ThermoTargetMiner as a proteome integral solubility alteration target database for prospective drugs against lung cancer DOI Creative Commons

Hezheng Lyu,

Hassan Gharibi, Bohdana Sokolova

et al.

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

Published: Aug. 6, 2024

Abstract Knowledge of the targets therapeutic compounds is vital for understanding their action mechanisms and side effects, but such valuable data seldom available. The multiple complementary techniques needed comprehensive target characterization must combine reliability with sufficient analysis throughput. Here, we leveraged Proteome Integral Solubility Alteration (PISA) assay to comprehensively characterize 67 approved drugs candidate against lung cancer. was performed on two cell lines representing different cancer phenotypes novel 77% tested molecules were found. Comparison protein solubility shifts in lysate vs. living cells highlighted directly interacting compounds. As PISA now joining arsenal fast reliable techniques, presented database, ThermoTargetMiner, will become a useful resource research.

Language: Английский

Hexokinase detachment from mitochondria drives the Warburg effect to support compartmentalized ATP production DOI Open Access
Kimberly S. Huggler,

Carlos A. Mellado Fritz,

Kyle M. Flickinger

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 8, 2025

ABSTRACT Hexokinase (HK) catalyzes the synthesis of glucose-6-phosphate, marking first committed step glucose metabolism. Most cancer cells express two homologous isoforms (HK1 and HK2) that can each bind to outer mitochondrial membrane (OMM). CRISPR screens across hundreds cell lines indicate both are dispensable for growth in traditional culture media. By contrast, HK2 deletion impairs Human Plasma-Like Medium (HPLM). Here, we find is required maintain sufficient cytosolic (OMM-detached) HK activity under conditions enhance HK1 binding OMM. Notably, OMM-detached rather than OMM-docked promotes “aerobic glycolysis” (Warburg effect), an enigmatic phenotype displayed by most proliferating cells. We show several proposed theories this cannot explain dependence instead severely glycolytic ATP production with little impact on total yield HPLM. Our results reveal a basis conditional essentiality suggest demand compartmentalized underlies Warburg effect.

Language: Английский

Citations

0
Discovery of RNA-Protein Molecular Clamps Using Proteome-Wide Stability Assays DOI
Stanley I. Goldstein, Alice C. Fan, Zihao Wang

et al.

Journal of Proteome Research, Journal Year: 2025, Volume and Issue: unknown

Published: March 12, 2025

Uncompetitive inhibition is an effective strategy for suppressing dysregulated enzymes and their substrates, but discovery of suitable ligands depends on often-unavailable structural knowledge serendipity. Hence, despite surging interest in mass spectrometry-based target identification, proteomic studies substrate-dependent engagement remain sparse. Herein, we describe a the ligand binding. Using proteome integral solubility alteration (PISA) assays, show that simple biochemical additives can enable detection RNA-protein-small molecule complexes native cell lysates. We apply our approach to rocaglates, molecules specifically clamp RNA eukaryotic translation initiation factor 4A (eIF4A), DEAD-box helicase 3X (DDX3X), potentially other members (DDX) family. To identify unexpected interactions, used class-specific thermal window compared ATP analog base dependencies key rocaglate-DDX interactions. report novel DDX targets high-profile rocaglates-including clinical candidate Zotatifin-and validate findings using limited proteolysis-mass spectrometry fluorescence polarization (FP) experiments. also provide insight into divergent DDX3X affinities between synthetic rocaglates. Taken together, study provides model screening uncompetitive inhibitors chemical proteomics uncovers actionable clamping targets, clearing path toward characterization molecular clamps associated helicases.

Language: Английский

Citations

0
Proteomic Discovery of RNA-Protein Molecular Clamps Using a Thermal Shift Assay with ATP and RNA (TSAR) DOI Creative Commons
Stanley I. Goldstein, Alice C. Fan,

Zi-Hao Wang

et al.

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

Published: April 19, 2024

Uncompetitive inhibition is an effective strategy for suppressing dysregulated enzymes and their substrates, but discovery of suitable ligands depends on often-unavailable structural knowledge serendipity. Hence, despite surging interest in mass spectrometry-based target identification, proteomic studies substrate-dependent engagement remain sparse. Herein, we describe the Thermal Shift Assay with ATP RNA (TSAR) as a template proteome-wide ligand binding. Using thermal shift assays, show that simple biochemical additives can facilitate detection native cell lysates. We apply our approach to rocaglates, family molecules specifically clamp eukaryotic translation initiation factor 4A (eIF4A), DEAD-box helicase 3X (DDX3X), potentially other members (DDX) helicases. To identify unexpected interactions, optimized class-specific denaturation window evaluated analog probe dependencies key rocaglate-DDX interactions. report novel DDX targets rocaglate clamping spectrum, confirm DDX3X common several widely studied analogs, provide insights into divergent affinities between synthetic rocaglates. independently validate high-profile including clinical candidate Zotatifin (

Language: Английский

Citations

2
Identification of Rocaglate Acyl Sulfamides as Selective Inhibitors of Glioblastoma Stem Cells DOI Creative Commons
Zihao Wang, Ritesh Thakare, Shalaka Chitale

et al.

