Chemical Proteomics Research Articles

Global Profiling Lysine Reactivity and Ligandability with Oxidant‐Triggered Bioconjugation Chemistry

Due to the high abundance and diverse functions of lysine residues, both in the interior and on the surface of proteins, the development of new methods to characterize their reactivity and ligandability could significantly expand the pool of druggable targets. To date, only a limited number of aminophilic electrophiles have been assessed for interactions with the lysine proteome, resulting in a substantial fraction remaining inaccessible to current probes. Here, to the best of our knowledge, we report the first oxidant‐triggered bioconjugation platform for in‐depth profiling of lysines. We quantified over 7000 covalently modifiable lysine residues, which significantly expands the coverage of ligandable lysines in the whole proteome. Chemical proteomics enabled the mapping of more than 100 endogenous kinases, thus providing a comprehensive landscape of ligandable catalytic lysines within the kinome. Moreover, we identified a suite of new ligandable lysines such as K60 of ENO1 and K31 of PPIA, offering insights for exploring new functional and targetable residues. These findings could provide valuable clues for the development of novel targeted covalent inhibitors (TCIs).

Global Profiling Lysine Reactivity and Ligandability with Oxidant-Triggered Bioconjugation Chemistry.

Due to the high abundance and diverse functions of lysine residues, both in the interior and on the surface of proteins, the development of new methods to characterize their reactivity and ligandability could significantly expand the pool of druggable targets. To date, only a limited number of aminophilic electrophiles have been assessed for interactions with the lysine proteome, resulting in a substantial fraction remaining inaccessible to current probes. Here, to the best of our knowledge, we report the first oxidant-triggered bioconjugation platform for in-depth profiling of lysines. We quantified over 7000 covalently modifiable lysine residues, which significantly expands the coverage of ligandable lysines in the whole proteome. Chemical proteomics enabled the mapping of more than 100 endogenous kinases, thus providing a comprehensive landscape of ligandable catalytic lysines within the kinome. Moreover, we identified a suite of new ligandable lysines such as K60 of ENO1 and K31 of PPIA, offering insights for exploring new functional and targetable residues. These findings could provide valuable clues for the development of novel targeted covalent inhibitors (TCIs).

Chemical tools to expand the ligandable proteome: Diversity-oriented synthesis-based photoreactive stereoprobes.

Chemical proteomics enables the global analysis of small molecule-protein interactions in native biological systems and has emerged as a versatile approach for ligand discovery. The range of small molecules explored by chemical proteomics has, however, remained limited. Here, we describe a diversity-oriented synthesis (DOS)-inspired library of stereochemically defined compounds bearing diazirine and alkyne units for UV light-induced covalent modification and click chemistry enrichment of interacting proteins, respectively. We find that these "photo-stereoprobes" interact in a stereoselective manner with hundreds of proteins from various structural and functional classes in human cells and demonstrate that these interactions can form the basis for high-throughput screening-compatible NanoBRET assays. Integrated phenotypic screening and chemical proteomics identified photo-stereoprobes that modulate autophagy by engaging the mitochondrial serine protease CLPP. Our findings show the utility of DOS-inspired photo-stereoprobes for expanding the ligandable proteome, furnishing target engagement assays, and facilitating the discovery and characterization of bioactive compounds in phenotypic screens.

A small molecule that reshapes the chromatin dynamics of FOXA1

In this issue, Won etal.1 report a covalent ligand binding the pioneer transcription factor FOXA1, altering its function and remodeling chromatin. This important finding highlights the potential of small molecules to modulate transcription factor activity and demonstrates the promise of chemical proteomics in discovering first-in-class ligands.

