A Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Assay Identifies Nilotinib as an Inhibitor of Inflammation in Acute Myeloid Leukemia DOI Creative Commons
José Luis Marín‐Rubio, Rachel E. Heap, María Emilia Dueñas

et al.

Journal of Medicinal Chemistry, Journal Year: 2022, Volume and Issue: 65(18), P. 12014 - 12030

Published: Sept. 12, 2022

Inflammatory responses are important in cancer, particularly the context of monocyte-rich aggressive myeloid neoplasm. We developed a label-free cellular phenotypic drug discovery assay to identify anti-inflammatory drugs human monocytes derived from acute leukemia (AML), by tracking several features ionizing only 2500 cells using matrix-assisted laser desorption/ionization-time flight (MALDI-TOF) mass spectrometry. A proof-of-concept screen showed that BCR-ABL inhibitor nilotinib, but not structurally similar imatinib, blocks inflammatory responses. In order (off-)targets we performed thermal proteome profiling (TPP). Unlike nilotinib and other later-generation inhibitors bind p38α inhibit p38α-MK2/3 signaling axis, which suppressed pro-inflammatory cytokine expression, cell adhesion, innate immunity markers activated AML. Thus, our study provides tool for new drugs, could contribute treatment inflammation neoplasms diseases.

Language: Английский

Matrix Thermal Shift Assay for Fast Construction of Multidimensional Ligand–Target Space DOI

Chengfei Ruan,

Yan Wang, Xiaolei Zhang

et al.

Analytical Chemistry, Journal Year: 2022, Volume and Issue: 94(17), P. 6482 - 6490

Published: April 20, 2022

Existing thermal shift-based mass spectrometry approaches are able to identify target proteins without chemical modification of the ligand, but they suffering from complicated workflows with limited throughput. Herein, we present a new method, termed matrix shift assay (mTSA), for fast deconvolution ligand-binding targets and binding affinities at proteome level. In mTSA, sample matrix, treated horizontally five different compound concentrations vertically technical replicates each condition, was denatured single temperature induce protein precipitation, then, data-independent acquisition employed quick quantification. Compared previous assays, analysis throughput mTSA significantly improved, costs as well efforts were reduced. More importantly, experiment design allowed simultaneous computation statistical significance fitting dose-response profiles, which can be combined enable more accurate identification proteins, reporting between ligand individual targets. Using pan-specific kinase inhibitor, staurosporine, demonstrated 36% improvement in screening sensitivity over traditional profiling (TPP) comparable latest two-dimensional TPP. Finally, successfully applied delineate landscape perfluorooctanesulfonic acid (PFOS), persistent organic pollutant that is hard perform on, revealed several potential might account toxicities PFOS.

Language: Английский

Citations

25
Stability-based approaches in chemoproteomics DOI Creative Commons
Amy L. George, María Emilia Dueñas, José Luis Marín‐Rubio

et al.

Expert Reviews in Molecular Medicine, Journal Year: 2024, Volume and Issue: 26

Published: Jan. 1, 2024

Abstract Target deconvolution can help understand how compounds exert therapeutic effects and accelerate drug discovery by helping optimise safety efficacy, revealing mechanisms of action, anticipate off-target identifying opportunities for expansion. Chemoproteomics, a combination chemical biology with mass spectrometry has transformed target deconvolution. This review discusses modification-free chemoproteomic approaches that leverage the change in protein thermodynamics induced small molecule ligand binding. Unlike modification-based methods relying on enriching specific targets, these offer proteome-wide evaluations, driven advancements sensitivity, increasing proteome coverage quantitation methods. Advances based denaturation/precipitation thermal or denaturation, protease degradation are evaluated, emphasising evolving landscape chemoproteomics its potential impact future drug-development strategies.

Language: Английский

Citations

5
Large-scale characterization of drug mechanism of action using proteome-wide thermal shift assays DOI Creative Commons
Jonathan G. Van Vranken, Jiaming Li,

Julian Mintseris

et al.

eLife, Journal Year: 2024, Volume and Issue: 13

Published: March 26, 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

Citations

4
GPMelt: A hierarchical Gaussian process framework to explore the dark meltome of thermal proteome profiling experiments DOI Creative Commons
Cécile Le Sueur, Magnus Rattray, Mikhail M. Savitski

et al.

