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Circulation. Genomic and Precision... Feb 2020Variants in ion channel genes have classically been studied in low throughput by patch clamping. Deep mutational scanning is a complementary approach that can...
BACKGROUND
Variants in ion channel genes have classically been studied in low throughput by patch clamping. Deep mutational scanning is a complementary approach that can simultaneously assess function of thousands of variants.
METHODS
We have developed and validated a method to perform a deep mutational scan of variants in , which encodes the major voltage-gated sodium channel in the heart. We created a library of nearly all possible variants in a 36 base region of in the S4 voltage sensor of domain IV and stably integrated the library into HEK293T cells.
RESULTS
In preliminary experiments, challenge with 3 drugs (veratridine, brevetoxin, and ouabain) could discriminate wild-type channels from gain- and loss-of-function pathogenic variants. High-throughput sequencing of the pre- and postdrug challenge pools was used to count the prevalence of each variant and identify variants with abnormal function. The deep mutational scan scores identified 40 putative gain-of-function and 33 putative loss-of-function variants. For 8 of 9 variants, patch clamping data were consistent with the scores.
CONCLUSIONS
These experiments demonstrate the accuracy of a high-throughput in vitro scan of variant function, which can be used to identify deleterious variants in and other ion channel genes.
Topics: Cardiotonic Agents; DNA Mutational Analysis; HEK293 Cells; Humans; Marine Toxins; Mutation; NAV1.5 Voltage-Gated Sodium Channel; Ouabain; Oxocins; Pharmacogenomic Testing; Veratridine
PubMed: 31928070
DOI: 10.1161/CIRCGEN.119.002786 -
Fitoterapia Sep 2019Veratrum californicum is a rich source of steroidal alkaloids, many of which have proven to be antagonists of the Hedgehog (Hh) signaling pathway that becomes aberrant...
Veratrum californicum is a rich source of steroidal alkaloids, many of which have proven to be antagonists of the Hedgehog (Hh) signaling pathway that becomes aberrant in over twenty types of cancer. These alkaloids first became known in the 1950's due to their teratogenic properties, which resulted in newborn and fetal lambs developing cyclopia as a result of pregnant ewes consuming Veratrum californicum. It was discovered that the alkaloids in V. californicum were concentrated in the root and rhizome of the plant with much lower amounts of the most active alkaloid, cyclopamine, present in the aerial plant, especially in the late growth season. Inspired by the limitations in analytical instrumentation and methods available to researchers at the time of the original investigation, we have used state-of-the-art instrumentation and modern analytical methods to quantitate four steroidal alkaloids based on study parameters including plant part, harvest location, and growth stage. The results of the current inquiry detail differences in alkaloid composition based on the study parameters, provide a detailed assessment for alkaloids that have been characterized previously (cyclopamine, veratramine, muldamine and isorubijervine), and identify at least six alkaloids that have not been previously characterized. This study provides insight into optimal harvest time, plant growth stage, harvest location, and plant part required to isolate, yet to be characterized, alkaloids of interest for exploration as Hh pathway antagonists with desirable medicinal properties.
Topics: Alkaloids; Hedgehog Proteins; Idaho; Molecular Structure; Phytochemicals; Plant Components, Aerial; Rhizome; Seasons; Steroids; Veratrum; Veratrum Alkaloids
PubMed: 31381957
DOI: 10.1016/j.fitote.2019.104281 -
The Turkish Journal of Gastroenterology... Mar 2020To investigate the effect and the possible mechanism of lanthanum citrate on the proliferation and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721...
BACKGROUND/AIMS
To investigate the effect and the possible mechanism of lanthanum citrate on the proliferation and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721 through the Hedgehog (Hh) signaling pathway.
MATERIALS AND METHODS
Different concentrations of lanthanum citrate and KAAD-cyclopamine (the Hh signaling pathway representative inhibitor) were used to treat SMMC-7721 cells. Cell proliferation was detected using Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assays. Cell apoptosis was detected using flow cytometry analysis of Annexin V-FITC/ propidium iodide (PI). The protein expressions of regulatory genes, such as cell cycle protein D1 (CyclinD1), cyclin-dependent kinase inhibitor 1 (p21), cysteinyl aspartate specific proteinase 3 (Caspase-3), B-cell lymphoma-2 (Bcl-2), glioma-associated oncogene homolog 1 (Gli1), and sonic hedgehog (Shh) were quantified using Western blot assays. The mRNA expressions of Gli1 and Shh were tested using quantitative real-time polymerase chain reaction (qRT-PCR) assays and the protein expressions of Gli1 and Shh were determined using immunofluorescence assays.
