Abstract 11415: Enhancing Direct Cardiac Reprogramming Efficiency Using Small Molecules

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Tamer M Mohamed ◽  
Nicole Stone ◽  
Emily Berry ◽  
Haixia Wang ◽  
Sheng Ding ◽  
...  
Keyword(s):  
Ejection Fraction ◽  
Small Molecules ◽  
High Throughput Screening ◽  
Cardiac Fibroblasts ◽  
Screening Method ◽  
Reprogramming Efficiency ◽  
Canonical Wnt ◽  
Wnt Inhibitors

The ability to directly trans-differentiate fibroblasts into cardiomyocytes through overexpression of three core transcription factors, Gata4, Mef2C &TBX5 (GMT), has been demonstrated by our group and others. Direct cardiac reprogramming has great potential to treat heart failure. However, clinical application remains limited due to inefficiencies in the reprogramming process and immaturity of the differentiated cardiomyocytes. In the current work, using a chemical approach we aim to increase the efficiency and the maturation of these directly reprogrammed cardiomyocytes. We have optimized a high-throughput screening method in 384-well plates using an alpha-MHC-GFP reporter to detect cardiac reprogramming in mouse cardiac fibroblasts. We screened 5500 compounds and identified 20 hits which have increased the reprogramming efficiency by 2-6 fold compared to controls. Among these top hits, 4 TGF-β inhibitors (DS 1-4) and 4 WNT inhibitors (DS 5-8) enhanced direct cardiac reprogramming. Further in depth investigations showed that the combination of the TGF-β inhibitor DS1 and the WNT inhibitor DS5 increased reprogramming efficiency 7 fold when added to the GMT-overexpressing fibroblasts on the first day of reprogramming. This combination of small molecules also enhanced the maturation of the reprogrammed cells, as we observed beating cells after only 1 week of reprogramming compared to 6–8 weeks without the small molecules. Activation of canonical WNT signaling or TGF-β signaling significantly abrogates the improvement in cardiac reprogramming by DS1 and DS5. These results indicate the specificity of the action of these inhibitors through the canonical WNT and TGF-β pathways. In a translational effort, we injected DS1, 2 and 5 intraperitoneally in mice for 2 weeks in combination with GMT following myocardial infarction (MI) and found a significant improvement in cardiac function as represented by an ejection fraction of 39.11% ±1.2% in the GMT + compounds group vs. 28.44% ± 3.98% in the GMT only group (p<0.05), 6 weeks after MI compared to an ejection fraction of 22.72% in the control untreated group. In conclusion, small molecules, especially the TGF-β inhibitors and WNT inhibitors, greatly enhanced direct reprogramming in vitro and in vivo.

scholarly journals A study of the mechanism of lncRNA-CR594175 in regulating proliferation and invasion of hepatocellular carcinoma cells in vivo and in vitro

2020 ◽  
Author(s):  
Quan Liu ◽  
Xuxu Yu ◽  
Minjie Yang ◽  
Xiangke Li ◽  
Xuejia Zhai ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
High Throughput Screening ◽  
Hepatoma Cell ◽  
Screening Method ◽  
Negative Regulation ◽  
Hepatoma Cell Line ◽  
Incidence And Mortality ◽  
Proliferation And Invasion

