Chemical Proteomics Research Articles

FAT-switch-based quantitative S-nitrosoproteomics reveals a key role of GSNOR1 in regulating ER functions

Reversible protein S-nitrosylation regulates a wide range of biological functions and physiological activities in plants. However, it is challenging to quantitively determine the S-nitrosylation targets and dynamics in vivo. In this study, we develop a highly sensitive and efficient fluorous affinity tag-switch (FAT-switch) chemical proteomics approach for S-nitrosylation peptide enrichment and detection. We quantitatively compare the global S-nitrosylation profiles in wild-type Arabidopsis and gsnor1/hot5/par2 mutant using this approach, and identify 2,121 S-nitrosylation peptides in 1,595 protein groups, including many previously unrevealed S-nitrosylated proteins. These are 408 S-nitrosylated sites in 360 protein groups showing an accumulation in hot5-4 mutant when compared to wild type. Biochemical and genetic validation reveal that S-nitrosylation at Cys337 in ER OXIDOREDUCTASE 1 (ERO1) causes the rearrangement of disulfide, resulting in enhanced ERO1 activity. This study offers a powerful and applicable tool for S-nitrosylation research, which provides valuable resources for studies on S-nitrosylation-regulated ER functions in plants.

Identification of mitochondrial proteins associated with cyanine 5 using a combination of affinity-based photocrosslinking with bio-orthogonal supramolecular latching

Visualization of mitochondria with cyanine dyes has been exploited to study the functions of mitochondria. However, proteins interacting with cyanine dyes have not been investigated clearly. Here, we report a chemical proteomics approach for the identification of mitochondrial proteins associated with cyanine dyes. This approach uses affinity-based photocrosslinking of Cy5-diazirine-alkyne (Cy5-DAyne) in combination with selective enrichment of target proteins by bio-orthogonal supramolecular latching based on unique host-guest interaction between cucurbit[7]uril (CB[7]) and its selected guest such as adamantylammonium (AdA). The enriched proteins then were analyzed using LC–MS/MS to provide a list of proteins closely associated with the Cy5 motif. This study suggests that the proteomic environment around the Cy5 motif is highly associated with energy production processes, including the respiratory chain, ATP synthesis, and fatty acid oxidization. Moreover, it demonstrates the potential of this chemical proteomic approach for the investigation of other organellar proteins using different selective dyes.

A Chemical Proteomics Approach to Discover Regulators of Innate Immune Signaling.

Innate immune pathways are tightly regulated to balance an appropriate response to infectious agents and tolerable levels of inflammation. Dysregulation of innate immune pathways can lead to severe autoinflammatory disorders or susceptibility to infections. Here, we aimed to identify kinases in common cellular pathways that regulate innate immune pathways by combining small-scale kinase inhibitor screening with quantitative proteomics. We found that inhibitors of kinases ATM, ATR, AMPK, and PLK1 reduced the induction of interferon-stimulated gene expression in response to innate immune pathway activation by poly(I:C) transfection. However, siRNA depletion of these kinases did not validate findings with kinase inhibitors, suggesting that off-target effects may explain their activities. We mapped the effects of kinase inhibitors to various stages in innate immune pathways. Determining the mechanisms by which kinase inhibitors antagonize these pathways may illuminate novel mechanisms of innate immune pathway control.

Discovery of chemical probes that perturb protein complexes using size exclusion chromatography and chemical proteomics

Most human proteins lack small-molecule ligands, rendering these proteins "undruggable." In this issue of Molecular Cell, Lazear etal.1 develop a novel chemical proteomic screening platform and discover new chemical probes targeting previously undruggable protein complexes.

Chemoproteomics Reveals the Pan-HER Kinase Inhibitor Neratinib To Target an Arabidopsis Epoxide Hydrolase Related to Phytohormone Signaling.

Plant phytohormone pathways are regulated by an intricate network of signaling components and modulators, many of which still remain unknown. Here, we report a forward chemical genetics approach for the identification of functional SA agonists in Arabidopsis thaliana that revealed Neratinib (Ner), a covalent pan-HER kinase inhibitor drug in humans, as a modulator of SA signaling. Instead of a protein kinase, chemoproteomics unveiled that Ner covalently modifies a surface-exposed cysteine residue of Arabidopsis epoxide hydrolase isoform 7 (AtEH7), thereby triggering its allosteric inhibition. Physiologically, the Ner application induces jasmonate metabolism in an AtEH7-dependent manner as an early response. In addition, it modulates PATHOGENESIS RELATED 1 (PR1) expression as a hallmark of SA signaling activation as a later effect. AtEH7, however, is not the exclusive target for this physiological readout induced by Ner. Although the underlying molecular mechanisms of AtEH7-dependent modulation of jasmonate signaling and Ner-induced PR1-dependent activation of SA signaling and thus defense response regulation remain unknown, our present work illustrates the powerful combination of forward chemical genetics and chemical proteomics for identifying novel phytohormone signaling modulatory factors. It also suggests that marginally explored metabolic enzymes such as epoxide hydrolases may have further physiological roles in modulating signaling.