RESULTS
The Annexin V-FITC and PI double staining results revealed that the 0.1 mM lanthanum citrate group and the 15 µM KAAD-cyclopamine group had both increased the apoptosis rate of SMMC-7721 cells. Both lanthanum citrate and KAAD-cyclopamine downregulated the protein expressions of CyclinD1, Bcl-2, Gli1, and Shh and upregulated the protein expressions of p21 and Caspase-3. Additionally, the immunofluorescence results revealed that the protein expressions of Gli1 and Shh were significantly decreased in both the lanthanum citrate group and the KAAD-cyclopamine group compared to the control group.
CONCLUSION
Lanthanum citrate inhibits proliferation and promotes apoptosis in HCC SMMC-7721 cells by suppressing the Hh signaling pathway.
Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cinnamates; Citrates; Hedgehog Proteins; Humans; Liver Neoplasms; Signal Transduction; Veratrum Alkaloids
PubMed: 32343239
DOI: 10.5152/tjg.2020.18800 -
Bioorganic & Medicinal Chemistry Jun 2021Constitutive activation of Hedgehog (Hh) pathway is intimately related with the occurrence and development of several malignancies, such as medulloblastoma (MB) and...
Constitutive activation of Hedgehog (Hh) pathway is intimately related with the occurrence and development of several malignancies, such as medulloblastoma (MB) and other tumors. Therefore, small molecular inhibitors of Hh pathway are urgently needed. In this study, three new steroidal alkaloids, ⊿ (20R, 24R) 23-oxo-24-methylsolacongetidine, ⊿ (20S, 24R) 23-oxo-24-methylsolacongetidine and veralinine 3-O-α-l-rhamnopyranosyl-(1 → 2)-β-D-glucopyranoside, together with six known alkaloids, 20-epi-verazine, verazine, protoverine 15-(l)-2'-methylbutyrate, jervine, veramarine and β1-chaconine, were isolated and determined from Veratrum grandiflorum Loes. The dual-luciferase bioassay indicated that all compounds exhibited significant inhibitions of Hh pathway with IC values of 0.72-14.31 μM against Shh-LIGHT 2 cells. To determine whether these Hh pathway inhibitors act with the Smoothened (Smo) protein, which is an important oncoprotein and target for this pathway, BODIPY-cyclopamine (BC) competitive binding assay was preferentially performed. Compared with BC alone, all compounds obviously reduced the fluorescence intensities of BC binding with Smo in Smo-overexpression HEK293T cells through fluorescence microscope and flow cytometer. By directly interacting with Smo, it revealed that they were actually novel natural Smo inhibitors. Then, their anti-tumor effects were investigated against the human MB cell line DAOY, which is a typical pediatric brain tumor cells line with highly expressed Hh pathway. Interestingly, most of compounds had slight proliferation inhibitions on DAOY cells after treatment for 24 h same as vismodegib, while β1-chaconine showed the strongest inhibitory effect on the growth of DAOY with IC value of 5.35 μM. In conclusion, our studies valuably provide several novel natural Smo inhibitors for potential targeting treatment of Hh-dependent tumors.
Topics: Alkaloids; Cell Line, Tumor; Cell Proliferation; HEK293 Cells; Humans; Medulloblastoma; Molecular Structure; Smoothened Receptor; Spectrum Analysis; Steroids; Veratrum
PubMed: 33910157
DOI: 10.1016/j.bmc.2021.116166 -
Journal of Pharmaceutical Analysis Oct 2019Jervine, a novel steroidal alkaloid from L., exhibits both antitumor effect and potential toxicity. The aim of study was to characterize the pharmacokinetic behaviors...
