Activity-based Protein Profiling Research Articles

TLR9-dependent dendritic cell maturation promotes IL-6-mediated upregulation of cathepsin X.

Cysteine cathepsins are lysosomal proteases subject to dynamic regulation within antigen-presenting cells during the immune response and associated diseases. To investigate the regulation of cathepsin X, a carboxy-mono-exopeptidase, during maturation of dendritic cells (DCs), we exposed immortalized mouse DCs to various Toll-like receptor agonists. Using a cathepsin X-selective activity-based probe, sCy5-Nle-SY, we observed a significant increase in cathepsin X activation upon TLR-9 agonism with CpG, and to a lesser extent with Pam3 (TLR1/2), FSL-1 (TLR2/6) and LPS (TLR4). Despite clear maturation of DCs in response to Poly I:C (TLR3), cathepsin X activity was only slightly increased by this agonist, suggesting differential regulation of cathepsin X downstream of TLR activation. We demonstrated that cathepsin X was upregulated at the transcriptional level in response to CpG. This occurred at late time points and was not dampened by NF-κB inhibition. Factors secreted from CpG-treated cells were able to provoke cathepsin X upregulation when applied to naïve cells. Among these factors was IL-6, which on its own was sufficient to induce transcriptional upregulation and activation of cathepsin X. IL-6 is highly secreted by DCs in response to CpG but much less so in response to poly I:C, and inhibition of the IL-6 receptor subunit glycoprotein 130 prevented CpG-mediated cathepsin X upregulation. Collectively, these results demonstrate that cathepsin X is differentially transcribed during DC maturation in response to diverse stimuli, and that secreted IL-6 is critical for its dynamic regulation.

Development and Application of Reversible and Irreversible Covalent Probes for Human and Mouse Cathepsin-K Activity Detection, Revealing Nuclear Activity.

Cathepsin-K (CTSK) is an osteoclast-secreted cysteine protease that efficiently cleaves extracellular matrices and promotes bone homeostasis and remodeling, making it an excellent therapeutic target. Detection of CTSK activity in complex biological samples using tailored tools such as activity-based probes (ABPs) will aid tremendously in drug development. Here, potent and selective CTSK probes are designed and created, comparing irreversible and reversible covalent ABPs with improved recognition components and electrophiles. The newly developed CTSK ABPs precisely detect active CTSK in mouse and human cells and tissues, from diseased and healthy states such as inflamed tooth implants, osteoclasts, and lung samples, indicating changes in CTSK's activity in the pathological samples. These probes are used to study how acidic pH stimulates mature CTSK activation, specifically, its transition from pro-form to mature form. Furthermore, this study reveals for the first time, why intact cells and cell lysate exhibit diverse CTSK activity while having equal levels of mature CTSK enzyme. Interestingly, these tools enabled the discovery of active CTSK in human osteoclast nuclei and in the nucleoli. Altogether, these novel probes are excellent research tools and can be applied in vivo to examine CTSK activity and inhibition in diverse diseases without immunogenicity hazards.

Glycyrrhizic acid ameliorates hepatic fibrosis by inhibiting oxidative stress via AKR7A2