A Turn-On Fluorescent Amino Acid Sensor Reveals Chloroquine's Effect on Cellular Amino Acids via Inhibiting Cathepsin L.

Maintaining homeostasis of metabolites such as amino acids is critical for cell survival. Dysfunction of nutrient balance can result in human diseases such as diabetes. Much remains to be discovered about how cells transport, store, and utilize amino acids due to limited research tools. Here we developed a novel, pan-amino acid fluorescent turn-on sensor, NS560. It detects 18 of the 20 proteogenic amino acids and can be visualized in mammalian cells. Using NS560, we identified amino acids pools in lysosomes, late endosomes, and surrounding the rough endoplasmic reticulum. Interestingly, we observed amino acid accumulation in large cellular foci after treatment with chloroquine, but not with other autophagy inhibitors. Using a biotinylated photo-cross-linking chloroquine analog and chemical proteomics, we identified Cathepsin L (CTSL) as the chloroquine target leading to the amino acid accumulation phenotype. This study establishes NS560 as a useful tool to study amino acid regulation, identifies new mechanisms of action of chloroquine, and demonstrates the importance of CTSL regulation of lysosomes.

Developing an Affinity-Based Chemical Proteomics Method to In Situ Capture Amorphous Aggregated Proteome and Profile Its Heterogeneity in Stressed Cells

Stress induced amorphous proteome aggregation is a hallmark for diseased cells, with the proteomic composition intimately associated with disease pathogenicity. Due to its particularly dynamic, reversible, and dissociable nature, as well as lack of specific recognition anchor, it is difficult to capture aggregated proteins in situ. In this work, we develop a chemical proteomics method (AggLink) to capture amorphous aggregated proteins in live stressed cells and identify the proteomic contents using LC-MS/MS. Our method relies on an affinity-based chemical probe (AggLink 1.0) that is optimized to selectively bind to and covalently label amorphous aggregated proteins in live stressed cells. Especially, chaotrope-compatible ligation enables effective enrichment of labeled aggregated proteins under urea denaturation and dissociation conditions. Compared to conventional fractionation-based method to profile aggregated proteome, our method showed improved enrichment selectivity, detection sensitivity, and identification accuracy. In HeLa cells, the AggLink method reveals the constituent heterogeneity of aggregated proteome induced by inhibition of pro-folding (HSP90) or pro-degradation (proteasome) pathway, which uncovers a synergistic strategy to reduce cancer cell viability. In addition, the unique fluorogenicity of our probe upon labeling aggregated proteome detects its cellular location and morphology. Together, the AggLink method may help to expand our knowledge of the previously nontargetable amorphous aggregated proteome.

Abstract 3839: Discovery of GSTZ1 as a novel target for drug refractory non-small cell lung cancer by using fragment-based chemical proteomics