Abstract Abstract Background Hepatocellular carcinoma (HCC) is one of the cancers of highest incidence and mortality worldwide. The proliferation and invasion of tumor cells are the main reason for poor prognosis after HCC surgery. Long non-coding RNA (lncRNA) has been shown to play a key role in the progression of HCC. LncRNA-CR594175 is one of the highly expressed lncRNAs in HCC tumors and their metastatic tumors that we have obtained by the High-throughput screening method.Methods To elucidate the role of lncRNA-CR594175 in regulating the proliferation and invasion of human hepatoma cell line, HepG2, we operated through lncRNA-CR594175 silencing to inhibit the progression of HCC, either through in vitro or in vivo experiments.Results We found that lncRNA-CR594175 was lower in adjacent non-cancerous tissues than in primary HCC, and was lower in primary HCC than in its metastasis. Silencing of lncRNA-CR594175 inhibited the proliferation and invasion of HepG2 cells and growth of subcutaneous tumors. The results revealed that lncRNA-CR594175, as a RNA sponge, broke the negative regulation of hsa-miR-142-3p on Catenin, beta-1 (CTNNB1), and once lncRNA-CR594175 was silenced, the hsa-miR142-3p regained its negative regulation on CTNNB1 which can promote HCC progression by activating the wnt pathway. Conclusions Our present study demonstrated for the first time that lncRNA-CR594175 silencing suppressed proliferation and invasion of HCC cells in vivo and in vitro by restoring the negative regulation of hsa-miR-142-3p on CTNNB1, laying a solid theoretical base for using lncRNA-CR594175 as genetic target therapy for HCC and offering a reasonable explanation for inactivation of miRNA in different tumors or in the tumor at different stages.

scholarly journals Suppression of Breast Cancer by Small Molecules That Block the Prolactin Receptor

Cancers ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2662
Author(s):  
Dana C. Borcherding ◽  
Eric R. Hugo ◽  
Sejal R. Fox ◽  
Eric M. Jacobson ◽  
Brian G. Hunt ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Small Molecules ◽  
High Throughput Screening ◽  
High Selectivity ◽  
Prolactin Receptor ◽  
Breast Cancer Patients ◽  
Clinical Phase ◽  
Anti Cancer

Prolactin (PRL) is a protein hormone which in humans is secreted by pituitary lactotrophs as well as by many normal and malignant non-pituitary sites. Many lines of evidence demonstrate that both circulating and locally produced PRL increase breast cancer (BC) growth and metastases and confer chemoresistance. Our objective was to identify and then characterize small molecules that block the tumorigenic actions of PRL in BC. We employed three cell-based assays in high throughput screening (HTS) of 51,000 small molecules and identified two small molecule inhibitors (SMIs), named SMI-1 and SMI-6. Both compounds bound to the extracellular domain (ECD) of the PRL receptor (PRLR) at 1–3 micromolar affinity and abrogated PRL-induced breast cancer cell (BCC) invasion and malignant lymphocyte proliferation. SMI-6 effectively reduced the viability of multiple BCC types, had much lower activity against various non-malignant cells, displayed high selectivity, and showed no apparent in vitro or in vivo toxicity. In athymic nude mice, SMI-6 rapidly and dramatically suppressed the growth of PRL-expressing BC xenografts. This report represents a pre-clinical phase of developing novel anti-cancer agents with the potential to become effective therapeutics in breast cancer patients.

A Revolution in Reprogramming: Small Molecules

2019 ◽  
Vol 19 (2) ◽  
pp. 77-90 ◽  
Author(s):  
Jin Zhou ◽  
Jie Sun
Keyword(s):  
Small Molecules ◽  
High Throughput Screening ◽  
Molecular Mechanisms ◽  
Genetic Factors ◽  
Cell Types ◽  
Cell Reprogramming ◽  
Cell Fates ◽  
Modern Biology ◽  
Reprogramming Efficiency

Transplantation of reprogrammed cells from accessible sources and in vivo reprogramming are potential therapies for regenerative medicine. During the last decade, genetic approaches, which mostly involved transcription factors and microRNAs, have been shown to affect cell fates. However, their potential carcinogenicity and other unexpected effects limit their translation into clinical applications. Recently, with the power of modern biology-oriented design and synthetic chemistry, as well as high-throughput screening technology, small molecules have been shown to enhance reprogramming efficiency, replace genetic factors, and help elucidate the molecular mechanisms underlying cellular plasticity and degenerative diseases. As a non-viral and non-integrating approach, small molecules not only show revolutionary capacities in generating desired exogenous cell types but also have potential as drugs that can restore tissues through repairing or reprogramming endogenous cells. Here, we focus on the recent progress made to use small molecules in cell reprogramming along with some related mechanisms to elucidate these issues.