BackgroundHepatic fibrosis is a reversible pathological phenomenon caused by the abnormal proliferation of connective tissues in the liver for self-repair after persistent liver injury. Among these tissues, the activation status of hepatic stellate cells (HSCs) is crucial. Glycyrrhizic acid (GA) agents have been proven to have excellent anti-fibrosis effects, but their targets are unclear. PurposeTo investigate the anti-hepatic fibrosis effect of GA and its target in activated HSCs. MethodsA mouse model of hepatic fibrosis was prepared with 20% carbon tetrachloride (CCl4) and GA was administered continuously for 4 weeks. Subsequently, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), type Ⅲ procollagen peptide (P III P), laminin (LN), hyaluronic acid (HA), and type Ⅳ collagen (Col Ⅳ) were measured. Liver tissues were subjected to hematoxylin and eosin (HE), Masson, and Sirius red staining and proteome sequencing analysis. Based on LX-2 cells, activity-based protein profiling (ABPP) was used to investigate the potential targets of GA, which was further validated by the cellular thermal shift assay (CETSA), immunofluorescence co-localization, molecular docking, small interfering RNA (siRNA) and western blot (WB) assays. ResultsIn vivo, GA significantly reduced serum ALT, AST, HA, P III P, Col IV, and LN levels. HE, Masson, and Sirius red staining showed that GA significantly ameliorated hepatic inflammatory response and collagen deposition in CCl4-treated mice. Proteome sequencing results showed that GA mainly regulated glutathione S-transferase family members involved in glutathione metabolism. In vitro, GA significantly inhibited LX-2 cell proliferation and reduced reactive oxygen species accumulation. ABPP suggested that aldo-keto reductase family 7 member A2 (AKR7A2) was the major binding protein of GA in LX-2 cells. CETSA, fluorescence co-localization, molecular docking, and surface plasmon resonance further validated GA binding to AKR7A2. The WB results showed that GA up-regulated AKR7A2 expression both in vitro and in vivo and was corroborated by siRNA experiments. ConclusionGA targeted AKR7A2 in LX-2 cells to defend against sustained oxidative stress injury, thereby inhibiting the proliferation of activated HSCs and reversing hepatic fibrosis.

Phosphinate Esters as Novel Warheads for Quenched Activity-Based Probes Targeting Serine Proteases.

Quenched activity-based probes (qABP) are invaluable tools to visualize aberrant protease activity. Unfortunately, most studies so far have only focused on cysteine proteases, and only a few studies describe the synthesis and use of serine protease qABPs. We recently used phosphinate ester electrophiles as a novel type of reactive group to construct ABPs for serine proteases. Here, we report on the construction of qABPs based on the phosphinate warhead, exemplified by probes for the neutrophil serine proteases. The most successful probes show sub-stoichiometric reaction with human neutrophil elastase, efficient fluorescence quenching, and rapid unquenching of fluorescence upon reaction with target proteases.

Activity-based NIR specific fluorescent probe reveals the abnormal elevation of prolyl endopeptidase in hippocampus during Alzheimer's disease progression

Although prolyl endopeptidase (PREP) has become one of the important proteins to investigate the pathogenesis of Alzheimer's Disease (AD), the activity of PREP in the brain of AD patients is still controversial. This study aims to construct a novel enzyme-activated near-infrared (NIR) fluorescence probe for in-situ detection and imaging of PREP in brain, as well as to reveal the relationship between the activity variation of PREP and the development of AD. For these purposes, based on PREP's unique endopeptidase activity catalyzing prolyl-bond hydrolysis and cleavage, four probes deriving from a good NIR fluorophore (ACM) were designed and synthesized. Observations indicated that the Z-GP-ACM exhibited superior selectivity and sensitivity in detecting PREP. In addition, molecular thermodynamic/kinetic calculations and enzymatic kinetics studies also showed that Z-GP-ACM was an excellent substrate for PREP. Subsequent experiments demonstrated that Z-GP-ACM can be used to measure and image endogenous PREP in living cells and mouse brain. We also revealed for the first time that PREP activity was abnormally increased in hippocampus of AD mice during the disease progression. All these results highlighted that Z-GP-ACM could be a promising tool for exploring the biological functions of endogenous PREP in living systems.

Environmental activity-based protein profiling for function-driven enzyme discovery from natural communities

