Hydroxylated cyclopamine analogues from Veratrum californicum and their hedgehog pathway inhibiting activity.

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.

Steroidal alkaloids from the roots of Veratrum stenophyllum.

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.

Pharmacokinetics of Veratramine and Jervine from Alcohol Extracts of Radix Veratri.

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⟶295.1m/z, Jervine quantitative ion pair was 426.2⟶114.1m/z, and Bullatine B (IS) quantitative ion pair was 438.2⟶420.1m/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 AUC0-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.

Residue 6.43 defines receptor function in class F GPCRs.

The class Frizzled of G protein-coupled receptors (GPCRs), consisting of ten Frizzled (FZD1-10) 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 P6.43. Functional comparison of FZD and FZD P6.43F 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, Asymmetric Synthesis of Jervine.

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.

Stereoselective Synthesis of (-)-Verazine and Congeners via a Cascade Ring-Switching Process of Furostan-26-acid.

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.

A Structure-Activity Relationship between the Veratrum Alkaloids on the Antihypertension and DNA Damage Activity in Mice.

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.

A NanoBRET-Based Binding Assay for Smoothened Allows Real-time Analysis of Ligand Binding and Distinction of Two Binding Sites for BODIPY-cyclopamine.

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.

Cryo-EM structure of oxysterol-bound human Smoothened coupled to a heterotrimeric Gi.

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 pathway1,2. 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 proteins3. 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 Gi protein. The structure reveals a ligand-binding site for 24(S),25-epoxycholesterol in the 7-transmembrane region, as well as a Gi-coupled activation mechanism of human SMO. Notably, the Gi protein presents a different arrangement from that of class-A GPCR-Gi complexes. Our work provides molecular insights into Hedgehog signal transduction and the activation of a class-F GPCR.

Teratogenic jervine increases the activity of doxorubicin in MCF-7/ADR cells by inhibiting ABCB1.

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.

The protective role of jervine against radiation-induced gastrointestinal toxicity.

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.

Development and validation of a rapid and sensitive UPLC-MS/MS assay for simultaneous quantification of paclitaxel and cyclopamine in mouse whole blood and tissue samples.

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.

Quantitative determination of six steroid alkaloids by sensitive hydrophilic interaction liquid chromatography electrospray ionization mass spectrometry and its application to pharmacokinetic study in rats.

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 (R2  > 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.

Sonic hedgehog drives layered double hydroxides-induced acute inflammatory landscape.

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 (Mg2Al-Cl and Zn2Al-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 Zn2Al-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.

Discovery of potent and novel smoothened antagonists via structure-based virtual screening and biological assays.

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 (IC50 < 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 IC50 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.

Structural Basis of Smoothened Activation in Hedgehog Signaling.

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.

Biomimetic nanoparticles delivered hedgehog pathway inhibitor to modify tumour microenvironment and improved chemotherapy for pancreatic carcinoma.

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.

Coadministration of Polymeric Conjugates of Docetaxel and Cyclopamine Synergistically Inhibits Orthotopic Pancreatic Cancer Growth and Metastasis.

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 1H-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.

Antitumor Activity of Isosteroidal Alkaloids from the Plants in the Genus Veratrum and Fritillaria.

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.

Discovery of a Novel Inhibitor of the Hedgehog Signaling Pathway through Cell-based Compound Discovery and Target Prediction.

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.