Polyphenol Tannin‐Induced Protein Precipitation for Drug Target Discovery by Chemical Proteomics

AbstractHigh‐throughput drug discovery is highly dependent on the targets available to accelerate the process of candidate screening. Unbiased drug target identification is of crucial importance to reveal the mechanisms of protein–drug interactions. In recent years, energetics‐based protein conformation methods have become an alternative strategy for protein target discovery of bioactive drugs. In this study, we present a novel tannin protein precipitation method (TAP) for identifying target proteins of small molecules using the effect on protein solubility of tannin. The feasibility of this method was validated using a drug model of methotrexate. It was found that the known target DHFR yields a significant solubility shift. This method was further investigated with Annexin A2, the target protein of protopanaxadiol (PPD)‐S by western blot, and by screening potential target proteins of PPD‐S in K562 cells by quantitative proteomics. Finally, the TAP method was employed to differentiate the target proteins of pair chiral molecules PPD‐S and PPD‐R, which can bring up novel insights into the mechanism of drug–protein interactions. This study not only provides an effective tool for drug research and development but also offers new ideas for understanding the mechanism of action of natural chiral bioactive compounds.

The impact of heating-induced lactosylation on the digestibility of lactotransferrin

Lactosylation of lysines occurs during the heating of dairy products, yet how lactosylation impact the lysine release during digestion remains largely unknown. This study examines the effect of lactosylation on the digestibility of lactotransferrin using chemical analysis, proteomics, and peptidomics. Under the applied heating conditions, lactotransferrin primarily undergoes early-stage Maillard reactions, producing lactulose-lysine. Furosine content increases with heating time and temperature, with time being more influential. And 23 out of 54 lysines in lactotransferrin were lactosylated. Following in-vitro infant digestion, free lysine levels in samples heated at 130 °C for 30s decreased by 25 % compared to unheated ones, likely due to lactosylation hindering protease cleavage. Intriguingly, lactosylated lysine was absent in peptides ranging from 5 to 17 amino acids but remained in larger peptides. The formation of large lactosylated peptides from heating impeded free lysine release. Further investigation is needed to determine if the human body can utilize these lactosylated peptides.

Quantitative chemical proteomics reveals that phenethyl isothiocyanate covalently targets BID to promote apoptosis

Naturally occurring isothiocyanates (ITCs) found in cruciferous vegetables, such as benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC), and sulforaphane (SFN), have attracted significant research interest for their promising anti-cancer activity in vitro and in vivo. While the induction of apoptosis is recognized to play a key role in the anti-cancer effects of ITCs, the specific protein targets and associated upstream events underlying ITC-induced apoptosis remain unknown. In this study, we present a set of chemical probes that are derived from BITC, PEITC, and SFN and equipped with bioorthogonal alkynyl handles to systematically profile the target proteins of ITCs in live cancer cells. Using a competition-based quantitative chemical proteomics approach, we identify a range of candidate target proteins of ITCs enriched in biological processes such as apoptosis. We show that BID, an apoptosis regulator of the Bcl-2 family, is covalently modified by ITCs on its N-terminal cysteines. Functional characterization demonstrates that covalent binding to N-terminal cysteines of BID by PEITC results in conformational changes of the protein and disruption of the self-inhibitory interaction between N- and C-terminal regions of BID, thus unleashing the highly active C-terminal segment to exert downstream pro-apoptotic effects. Consistently, PEITC promotes the cleavage and mitochondrial translocation of BID, leading to a strong induction of apoptosis. We further show that mutation of N-terminal cysteines impairs the N- and C-terminal interaction of BID, relieving the self-inhibition and enhancing its apoptotic activity. Overall, our chemical proteomics profiling and functional studies not only reveal BID as the principal target of PEITC in mediating upstream events for the induction of apoptosis, but also uncover a novel molecular mechanism involving N-terminal cysteines within the first helix of BID in regulating its pro-apoptotic potential.

Identification of Aberrant Expression of Gemcitabine-Targeting Proteins in Drug-Resistant Cells Using an Activity-Based Gemcitabine Probe.