BackgroundMicrobial communities are important drivers of global biogeochemical cycles, xenobiotic detoxification, as well as organic matter decomposition. Their major metabolic role in ecosystem functioning is ensured by a unique set of enzymes, providing a tremendous yet mostly hidden enzymatic potential. Exploring this enzymatic repertoire is therefore not only relevant for a better understanding of how microorganisms function in their natural environment, and thus for ecological research, but further turns microbial communities, in particular from extreme habitats, into a valuable resource for the discovery of novel enzymes with potential applications in biotechnology. Different strategies for their uncovering such as bioprospecting, which relies mainly on metagenomic approaches in combination with sequence-based bioinformatic analyses, have emerged; yet accurate function prediction of their proteomes and deciphering the in vivo activity of an enzyme remains challenging.ResultsHere, we present environmental activity-based protein profiling (eABPP), a multi-omics approach that extends genome-resolved metagenomics with mass spectrometry-based ABPP. This combination allows direct profiling of environmental community samples in their native habitat and the identification of active enzymes based on their function, even without sequence or structural homologies to annotated enzyme families. eABPP thus bridges the gap between environmental genomics, correct function annotation, and in vivo enzyme activity. As a showcase, we report the successful identification of active thermostable serine hydrolases from eABPP of natural microbial communities from two independent hot springs in Kamchatka, Russia.ConclusionsBy reporting enzyme activities within an ecosystem in their native state, we anticipate that eABPP will not only advance current methodological approaches to sequence homology-guided enzyme discovery from environmental ecosystems for subsequent biocatalyst development but also contributes to the ecological investigation of microbial community interactions by dissecting their underlying molecular mechanisms.

Exploring Protein Bioconjugation: A Redox-Based Strategy for Tryptophan Targeting.

Amino acid bioconjugation technology has emerged as a pivotal tool for linking small-molecule fragments with proteins, antibodies, and even cells. The study in Nature by Chang and Toste introduces a redox-based strategy for tryptophan bioconjugation, employing N-sulfonyloxaziridines as oxidative cyclization reagents, demonstrating high efficiency comparable to traditional click reactions. Meanwhile, this tool provides feasible methods for investigating the mechanisms underlying functional tryptophan-related biochemical processes, paving the way for protein function exploration, activity-based proteomics for functional amino acid identification and characterization, and even the design of covalent inhibitors.

Chemoproteomics Identifies State-Dependent and Proteoform-Selective Caspase-2 Inhibitors.

Caspases are a highly conserved family of cysteine-aspartyl proteases known for their essential roles in regulating apoptosis, inflammation, cell differentiation, and proliferation. Complementary to genetic approaches, small-molecule probes have emerged as useful tools for modulating caspase activity. However, due to the high sequence and structure homology of all 12 human caspases, achieving selectivity remains a central challenge for caspase-directed small-molecule inhibitor development efforts. Here, using mass spectrometry-based chemoproteomics, we first identify a highly reactive noncatalytic cysteine that is unique to caspase-2. By combining both gel-based activity-based protein profiling (ABPP) and a tobacco etch virus (TEV) protease activation assay, we then identify covalent lead compounds that react preferentially with this cysteine and afford a complete blockade of caspase-2 activity. Inhibitory activity is restricted to the zymogen or precursor form of monomeric caspase-2. Focused analogue synthesis combined with chemoproteomic target engagement analysis in cellular lysates and in cells yielded both pan-caspase-reactive molecules and caspase-2 selective lead compounds together with a structurally matched inactive control. Application of this focused set of tool compounds to stratify the functions of the zymogen and partially processed (p32) forms of caspase-2 provide evidence to support that caspase-2-mediated response to DNA damage is largely driven by the partially processed p32 form of the enzyme. More broadly, our study highlights future opportunities for the development of proteoform-selective caspase inhibitors that target nonconserved and noncatalytic cysteine residues.

Detection of Sulfoquinovosidase Activity in Cell Lysates Using Activity‐Based Probes

AbstractThe sulfolipid sulfoquinovosyl diacylglycerol (SQDG), produced by plants, algae, and cyanobacteria, constitutes a major sulfur reserve in the biosphere. Microbial breakdown of SQDG is critical for the biological utilization of its sulfur. This commences through release of the parent sugar, sulfoquinovose (SQ), catalyzed by sulfoquinovosidases (SQases). These vanguard enzymes are encoded in gene clusters that code for diverse SQ catabolic pathways. To identify, visualize and isolate glycoside hydrolase CAZY‐family 31 (GH31) SQases in complex biological environments, we introduce SQ cyclophellitol‐aziridine activity‐based probes (ABPs). These ABPs label the active site nucleophile of this enzyme family, consistent with specific recognition of the SQ cyclophellitol‐aziridine in the active site, as evidenced in the 3D structure of Bacillus megaterium SQase. A fluorescent Cy5‐probe enables visualization of SQases in crude cell lysates from bacteria harbouring different SQ breakdown pathways, whilst a biotin‐probe enables SQase capture and identification by proteomics. The Cy5‐probe facilitates monitoring of active SQase levels during different stages of bacterial growth which show great contrast to more traditional mRNA analysis obtained by RT‐qPCR. Given the importance of SQases in global sulfur cycling and in human microbiota, these SQase ABPs provide a new tool with which to study SQase occurrence, activity and stability.

