An optimized InCell Western screening technique identifies hexachlorophene as a novel potent TDP43 targeting drug

Narayan, Malathi; Peralta, Diego A.; Gibson, Chelsea; Zitnyar, Ashley; Jinwal, Umesh K.

doi:10.1016/j.jbiotec.2015.04.012

Cited by 6 publications

(6 citation statements)

References 18 publications

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“…Hexachlorophene (2,2 -methylenebis(3,4,6trichlorophenol)-3,4,6-trichloro-2-[(2,3,5-trichloro-6hydroxyphenyl)methyl]phenol) is an organochlorine compound that was once widely used as a disinfectant (a common use being the skin cleansing of newborns), until it was withdrawn due to safety issues [40]. Hexachlorophene is known to have several pharmacological activities, including the inhibition of TAR DNA binding protein [41], inhibition of coronavirus entry [42] and inhibition of amyloid assembly [43]. Hexachlorophene is also known as a glutaminase inhibitor [44] and as an inhibitor of the beta-catenin pathway [45].…”

Section: Discussionmentioning

confidence: 99%

Screening of a composite library of clinically used drugs and well-characterized pharmacological compounds for cystathionine β-synthase inhibition identifies benserazide as a drug potentially suitable for repurposing for the experimental therapy of colon cancer

Druzhyna

Szczesny

Oláh

et al. 2016

Pharmacological Research

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Cystathionine-β-synthase (CBS) has been recently identified as a drug target for several forms of cancer. Currently no potent and selective CBS inhibitors are available. Using a composite collection of 8871 clinically used drugs and well-annotated pharmacological compounds (including the LOPAC library, the FDA Approved Drug Library, the NIH Clinical Collection, the New Prestwick Chemical Library, the US Drug Collection, the International Drug Collection, the `Killer Plates' collection and a small custom collection of PLP-dependent enzyme inhibitors), we conducted an in vitro screen in order to identify inhibitors for CBS using a primary 7-azido-4-methylcoumarin (AzMc) screen to detect CBS-derived hydrogen sulfide (H2S) production. Initial hits were subjected to counterscreens using the methylene blue assay (a secondary assay to measure H2S production) and were assessed for their ability to quench the H2S signal produced by the H2S donor compound GYY4137. Four compounds, hexachlorophene, tannic acid, aurintricarboxylic acid and benserazide showed concentration-dependent CBS inhibitory actions without scavenging H2S released from GYY4137, identifying them as direct CBS inhibitors. Hexachlorophene (IC50: ~60 μM), tannic acid (IC50: ~40 μM) and benserazide (IC50: ~30 μM) were less potent CBS inhibitors than the two reference compounds AOAA (IC50: ~3 μM) and NSC67078 (IC50: ~1 μM), while aurintricarboxylic acid (IC50: ~3 μM) was equipotent with AOAA. The second reference compound NSC67078 not only inhibited the CBS-induced AzMC fluorescence signal (IC50: ~1 μM), but also inhibited with the GYY4137-induced AzMC fluorescence signal with (IC50 of ~6 μM) indicative of scavenging/non-specific effects. Hexachlorophene (IC50: ~6 μM), tannic acid (IC50: ~20 μM), benserazide (IC50: ~20 μM), and NSC67078 (IC50: ~0.3 μM) inhibited HCT116 colon cancer cells proliferation with greater potency than AOAA (IC50: ~300 μM). In contrast, although a CBS inhibitor in the cell-free assay, aurintricarboxylic acid failed to inhibit HCT116 proliferation at lower concentrations, and stimulated cell proliferation at 300 μM. Copper-containing compounds present in the libraries, were also found to be potent inhibitors of recombinant CBS; however this activity was due to the CBS inhibitory effect of copper ions themselves. However, copper ions, up to 300 μM, did not inhibit HCT116 cell proliferation. Benserazide was only a weak inhibitor of the activity of the other H2S-generating enzymes CSE and 3-MST activity (16% and 35% inhibition at 100 μM, respectively) in vitro. Benserazide suppressed HCT116 mitochondrial function and inhibited proliferation of the high CBS-expressing colon cancer cell line HT29, but not the low CBS-expressing line, LoVo. The major benserazide metabolite 2,3,4-trihydroxybenzylhydrazine also inhibited CBS activity and suppressed HCT116 cell proliferation in vitro. In an in vivo study of nude mice bearing human colon cancer cell xenografts, benserazide (50 mg/kg/day s.q.) prevented tumor growth. In silico docking simulations...

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Section: Discussionmentioning

confidence: 99%

Screening of a composite library of clinically used drugs and well-characterized pharmacological compounds for cystathionine β-synthase inhibition identifies benserazide as a drug potentially suitable for repurposing for the experimental therapy of colon cancer

Druzhyna

Szczesny

Oláh

et al. 2016

Pharmacological Research

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“…It also reduced the number of cells with cytosolic TDP-43 inclusions and its cleavage in a FTLD-U mouse model where this protein is expressed in the forebrain (Wang, Gou, et al, 2012). In addition, rapamycin-treated VCP R155H/+ mice demonstrated significant improvement in muscle performance, quadriceps histological analysis, and rescue of ubiquitin, and TDP-43 pathology and defective autophagy (Nalbandian et al, 2015). This effect of rapamycin is also evolutionarily conserved as this compound was also capable of reducing Drosophila TDP-43 toxicity in an overexpressing fly model (Cheng, Lin, & Shen, 2015).…”