Gemcitabine-based monotherapy or combination therapy has become the standard treatment for locally advanced and metastatic pancreatic cancer. However, the emergence of resistance within weeks of treatment severely compromises therapeutic efficacy. The intricate biological process of gemcitabine resistance in pancreatic cancer presents a complex challenge, as the underlying mechanisms remain unclear. Identifying the target protein of gemcitabine is crucial for studying its drug-resistance mechanism. An activity-based probe is a powerful tool for studying drug target proteins, but the current lack of activity-based gemcitabine probes with robust biological activity hinders research on gemcitabine. In this study, we developed three active probes based on gemcitabine, among which Gem-3 demonstrated excellent stability and labeling efficacy. We utilized Gem-3 in conjunction with chemical proteomics to identify intracellular target proteins. We identified 79 proteins that interact with gemcitabine, most of which were previously unknown and represented various functional classes. Additionally, we validated the increased expression of IFIT3 and MARCKS in drug-resistant cells, along with the activation of the NF-κB signaling pathway. These findings substantially contribute to our comprehension of gemcitabine's target proteins and further our understanding of the mechanisms driving gemcitabine resistance in pancreatic cancer cells.

Cracking Lysine Crotonylation (Kcr): Enlightening a Promising Post-Translational Modification.

Lysine crotonylation (Kcr) is a recently discovered post-translational modification (PTM). Both histone and non-histone Kcr-proteins have been associated with numerous diseases including cancer, acute kidney injury, HIV latency, and cardiovascular disease. Histone Kcr enhances gene expression to a larger extend than the extensively studied lysine acetylation (Kac), suggesting Kcr as a novel potential therapeutic target. Although numerous scientific reports on crotonylation were published in the last years, relevant knowledge gaps concerning this PTM and its regulation still remain. To date, only few selective Kcr-interacting proteins have been identified and selective methods for the enrichment of Kcr-proteins in chemical proteomics analysis are still lacking. The development of new techniques to study this underexplored PTM could then clarify its function in health and disease and hopefully accelerate the development of new therapeutics for Kcr-related disease. Herein we briefly review what is known about the regulation mechanisms of Kcr and the current methods used to identify Kcr-proteins and their interacting partners. This report aims to highlight the significant potential of Kcr as a therapeutic target and to identify the existing scientific gaps that new research must address.

6-Gingerol Inhibits De Novo Lipogenesis by Targeting Stearoyl-CoA Desaturase to Alleviate Fructose-Induced Hepatic Steatosis.

Metabolic-associated fatty liver disease (MAFLD), also known as non-alcoholic fatty liver disease (NAFLD), is a worldwide liver disease without definitive or widely used therapeutic drugs in clinical practice. In this study, we confirm that 6-gingerol (6-G), an active ingredient of ginger (Zingiber officinale Roscoe) in traditional Chinese medicine (TCM), can alleviate fructose-induced hepatic steatosis. It was found that 6-G significantly decreased hyperlipidemia caused by high-fructose diets (HFD) in rats, and reversed the increase in hepatic de novo lipogenesis (DNL) and triglyceride (TG) levels induced by HFD, both in vivo and in vitro. Mechanistically, chemical proteomics and cellular thermal shift assay (CETSA)-proteomics approaches revealed that stearoyl-CoA desaturase (SCD) is a direct binding target of 6-G, which was confirmed by further CETSA assay and molecular docking. Meanwhile, it was found that 6-G could not alter SCD expression (in either mRNA or protein levels), but inhibited SCD activity (decreasing the desaturation levels of fatty acids) in HFD-fed rats. Furthermore, SCD deficiency mimicked the ability of 6-G to reduce lipid accumulation in HF-induced HepG2 cells, and impaired the improvement in hepatic steatosis brought about by 6-G treatment in HFD supplemented with oleic acid diet-induced SCD1 knockout mice. Taken together, our present study demonstrated that 6-G inhibits DNL by targeting SCD to alleviate fructose diet-induced hepatic steatosis.

Chemical Proteomics Approaches for Screening Small Molecule Inhibitors Covalently Binding to SARS-Cov-2.