PLoS Computational Biology, Journal Year: 2024, Volume and Issue: 20(9), P. e1011632 - e1011632

Published: Sept. 27, 2024

Thermal proteome profiling (TPP) is a wide technology that enables unbiased detection of protein drug interactions as well changes in post-translational state proteins between different biological conditions. Statistical analysis temperature range TPP (TPP-TR) datasets relies on comparing melting curves, describing the amount non-denatured function temperature, conditions (e.g. presence or absence drug). However, state-of-the-art models are restricted to sigmoidal behaviours while unconventional representing up 50% TPP-TR datasets, have recently been shown carry important information. We present novel statistical framework, based hierarchical Gaussian process and named GPMelt, make with respect profiles proteins. GPMelt scales multiple conditions, extension model deeper hierarchies (i.e. additional sub-levels) allows deal complex protocols. Collectively, our framework extends for both peptide level offering access thousands previously excluded curves thus substantially increasing coverage ability uncover new biology.

Language: Английский

Citations

3
Proximity Labeling, Quantitative Proteomics, and Biochemical Studies Revealed the Molecular Mechanism for the Inhibitory Effect of Indisulam on the Proliferation of Gastric Cancer Cells DOI
Jiaqi Lü,

Honglv Jiang,

Dan Li

et al.

Journal of Proteome Research, Journal Year: 2021, Volume and Issue: 20(9), P. 4462 - 4474

Published: Aug. 23, 2021

Indisulam exhibits antitumor activity against several cancer cells. Although the DCAF15-indisulam-RBM39 axis has been well documented in inhibition of cell growth, it is unknown whether RBM39 degradation alone mechanism action indisulam. Here, we verified inhibitory effect indisulam on proliferation gastric cells and its dependence DCAF15. Proximity-dependent biotin labeling with TurboID quantitative proteomics revealed that indeed promoted interaction between DCAF15 RBM39. Immunoblotting immunofluorescence also ubiquitin-mediated colocalized nucleus. knockdown almost completely abolished indisulam-mediated reduction. Further eliminated upon treatment. tumor tissues confirmed downregulation by Database analysis unveiled was highly expressed high expression significantly shortened survival time patients. Taken together, demonstrated enhanced ubiquitination promoting DCAF15, thus inhibiting This work may provide valuable information for drug discovery through proteolysis targeting chimeras. MS data were deposited ProteomeXchange (Dataset identifier: PXD024168).

Language: Английский

Citations

21
ProSAP: a GUI software tool for statistical analysis and assessment of thermal stability data DOI
Hongchao Ji, Xue Lu, Zhenxiang Zheng

et al.

Briefings in Bioinformatics, Journal Year: 2022, Volume and Issue: 23(3)

Published: March 2, 2022

Abstract The Cellular Thermal Shift Assay (CETSA) plays an important role in drug-target identification, and statistical analysis is a crucial step significantly affecting conclusion. We put forward ProSAP (Protein Stability Analysis Pod), open-source, cross-platform user-friendly software tool, which provides multiple methods for thermal proteome profiling (TPP) analysis, nonparametric (NPA), integral solubility alteration isothermal shift assay (iTSA). For testing the performance of ProSAP, we processed several datasets compare different algorithms. Overall, TPP more accurate with fewer false positive targets, but NPA are flexible free from parameters. iTSA, edgeR DESeq2 identify true targets than t-test Limma, when it comes to ranking, four show not much difference. available at https://github.com/hcji/ProSAP https://zenodo.org/record/5763315.

Language: Английский

Citations

15
Improved in situ characterization of protein complex dynamics at scale with thermal proximity co-aggregation DOI Creative Commons
Siyuan Sun, Zhenxiang Zheng, Jun Wang

et al.