Detection of Sulfoquinovosidase Activity in Cell Lysates Using Activity-Based Probes.

The sulfolipid sulfoquinovosyl diacylglycerol (SQDG), produced by plants, algae, and cyanobacteria, constitutes a major sulfur reserve in the biosphere. Microbial breakdown of SQDG is critical for the biological utilization of its sulfur. This commences through release of the parent sugar, sulfoquinovose (SQ), catalyzed by sulfoquinovosidases (SQases). These vanguard enzymes are encoded in gene clusters that code for diverse SQ catabolic pathways. To identify, visualize and isolate glycoside hydrolase CAZY-family 31 (GH31) SQases in complex biological environments, we introduce SQ cyclophellitol-aziridine activity-based probes (ABPs). These ABPs label the active site nucleophile of this enzyme family, consistent with specific recognition of the SQ cyclophellitol-aziridine in the active site, as evidenced in the 3D structure of Bacillus megaterium SQase. A fluorescent Cy5-probe enables visualization of SQases in crude cell lysates from bacteria harbouring different SQ breakdown pathways, whilst a biotin-probe enables SQase capture and identification by proteomics. The Cy5-probe facilitates monitoring of active SQase levels during different stages of bacterial growth which show great contrast to more traditional mRNA analysis obtained by RT-qPCR. Given the importance of SQases in global sulfur cycling and in human microbiota, these SQase ABPs provide a new tool with which to study SQase occurrence, activity and stability.

Reversibly Reactive Affinity Selection-Mass Spectrometry Enables Identification of Covalent Peptide Binders.

Covalent peptide binders have found applications as activity-based probes and as irreversible therapeutic inhibitors. Currently, there is no rapid, label-free, and tunable affinity selection platform to enrich covalent reactive peptide binders from synthetic libraries. We address this challenge by developing a reversibly reactive affinity selection platform termed ReAct-ASMS enabled by tandem high-resolution mass spectrometry (MS/MS) to identify covalent peptide binders to native protein targets. It uses mixed disulfide-containing peptides to build reversible peptide-protein conjugates that can enrich for covalent variants, which can be sequenced by MS/MS after reduction. Using this platform, we identified covalent peptide binders against two oncoproteins, human papillomavirus 16 early protein 6 (HPV16 E6) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 protein (Pin1). The resulting peptide binders efficiently and selectively cross-link Cys58 of E6 at 37 °C and Cys113 of Pin1 at room temperature, respectively. ReAct-ASMS enables the identification of highly selective covalent peptide binders for diverse molecular targets, introducing an applicable platform to assist preclinical therapeutic development pipelines.

Mechanistic insights into CrCEP1: A dual-function cysteine protease with endo- and transpeptidase activity

Proteases, essential regulators of plant stress responses, remain enigmatic in their precise functional roles. By employing activity-based probes for real-time monitoring, this study aimed to delve into protease activities in Chlamydomonas reinhardtii exposed to oxidative stress induced by hydrogen peroxide. However, our work revealed that the activity-based probes strongly labelled three non-proteolytic proteins—PsbO, PsbP, and PsbQ—integral components of photosystem II's oxygen-evolving complex. Subsequent biochemical assays and mass spectrometry experiments revealed the involvement of CrCEP1, a previously uncharacterized papain-like cysteine protease, as the catalyst of this labelling reaction. Further experiments with recombinant CrCEP1 and PsbO proteins replicated the reaction in vitro. Our data unveiled that endopeptidase CrCEP1 also has transpeptidase activity, ligating probes and peptides to the N-termini of Psb proteins, thereby expanding the repertoire of its enzymatic activities. The hitherto unknown transpeptidase activity of CrCEP1, working in conjunction with its proteolytic activity, unveils putative complex and versatile roles for proteases in cellular processes during stress responses.