Although various strategies have been used to prevent and treat SARS-CoV-2, the spread and evolution of SARS-CoV-2 is still progressing rapidly. The emerging variants Omicron and its sublineage have a greater ability to spread and escape nearly all current monoclonal antibodies treatments, highlighting an urgent need to develop therapeutics targeting current and emerging Omicron variants or recombinants with breadth and potency. Here, some small molecule drugs are rapidly identified that could covalently binding to receptor binding domain (RBD) protein of Omicron through the combined application of artificial intelligence (AI) and activity-based protein profiling (ABPP) technology. The surface plasmon resonance (SPR) and pseudo-virus neutralization experiments further reveal that an FDA-approved drug gallic acid has robust neutralization potency against Omicron pseudo-virus with the IC50 values of 23.56 × 10-6 m. Taken together, a platform combining AI intelligence, biochemical, SPR, molecular docking, and pseudo-virus-based screening for rapid identification and evaluation of potential anti-SARS-CoV-2 small molecule drugs is established and the effectiveness of the platform is validated.

Benzo(a)anthracene Targeting SLC1A5 to Synergistically Enhance PAH Mixture Toxicity.

Human exposure to polycyclic aromatic hydrocarbons (PAHs) as mutagenic and carcinogenic pollutants in the environment often occurs in the form of mixtures. Although the mixture effects of PAHs have been previously recognized, the toxicological mechanisms to explain them still remain quite unclear. This study combined metabolomics and chemical proteomics methods to comprehensively understand the mixture effects of a PAH mixture including benzo(a)anthracene (BaA), benzo(b)fluoranthene (BbF), benzo(a)pyrene (BaP), and chrysene (CHR). Among them, BaA has shown a strong synergistic effect with other PAHs. Interestingly, BaA alone is not a potent oxidative stress inducer in liver cells but dose-dependently amplifies oxidative damage caused by the PAH mixture. Global metabolomics analysis results revealed damage to the antioxidant glutathione synthesis, which was caused by the glutamine depletion caused by BaA in the mixture. Subsequently, the label-free chemical proteomics and cellular thermal shift analysis (CETSA) demonstrated that the PAH mixture altered the thermal shift of glutamine transporter SLC1A5. Furthermore, Western blotting and the isothermal titration calorimetry (ITC) interaction measurements showed nanomolar KD values between BaA and SLC1A5. Overall, this study showed that BaA synergistically contributed to PAH mixture induced oxidative damage by targeting SLC1A5 to inhibit glutamate transport into cells, resulting in the inhibition of glutathione synthesis.

End-to-End Throughput Chemical Proteomics for Photoaffinity Labeling Target Engagement and Deconvolution.

Photoaffinity labeling (PAL) methodologies have proven to be instrumental for the unbiased deconvolution of protein-ligand binding events in physiologically relevant systems. However, like other chemical proteomic workflows, they are limited in many ways by time-intensive sample manipulations and data acquisition techniques. Here, we describe an approach to address this challenge through the innovation of a carboxylate bead-based protein cleanup procedure to remove excess small-molecule contaminants and couple it to plate-based, proteomic sample processing as a semiautomated solution. The analysis of samples via label-free, data-independent acquisition (DIA) techniques led to significant improvements on a workflow time per sample basis over current standard practices. Experiments utilizing three established PAL ligands with known targets, (+)-JQ-1, lenalidomide, and dasatinib, demonstrated the utility of having the flexibility to design experiments with a myriad of variables. Data revealed that this workflow can enable the confident identification and rank ordering of known and putative targets with outstanding protein signal-to-background enrichment sensitivity. This unified end-to-end throughput strategy for processing and analyzing these complex samples could greatly facilitate efficient drug discovery efforts and open up new opportunities in the chemical proteomics field.

Labeled and Label-Free Target Identifications of Natural Products.

Target identification, employing chemical proteomics, constitutes a continuous challenging endeavor in the drug development of natural products (NPs). Understanding their targets is crucial for deciphering their mechanisms and developing potential probes or drugs. Identifications fall into two main categories: labeled and label-free techniques. Labeled methods use the molecules tagged with markers such as biotin or fluorescent labels to easily detect interactions with target proteins. Thorough structure-activity relationships are essential before labeling to avoid changes in the biological activity or binding specificity. In contrast, label-free technologies identify target proteins without modifying natural products, relying on changes in the stability, thermal properties, or precipitation in the presence or absence of these products. Each approach has its advantages and disadvantages, offering a comprehensive understanding of the mechanisms and therapeutic potential of the NPs. Here, we summarize target identification techniques for natural molecules, highlight case studies of notable NPs, and explore future applications and directions.