ACS Central Science, Journal Year: 2024, Volume and Issue: 10(8), P. 1640 - 1656

Published: Aug. 8, 2024

Glioblastoma (GBM) is the most aggressive and frequently occurring type of malignant brain tumor in adults. The initiation, progression, recurrence tumors are known to be driven by a small subpopulation cells as tumor-initiating or cancer stem (CSCs). GBM CSCs play pivotal role orchestrating drug resistance relapse. As prospective avenue for intervention, targeted suppression holds considerable promise. In this study, we found that rocaglates, compounds which inhibit translation

Language: Английский

Citations

2
Lineage-specific proteome remodeling of diverse lung cancer cells by targeted epigenetic inhibitors DOI Creative Commons
Chuwei Lin,

Catherine M Sniezek,

Christopher D. McGann

et al.

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

Published: May 28, 2024

Summary Epigenetic inhibitors exhibit powerful antiproliferative and anticancer activities. However, cellular responses to small-molecule epigenetic inhibition are heterogenous dependent on factors such as the genetic background, metabolic state, on-/off-target engagement of individual compounds. The molecular study extent this heterogeneity often measures changes in a single cell line or using small number To more comprehensively profile effects perturbations their influence these heterogeneous responses, we present resource based quantification chromatin, proteome, transcriptome remodeling due histone deacetylase (HDACi) non-isogenic lines. Through quantitative profiling 10,621 proteins, data reveal coordinated HDACi treated cancer cells. HDACi-regulated proteins differ greatly across lines with consistent (JUN, MAP2K3, CDKN1A) divergent (CCND3, ASF1B, BRD7) cell-state effectors. Together provide valuable insight into cell-type driven that must be taken consideration when monitoring culture models

Language: Английский

Citations

1
The proteomic landscape and temporal dynamics of mammalian gastruloid development DOI Creative Commons
Riddhiman K. Garge,

Valerie Lynch,

Rose Fields

et al.

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

Published: Sept. 7, 2024

Gastrulation is the highly coordinated process by which early embryo breaks symmetry, establishes germ layers and a body plan, sets stage for organogenesis. As mammalian development challenging to study

Language: Английский

Citations

1
Exploring Mechanisms of Action in Combinatorial Therapy through Stability/Solubility Alterations: Advancing AML Treatment DOI Creative Commons
Elham Gholizadeh, Ehsan Zangene, Uladzislau Vadadokhau

et al.

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

Published: Nov. 8, 2024

Abstract Acute myeloid leukemia (AML) is an aggressive blood cancer with a poor prognosis. Although treatments like allogeneic hematopoietic stem cell transplantation and high-dose chemotherapy can potentially cure younger patients in some cases, challenges such as relapse treatment-related toxicities remain significant. Combination therapy has been cornerstone AML treatment, offering enhanced efficacy by leveraging the synergistic effects of multiple agents. However, high toxicity levels genetic heterogeneity complicate identification effective universally applicable drug regimens. To address these challenges, we introduce CoPISA workflow (Proteome Integral Solubility/Stability Alteration Analysis for Combinations), innovative method designed to study drug-target interactions specifically within combination therapies. detects changes protein solubility/stability that occur only when two drugs are used together, revealing cooperative mechanisms single-drug cannot achieve. We applied this highly low-toxicity combinations AML, previously introduced our group: LY3009120-sapanisertib (LS) ruxolitinib-ulixertinib (RU). utilizes advanced proteomic tools investigate both primary secondary target effects, providing deeper understanding how therapies influence signaling pathways overcome resistance. Furthermore, propose novel concept termed “conjunctional inhibition”, where combined action induces biological responses be achieved individual This approach introduces transformation designing combinatorial offers new directions more other complex diseases.

Language: Английский

Citations

1
ThermoTargetMiner as a proteome integral solubility alteration target database for prospective drugs against lung cancer DOI Creative Commons

Hezheng Lyu,

Hassan Gharibi, Bohdana Sokolova

et al.

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

Published: Aug. 6, 2024

Abstract Knowledge of the targets therapeutic compounds is vital for understanding their action mechanisms and side effects, but such valuable data seldom available. The multiple complementary techniques needed comprehensive target characterization must combine reliability with sufficient analysis throughput. Here, we leveraged Proteome Integral Solubility Alteration (PISA) assay to comprehensively characterize 67 approved drugs candidate against lung cancer. was performed on two cell lines representing different cancer phenotypes novel 77% tested molecules were found. Comparison protein solubility shifts in lysate vs. living cells highlighted directly interacting compounds. As PISA now joining arsenal fast reliable techniques, presented database, ThermoTargetMiner, will become a useful resource research.

Language: Английский

Citations

0