scholarly journals New mechanisms of radioiodide uptake revealed via a novel high throughput drug screening approach in thyroid cancer

2020 ◽  
Author(s):  
Martin L. Read ◽  
Katie Brookes ◽  
Caitlin E.M. Thornton ◽  
Alice Fletcher ◽  
Mohammed Alshahrani ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Cancer Genes ◽  
Iodide Uptake ◽  
Approved Drugs ◽  
The Impact ◽  
Fda Approved Drugs

ABSTRACTNew combinatorial drug strategies are urgently needed to improve radioiodide (RAI) uptake and efficiently ablate thyroid cancer cells, thereby addressing recurrent and metastatic disease. Cellular iodide uptake is accomplished solely by the sodium iodide symporter (NIS), but the complexity of NIS functional regulation and a lack of amenable high-throughput screening assays has impeded progress. We utilised mutated yellow fluorescent protein (YFP) as a surrogate biosensor of intracellular iodide for ∼1200 FDA-approved drugs, allowing us to appraise the impact of 73 leading compounds at 10 doses on 125I uptake in thyroid cancer cell lines. Subsequent mechanistic analysis suggests three predominant modes of drug action: Firstly, a number of drugs inhibited specific regulation of NIS function by the protein VCP. Secondly, some drugs enhanced transcriptional or post-transcriptional regulation of NIS expression. Thirdly, several drugs strongly implicated proteasomal degradation and the unfolded protein response in the cellular processing of NIS. Exploiting these mechanistic insights, multiple compounds gave striking increases in radioiodide uptake when combined with the drug SAHA. Importantly, our new drug combination strategies were also effective in human primary thyrocytes, suggesting they target endogenous NIS physiology. In patients with papillary thyroid cancer, genes involved in proteostasis were remarkably altered and predicted significantly worse outcome, but only in those patients who received RAI therapy. Collectively, we therefore propose a new model of intracellular NIS processing, and identify key nodes which may now be druggable in patients with aggressive thyroid cancer.SUMMARYOur data identify FDA-approved drugs that enhance radioiodide uptake outside of the canonical pathways of NIS processing, leading to a new mechanistic understanding of endogenous NIS function which is subverted in cancer.

scholarly journals Development and utilization of human decidualization reporter cell line uncovers new modulators of female fertility

2019 ◽  
Vol 116 (39) ◽  
pp. 19541-19551
Author(s):  
Meade Haller ◽  
Yan Yin ◽  
Liang Ma
Keyword(s):  
Cell Line ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Patient Specific ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
A Genome ◽  
Decidual Cells

Failure of embryo implantation accounts for a significant percentage of female infertility. Exquisitely coordinated molecular programs govern the interaction between the competent blastocyst and the receptive uterus. Decidualization, the rapid proliferation and differentiation of endometrial stromal cells into decidual cells, is required for implantation. Decidualization defects can cause poor placentation, intrauterine growth restriction, and early parturition leading to preterm birth. Decidualization has not yet been systematically studied at the genetic level due to the lack of a suitable high-throughput screening tool. Herein we describe the generation of an immortalized human endometrial stromal cell line that uses yellow fluorescent protein under the control of the prolactin promoter as a quantifiable visual readout of the decidualization response (hESC-PRLY cells). Using this cell line, we performed a genome-wide siRNA library screen, as well as a screen of 910 small molecules, to identify more than 4,000 previously unrecognized genetic and chemical modulators of decidualization. Ontology analysis revealed several groups of decidualization modulators, including many previously unappreciated transcription factors, sensory receptors, growth factors, and kinases. Expression studies of hits revealed that the majority of decidualization modulators are acutely sensitive to ovarian hormone exposure. Gradient treatment of exogenous factors was used to identify EC50 values of small-molecule hits, as well as verify several growth factor hits identified by the siRNA screen. The high-throughput decidualization reporter cell line and the findings described herein will aid in the development of patient-specific treatments for decidualization-based recurrent pregnancy loss, subfertility, and infertility.

Abstract 100: High-throughput Screening of FDA-approved Drugs Identifies Novel Inhibitors of Macropinocytosis

2018 ◽  
Vol 38 (Suppl_1) ◽  
Author(s):  
Hui-Ping Lin ◽  
Pushpankur Ghoshal ◽  
Bhupesh Singla ◽  
Jessica L Faulkner ◽  
Mary C Shaw ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Approved Drugs ◽  
Fda Approved Drugs

scholarly journals High-throughput screening of FDA-approved drugs using oxygen biosensor plates reveals secondary mitofunctional effects

Mitochondrion ◽  
2014 ◽  
Vol 17 ◽  
pp. 116-125 ◽  
Author(s):  
Sunil Sahdeo ◽  
Alexey Tomilov ◽  
Kelly Komachi ◽  
Christine Iwahashi ◽  
Sandipan Datta ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Approved Drugs ◽  
Fda Approved Drugs

scholarly journals A High-Throughput Screening Approach To Repurpose FDA-Approved Drugs for Bactericidal Applications againstStaphylococcus aureusSmall-Colony Variants

