CETSA and thermal proteome profiling strategies for target identification and drug discovery of natural products

Phytomedicine, Journal Year: 2023, Volume and Issue: 116, P. 154862 - 154862

Published: May 20, 2023

Language: Английский

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Current Advances in CETSA

Frontiers in Molecular Biosciences, Journal Year: 2022, Volume and Issue: 9

Published: June 9, 2022

Knowing that the drug candidate binds to its intended target is a vital part of discovery. Thus, several labeled and label-free methods have been developed study engagement. In recent years, cellular thermal shift assay (CETSA) with variations has widely adapted discovery workflows. Western blot-based CETSA used primarily validate binding molecule protein whereas based on bead chemistry detection (CETSA HT) screen molecular libraries find novel molecules pre-determined target. Mass spectrometry-based also known as proteome profiling (TPP) emerged powerful tool for deconvolution finding partners old molecules. With this technology, it possible probe shifts among over 7,000 proteins from one sample identify wanted but unwanted off-targets cause adverse effects. addition, proteome-wide method can provide information biological process initiated by ligand binding. The continued development mass spectrometry labeling reagents, such isobaric tandem tag technology (TMT) continues increase throughput MS, allowing use structure-activity relationship (SAR) studies limited number review, we discussed differences between different engagement, our focus was advances in method.

Language: Английский

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Deep thermal profiling for detection of functional proteoform groups

Nature Chemical Biology, Journal Year: 2023, Volume and Issue: 19(8), P. 962 - 971

Published: March 20, 2023

Abstract The complexity of the functional proteome extends considerably beyond coding genome, resulting in millions proteoforms. Investigation proteoforms and their roles is important to understand cellular physiology its deregulation diseases but challenging perform systematically. Here we applied thermal profiling with deep peptide coverage detect proteoform groups acute lymphoblastic leukemia cell lines different cytogenetic aberrations. We detected 15,846 proteoforms, capturing differently spliced, cleaved post-translationally modified proteins expressed from 9,290 genes. identified differential co-aggregation pairs established links disease biology. Moreover, systematically made use measured biophysical states find specific biomarkers drug sensitivity. Our approach, thus, provides a powerful unique tool for systematic detection annotation groups.

Language: Английский

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Comparison of Quantitative Mass Spectrometric Methods for Drug Target Identification by Thermal Proteome Profiling

Journal of Proteome Research, Journal Year: 2023, Volume and Issue: 22(8), P. 2629 - 2640

Published: July 13, 2023

Thermal proteome profiling (TPP) provides a powerful approach to studying proteome-wide interactions of small therapeutic molecules and their target off-target proteins, complementing phenotypic-based drug screens. Detecting differences in thermal stability due engagement requires high quantitative accuracy consistent detection. Isobaric tandem mass tags (TMTs) are used multiplex samples increase quantification precision TPP analysis by data-dependent acquisition (DDA). However, advances data-independent (DIA) can provide higher sensitivity protein coverage with reduced costs sample preparation steps. Herein, we explored the performance different DIA-based label-free approaches compared TMT-DDA for shift quantitation. Acute myeloid leukemia cells were treated losmapimod, known inhibitor MAPK14 (p38α). Label-free DIA approaches, particularly library-free mode DIA-NN, comparable ability detect losmapimod one its downstream targets, MAPKAPK3. Using quantitation is cost-effective alternative labeled pipeline.

Language: Английский

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Novel WRN Helicase Inhibitors Selectively Target Microsatellite-Unstable Cancer Cells

Cancer Discovery, Journal Year: 2024, Volume and Issue: 14(8), P. 1457 - 1475

Published: April 6, 2024

Microsatellite-unstable (MSI) cancers require WRN helicase to resolve replication stress due expanded DNA (TA)n dinucleotide repeats. is a promising synthetic lethal target for MSI tumors, and inhibitors are in development. In this study, we used CRISPR-Cas9 base editing map residues critical cells, validating the domain as primary drug target. Fragment-based screening led development of potent highly selective covalent inhibitors. These compounds selectively suppressed model growth vitro vivo by mimicking loss, inducing double-strand breaks at TA repeats damage. Assessment biomarkers preclinical models linked TA-repeat expansions mismatch repair alterations compound activity. Efficacy was confirmed immunotherapy-resistant organoids patient-derived xenograft models. The discovery potent, provides proof concept targeting cancer tools dissect biology. Significance: We report characterization treatment, with biomarker analysis evaluation efficacy immunotherapy-refractory findings pave way translate inhibition into therapies provide investigate See related commentary Wainberg, p. 1369.