Section: Autophagy and Proteasome Systemsmentioning

confidence: 84%

“…This is a lysosomal inhibitor that reverses autophagy by accumulating in lysosomes. It has been reported that chloroquine-treated VCP R155H/+ mice revealed progressive muscle weakness, cytoplasmic accumulation of TDP-43, ubiquitin-positive inclusion bodies and increased LC3-I/II, p62/SQSTM1, and optineurin expression levels (Nalbandian, Llewellyn, Nguyen, Yazdi, & Kimonis, 2015).…”

Section: Autophagy and Proteasome Systemsmentioning

confidence: 99%

Targeting TDP‐43 proteinopathy with drugs and drug‐like small molecules

Buratti

2020

British J Pharmacology

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Following the discovery of the involvement of the ribonucleoprotein TDP‐43 in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), a major research focus has been to develop treatments that can prevent or alleviate these disease conditions. One pharmacological approach has been to use TDP‐43‐based disease models to test small molecules and drugs already known to have some therapeutic effect in a variety of neurodegenerative conditions. In parallel, various disease models have been used to perform high‐throughput screens of drugs and small compound libraries. The aim of this review will be to provide a general overview of the compounds that have been described to alter pathological characteristics of TDP‐43. These include expression levels, cytoplasmic mis‐localization, post‐translational modifications, cleavage, stress granule recruitment and aggregation. In parallel, this review will also address the use of compounds that modify the autophagic/proteasome systems that are known to target TDP‐43 misfolding and aggregation. LINKED ARTICLES This article is part of a themed issue on Neurochemistry in Japan. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.6/issuetoc

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“…We first characterised 107 clones of cells by immunostaining wells with a STAT3 -specific antibody, as well as a total cell stain used to normalise for differences in cell confluence in a high-throughput Li-Cor In-Cell Western (Fig. 2B) (39). Based on ratios of fluorescence intensity in channels corresponding to STAT3 abundance and total cell staining, we identified gene-edited clones with reduced STAT3 immunostaining relative to non-edited controls (Fig.…”

Section: Resultsmentioning

confidence: 99%

GenEditID: an open-access platform for the high-throughput identification of CRISPR edited cell clones

Xue

Tung

Siersbæk

et al. 2019

Preprint

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25CRISPR-Cas9-based gene editing is a powerful tool to reveal genotype-phenotype 26 relationships, but identifying cell clones carrying desired edits remains challenging. To 27 address this issue we developed GenEditID, a flexible, open-access platform for sample 28 tracking, analysis and integration of multiplexed deep sequencing and proteomic data, and 29 intuitive plate-based data visualisation to facilitate gene edited clone identification. To 30 demonstrate the scalability and sensitivity of this method, we identified KO clones in parallel 31 from multiplexed targeting experiments, and optimised conditions for single base editing 32 using homology directed repair. GenEditID enables non-specialist groups to expand their 33 gene targeting efforts, facilitating the study of genetically complex human disease. 34 35 KEYWORDS 36 CRISPR-Cas9; gene editing; GWAS; Illumina sequencing; multiplexed; pluripotent stem cell, 37 LIMS, In-Cell Western 38 39 BACKGROUND 40In the last decade, there has been an explosion of data from the sequencing of human 41 populations, including genome-wide association studies (GWAS) based on DNA microarrays 42 and increasingly also whole exomes and whole genomes (1). These studies have revealed 43 thousands of replicable genetic associations for complex diseases such as diabetes, obesity, 44Alzheimer's Disease and breast cancer (2-4). However, mechanistically determining how 45 these genetic associations contribute to disease remains challenging. Causal evidence 46 requires careful functional follow-up experiments in model cellular systems, organisms and 47 eventually in humans, but the traditional approach of characterising one gene at a time 48 cannot keep pace with the rate of genetic discovery. Furthermore, many associated variants 49 are non-coding, so the genetic elements responsible for conferring disease risk are often 50 unclear (5, 6). This issue is exemplified by the fat mass and obesity associated (FTO) locus, 51 3 in which intronic SNPs are strongly associated with obesity, largely due to increased food 52 intake (7-10) irrespective of gender, age or ethnicity (11, 12). Despite intense study, the 53 identify of the genetic elements that mediate SNP-associated phenotypes remains 54 controversial. Some studies suggest that effect on appetite might not be driven by the FTO 55 gene itself as initially thought, but instead by the nearby genes retinitis pigmentosa GTPase 56 regulator-interacting protein-1 like (RPGRIP1L) (13-15), or by iroquois homeobox 3 (IRX3) 57 and iroquois homeobox 5 (IRX5) (16, 17). Two powerful tools have recently emerged to help 58 meet the challenge of uncovering disease mechanisms from the translating the growing 59 wealth of genetic data: human pluripotent stem cells (hPSCs) and the CRISPR-Cas9 system 60 (18, 19). 62hPSCs facilitate human disease modelling since they can be indefinitely maintained in a 63 pluripotent state and can theoretically be differentiated into any cell type in the body, 64including disease-relevant cell populations (20, 21). For ex...

show abstract

An optimized InCell Western screening technique identifies hexachlorophene as a novel potent TDP43 targeting drug

Cited by 6 publications

References 18 publications

Screening of a composite library of clinically used drugs and well-characterized pharmacological compounds for cystathionine β-synthase inhibition identifies benserazide as a drug potentially suitable for repurposing for the experimental therapy of colon cancer

Screening of a composite library of clinically used drugs and well-characterized pharmacological compounds for cystathionine β-synthase inhibition identifies benserazide as a drug potentially suitable for repurposing for the experimental therapy of colon cancer

Targeting TDP‐43 proteinopathy with drugs and drug‐like small molecules

GenEditID: an open-access platform for the high-throughput identification of CRISPR edited cell clones

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