscale drug delivery system, e.g. PEG-b-PCL micelles., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A convenient and efficient one-pot synthesis of cyclopamine from peimisine is described. The key steps involve one-pot hydrazination and subsequent Bamford-Stevens reaction. The mild reaction conditions, high overall yield as well as an easy purification indicate this process can potentially be used for the scale-up preparation of cyclopamine., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Hedgehog (Hh) and epidermal growth factor receptor (EGFR) signaling are involved in pancreatic cancer progression. Targeting these pathways simultaneously with cyclopamine (Hh inhibitor) and gefitinib (EGFR inhibitor) is a promising approach for treating pancreatic cancer. However, the major limitation for effective clinical translation of these molecules is their low aqueous solubility. We have previously demonstrated that methoxy polyethyleneglycol-b-poly(carbonate-co-lactic acid) {mPEG-b-P(CB-co-LA)} copolymer solubilizes hydrophobic anticancer drugs and has the potential to deliver to tumors by an enhanced permeability and retention (EPR) effect. In this study, using the nanoprecipitation method, cyclopamine and gefitinib were efficiently loaded into mPEG-b-P(CB-co-LA) micelles with encapsulation efficiencies of 94.4 and 88.6%, respectively. These micelles had a narrow particle size distribution with a mean particle size of 54.3 nm and a PDI of 0.14. Combination therapy showed a synergistic effect against L3.6pl cells but an additive effect against MIA PaCa-2 cells. Caspase 3/7 activity was also increased when this combination therapy was used, indicating apoptotic cell death. Gene and protein expression analysis indicated cross-talk between Hh and EGFR signaling. Furthermore, the combination decreased tumor growth rate in L3.6pl-derived xenograft mouse tumors. These data suggest the applicability of our micellar system to effectively load and deliver cyclopamine and gefitinib for combination chemotherapy.

Background: The Hedgehog Signaling Pathway is one of signaling pathways that are very important to embryonic development. The participation of inhibitors in the Hedgehog Signal Pathway can control cell growth and death, and searching novel inhibitors to the functioning of the pathway are in a great demand. As the matter of fact, effective inhibitors could provide efficient therapies for a wide range of malignancies, and targeting such pathway in cells represents a promising new paradigm for cell growth and death control. Current research mainly focuses on the syntheses of the inhibitors of cyclopamine derivatives, which bind specifically to the Smo protein, and can be used for cancer therapy. While quantitatively structure-activity relationship (QSAR) studies have been performed for these compounds among different cell lines, none of them have achieved acceptable results in the prediction of activity values of new compounds. In this study, we proposed a novel collaborative QSAR model for inhibitors of the Hedgehog Signaling Pathway by integration the information from multiple cell lines. Such a model is expected to substantially improve the QSAR ability from single cell lines, and provide useful clues in developing clinically effective inhibitors and modifications of parent lead compounds for target on the Hedgehog Signaling Pathway., Results: In this study, we have presented: (1) a collaborative QSAR model, which is used to integrate information among multiple cell lines to boost the QSAR results, rather than only a single cell line QSAR modeling. Our experiments have shown that the performance of our model is significantly better than single cell line QSAR methods; and (2) an efficient feature selection strategy under such collaborative environment, which can derive the commonly important features related to the entire given cell lines, while simultaneously showing their specific contributions to a specific cell-line. Based on feature selection results, we have proposed several possible chemical modifications to improve the inhibitor affinity towards multiple targets in the Hedgehog Signaling Pathway., Conclusions: Our model with the feature selection strategy presented here is efficient, robust, and flexible, and can be easily extended to model large-scale multiple cell line/QSAR data. The data and scripts for collaborative QSAR modeling are available in the Additional file 1.

The Veratrum alkaloid cyclopamine, an inhibitor of cancer stem cell growth, was used as a representative scaffold to evaluate the inhibitory impact of glycosylation with a group of nonmetabolic saccharides, such as d-threose. In a five-step divergent process, a 32-member glycoside library was created and assayed to determine that glycosides of such sugars notably improved the GI50 value of cyclopamine while metabolic sugars, such as d-glucose, did not.

Ethnopharmacological Relevance: Veratrum patulum L. (Liliaceae) is used as one source of the Chinese traditional drug "Lilu" which has been used in the treatment of aphasia arising from apoplexy, wind type dysentery, headache, etc. for thousands of years., Aim of the Study: To evaluate the antithrombotic effect of Veratrum patulum L. (Liliaceae) total steroidal alkaloids (VpA) and explore the potential mechanisms., Materials and Methods: The antithrombotic effect of VpA was evaluated in two experimental thrombosis models in rat. The potential antithrombotic mechanisms of VpA were explored by determining the coagulation parameters and platelet aggregation induced by various agonists. Cutting-tail rat model was used to evaluate the influence of VpA on bleeding time., Results: VpA significantly inhibit the formation of arterial and venous thrombosis and showed different inhibitory effect on the platelet aggregation induced by various agonists with the following potency order: collagen>ADP>thrombin. VpA showed no influence on the coagulation parameters in rat. But VpA decreased the cutting-tail bleeding time in rat., Conclusion: VpA possess antithrombotic effect on venous and arterial thrombosis. The antithrombotic effect of VpA is due to its inhibition to platelet aggregation, especially induced by collagen., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

A Veratrum piperidine chiron was prepared over 11 steps (7.9% yield) from (-)-citronellal. Three methods for the installation of the propargylic side chain onto a cyclic enamide are presented.