scholarly journals High Throughput Screening Method for Identification of New Lipofection Reagents

2001 ◽  
Vol 6 (4) ◽  
pp. 245-254 ◽  
Author(s):  
Anne E. Regelin ◽  
Erhard Fernholz ◽  
Harald F. Krug ◽  
Ulrich Massing
Keyword(s):  
High Throughput Screening ◽  
Screening Method ◽  
Genetic Material ◽  
Cell Types ◽  
Cationic Lipids ◽  
Adherent Cell ◽  
Gene Assay

Lipofection, the transfer of genetic material into cells by means of cationic lipids, is of growing interest for in vitro and in vivo approaches. In order to identify ideal lipofection reagents in a HTS, we have developed an automated lipofection method for the transfer of reporter genes into cells and for determination of the lipofection results. The method has specifically been designed and optimized for 96-well microtiter plates and can successfully be carried out by a pipetting robot with accessory equipment. It consists of two separate parts: (1) pretransfection (preparation of liposomes, formation of lipoplexes, and lipoplex transfer to the cells) and (2) posttransfection (determination of the reporter enzyme activity and the protein content of the transfected cells). Individual steps of the lipofection method were specifically optimized—for example, lipoplex formation and incubation time as well as cell lysis, cell cultivating, and the reporter gene assay. The HTS method facilitates characterization of the transfection properties (efficiency and cytotoxicity) of large numbers of (cationic) lipids in various adherent cell types.

scholarly journals A study of the mechanism of lncRNA-CR594175 in regulating proliferation and invasion of hepatocellular carcinoma cells in vivo and in vitro

2020 ◽  
Vol 15 (1) ◽  
Author(s):  
Quan Liu ◽  
Xuxu Yu ◽  
Minjie Yang ◽  
Xiangke Li ◽  
Xuejia Zhai ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
High Throughput Screening ◽  
Hepatoma Cell ◽  
Screening Method ◽  
Negative Regulation ◽  
Hepatoma Cell Line ◽  
Incidence And Mortality ◽  
Proliferation And Invasion

Abstract Background Hepatocellular carcinoma (HCC) is one of the cancers of highest incidence and mortality worldwide. The proliferation and invasion of tumor cells are the main reason for poor prognosis after HCC surgery. Long non-coding RNA (lncRNA) has been shown to play a key role in the progression of HCC. LncRNA-CR594175 is one of the highly expressed lncRNAs in HCC tumors and their metastatic tumors that we have obtained by the High-throughput screening method. Methods To elucidate the role of lncRNA-CR594175 in regulating the proliferation and invasion of human hepatoma cell line, HepG2, we operated through lncRNA-CR594175 silencing to inhibit the progression of HCC, either through in vitro or in vivo experiments. Results We found that lncRNA-CR594175 was lower in adjacent non-cancerous tissues than in primary HCC, and was lower in primary HCC than in its metastasis. Silencing of lncRNA-CR594175 inhibited the proliferation and invasion of HepG2 cells and growth of subcutaneous tumors. The results revealed that lncRNA-CR594175, as a RNA sponge, broke the negative regulation of hsa-miR-142-3p on Catenin, beta-1 (CTNNB1), and once lncRNA-CR594175 was silenced, the hsa-miR142-3p regained its negative regulation on CTNNB1 which can promote HCC progression by activating the wnt pathway. Conclusions Our present study demonstrated for the first time that lncRNA-CR594175 silencing suppressed proliferation and invasion of HCC cells in vivo and in vitro by restoring the negative regulation of hsa-miR-142-3p on CTNNB1, laying a solid theoretical base for using lncRNA-CR594175 as genetic target therapy for HCC and offering a reasonable explanation for inactivation of miRNA in different tumors or in the tumor at different stages.