Nature Communications, Journal Year: 2023, Volume and Issue: 14(1)

Published: Nov. 24, 2023

Cellular activities are carried out vastly by protein complexes but large repertoire of remains functionally uncharacterized which necessitate new strategies to delineate their roles in various cellular processes and diseases. Thermal proximity co-aggregation (TPCA) is readily deployable characterize complex dynamics situ at scale. We develop a version termed Slim-TPCA that uses fewer temperatures increasing throughputs over 3X, with scoring metrics statistical evaluation result minimal compromise coverage detect more relevant complexes. Less samples needed, batch effects minimized while cost reduced two orders magnitude. applied profile K562 cells under different duration glucose deprivation. More found dissociated, accordance the expected downregulation most activities, include 55S ribosome respiratory mitochondria revealing utility TPCA study organelles. Protein transport degradation increasingly assembled unveiling involvement metabolic reprogramming during In summary, an efficient strategy for characterization scale across conditions, available as Python package https://pypi.org/project/Slim-TPCA/ .

Language: Английский

Citations

8
Network integration of thermal proteome profiling with multi-omics data decodes PARP inhibition DOI Creative Commons
Mira Lea Burtscher,

Stephan Gade,

Martín Garrido‐Rodriguez

et al.

Molecular Systems Biology, Journal Year: 2024, Volume and Issue: 20(4), P. 458 - 474

Published: March 7, 2024

Abstract Complex disease phenotypes often span multiple molecular processes. Functional characterization of these processes can shed light on mechanisms and drug effects. Thermal Proteome Profiling (TPP) is a mass-spectrometry (MS) based technique assessing changes in thermal protein stability that serve as proxies functional changes. These unique insights TPP complement those obtained by other omics technologies. Here, we show how be integrated with phosphoproteomics transcriptomics network-based approach using COSMOS, multi-omics integration framework, to provide an view transcription factors, kinases proteins altered stability. This allowed us recover consequences Poly (ADP-ribose) polymerase (PARP) inhibition ovarian cancer cells cell cycle DNA damage response well interferon hippo signaling. We found offers complementary perspective data modalities, its obtain more complete overview PARP inhibition. anticipate this strategy used integrate proteomics study

Language: Английский

Citations

3
Streamlined analysis of drug targets by proteome integral solubility alteration indicates organ-specific engagement DOI Creative Commons
Tanveer S. Batth, Marie Locard‐Paulet, Nadezhda T. Doncheva

et al.

Nature Communications, Journal Year: 2024, Volume and Issue: 15(1)

Published: Oct. 16, 2024

Proteins are the primary targets of almost all small molecule drugs. However, even most selectively designed drugs can potentially target several unknown proteins. Identification potential drug facilitate design new and repurposing existing ones. Current state-of-the-art proteomics methodologies enable screening thousands proteins against a limited number molecules. Here we report development label-free quantitative approach that enables proteome-wide organic molecules in scalable, reproducible, rapid manner by streamlining proteome integral solubility alteration (PISA) assay. We used rat organs ex-vivo to determine organ specific medical enzyme inhibitors identify for common such as Ibuprofen. Finally, global profiling revealed overarching trends how affect through either direct or indirect protein interactions.

Language: Английский

Citations

3
A Comparison of Two Stability Proteomics Methods for Drug Target Identification in OnePot 2D Format DOI
Yingrong Xu, Graham M. West,

Mario Abdelmessih

et al.

ACS Chemical Biology, Journal Year: 2021, Volume and Issue: 16(8), P. 1445 - 1455

Published: Aug. 10, 2021

Stability proteomics techniques that do not require drug modifications have emerged as an attractive alternative to affinity purification methods in target engagement studies. Two representative include the chemical-denaturation-based SPROX (Stability of Proteins from Rates Oxidation), which utilizes peptide-level quantification and thermal-denaturation-based TPP (Thermal Proteome Profiling), protein-level quantification. Recently, "OnePot" strategy was adapted for both increase throughput. When combined with 2D setup measures denaturation dose dimensions, OnePot format offers improved analysis specificity higher resource efficiency. However, a systematic evaluation comparison between are still lacking. Here, we performed identify protein targets well-studied pan-kinase inhibitor staurosporine K562 lysate, curve-fitting formats. We found provided ∼10× throughput, achieved ∼1.6× coverage involves more straightforward data analysis. also compared current "gold-standard" stability technique format. The is ∼1.5 fold SPROX; however, domain-level information, identifies comparable numbers kinase hits, has signal (R value), requires ∼3× less MS time. Unique hits encompass higher-molecular-weight proteins, unique atypical kinases. discuss hit stratification prioritization strategies promote efficiency followup.

Language: Английский

Citations

20