Parascedosporium putredinis NO1 tailors its secretome for different lignocellulosic substrates.

Parascedosporium putredinis NO1 is a plant biomass-degrading ascomycete with a propensity to target the most recalcitrant components of lignocellulose. Here we applied proteomics and activity-based protein profiling (ABPP) to investigate the ability of P. putredinis NO1 to tailor its secretome for growth on different lignocellulosic substrates. Proteomic analysis of soluble and insoluble culture fractions following the growth of P. putredinis NO1 on six lignocellulosic substrates highlights the adaptability of the response of the P. putredinis NO1 secretome to different substrates. Differences in protein abundance profiles were maintained and observed across substrates after bioinformatic filtering of the data to remove intracellular protein contamination to identify the components of the secretome more accurately. These differences across substrates extended to carbohydrate-active enzymes (CAZymes) at both class and family levels. Investigation of abundant activities in the secretomes for each substrate revealed similar variation but also a high abundance of "unknown" proteins in all conditions investigated. Fluorescence-based and chemical proteomic ABPP of secreted cellulases, xylanases, and β-glucosidases applied to secretomes from multiple growth substrates for the first time confirmed highly adaptive time- and substrate-dependent glycoside hydrolase production by this fungus. P. putredinis NO1 is a promising new candidate for the identification of enzymes suited to the degradation of recalcitrant lignocellulosic feedstocks. The investigation of proteomes from the biomass bound and culture supernatant fractions provides a more complete picture of a fungal lignocellulose-degrading response. An in-depth understanding of this varied response will enhance efforts toward the development of tailored enzyme systems for use in biorefining.IMPORTANCEThe ability of the lignocellulose-degrading fungus Parascedosporium putredinis NO1 to tailor its secreted enzymes to different sources of plant biomass was revealed here. Through a combination of proteomic, bioinformatic, and fluorescent labeling techniques, remarkable variation was demonstrated in the secreted enzyme response for this ascomycete when grown on multiple lignocellulosic substrates. The maintenance of this variation over time when exploring hydrolytic polysaccharide-active enzymes through fluorescent labeling, suggests that this variation results from an actively tailored secretome response based on substrate. Understanding the tailored secretomes of wood-degrading fungi, especially from underexplored and poorly represented families, will be important for the development of effective substrate-tailored treatments for the conversion and valorization of lignocellulose.

Inhibition of ALDOA‐ and ENO1‐Mediated Glycolysis of Oridonin as a Novel Antitumor Strategy of Non‐Small‐Cell Lung Cancer

AbstractNon‐small‐cell lung cancer (NSCLC) is the most common type of lung cancer. Oridonin (ORI), the main pharmacodynamic component of the Chinese herb Herba Rabdosiae with anticancer activity is reported to inhibit various types of cancer including NSCLC. However, its direct target proteins acting on NSCLC still require further investigation. Here, the direct protein profile is analyzed and the key targets of ORI are identified by combining activity‐based protein profiling (ABPP) and the cellular thermal shift assay (CETSA). As a result, ORI can significantly promote Lewis Lung Carcinoma (LLC) cell apoptosis, decrease mitochondrial membrane potential, and increase reactive oxygen species accumulation. In addition, both ABPP and CETSA indicate that ORI directly binds to key proteins of the glycolysis pathway, including Aldolase A (ALDOA), Enolase 1 (ENO1), pyruvate kinase M2, and lactate dehydrogenase. Based on the LLC mouse model, ORI markedly inhibits the growth of lung tumors, increases the serum levels of antioxidant enzymes catalase, superoxide dismutase, and glutathione peroxidase, and reduces the production of malondialdehyde. To sum up, ORI can be regarded as a novel antitumor strategy of NSCLC by inhibiting ALDOA‐ and ENO1‐mediated glycolysis.