Jervine, a novel steroidal alkaloid from L., exhibits both antitumor effect and potential toxicity. The aim of study was to characterize the pharmacokinetic behaviors and enterohepatic circulation of jervine in rats. A rapid and simple ultra-high performance liquid chromatography-tandem mass spectrometric method was developed and validated for quantification of jervine and alpinetin (internal standard) in rat plasma. After extraction from rat plasma by a simple protein-precipitation method, the analyte was separated on a C column (2.1 mm × 50 mm, 1.7 μm) using water with 0.1% formic acid and acetonitrile as the mobile phase delivered at a flow rate of 0.4 mL/min. Jervine and alpinetin were determined in the positive mode with multiple reaction monitoring (MRM) of the ion transitions at 426.3 → 108.8 and 271.0 → 166.9, respectively. Molecular docking method was used to investigate the binding of jervine to p-glycoprotein and dehydroepiandrosterone sulfotransferase. The method was well validated within acceptance limits including specificity, matrix effect, recovery, precision, accuracy, and stability, and was successfully applied to the pharmacokinetic study of jervine after oral and intravenous administration to rats. Jervine presented a small volume of distribution, fast absorption, high oral bioavailability, and enterohepatic circulation. The enterohepatic circulation was first observed in veratrum alkaloids, and was further investigated by molecular docking studies, which was related to the binding of jervine to p-glycoprotein and dehydroepiandrosterone sulfotransferase. The pharmacokinetic properties and enterohepatic circulation of jervine in rats provided a significant basis for the drug-drug interaction and toxicity study in the future.
PubMed: 31929946
DOI: 10.1016/j.jpha.2019.04.004 -
Phytochemistry Mar 2021Fritillaria cirrhosa D. Don (Liliaceae, syn. Fritillaria roylei Hook.) is a critically endangered medicinal herb of immense importance due to its pharmaceutical...
Comparative transcriptome analysis infers bulb derived in vitro cultures as a promising source for sipeimine biosynthesis in Fritillaria cirrhosa D. Don (Liliaceae, syn. Fritillaria roylei Hook.) - High value Himalayan medicinal herb.
Fritillaria cirrhosa D. Don (Liliaceae, syn. Fritillaria roylei Hook.) is a critically endangered medicinal herb of immense importance due to its pharmaceutical bioactive compound, especially sipeimine, used for the treatment of chronic respiratory disorders. However, the industrial demand for sipeimine solely depends on its endangered natural habitat. Therefore; there is an utmost need for its biodiversity conservation as well as for the sustainable utilization of phytochemicals. Plant cell culture and transcriptomics-based molecular bioprospection of key regulatory genes involved in sipeimine biosynthesis as such will play a crucial role in exploring the unexplored traits, that are in supply crisis or nearly in extinction stage. De novo comparative transcriptome sequencing of the bulb (in vivo), callus, and regenerated plantlets (in vitro) resulted in more than 150 million high-quality paired-end clean reads that assembled into final 31,428 transcripts. Functional annotation and unigenes classification with multiple public databases such as KEGG, Refseq, Uniprot, TAIR, GO, and COG, etc. along with chemical structures and functional biocatalytic activity analysis of different steroidal alkaloids facilitated the identification of 30 unigenes specific to sipeimine biosynthesis. Additionally, ABC transporters and TFs like bHLH, MYC, MYB, and WRKY suggests their possible role in metabolite translocation and regulation in vivo as well as in vitro tissues. Differential gene expression and quantitative analysis revealed that the MVA pathway probably the predominant route for 5C intermediate (IPP & DMAPP) biosynthesis. Further, the genes involved in the downstream biosynthesis pathway viz. SQLE, CAS1, SMT1, SMO1, SMO2, SC5DL, DHCR7, DHCR24, CYP710A, 3β-HSD, CYP90D2, and CYP374A6 shown similar expression pattern with RNA-Seq and qRT-PCR findings. The positive correlation between higher expression of proposed biosynthetic pathway genes and relatively higher accumulation of sipeimine in differentiated naturally grown bulb tissues (in vivo), undifferentiated cells (callus), and de-differentiated tissues i.e. regenerated plantlets (in vitro) has been evident from the present study. Comprehensive genomic resources created in F. cirrhosa will provide strong evidence of bulb derived in vitro culture as an alternative promising source for steroidal alkaloids biosynthesis and metabolite upscaling through genetic and metabolic engineering.