Language: Английский

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High-throughput drug target discovery using a fully automated proteomics sample preparation platform

Chemical Science, Journal Year: 2024, Volume and Issue: 15(8), P. 2833 - 2847

Published: Jan. 1, 2024

Drug development is plagued by inefficiency and high costs due to issues such as inadequate drug efficacy unexpected toxicity. Mass spectrometry (MS)-based proteomics, particularly isobaric quantitative offers a solution unveil resistance mechanisms unforeseen side effects related off-targeting pathways. Thermal proteome profiling (TPP) has gained popularity for target identification at the scale. However, it involves experiments with multiple temperature points, resulting in numerous samples considerable variability large-scale TPP analysis. We propose high-throughput discovery workflow that integrates single-temperature TPP, fully automated proteomics sample preparation platform (autoSISPROT), data independent acquisition (DIA) quantification. The autoSISPROT enables simultaneous processing of 96 less than 2.5 hours, achieving protein digestion, desalting, optional TMT labeling (requires an additional 1 hour) 96-channel all-in-tip operations. results demonstrated excellent performance >94% digestion efficiency, >98% >0.9 intra- inter-batch Pearson correlation coefficients. By automatically 87 samples, we identified both known targets potential off-targets 20 kinase inhibitors, affording over 10-fold improvement throughput compared classical TPP. This target/off-target identification.

Language: Английский

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Drug Target Identification in Tissues by Thermal Proteome Profiling

The Annual Review of Pharmacology and Toxicology, Journal Year: 2021, Volume and Issue: 62(1), P. 465 - 482

Published: Sept. 9, 2021

Drug target deconvolution can accelerate the drug discovery process by identifying a drug's targets (facilitating medicinal chemistry efforts) and off-targets (anticipating toxicity effects or adverse reactions). Multiple mass spectrometry–based approaches have been developed for this purpose, but thermal proteome profiling (TPP) remains to date only one that does not require compound modification be used identify intracellular in living cells. TPP is based on principle stability of protein affected its interactions. Recent developments approach expanded applications beyond drugs cell cultures studying protein-drug interactions biological phenomena tissues. These open up possibility treatment mechanisms disease holistic fashion, which result design better lead understanding fundamental biology.

Language: Английский

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Assessing target engagement using proteome-wide solvent shift assays

eLife, Journal Year: 2021, Volume and Issue: 10

Published: Dec. 8, 2021

Recent advances in mass spectrometry (MS) have enabled quantitative proteomics to become a powerful tool the field of drug discovery, especially when applied toward proteome-wide target engagement studies. Similar temperature gradients, increasing concentrations organic solvents stimulate unfolding and precipitation cellular proteome. This property can be influenced by physical association with ligands other molecules, making individual proteins more or less susceptible solvent-induced denaturation. Herein, we report development solvent shift assays combining principles (Zhang et al., 2020) modern proteomics. Using this approach, developed proteome profiling (SPP), which is capable establishing through analysis SPP denaturation curves. We readily identified specific targets compounds known mechanisms action. As further efficiency boost, concept area under curve develop integral solubility alteration (solvent-PISA) demonstrate that approach serve as reliable surrogate for SPP. propose alternative methods, like thermal profiling, it will possible increase absolute number high-quality melting curves are attainable either individually, thereby fraction screened evidence ligand binding.

Language: Английский

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Discovery and Structural Characterization of Small Molecule Binders of the Human CTLH E3 Ligase Subunit GID4

Journal of Medicinal Chemistry, Journal Year: 2022, Volume and Issue: 65(19), P. 12725 - 12746

Published: Sept. 19, 2022

Targeted protein degradation (TPD) strategies exploit bivalent small molecules to bridge substrate proteins an E3 ubiquitin ligase induce degradation. Few E3s have been explored as effectors due a dearth of E3-binding molecules. We show that genetically induced recruitment the GID4 subunit CTLH complex induces An NMR-based fragment screen followed by structure-guided analog elaboration identified two binders GID4, 16 and 67, with Kd values 110 17 μM in vitro. A parallel DNA-encoded library (DEL) five best which, 88, had 5.6 vitro EC50 558 nM cells strong selectivity for GID4. X-ray co-structure determination revealed basis GID4–small molecule interactions. These results position GID4-CTLH TPD provide candidate scaffolds high-affinity moieties bind

Language: Английский

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Large-scale characterization of drug mechanism of action using proteome-wide thermal shift assays

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Jan. 27, 2024

In response to an ever-increasing demand of new small molecules therapeutics, numerous chemical and genetic tools have been developed interrogate compound mechanism action. Owing its ability approximate compound-dependent changes in thermal stability, the proteome-wide shift assay has emerged as a powerful tool this arsenal. The most recent iterations drastically improved overall efficiency these assays, providing opportunity screen compounds at previously unprecedented rate. Taking advantage advance, we quantified more than one million stability measurements multiple classes therapeutic (96 living cells 70 lysates). When interrogating dataset whole, approximately 80% (with quantifiable targets) caused significant change annotated target. There was also wealth evidence portending off-target engagement despite extensive use laboratory and/or clinic. Finally, combined application cell- lysate-based aided classification primary (direct ligand binding) secondary (indirect) stability. Overall, study highlights value assays drug development process by affording unbiased reliable assessment

Language: Английский

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