A widely-applicable high-throughput cellular thermal shift assay (CETSA) using split Nano Luciferase

Martínez, Natàlia; Asawa, Rosita R.; Cyr, Matthew; Zakharov, Alexey; Urban, Daniel J.; Roth, Jacob S.; Wallgren, Eric; Klumpp‐Thomas, Carleen; Coussens, Nathan P.; Rai, Ganesha; Yang, Shyh‐Ming; Hall, Matthew D.; Marugán, Juan; Simeonov, Anton; Henderson, Mark J.

doi:10.1038/s41598-018-27834-y

Cited by 78 publications

(101 citation statements)

References 37 publications

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“…To identify new therapeutic options for PKD, we screened 8,814 unique compounds using the screening paradigm described above. The small molecule collections we chose included the NIH Chemical Genomics Center (NCGC) Pharmaceutical Collection (NPC), Mechanism of Interrogation Plate (MIPE), the NCATS Pharmacologically Active Chemical Toolbox (NPACT), and the Kinase Inhibitor collections, comprised of approved and investigational drugs as well as highly-annotated tool compounds with varying degrees of validation as therapeutics 10,11,[30][31][32][33] . For all compounds tested, we derived CRCs in all four cell lines and we used ΔAUC values to identify compounds with robust bioactivity profiles against Pkd1-null cells but minimal or less pronounced effects in wt cells (activity cutoffs are described in Materials and Methods and assay performance is described in Supplementary Table 1).…”

Section: Resultsmentioning

confidence: 99%

“…Chemical classes include synthetic small molecules as well as microbial-and plant-derived purified natural products (https://ncats.nih.gov/ preclinical/core/compound/npact). The Kinase Inhibitor collection contains 977 clinical and pre-clinical-stage compounds that inhibit one or more kinases 33 . The MIPE (Mechanism Interrogation Plate) collection contains approved and investigational 2,480 oncology-focused agents 30-32 . cell lines and culture conditions.…”

Section: Methods Compound Libraries the Nih Chemical Genomics Centermentioning

confidence: 99%

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A high-throughput screening platform for Polycystic Kidney Disease (PKD) drug repurposing utilizing murine and human ADPKD cells

Asawa

Danchik

Zahkarov

et al. 2020

Sci Rep

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Autosomal dominant polycystic kidney disease (ADpKD) is one of the most common inherited monogenic disorders, characterized by a progressive decline in kidney function due in part to the formation of fluid-filled cysts. While there is one FDA-approved therapy, it is associated with potential adverse effects, and all other clinical interventions are largely supportive. Insights into the cellular pathways underlying ADpKD have revealed striking similarities to cancer. Moreover, several drugs originally developed for cancer have shown to ameliorate cyst formation and disease progression in animal models of ADpKD. these observations prompted us to develop a high-throughput screening platform of cancer drugs in a quest to repurpose them for ADPKD. We screened ~8,000 compounds, including compounds with oncological annotations, as well as FDA-approved drugs, and identified 155 that reduced the viability of Pkd1-null mouse kidney cells with minimal effects on wild-type cells. We found that 109 of these compounds also reduced in vitro cyst growth of Pkd1-null cells cultured in a 3D matrix. Moreover, the result of the cyst assay identified therapeutically relevant compounds, including agents that interfere with tubulin dynamics and reduced cyst growth without affecting cell viability. Because it is known that several ADpKD therapies with promising outcomes in animal models failed to be translated to human disease, our platform also incorporated the evaluation of compounds in a panel of primary ADPKD and normal human kidney (NHK) epithelial cells. Although we observed differences in compound response amongst ADPKD and NHK cell preparation, we identified 18 compounds that preferentially affected the viability of most ADPKD cells with minimal effects on NHK cells. Our study identifies attractive candidates for future efficacy studies in advanced pre-clinical models of ADPKD. Autosomal dominant polycystic kidney disease (ADPKD) has a prevalence of 1 in 500-1,000 individuals and is the most common inherited kidney disease, accounting for 6-9% of patients on renal replacement therapy. ADPKD is caused mainly by mutations in PKD1 and to a lesser extent in PKD2, GANAB and DNAJB11 genes. The disease is characterized by a progressive decline in kidney function due to the formation of fluid-filled cysts as well as activation of inflammatory and proliferative pathways, typically leading to end-stage renal disease by the fifth or sixth decade of life 1. Although cyst formation in the kidneys is the hallmark of ADPKD, other epithelial organs including the liver and pancreas are also commonly affected 2,3. With the 2018 FDA approval of Tolvaptan, a vasopressin V 2-receptor antagonist, there is now one therapy available to slow disease progression; however, the drug was only approved for patients at risk of rapid disease progression due to its potential side effects. Nevertheless, most interventions focus on alleviating disease-related symptoms.