Multiplexed Target Profiling with Integrated Chemical Genomics and Chemical Proteomics.

Target identification is crucial for elucidating the mechanisms of bioactive molecules in drug discovery. However, traditional methods assess compounds individually, making it challenging to efficiently examine multiple compounds in parallel, especially for structurally diverse compounds. This study reports a novel strategy called chemical genomics-facilitated chemical proteomics (CGCP) for multiplexing the target identification of bioactive small molecules. CGCP correlates compounds' perturbation of global transcription, or chemical genomic profiles, with their reactivity toward target proteins, enabling simultaneous identification of targets. We demonstrated the utility of CGCP by studying the targets of celastrol (Cel) and four other electrophilic compounds with varying levels of similarity to Cel based on their chemical genomic profiles. We identified multiple novel targets and binding sites shared by the compounds in a single experiment. CGCP enabled multiplexity and improved the efficiency of target identification for structurally distinct compounds, indicating its potential to accelerate drug discovery.

Chemoproteomics of Marine Natural Product Naamidine J Unveils CSE1L as a Therapeutic Target in Acute Lung Injury.

Acute lung injury is a devastating illness characterized by severe inflammation mediated by aberrant activation of macrophages, resulting in significant morbidity and mortality, highlighting the urgent need for novel pharmacological targets and drug candidates. In this study, we identified a novel target for regulating inflammation in macrophages and acute lung injury via chemical proteomics and genetics based on a marine alkaloid, naamidine J (NJ). The structures of NJ-related naamidine alkaloids were first confirmed or revised by a combination of quantum chemical calculations and X-ray diffraction analysis. NJ was found as a potential anti-inflammatory agent by screening our compound library, and CSE1L was identified by chemoproteomics as a main cellular target of NJ to inhibit inflammation in macrophages and protect against acute lung injury. Mechanistically, we demonstrated that NJ directly interacted with CSE1L on the sites of His745 and Phe903 and then inhibited the nuclear translocation and transcriptional activity of transcription factor SP1, thereby suppressing inflammation in macrophages and ameliorating acute lung injury. Taken together, these findings have uncovered a novel pharmacological target for the treatment of acute lung injury and have also provided a potential druggable pocket of CSE1L and a lead compound or an available chemical tool from marine sources for investigating CSE1L function and developing novel drug candidates against acute lung injury.

A Kinetic Scout Approach Accelerates Targeted Protein Degrader Development.

Bifunctional molecules such as targeted protein degraders induce proximity to promote gain-of-function pharmacology. These powerful approaches have gained broad traction across academia and the pharmaceutical industry, leading to an intensive focus on strategies that can accelerate their identification and optimization. We and others have previously used chemical proteomics to map degradable target space, and these datasets have been used to develop and train multiparameter models to extend degradability predictions across the proteome. In this study, we now turn our attention to develop generalizable chemistry strategies to accelerate the development of new bifunctional degraders. We implement lysine-targeted reversible-covalent chemistry to rationally tune the binding kinetics at the protein-of-interest across a set of 25 targets. We define an unbiased workflow consisting of global proteomics analysis, IP/MS of ternary complexes and the E-STUB assay, to mechanistically characterize the effects of ligand residence time on targeted protein degradation and formulate hypotheses about the rate-limiting step of degradation for each target. Our key finding is that target residence time is a major determinant of degrader activity, and this can be rapidly and rationally tuned through the synthesis of a minimal number of analogues to accelerate early degrader discovery and optimization efforts.

An allosteric cyclin E-CDK2 site mapped by paralog hopping with covalent probes.