Topics: Cevanes; Fritillaria; Gene Expression Profiling; Gene Expression Regulation, Plant; Liliaceae; Plants, Medicinal; Transcriptome
PubMed: 33370713
DOI: 10.1016/j.phytochem.2020.112631 -
Pharmaceutical Biology Dec 2021Peimine and paeoniflorin can be combined for the treatment of cough in paediatrics. The interaction during the co-administration could dramatically affect the...
CONTEXT
Peimine and paeoniflorin can be combined for the treatment of cough in paediatrics. The interaction during the co-administration could dramatically affect the bioavailability of drugs.
OBJECTIVE
The interaction between peimine and paeoniflorin was investigated in this study.
MATERIALS AND METHODS
The pharmacokinetics of paeoniflorin (20 mg/kg) with or without the coadministration of peimine (5 mg/kg for 10 days before paeoniflorin) was orally investigated in Sprague-Dawley rats ( = 6). The group without the peimine was set as the control group. The metabolic stability of paeoniflorin was studied in rat liver with microsomes. The effect of peimine on the absorption of paeoniflorin was investigated with Caco-2 cell monolayers.
RESULTS
The (244.98 ± 10.95 vs. 139.18 ± 15.14 μg/L) and AUC (3295.92 ± 263.02 vs. 139.18 ± 15.14 h·μg/L) of paeoniflorin was increased by peimine. The was prolonged from 5.33 ± 1.65 to 14.21 ± 4.97 h and the clearance was decreased from 15.43 ± 1.75 to 4.12 ± 0.57 L/h/kg. Consistently, peimine increased the metabolic stability of paeoniflorin with rat liver microsomes with the increased (56.78 ± 2.62 vs. 26.33 ± 3.15 min) and the decreased intrinsic clearance (24.42 ± 3.78 vs. 52.64 ± 4.47 μL/min/mg protein). Moreover, the transportation of paeoniflorin was also inhibited by peimine as the efflux ratio decreased from 3.06 to 1.63.
DISCUSSION AND CONCLUSIONS
Peimine increased the systemic exposure of paeoniflorin through inhibiting the activity of CYP3A4 and P-gp. These results provide a reference for further studies in a broader population.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; Area Under Curve; Caco-2 Cells; Cevanes; Cytochrome P-450 CYP3A; Drug Interactions; Glucosides; Half-Life; Humans; Male; Microsomes, Liver; Monoterpenes; Rats; Rats, Sprague-Dawley
PubMed: 33721550
DOI: 10.1080/13880209.2021.1875013 -
International Journal of Molecular... May 2022Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive subtype of kidney cancer, with high mortality rates worldwide. The sonic hedgehog (SHH)...
Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive subtype of kidney cancer, with high mortality rates worldwide. The sonic hedgehog (SHH) molecular cascade is altered in various malignancies in tumorigenesis, and several SHH pathway inhibitors have been considered as potential anticancer drugs. The aim of the present study was to determine the expression profile of SHH signaling components and their target genes in ccRCC. Additionally, the present study examined the effects of SHH pathway inhibitory drugs (RU‑SKI43, cyclopamine and GLI‑antagonist 61) on cell viability, cell cycle progression, expression levels of SHH target genes and migration ability in 786‑O, ACHN and HK2 cells. The study also included paired tumor and normal samples from 62 patients with ccRCC. The mRNA levels in clinical samples and cell lines were measured via reverse transcription‑quantitative PCR. Cell viability was examined using a sulforhodamine B assay. Flow cytometry was used to investigate cell cycle progression and the migratory rate of cells was assessed using a wound healing assay. High mRNA levels of , smoothened (), glioma‑associated zinc finger protein (), BCL2 apoptosis regulator (), MYC proto‑oncogene (), vascular endothelial growth factor A () and cyclin D1 () were observed in the tumor tissues, especially in early ccRCC, according to the TNM stage or World Health Organization/International Society of Urological Pathology (ISUP) grade. High expression levels of , as well as low mRNA expression, were associated with short overall survival, and increased expression was an independent prognostic factor of a poor outcome in patients with advanced ISUP grade (Cox hazard ratio test). Cyclopamine treatment was found to arrest 786‑O cells in the G/M phase and decreased the expression levels of , , and . RU‑SKI43 inhibited cell migration and decreased the expression levels of , and in ACHN cells. Overall, the results of the present study suggested that SHH signaling may be involved in the early development of ccRCC, and the expression levels of and may serve as prognostic factors of this disease. Cyclopamine and RU‑SKI43 appear to be potential anti‑renal cell carcinoma drugs; however, this hypothesis requires verification by further studies.