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Section: Resultsmentioning

confidence: 99%

Section: Methods Compound Libraries the Nih Chemical Genomics Centermentioning

confidence: 99%

A high-throughput screening platform for Polycystic Kidney Disease (PKD) drug repurposing utilizing murine and human ADPKD cells

Asawa

Danchik

Zahkarov

et al. 2020

Sci Rep

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“…Nluc has already been successfully used in similar applications which monitor protein aggregation. Such applications fuse either full length Nluc (Dart, Machleidt et al 2018) or a split Nluc (Martinez, Asawa et al 2018) fusions to the protein of interest and monitor the decrease in luminescence activity to measure aggregation of the protein of interest after thermal denaturation. In contrast, the ThermoBRET assay described here captures the initial conformational unfolding events which expose maleimide reactive cysteine residues in the protein of interest.…”

Section: Discussionmentioning

confidence: 99%

ThermoBRET: a ligand-engagement nanoscale thermostability assay applied to GPCRs

Hoare

Kaur

Dijon

et al. 2020

Preprint

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Sensitive assays to measure the thermostability of membrane proteins are important tools in protein purification optimisation and drug discovery. Here, we present a ThermoBRET method to quantify the relative thermostability of G protein coupled receptors (GPCRs), using cannabinoid receptor 2 (CB2) as an example. This method applies the principles of Bioluminescence Resonance Energy Transfer (BRET) between Nanoluciferase (Nluc) and a thiol-reactive fluorescent dye that covalently binds cysteines in the GPCR transmembrane domain, exposed by unfolding. We demonstrate that the melting point (Tm) of Nluc-fused GPCRs can be determined in non-purified detergent solubilised membrane preparations, revealing differences in thermostability for different detergent solubilising conditions and in the presence of stabilising ligands. In addition, we extended the range of the assay by developing the thermostable tsNLuc by incorporating mutations from the fragments of split-Nluc (Tm of 87 °C vs 59 °C). ThermoBRET allows the high-throughput determination of GPCR thermostability which will be useful for protein purification optimisation strategies and as part of a drug discovery screening platform.

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“…For example, an LDHA inhibitor was examined with heat applied for 5-50 min, with a corresponding 35-fold right-shift in EC 50 , but compound rank order was maintained. 39 In this context, it is important to note that the rate of heating may also influence the measured effect of different compounds. Drawing from the wealth of knowledge accumulated during studies of targets and compounds using biochemical thermal shift methods, the behavior of p38a inhibitor classes with differing enthalpic and entropic contributions to p38α binding was examined.…”

Section: Interpretating Experimental Outcomesmentioning

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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A widely-applicable high-throughput cellular thermal shift assay (CETSA) using split Nano Luciferase

Cited by 78 publications

References 37 publications

A high-throughput screening platform for Polycystic Kidney Disease (PKD) drug repurposing utilizing murine and human ADPKD cells

A high-throughput screening platform for Polycystic Kidney Disease (PKD) drug repurposing utilizing murine and human ADPKD cells

ThermoBRET: a ligand-engagement nanoscale thermostability assay applied to GPCRs

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

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