An activity-based probe for antimicrobial target DXP synthase, a thiamin diphosphate-dependent enzyme

This work reports an alkyl acetylphosphonate (alkylAP) activity-based probe (ABP) for 1-deoxy-d-xylulose 5-phosphate synthase DXPS, a promising antimicrobial target. This essential thiamin diphosphate (ThDP)-dependent enzyme operates at a branchpoint in bacterial central metabolism and is believed to play key roles in pathogen adaptation during infection. How different bacterial pathogens harness DXPS activity to adapt and survive within host environments remains incompletely understood, and tools for probing DXPS function in different contexts of infection are lacking. Here, we have developed alkylAP-based ABP 1, designed to react with the ThDP cofactor on active DXPS to form a stable C2α-phosphonolactylThDP adduct which subsequently crosslinks to the DXPS active site upon photoactivation. ABP 1 displays low micromolar potency against DXPS and dose-dependent labeling of DXPS that is blocked by alkylAP-based inhibitors. The probe displays selectivity for DXPS over ThDP-dependent enzymes and is capable of detecting active DXPS in a complex proteome. These studies represent an important advance toward development of tools to probe DXPS function in different contexts of bacterial infection, and for drug discovery efforts on this target.

Development of Inhibitors, Probes, and PROTAC Provides a Complete Toolbox to Study PARK7 in the Living Cell.

The integration of diverse chemical tools like small-molecule inhibitors, activity-based probes (ABPs), and proteolysis targeting chimeras (PROTACs) advances clinical drug discovery and facilitates the exploration of various biological facets of targeted proteins. Here, we report the development of such a chemical toolbox for the human Parkinson disease protein 7 (PARK7/DJ-1) implicated in Parkinson's disease and cancers. By combining structure-guided design, miniaturized library synthesis, and high-throughput screening, we identified two potent compounds, JYQ-164 and JYQ-173, inhibiting PARK7 in vitro and in cells by covalently and selectively targeting its critical residue, Cys106. Leveraging JYQ-173, we further developed a cell-permeable Bodipy probe, JYQ-196, for covalent labeling of PARK7 in living cells and a first-in-class PARK7 degrader JYQ-194 that selectively induces its proteasomal degradation in human cells. Our study provides a valuable toolbox to enhance the understanding of PARK7 biology in cellular contexts and opens new opportunities for therapeutic interventions.

Multikinase inhibitors modulate non-constitutive proteasome expression in colorectal cancer cells.

Introduction: Proteasomes are multi-subunit protein complexes responsible for protein degradation in cells. Immunoproteasomes and intermediate proteasomes (together non-constitutive proteasomes) are specific forms of proteasomes frequently associated with immune response, antigen presentation, inflammation and stress. Expression of non-constitutive proteasome subunits has a prognostic value in several types of cancer. Thus, factors that modulate non-constitutive proteasome expression in tumors are of particular interest. Multikinase inhibitors (MKIs) demonstrate promising results in treatment of cancer. At the same time, their immunomodulatory properties and effects on non-constitutive proteasome expression in colorectal cancer cells are poorly investigated. Methods: Proteasome subunit expression in colorectal cancer was evaluated by bioinformatic analysis of available datasets. Two colorectal cancer cell lines, expressing fluorescent non-constitutive proteasomes were treated with multikinase inhibitors: regorafenib and sorafenib. The proteasome subunit expression was assessed by real-time PCR, Western blotting and flow cytometry. The proteasome activity was studied using proteasome activity-based probe and fluorescent substrates. Intracellular proteasome localization was revealed by confocal microscopy. Reactive oxygen species levels following treatment were determined in cells. Combined effect of proteasome inhibition and treatment with MKIs on viability of cells was estimated. Results: Expression of non-constitutive proteasomes is increased in BRAF-mutant colorectal tumors. Regorafenib and sorafenib stimulated the activity and synthesis of non-constitutive proteasomes in examined cell lines. MKIs induced oxidative stress and redistribution of proteasomes within cells. Sorafenib stimulated formation of cytoplasmic aggregates, containing proteolyticaly active non-constitutive proteasomes, while regorafenib had no such effect. MKIs caused no synergistic action when were combined with the proteasome inhibitor. Discussion: Obtained results indicate that MKIs might affect the crosstalk between cancer cells and immune cells via modulation of intracellular proteasome pool. Observed phenomenon should be considered when MKI-based therapy is applied.