Topics: Carcinoma, Renal Cell; Hedgehog Proteins; Humans; Kidney Neoplasms; Vascular Endothelial Growth Factor A; Veratrum Alkaloids; Zinc Finger Protein GLI1
PubMed: 35266008
DOI: 10.3892/ijmm.2022.5114 -
Plants (Basel, Switzerland) Feb 2020L. (Liliaceae), a monocotyledonous species distributed throughout the Changbai mountains of Northeast China, is pharmaceutically important, due to the capacity to...
L. (Liliaceae), a monocotyledonous species distributed throughout the Changbai mountains of Northeast China, is pharmaceutically important, due to the capacity to produce the anticancer drug cyclopamine. An efficient transformation system of mediated with is presented. Murashige and Skoog (MS) medium containing 8 mg/L picloram was used to induce embryogenic calli from immature embryos with 56% efficiency. LBA4404 carrying the bar gene driven by the cauliflower mosaic virus 35S promoter was employed for embryogenic callus inoculation. cell density OD660 = 0.8 for inoculation, half an hour infection period, and three days of co-culture duration were found to be optimal for callus transformation. Phosphinothricin (PPT, 16 mg/L) was used as the selectable agent, and a transformation efficiency of 15% (transgenic plants/100 infected calli) was obtained. The transgenic nature of the regenerated plants was confirmed by PCR and Southern blot analysis, and expression of the bar gene was detected by RT-PCR and Quick PAT/bar strips. The steroid alkaloids cyclopamine, jervine, and veratramine were detected in transgenic plants, in non-transformed and control plants collected from natural sites. The transformation system constitutes a prerequisite for the production of the pharmaceutically important anticancer drug cyclopamine by metabolic engineering of .
PubMed: 32033134
DOI: 10.3390/plants9020191 -
Scientific Reports Jul 2020Voltage-gated Na (Na) channels regulate homeostasis in bacteria and control membrane electrical excitability in mammals. Compared to their mammalian counterparts,...
Voltage-gated Na (Na) channels regulate homeostasis in bacteria and control membrane electrical excitability in mammals. Compared to their mammalian counterparts, bacterial Na channels possess a simpler, fourfold symmetric structure and have facilitated studies of the structural basis of channel gating. However, the pharmacology of bacterial Na remains largely unexplored. Here we systematically screened 39 Na modulators on a bacterial channel (NaChBac) and characterized a selection of compounds on NaChBac and a mammalian channel (human Na1.7). We found that while many compounds interact with both channels, they exhibit distinct functional effects. For example, the local anesthetics ambroxol and lidocaine block both Na1.7 and NaChBac but affect activation and inactivation of the two channels to different extents. The voltage-sensing domain targeting toxin BDS-I increases Na1.7 but decreases NaChBac peak currents. The pore binding toxins aconitine and veratridine block peak currents of Na1.7 and shift activation (aconitine) and inactivation (veratridine) respectively. In NaChBac, they block the peak current by binding to the pore residue F224. Nonetheless, aconitine has no effect on activation or inactivation, while veratridine only modulates activation of NaChBac. The conservation and divergence in the pharmacology of bacterial and mammalian Na channels provide insights into the molecular basis of channel gating and will facilitate organism-specific drug discovery.
Topics: Aconitine; Anesthetics, Local; Bacterial Proteins; Drug Interactions; Electrophysiological Phenomena; HEK293 Cells; Humans; Ion Channel Gating; NAV1.7 Voltage-Gated Sodium Channel; Small Molecule Libraries; Sodium Channels; Toxins, Biological; Veratridine; Voltage-Gated Sodium Channel Agonists
PubMed: 32612253
DOI: 10.1038/s41598-020-67761-5