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Ryan P. Trombetta ◽  
Paul M. Dunman ◽  
Edward M. Schwarz ◽  
Stephen L. Kates ◽  
Hani A. Awad
Keyword(s):  
High Throughput ◽  
Bactericidal Activity ◽  
High Throughput Screening ◽  
Drug Repurposing ◽  
Growth Patterns ◽  
Content Type ◽  
Airway Infections ◽  
Approved Drugs ◽  
Conventional Antibiotics ◽  
Fda Approved Drugs

ABSTRACTDrug repurposing offers an expedited and economical route to develop new clinical therapeutics in comparison to traditional drug development. Growth-based high-throughput screening is concomitant with drug repurposing and enables rapid identification of new therapeutic uses for investigated drugs; however, this traditional method is not compatible with microorganisms with abnormal growth patterns such asStaphylococcus aureussmall-colony variants (SCV). SCV subpopulations are auxotrophic for key compounds in biosynthetic pathways, which result in low growth rate. SCV formation is also associated with reduced antibiotic susceptibility, and the SCV’s ability to revert to the normal cell growth state is thought to contribute to recurrence ofS. aureusinfections. Thus, there is a critical need to identify antimicrobial agents that are potent against SCV in order to effectively treat chronic infections. Accordingly, here we describe adapting an adenylate kinase (AK)-based cell death reporter assay to identify members of a Food and Drug Administration (FDA)-approved drug library that display bactericidal activity againstS. aureusSCV. Four library members, daunorubicin, ketoconazole, rifapentine, and sitafloxacin, exhibited potent SCV bactericidal activity against a stableS. aureusSCV. Further investigation showed that sitafloxacin was potent against methicillin-susceptible and -resistantS. aureus, as well asS. aureuswithin an established biofilm. Taken together, these results demonstrate the ability to use the AK assay to screen small-molecule libraries for SCV bactericidal agents and highlight the therapeutic potential of sitafloxacin to be repurposed to treat chronicS. aureusinfections associated with SCV and/or biofilm growth states.IMPORTANCEConventional antibiotics fail to successfully treat chronic osteomyelitis, endocarditis, and device-related and airway infections. These recurring infections are associated with the emergence of SCV, which are recalcitrant to conventional antibiotics. Studies have investigated antibiotic therapies to treat SCV-related infections but have had little success, emphasizing the need to identify novel antimicrobial drugs. However, drug discovery is a costly and time-consuming process. An alternative strategy is drug repurposing, which could identify FDA-approved and well-characterized drugs that could have off-label utility in treating SCV. In this study, we adapted a high-throughput AK-based assay to identify 4 FDA-approved drugs, daunorubicin, ketoconazole, rifapentine, and sitafloxacin, which display antimicrobial activity againstS. aureusSCV, suggesting an avenue for drug repurposing in order to effectively treat SCV-related infections. Additionally, this screening paradigm can easily be adapted for other drug/chemical libraries to identify compounds bactericidal against SCV.

scholarly journals OR28-03 Drug Repurposing Identifies Inhibitors of the Proteostasis Network to Augment Radioiodine Uptake in Combinatorial Approaches Targeting Thyroid Cancer

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Martin L Read ◽  
Katie Brookes ◽  
Alice Fletcher ◽  
Caitlin E M Thornton ◽  
Mohammed Alshahrani ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Cancer Cells ◽  
High Throughput ◽  
High Throughput Screening ◽  
Drug Repurposing ◽  
Iodide Uptake ◽  
Thyroid Cells ◽  
Radioiodine Uptake ◽  
Repurposed Drugs ◽  
Thyroid Cancer Cells

Abstract New combinatorial drug strategies are urgently needed to improve radioiodine (RAI) uptake and efficiently ablate thyroid cancer cells, thereby reducing the risk of recurrent disease. Drug repurposing offers the promise of identifying already approved compounds capable of inducing sodium iodide symporter (NIS) function to enhance iodide uptake. However, a lack of thyroid cell-based assays amenable to high-throughput screening has limited progress. We utilised the mutated yellow fluorescent protein (YFP) as a surrogate biosensor of intracellular iodide and screened the Prestwick Chemical Library (1200 drugs; 95% approved) for quenching of YFP fluorescence. This allowed us to identify putative candidate drugs which increased iodide uptake >2 SD above mean. Categorisation of these revealed a high proportion of drugs that modulate the proteostasis network (19/48; ~40%), including key processes in protein homeostasis such as endoplasmic reticulum-associated protein degradation (ERAD) and autophagy. Secondary screening validated the activity of proteostasis modulators in enhancing iodide uptake after ranking 73 leading compounds based on their pharmacologic (AUC, EMAX and EC50) and specificity of response (NIS+ve vs NIS-ve YFP-thyroid cells) at ten different drug doses (0.1 to 50 μM). Of importance, several repurposed drugs (e.g. ebastine, Prestwick N, Prestwick C and clotrimazole) in combination with the HDAC inhibitor vorinostat induced a robust enhancement in RAI uptake in thyroid cancer cells (TPC-1 and 8505C NIS+ve cells, up to 11-fold vs DMSO, P<0.001), which was significantly greater than using vorinostat alone (up to 3-fold, P<0.01). For clotrimazole, we designed 7 new chemical derivatives, 3 of which showed enhanced aqueous solubility and retained the ability to significantly enhance RAI uptake. TaqMan RT-PCR revealed that, in contrast to vorinostat, our repurposed drugs failed to alter NIS mRNA expression, highlighting post-transcriptional mechanisms. Critically, 11 repurposed drugs induced significant gains in RAI uptake in human primary thyroid cells (up to 4.1-fold; P<0.05), the most physiological setting for NIS function. In conclusion, we performed high-throughput screening and identified proteostasis modulators, as well as other repurposed drugs, that markedly enhance radioiodine uptake. Further clinical investigation of these drugs might offer new combinatorial approaches, especially with existing therapies, to improve the treatment of thyroid cancer.