Identification of Acyl-Protein Thioesterase-1 as a Polysorbate-Degrading Host Cell Protein in a Monoclonal Antibody Formulation Using Activity-Based Protein Profiling

Polysorbate (PS) degradation in monoclonal antibody (mAb) formulations poses a significant challenge in the biopharmaceutical industry. PS maintains protein stability during drug product's shelf life but is vulnerable to breakdown by low-abundance residual host cell proteins (HCPs), particularly hydrolytic enzymes such as lipases and esterases. In this study, we used activity-based protein profiling (ABPP) coupled with mass spectrometry to identify acyl-protein thioesterase-1 (APT-1) as a polysorbate-degrading HCP in one case of mAb formulation with stability problems. We validated the role of APT1 by matching the polysorbate degradation fingerprint in the mAb formulation with that of a recombinant APT1 protein. Furthermore, we found an agreement between APT1 levels and PS degradation rates in the mAb formulation, and we successfully halted PS degradation using APT1-specific inhibitors ML348 and ML211. APT1 was found to co-purify with a specific mAb via hitchhiking mechanism. Our work provides a streamlined approach to identifying critical HCPs in PS degradation, supporting quality-by-design principles in pharmaceutical development.

Detection of Active SARS-CoV-2 3CL Protease in Infected Cells Using Activity-Based Probes with a 2,6-Dichlorobenzoyloxymethyl Ketone Reactive Warhead.

The 3CL protease (3CLpro) is a viral cysteine protease of SARS-CoV-2 and is responsible for the main processing of the viral polyproteins involved in viral replication and proliferation. Despite the importance of 3CLpro as a drug target, the intracellular dynamics of active 3CLpro, including its expression and subcellular localization in SARS-CoV-2-infected cells, are poorly understood. Herein, we report an activity-based probe (ABP) with a clickable alkyne and an irreversible warhead for the SARS-CoV-2 3CL protease. We designed and synthesized two ABPs that contain a chloromethyl ketone (probe 2) or 2,6-dichlorobenzoyloxymethyl ketone (probe 3) reactive group at the P1' site. Labeling of recombinant 3CLpro by the ABPs in the purified and proteome systems revealed that probe 3 displayed ligand-directed and selective labeling against 3CLpro. Labeling of transiently expressed active 3CLpro in COS-7 cells also validated the good target selectivity of probe 3 for 3CLpro. We finally demonstrated that endogenously expressed 3CLpro in SARS-CoV-2-infected cells can be detected by fluorescence microscopy imaging using probe 3, suggesting that active 3CLpro at 5 h postinfection is localized in the juxtanuclear region. To the best of our knowledge, this is the first report investigating the subcellular localization of active 3CLpro by using ABPs. We believe that probe 3 will be a useful chemical tool for acquiring important biological knowledge of active 3CLpro in SARS-CoV-2-infected cells.

Bile Salt Hydrolase Activity-Based Probes for Monitoring Gut Microbial Bile Acid Metabolism.

Bile acids are bioactive metabolites that are biotransformed into secondary bile acids by the gut microbiota, a vast consortium of microbes that inhabit the intestines. The first step in intestinal secondary bile acid metabolism is carried out by a critical enzyme, bile salt hydrolase (BSH), that catalyzes the gateway reaction that precedes all subsequent microbial metabolism of these important metabolites. As gut microbial metabolic activity is difficult to probe due to the complex nature of the gut microbiome, approaches are needed to profile gut microbiota-associated enzymes such as BSH. Here, we develop a panel of BSH activity-based probes (ABPs) to determine how changes in diurnal rhythmicity of gut microbiota-associated metabolism affects BSH activity and substrate preference. This panel of covalent probes enables determination of BSH activity and substrate specificity from multiple gut anerobic bacteria derived from the human and mouse gut microbiome. We found that both gut microbiota-associated BSH activity and substrate preference is rhythmic, likely due to feeding patterns of the mice. These results indicate that this ABP-based approach can be used to profile changes in BSH activity in physiological and disease states that are regulated by circadian rhythms.