scholarly journals High-Throughput Screening and Identification of Human Adenovirus Type 5 Inhibitors

2021 ◽  
Vol 11 ◽  
Author(s):  
Xiaojing Wen ◽  
Li Zhang ◽  
Shan Zhao ◽  
Qiang Liu ◽  
Wenyi Guan ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Tissue Sections ◽  
Screening And Identification ◽  
New Treatment

Human adenovirus infections can develop into diffuse multi-organ diseases in young children and immunocompromised patients, and severe cases can lead to death. However, there are no approved antiviral drugs available to treat adenovirus diseases. In this study, a chemiluminescence-based, high-throughput screening (HTS) assay was developed and applied to screen human adenovirus 5(HAdV5)inhibitors from 1,813 approved drug library and 556 traditional Chinese medicine-sourced small-molecule compounds. We identified three compounds with in vitro anti-HAdV5 activities in the low-micromolar range (EC50 values 0.3-4.5 μM, selectivity index values 20-300) that also showed inhibitory effects on HAdV3. Cardamomin (CDM) had good anti-HAdV5 activity in vitro. Furthermore, three dilutions of CDM (150, 75, and 37.5 mg/kg/d) administered to BALB/c mouse models inhibited HAdV5-fluc infection at 1 day post-infection by 80% (p &lt; 0.05), 76% (p &lt; 0.05), and 58% (p &lt; 0.05), respectively. HE-staining of pathological tissue sections of mice infected with a wildtype adenoviral strain showed that CDM had a protective effect on tissues, especially in the liver, and greatly inhibited virus-induced necrosis of liver tissue. Thus, CDM inhibits adenovirus replication in vivo and in vitro. This study established a high-throughput screening method for anti-HAdV5 drugs and demonstrated CDM to be a candidate for HAdV5 therapy, potentially providing a new treatment for patients infected with adenoviruses.

scholarly journals A study of the mechanism of lncRNA-CR594175 in regulating proliferation and invasion of hepatocellular carcinoma cells in vivo and in vitro

2020 ◽  
Author(s):  
Quan Liu ◽  
Xuxu Yu ◽  
Minjie Yang ◽  
Xiangke Li ◽  
Xuejia Zhai ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Hepg2 Cells ◽  
High Throughput Screening ◽  
Target Therapy ◽  
Screening Method ◽  
Negative Regulation ◽  
Incidence And Mortality ◽  
Proliferation And Invasion

Abstract Background Hepatocellular carcinoma (HCC) is one of the cancers of highest incidence and mortality worldwide. The proliferation and invasion of tumor cells are the main reason for poor prognosis after HCC surgery. Long non-coding RNA (lncRNA) has been shown to play a key role in the progression of HCC. LncRNA-CR594175 is one of the highly expressed lncRNAs in HCC tumors and their metastatic tumors that we have obtained by the High-throughput screening method.Methods To elucidate the role of CR594175 in regulating the proliferation and invasion of HepG2 cells, and to initially try to use a genetic engineering operation plan-CR594175 silence to inhibit the HCC progression through functional experiments in vitro and subcutaneous tumor-bearing experiments.Results We found that lncRNA-CR594175 was lower in adjacent noncancerous tissues than in primary HCC, and was lower in primary HCC than in their metastases. SilencingCR594175 inhibited the proliferation and invasion of HepG2 cells and growth of subcutaneous tumors. The mechanism research revealed that CR594175, as a RNA sponge, broke the negative regulation of hsa-miR-142-3p on CTNNB1 (Catenin, beta-1), and once CR594175 was silenced, the hsa-miR142-3p regained its negative regulation on CTNNB1 which can promote HCC progression by activating the wnt pathway. Conclusions Our present study demonstrates for the first time that CR594175 silencing suppressed proliferation and invasion of HCC cells in vivo and in vitro by restoring the negative regulation of hsa-miR-142-3p on CTNNB1, laying a solid theoretic base for using lncRNA-CR594175 as genetic target therapy for HCC and offering a reasonable explanation for inactivation of miRNA in different tumors or tumor at different stages.