Abstract A subset of non-small cell lung cancer (NSCLC) patients respond poorly to clinical drugs targeting defined oncogenic driver mutations. To improve the efficacy of these drugs in drug refractory NSCLC, the identification of supporting therapeutic targets and discovery of the binding molecules are urgently needed. Photoreactive fragment-like probes can discover target proteins that constitute novel cellular vulnerabilities and provide the chemical basis for drug discovery. However, the rational design of the fragment probe library, target prioritization, and sample throughput are still challenging. We applied a structurally diverse panel of BioCore fragments fully functionalized with orthogonal diazirine and alkyne moieties. These probes were used for protein crosslinking in live cells and subsequent target identification through label-free quantitative LC-MS/MS analysis. High-confidence targets were queried against a pharmacogenomic database (DepMap) and prioritized through multiple cross-comparison with other probes. The top-ranked probe-binding target was validated using a competitive affinity assay, an enzymatic activity assay, and RNA interference. Proteome-wide tyrosine reactivity was profiled using sulfur-triazole exchange chemistry (SuTEx). The tyrosine phosphorylation of proteins was investigated using a pY-100 antibody and western blotting. Using sotorasib-refractory KRAS G12C H1792 lung cancer cells, we identified 932 probe-enriched proteins from panel-wide cross-comparisons suggesting the high potential of exploring the ligandable proteome. We also performed intensive cross-comparison analysis and identified 31 unique high-confidence targets, with glutathione S-transferase zeta 1 (GSTZ1) identified as a unique target of probe 17. We found that high expression of GSTZ1 was significantly associated with poorer NSCLC patient survival. Probe 17 was validated to physically bind to GSTZ1 and inhibit the enzymatic activity of GSTZ1. In addition, GSTZ1 gene knockdown sensitized drug-refractory NSCLC cells with KRAS G12C, FGFR1 amplification, and DDR2 mutation to clinical targeted drugs and induced more cell apoptosis in combination with these targeted drugs. SuTEx proteomics suggests modulation of drug resistance pathways leading to the identification of altered KRAS and FGFR1 tyrosine phosphorylation by GSTZ1, which provides a functional insight into the mechanism of drug sensitization. We developed a chemical biology workflow for the simultaneous discovery of high-confidence targets and their binding probe molecules, such as probe 17 and GSTZ1. GSTZ1 was found to cooperate with oncogenic alterations in supporting refractory NSCLC cell survival signaling, which may form the biological basis for developing novel GSTZ1 inhibitors to improve the therapeutic efficacy of oncogene-directed targeted drugs. Citation Format: Yi Liao, Sean Chin Chan, Eric A. Welsh, Bin Fang, Luxin Sun, Ernst Schönbrunn, John M. Koomen, Derek R. Duckett, Eric B. Haura, Andrii Monastyrskyi, Uwe Rix. Discovery of GSTZ1 as a novel target for drug refractory non-small cell lung cancer by using fragment-based chemical proteomics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3839.

Avidity-Based Method for the Efficient Generation of Monoubiquitinated Recombinant Proteins.

Monoubiquitination of proteins governs diverse physiological processes, and its dysregulation is implicated in multiple pathologies. The difficulty of preparing sufficient material often complicates the biophysical studies of monoubiquitinated recombinant proteins. Here we describe a robust avidity-based method that overcomes this problem. As a proof-of-concept, we produced milligram quantities of two monoubiquitinated targets, Parkinson's protein α-synuclein and ESCRT-protein ALIX, using NEDD4-family E3 ligases. Monoubiquitination hotspots were identified by quantitative chemical proteomics. Using FRAP and dye-binding assays, we uncovered strikingly opposite effects of monoubiquitination on the phase separation and fibrillization properties of these two amyloidogenic proteins, reflecting differences in their intermolecular interactions, thereby providing unique insights into the impact of monoubiquitination on protein aggregation.

Hierarchical multi-omics data integration and modeling predict cell-specific chemical proteomics and drug responses

Drug-induced phenotypes result from biomolecular interactions across various levels of a biological system.Characterization of pharmacological actions therefore requires integration of multi-omics data. Proteomics profiles, which may more directly reflect disease mechanisms and biomarkers than transcriptomics, have not been widely exploited due to data scarcity and frequent missing values. A computational method for inferring drug-induced proteome patterns would therefore enable progress in systems pharmacology. To predict the proteome profiles and corresponding phenotypes of an uncharacterized cell or tissue type that has been disturbed by an uncharacterized chemical, we developed an end-to-end deep learning framework: TransPro. TransPro hierarchically integrated multi-omics data, in line with the central dogma of molecular biology. Our in-depth assessments of TransPro's predictions of anti-cancer drug sensitivity and drug adverse reactions reveal that TransPro's accuracy is on par with that of experimental data. Hence,TransPro may facilitate the imputation of proteomics data and compound screening in systems pharmacology.

Pepstatin-Based Probes for Photoaffinity Labeling of Aspartic Proteases and Application to Target Identification.

Aspartic proteases are a small class of proteases implicated in a wide variety of human diseases. Covalent chemical probes for photoaffinity labeling (PAL) of these proteases are underdeveloped. We here report a full on-resin synthesis of clickable PAL probes based on the natural product inhibitor pepstatin incorporating a minimal diazirine reactive group. The position of this group in the inhibitor determines the labeling efficiency. The most effective probes sensitively detect cathepsin D, a biomarker for breast cancer, in cell lysates. Moreover, through chemical proteomics experiments and deep learning algorithms, we identified sequestosome-1, an important player in autophagy, as a direct interaction partner and substrate of cathepsin D.