More than half of the ~20,000 protein-encoding human genes have paralogs. Chemical proteomics has uncovered many electrophile-sensitive cysteines that are exclusive to subsets of paralogous proteins. Here we explore whether such covalent compound-cysteine interactions can be used to discover ligandable pockets in paralogs lacking the cysteine. Leveraging the covalent ligandability of C109 in the cyclin CCNE2, we substituted the corresponding residue in paralog CCNE1 to cysteine (N112C) and found through activity-based protein profiling that this mutant reacts stereoselectively and site-specifically with tryptoline acrylamides. We then converted the tryptoline acrylamide-CCNE1-N112C interaction into in vitro NanoBRET (bioluminescence resonance energy transfer) and in cellulo activity-based protein profiling assays capable of identifying compounds that reversibly inhibit both the N112C mutant and wild-type CCNE1:CDK2 (cyclin-dependent kinase 2) complexes. X-ray crystallography revealed a cryptic allosteric pocket at the CCNE1:CDK2 interface adjacent to N112 that binds the reversible inhibitors. Our findings, thus, show how electrophile-cysteine interactions mapped by chemical proteomics can extend the understanding of protein ligandability beyond covalent chemistry.

Distinct modes of coupling between VCP, an essential unfoldase, and deubiquitinases.

Errors in proteostasis, which requires regulated degradation and recycling of diverse proteins, are linked to aging, cancer and neurodegenerative disease (1). In particular, recycling proteins from multiprotein complexes, organelles and membranes is initiated by ubiquitylation, extraction and unfolding by the essential mechanoenzyme VCP (2-4), and ubiquitin removal by deubiquitinases (DUBs), a class of ∼100 ubiquitin-specific proteases in humans (5, 6). As VCP's substrate recognition requires ubiquitylation, the removal of ubiquitins from substrates for recycling must follow extraction and unfolding. How the activities of VCP and different DUBs are coordinated for protein recycling or other fates is unclear. Here, we employ a photochemistry-based approach to profile proteome-wide domain-specific VCP interactions in living cells (7). We identify DUBs that bind near the entry, exit, or both sites of VCP's central pore, the channel for ATP-dependent substrate translocation (8-10). From this set of DUBs, we focus on VCPIP1, required for organelle assembly and DNA repair (11-13), that our chemical proteomics workflow indicates binds the central pore's entry and exit sites. We determine a ∼3Å cryo-EM structure of the VCP-VCPIP1 complex and find up to 3 VCPIP1 protomers interact with the VCP hexamer. VCPIP1's UBX-L domain binds VCP's N-domain in a 'down' conformation, linked to VCP's ADP-bound state (2, 14), and the deubiquitinase domain is positioned at the central pore's exit site, poised to remove ubiquitin following substrate unfolding. We find that VCP stimulates VCPIP1's DUB activity and use mutagenesis and single-molecule mass photometry assays to test the structural model. Together, our data suggest that DUBs bind VCP at distinct sites and reveal how the two enzyme activities can be coordinated to achieve specific downstream outcomes for ubiquitylated proteins.

Natural Product Virtual-Interact-Phenotypic Target Characterization: A Novel Approach Demonstrated with Salvia Miltiorrhiza Extract

Natural products (NPs) have historically been a fundamental source for drug discovery. Yet the complex nature of NPs presents substantial challenges in pinpointing bioactive constituents, and corresponding targets. In the present study, an innovative Natural Product Virtual screening-Interaction-Phenotype (NP-VIP) strategy that integrates virtual screening, chemical proteomics, and metabolomics to identify and validate the bioactive targets of NPs. This approach reduces false positive results and enhances the efficiency of target identification. Salvia miltiorrhiza (SM), a herb with recognized therapeutic potential against ischemic stroke (IS), was used to illustrate the workflow. Utilizing virtual screening, chemical proteomics, and metabolomics, potential therapeutic targets for SM in the IS treatment were identified, totaling 29, 100, and 78, respectively. Further analysis via the NP-VIP strategy highlighted five high-confidence targets, including Poly [ADP-ribose] polymerase 1 (PARP1), signal transducer and activator of transcription 3 (STAT3), amyloid precursor protein (APP), glutamate-ammonia ligase (GLUL), and glutamate decarboxylase 67 (GAD67). These targets were subsequently validated and found to play critical roles in the neuroprotective effects of SM. The study not only underscores the importance of SM in treating IS but also sets a precedent for NP research, proposing a comprehensive approach that could be adapted for broader pharmacological explorations.