Positioning High-Throughput CETSA in Early Drug Discovery through Screening against B-Raf and PARP1

Shaw, J.C.; Dale, Ian L.; Hemsley, Paul; Leach, Lindsey; Dekki, Nancy; Orme, Jonathan P.; Talbot, Verity; Narvaez, A.J.; Bista, Michal; Molina, Daniel Martinez; Dabrowski, Michael; Main, Martin J.; Gianni, Davide

doi:10.1177/2472555218813332

Cited by 38 publications

(25 citation statements)

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“…The present study aimed to establish such a strategy, using the small molecule melatonin to improve AdMSC cell therapy. Such small cell-permeable molecules can provide robust and reproducible results, and affect signaling pathways (22). Additionally, the present study investigated whether melatonin treatment could improve the proliferative activity and anti-inflammatory effects of MSCs, and whether it could regulate the tri-lineage differentiation potential of MSCs compared with normal culture conditions.…”

Section: Introductionmentioning

confidence: 99%

Biological effects of melatonin on human adipose‑derived mesenchymal stem cells

et al. 2019

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Mesenchymal stem cells (MScs) are capable of differentiating into other cell types and exhibit immunomodulatory effects. MScs are affected by several intrinsic and extrinsic signaling modulators, including growth factors, cytokines, extracellular matrix and hormones. Melatonin, produced by the pineal gland, is a hormone that regulates sleep cycles. Recent studies have shown that melatonin improves the therapeutic effects of stem cells. The present study aimed to investigate whether melatonin enhances the biological activities of human adipose-derived MScs. The results demonstrated that treatment with melatonin promoted cell proliferation by inducing SRY-box transcription factor 2 gene expression and preventing replicative senescence. In addition, melatonin exerted anti-adipogenic effects on MScs. PcR analysis revealed that the expression of the ccAAT enhancer binding protein a gene, a key transcription factor in adipogenesis, was decreased following melatonin treatment, resulting in reduced adipogenic differentiation in an in vitro assay. The present study also examined the effect of melatonin on the immunomodulatory response using a co-culture system of human peripheral blood mononuclear cells and MScs. Activated T cells were strongly inhibited following melatonin exposure compared with those in the control group. Finally, the favorable effects of melatonin on MSCs were confirmed using luzindole, a selective melatonin receptor antagonist. The proliferation-promoting, anti-inflammatory effects of melatonin suggested that melatonin-treated MScs may be used for effective cell therapy.

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Section: Introductionmentioning

confidence: 99%

Biological effects of melatonin on human adipose‑derived mesenchymal stem cells

et al. 2019

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“…AlphaScreen CETSA has been reported in several studies. Shaw et al 15 examined B-Raf target engagement using HT-CETSA and identified 13 compounds from a set of 896 kinase inhibitors that thermally stabilized the protein. All 13 compounds had prior evidence of B-Raf binding and were tested at multiple concentrations to rank-order the inhibitor potencies.…”

Section: Ht-cetsa: Endogenous Proteinmentioning

confidence: 99%

High-Throughput Cellular Thermal Shift Assays in Research and Drug Discovery

et al. 2020

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Thermal shift assays (TSAs) can reveal changes in protein structure, due to a resultant change in protein thermal stability. Since proteins are often stabilized upon binding of ligand molecules, these assays can provide a readout for protein target engagement. TSA has traditionally been applied using purified proteins and more recently has been extended to study target engagement in cellular environments with the emergence of cellular thermal shift assays (CETSAs). The utility of CETSA in confirming molecular interaction with targets in a more native context, and the desire to apply this technique more broadly, has fueled the emergence of higher-throughput techniques for CETSA (HT-CETSA). Recent studies have demonstrated that HT-CETSA can be performed in standard 96-, 384-, and 1536-well microtiter plate formats using methods such as beta-galactosidase and NanoLuciferase reporters and AlphaLISA assays. HT-CETSA methods can be used to select and characterize compounds from high-throughput screens and to prioritize compounds in lead optimization by facilitating dose–response experiments. In conjunction with cellular and biochemical activity assays for targets, HT-CETSA can be a valuable addition to the suite of assays available to characterize molecules of interest. Despite the successes in implementing HT-CETSA for a diverse set of targets, caveats and challenges must also be recognized to avoid overinterpretation of results. Here, we review the current landscape of HT-CETSA and discuss the methodologies, practical considerations, challenges, and applications of this approach in research and drug discovery. Additionally, a perspective on potential future directions for the technology is presented.

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“…Parallel developments in this field have also afforded microplate-based high-throughput variants of CETSA to expand applications to screening, structure–activity relationship (SAR), and mechanism-of-action (MoA) profiling. 9,30–35 Our significant interest in the SAR and MoA areas triggered a further annotation of the literature with regard to their experimental details, with an emphasis on aspects that affect apparent compound potency. This includes the transient heat pulse design, whether compound was washed away prior to heating (e.g., through trypsinization), the inclusion of more than one compound to understand whether observed data agree with other measures of SAR, and whether the compounds were tested at multiple concentrations.…”

Section: Literature Annotationmentioning

confidence: 99%