Chemical proteomic profiling with photoaffinity labeling strategy identifies antimalarial targets of artemisinin

Present research on the antimalarial mechanisms of artemisinin (ART) is mainly focused on covalent drug binding targets alkylated by free radicals, while non-covalent binding targets have rarely been reported. Here, we developed a novel photoaffinity probe of ART to globally capture and identify the antimalarial target proteins of ART through chemical proteomics. The results demonstrated that ART can bind to parasite proteins by both covalent and non-covalent modification, and these may jointly contribute to the antimalarial effects. Our work enriches the research on the antimalarial targets of ART, and provides a new perspective for further exploring the antimalarial mechanism of ART.

Remodeling oncogenic transcriptomes by small molecules targeting NONO.

Much of the human proteome is involved in mRNA homeostasis, but most RNA-binding proteins lack chemical probes. Here we identify electrophilic small molecules that rapidly and stereoselectively decrease the expression of transcripts encoding the androgen receptor and its splice variants in prostate cancer cells. We show by chemical proteomics that the compounds engage C145 of the RNA-binding protein NONO. Broader profiling revealed that covalent NONO ligands suppress an array of cancer-relevant genes and impair cancer cell proliferation. Surprisingly, these effects were not observed in cells genetically disrupted for NONO, which were instead resistant to NONO ligands. Reintroduction of wild-type NONO, but not a C145S mutant, restored ligand sensitivity in NONO-disrupted cells. The ligands promoted NONO accumulation in nuclear foci and stabilized NONO-RNA interactions, supporting a trapping mechanism that may prevent compensatory action of paralog proteins PSPC1 and SFPQ. These findings show that NONO can be co-opted by covalent small molecules to suppress protumorigenic transcriptional networks.

Different chemical proteomic approaches to identify the targets of lapatinib

The process of identifying the protein targets and off-targets of a biologically active compound is of great importance in modern drug discovery. Various chemical proteomics approaches have been established for this purpose. To compare the different approaches, and to understand which method would provide the best results, we have chosen the EGFR inhibitor lapatinib as an example molecule. Lapatinib derivatives were designed using linkers with motifs, including amino (amidation), alkyne (click chemistry) and the diazirine group (photo-affinity). These modified lapatinib analogues were validated for their ability to inhibit EGFR activity in vitro and were shown to pull down purified recombinant EGFR protein. In all of the approaches evaluated here, we identified EGFR as the main protein target from the lysate of immortalised cell line expressing EGFR, thus validating its potential use to identify unknown protein targets. Taken together, the results reported here give insight into the cellular activities of lapatinib.

The Promises of Proteomics and Metabolomics for Unravelling the Mechanism and Side Effect Landscape of Beta-Adrenoceptor Antagonists in Cardiovascular Therapeutics.

Cardiovascular medicine witnessed notable advances for the past decade. Multiomics research offers a new lens for precision/personalized medicine for existing and emerging drugs used in the cardiovascular clinic. Beta-blockers are vital in treating hypertension and chronic heart failure. However, clinical use of beta-blockers is also associated with side effects and person-to-person variations in their pharmacokinetics and pharmacodynamics. A comprehensive understanding of the mechanisms that underpin the side effect landscape of beta-blockers is imperative to optimize their therapeutic value. In addition, current research emphasizes the circadian clock's vital roles in regulating pharmacological parameters. Administration of the beta-blockers at specific dosing times could potentially improve their effectiveness and reduce their toxic effects. The rapid development of mass spectrometry technologies with chemical proteomics and thermal proteome profiling methods has also substantially advanced our understanding of underlying side effects mechanisms by unbiased deconvolution of drug targets and off-targets. Metabolomics is steadily demonstrating its utility for conducting mechanistic and toxicological analyses of pharmacological compounds. This article discusses the promises of cutting-edge proteomics and metabolomics approaches to investigate the molecular targets, mechanism of action, adverse effects, and dosing time dependency of beta-blockers.

ACR-Based Probe for the Quantitative Profiling of Histidine Reactivity in the Human Proteome.

The quantitative profiling of residue reactivity in proteins promotes the discovery of covalent druggable targets for precise therapy. Histidine (His) residues, accounting for more than 20% of the active sites in enzymes, have not been systematically characterized for their reactivity, due to lack of labeling probes. Herein, we report a chemical proteomics platform for the site-specific quantitative analysis of His reactivity by combination of acrolein (ACR) labeling and reversible hydrazine chemistry enrichment. Based on this platform, in-depth characterization of His residues was conducted for the human proteome, in which the rich content of His residues (>8200) was quantified, including 317 His hyper-reactive residues. Intriguingly, it was observed that the hyper-reactive residues were less likely to be the sites for phosphorylation, and the possible mechanism of this antagonistic effect still needs to be evaluated in further research. Based on the first comprehensive map of His residue reactivity, many more residues could be adopted as the bindable sites to disrupt the activities of a diverse number of proteins; meanwhile, ACR derivatives could also be used as a novel reactive warhead in the development of covalent inhibitors.

