Reproducibility and accuracy of microscale thermophoresis in the NanoTemper Monolith: a multi laboratory benchmark study

SummaryIntroduction Differentiation therapy is a promising strategy for cancer treatment. The translationally controlled tumor protein (TCTP) is an encouraging target in this context. By now, this field of research is still at its infancy, which motivated us to perform a large-scale screening for the identification of novel ligands of TCTP. We studied the binding mode and the effect of TCTP blockade on the cell cycle in different cancer cell lines. Methods Based on the ZINC-database, we performed virtual screening of 2,556,750 compounds to analyze the binding of small molecules to TCTP. The in silico results were confirmed by microscale thermophoresis. The effect of the new ligand molecules was investigated on cancer cell survival, flow cytometric cell cycle analysis and protein expression by Western blotting and co-immunoprecipitation in MOLT-4, MDA-MB-231, SK-OV-3 and MCF-7 cells. Results Large-scale virtual screening by PyRx combined with molecular docking by AutoDock4 revealed five candidate compounds. By microscale thermophoresis, ZINC10157406 (6-(4-fluorophenyl)-2-[(8-methoxy-4-methyl-2-quinazolinyl)amino]-4(3H)-pyrimidinone) was identified as TCTP ligand with a KD of 0.87 ± 0.38. ZINC10157406 revealed growth inhibitory effects and caused G0/G1 cell cycle arrest in MOLT-4, SK-OV-3 and MCF-7 cells. ZINC10157406 (2 × IC50) downregulated TCTP expression by 86.70 ± 0.44% and upregulated p53 expression by 177.60 ± 12.46%. We validated ZINC10157406 binding to the p53 interaction site of TCTP and replacing p53 by co-immunoprecipitation. Discussion ZINC10157406 was identified as potent ligand of TCTP by in silico and in vitro methods. The compound bound to TCTP with a considerably higher affinity compared to artesunate as known TCTP inhibitor. We were able to demonstrate the effect of TCTP blockade at the p53 binding site, i.e. expression of TCTP decreased, whereas p53 expression increased. This effect was accompanied by a dose-dependent decrease of CDK2, CDK4, CDK, cyclin D1 and cyclin D3 causing a G0/G1 cell cycle arrest in MOLT-4, SK-OV-3 and MCF-7 cells. Our findings are supposed to stimulate further research on TCTP-specific small molecules for differentiation therapy in oncology.

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Novel Method for Quantifying AhR-Ligand Binding Affinities Using Microscale Thermophoresis

Biosensors ◽

10.3390/bios11030060 ◽

2021 ◽

Vol 11 (3) ◽

pp. 60

Author(s):

Anne Stinn ◽

Jens Furkert ◽

Stefan H. E. Kaufmann ◽

Pedro Moura-Alves ◽

Michael Kolbe

Keyword(s):

Ligand Binding ◽

Environmental Pollutants ◽

Screening Strategy ◽

Pharmacological Intervention ◽

Binding Affinities ◽

Free System ◽

Microscale Thermophoresis ◽

Aryl Hydrocarbon ◽

Promising Target ◽

A Cell

The aryl hydrocarbon receptor (AhR) is a highly conserved cellular sensor of a variety of environmental pollutants and dietary-, cell- and microbiota-derived metabolites with important roles in fundamental biological processes. Deregulation of the AhR pathway is implicated in several diseases, including autoimmune diseases and cancer, rendering AhR a promising target for drug development and host-directed therapy. The pharmacological intervention of AhR processes requires detailed information about the ligand binding properties to allow specific targeting of a particular signaling process without affecting the remaining. Here, we present a novel microscale thermophoresis-based approach to monitoring the binding of purified recombinant human AhR to its natural ligands in a cell-free system. This approach facilitates a precise identification and characterization of unknown AhR ligands and represents a screening strategy for the discovery of potential selective AhR modulators.

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A Benchmark Study of Complementary-Field Effect Transistor (CFET) Process Integration Options: Comparing Bulk vs. SOI vs. DSOI starting substrates

2019 IEEE SOI-3D-Subthreshold Microelectronics Technology Unified Conference (S3S) ◽

10.1109/s3s46989.2019.9320683 ◽

2019 ◽

Author(s):

Benjamin Vincent ◽

J. Ervin ◽

J. Boemmels ◽

J. Ryckaert

Keyword(s):

Field Effect ◽

Field Effect Transistor ◽

Process Integration ◽

Benchmark Study ◽

Effect Transistor

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Pulsed response theory prediction of ZnO nanocluster polarizabilities: A benchmark study

Chemical Physics Letters ◽

10.1016/j.cplett.2021.138746 ◽

2021 ◽

pp. 138746

Author(s):

I.V.A.K. Reddy ◽

Alexander Baev ◽

Paras N. Prasad ◽

Hans Agren

Keyword(s):

Response Theory ◽

Benchmark Study

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Adaptive selection of classifiers for bug prediction: A large-scale empirical analysis of its performances and a benchmark study

Science of Computer Programming ◽

10.1016/j.scico.2021.102611 ◽

2021 ◽

Vol 205 ◽

pp. 102611 ◽

Cited By ~ 1

Author(s):

Fabiano Pecorelli ◽

Dario Di Nucci

Keyword(s):

Empirical Analysis ◽

Large Scale ◽

Adaptive Selection ◽

Benchmark Study ◽

Selection Of

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