scholarly journals Optimizing the Expression of Recombinant αβγ GABAA Receptors in HEK293 Cells for High-Throughput Screening

2008 ◽  
Vol 14 (1) ◽  
pp. 86-91 ◽  
Author(s):  
Daniel Gilbert ◽  
Abolghasem Esmaeili ◽  
Joseph W. Lynch
Keyword(s):  
High Throughput ◽  
Gabaa Receptors ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Effective Means ◽  
Hek293 Cells ◽  
Gaba A ◽  
Protein Assay ◽  
Lead Compounds

Despite being important clinical targets, it is not straightforward to reliably express recombinant trimeric αβγ GABA-A receptors (GABAARs) for high-throughput screening. This study therefore sought to devise a simple and reliable means of transiently expressing α1β1γ1 and α1β1γ2 GABAARs in HEK293 cells. Expression efficiencies resulting from 5 different transfection strategies were assessed by flow cytometry and pharmacological analysis using an anion-sensitive yellow fluorescent protein-based assay. PolyFect™ and Effectene™, employed according to the manufacturers' instructions, conferred the strongest and most reliable expression of trimeric αβγ GABAARs. Functional analysis via the yellow fluorescent protein assay revealed dramatic differences in the pharmacological properties of γ1- and γ2-containing receptors, consistent with previous electrophysiological characterizations. The authors conclude that this method of expressing and screening recombinant GABAARs provides an effective means of discovering novel GABAAR modulators for use as therapeutic lead compounds and pharmacological probes. ( Journal of Biomolecular Screening 2009:86-91)

A yellow fluorescent protein-based assay for high-throughput screening of glycine and GABAA receptor chloride channels

2005 ◽  
Vol 380 (3) ◽  
pp. 340-345 ◽  
Author(s):  
Wade Kruger ◽  
Daniel Gilbert ◽  
Rebecca Hawthorne ◽  
Deanne H. Hryciw ◽  
Stephan Frings ◽  
...  
Keyword(s):  
Gabaa Receptor ◽  
High Throughput ◽  
High Throughput Screening ◽  
Chloride Channels ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein

scholarly journals A High-throughput Screening Method to Identify Proteins Involved in Unfolded Protein Response Signaling in Plants

2019 ◽  
Author(s):  
André Alcântara ◽  
Denise Seitner ◽  
Fernando Navarrete ◽  
Armin Djamei
Keyword(s):  
Unfolded Protein Response ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Screening Method ◽  
Screening Assay ◽  
Unfolded Protein ◽  
High Throughput Screening Assay ◽  
Protein Response

AbstractBackgroundThe unfolded protein response (UPR) is a highly conserved process in eukaryotic organisms that plays a crucial role in adaptation and development. While the most ubiquitous components of this pathway have been characterized, current efforts are focused on identifying and characterizing other UPR factors that play a role in specific conditions, such as developmental changes, abiotic cues, and biotic interactions. Considering the central role of protein secretion in plant pathogen interactions, there has also been a recent focus on understanding how pathogens manipulate their host’s UPR to facilitate infection.ResultsWe developed a high-throughput screening assay to identify proteins that interfere with UPR signalingin planta. A set of 35 genes from a library of secreted proteins from the maize pathogenUstilago maydiswere transiently co-expressed with a reporter construct that upregulates enhanced yellow fluorescent protein (eYFP) expression upon UPR stress inNicotiana benthamianaplants. After UPR stress induction, leaf discs were placed in 96 well plates and eYFP expression was measured. This allowed us to identify a previously undescribed fungal protein that inhibits plant UPR signaling, which was then confirmed using the classical but more laborious qRT-PCR method.ConclusionsWe have established a rapid and reliable fluorescence-based method to identify heterologously expressed proteins involved in UPR stress in plants. This system can be used for initial screens with libraries of proteins and potentially other molecules to identify candidates for further validation and characterization.

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