Benjamin F. Cravatt III – Chemical Proteomics Trailblazer

AbstractThe advances in large‐scale bioanalytical methods and computational approaches have afforded tremendous insight into fundamental mechanisms of health and disease as well as provided new opportunities for therapeutic development. However, the majority of proteins in humans remain undrugged or lack chemical probes to explore their functions in vivo. The pioneering studies on activity‐based protein profiling (ABPP) by Ben Cravatt and his lab members have provided a systematic and generalizable approach to address this major challenge in chemical biology and drug discovery. Notably, the Cravatt laboratory paved the way for employing activity‐ and reactivity‐based chemical probes with quantitative proteomics to identify small molecule protein targets and discover unpredicted protein hot spots for native and synthetic ligands. Importantly, these chemical proteomic methods have revealed new protein targets and mechanisms of action for drug discovery and have been widely adopted by many other laboratories around the world to discover new biology and pharmacology. The recognition of Ben Cravatt as a co‐recipient of the 2022 Wolf Prize in Chemistry for this trailblazing work in chemical proteomics is most well‐deserved.

Effect of Dl-3-n-butylphthalide on mitochondrial Cox7c in models of cerebral ischemia/reperfusion injury.

Several studies have demonstrated the protective effect of dl-3-n-Butylphthalide (NBP) against cerebral ischemia, which may be related to the attenuation of mitochondrial dysfunction. However, the specific mechanism and targets of NBP in cerebral ischemia/reperfusion remains unclear. In this study, we used a chemical proteomics approach to search for targets of NBP and identified cytochrome C oxidase 7c (Cox7c) as a key interacting target of NBP. Our findings indicated that NBP inhibits mitochondrial apoptosis and reactive oxygen species (ROS) release and increases ATP production through upregulation of Cox7c. Subsequently, mitochondrial respiratory capacity was improved and the HIF-1α/VEGF pathway was upregulated, which contributed to the maintenance of mitochondrial membrane potential and blood brain barrier integrity and promoting angiogenesis. Therefore, our findings provided a novel insight into the mechanisms underlying the neuroprotective effects of NBP, and also proposed for the first time that Cox7c exerts a critical role by protecting mitochondrial function.

Identification of a novel heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) ligand that disrupts HnRNPA2B1/nucleic acid interactions to inhibit the MDMX-p53 axis in gastric cancer

Gastric carcinoma is a highly malignant tumor that still lacks effective molecular targets. Heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) is an essential oncogenic driver overexpressed in various cancers. The potential role of hnRNPA2B1 in oncotherapy has not been revealed because of the absence of active chemical molecules. In this study, we identified the pseudourea derivative XI-011 as a novel hnRNPA2B1 ligand using chemical proteomics. An interaction study indicated that XI-011 could bind the nucleotide-binding domain to disrupt the recruitment of hnRNPA2B1 to the promoter and untranslated region of the murine double minute X (MDMX) gene, thereby inhibiting its transcription. In addition, chemical targeting of hnRNPA2B1 recovered inactivated p53 and enhanced the therapeutic efficacy of apatinib in vivo. This work presented a novel strategy to restore p53 activity for the treatment of gastric cancers via chemically targeting hnRNPA2B1.

DT-010 Exerts Cardioprotective Effects by Regulating the Crosstalk between the AMPK/PGC-1α Pathway and ERp57.

The AMP-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) pathway performs a crucial role in energy metabolism and mitochondrial network. Our previous study found that DT-010, a novel danshensu (DSS) and tetramethylpyrazine (TMP) conjugate, had significant cardioprotective properties in vitro and in vivo. We also reported that ERp57 served as a major target of DSS using the chemical proteomics approach. In this article, we focus on exploring the interrelationship between the regulation of the AMPK/PGC-1α pathway and promoting ERp57 expression induced by DT-010 in tert-butylhydroperoxide- (t-BHP-) induced H9c2 cell injury. The results showed that DT-010 activated the AMPK/PGC-1α pathway and increased ERp57 protein expression. Importantly, the above phenomenon as well as the mitochondrial function can be partially reversed by siRNA-mediated ERp57 suppression. Meanwhile, silencing AMPK significantly inhibited the ERp57 expression induced by DT-010. In addition, molecular docking and kinase assay in vitro revealed that DT-010 had no direct regulation effects on AMPK activity. Taken together, DT-010 exerted cardioprotective effects by regulating the crosstalk of AMPK/PGC-1α pathway and ERp57, representing a potential therapeutic agent for ischemic heart disease.