scholarly journals High-Throughput Chemical Screening for Antivirulence Developmental Phenotypes in Trypanosoma brucei

2014 ◽  
Vol 13 (3) ◽  
pp. 412-426 ◽  
Author(s):  
Paula MacGregor ◽  
Alasdair Ivens ◽  
Steven Shave ◽  
Iain Collie ◽  
David Gray ◽  
...  
Keyword(s):  
Small Molecules ◽  
Trypanosoma Brucei ◽  
High Throughput ◽  
High Throughput Screening ◽  
Morphological Changes ◽  
Specific Gene ◽  
African Trypanosomes ◽  
Chemical Screening

ABSTRACT In the bloodstream of mammalian hosts, the sleeping sickness parasite, Trypanosoma brucei , exists as a proliferative slender form or a nonproliferative, transmissible, stumpy form. The transition between these developmental forms is controlled by a density-dependent mechanism that is important for the parasite's infection dynamics, immune evasion via ordered antigenic variation, and disease transmissibility. However, stumpy formation has been lost in most laboratory-adapted trypanosome lines, generating monomorphic parasites that proliferate uncontrolled as slender forms in vitro and in vivo . Nonetheless, these forms are readily amenable to cell culture and high-throughput screening for trypanocidal lead compounds. Here, we have developed and exploited a high-throughput screen for developmental phenotypes using a transgenic monomorphic cell line expressing a reporter under the regulation of gene control signals from the stumpy-specific molecule PAD1. Using a whole-cell fluorescence-based assay to screen over 6,000 small molecules from a kinase-focused compound library, small molecules able to activate stumpy-specific gene expression and proliferation arrest were assayed in a rapid assay format. Independent follow-up validation identified one hit able to induce modest, yet specific, changes in mRNA expression indicative of a partial differentiation to stumpy forms in monomorphs. Further, in pleomorphs this compound induced a stumpy-like phenotype, entailing growth arrest, morphological changes, PAD1 expression, and enhanced differentiation to procyclic forms. This not only provides a potential tool compound for the further understanding of stumpy formation but also demonstrates the use of high-throughput screening in the identification of compounds able to induce specific phenotypes, such as differentiation, in African trypanosomes.

Conjugates of Classical DNA/RNA Binder with Nucleobase: Chemical, Biochemical and Biomedical Applications

2019 ◽  
Vol 26 (30) ◽  
pp. 5609-5624
Author(s):  
Dijana Saftić ◽  
Željka Ban ◽  
Josipa Matić ◽  
Lidija-Marija Tumirv ◽  
Ivo Piantanida
Keyword(s):  
Molecular Weight ◽  
Small Molecules ◽  
Biomedical Applications ◽  
Protein Targets ◽  
New Class ◽  
Rna Targets ◽  
Covalently Linked ◽  
Structural Aspects

: Among the most intensively studied classes of small molecules (molecular weight < 650) in biomedical research are small molecules that non-covalently bind to DNA/RNA, and another intensively studied class is nucleobase derivatives. Both classes have been intensively elaborated in many books and reviews. However, conjugates consisting of DNA/RNA binder covalently linked to nucleobase are much less studied and have not been reviewed in the last two decades. Therefore, this review summarized reports on the design of classical DNA/RNA binder – nucleobase conjugates, as well as data about their interactions with various DNA or RNA targets, and even in some cases protein targets are involved. According to these data, the most important structural aspects of selective or even specific recognition between small molecule and target are proposed, and where possible related biochemical and biomedical aspects were discussed. The general conclusion is that this, rather new class of molecules showed an amazing set of recognition tools for numerous DNA or RNA targets in the last two decades, as well as few intriguing in vitro and in vivo selectivities. Several lead research lines show promising advancements toward either novel, highly selective markers or bioactive, potentially druggable molecules.

Sign in / Sign up

Export Citation Format

Share Document