Fluorophosphonate-Based Degrader Identifies Degradable Serine Hydrolases by Quantitative Proteomics

Novel chemical biology probes linking a serine hydrolase-directed fluorophosphonate warhead and cereblon-binding pomalidomide were assessed for the degradation of serine hydrolases. A quantitative proteomics approach to detect degraded proteins revealed that, despite the engagement of ∼40 serine hydrolases, degradation was achieved for only a single serine hydrolase, lysophospholipase II (LYPLA2).     

4-Oxo-β-Lactams as Novel Inhibitors for Rhomboid Proteases

Intramembrane serine proteases (rhomboid proteases) are involved in a variety of biological processes and are implicated in several diseases. Here, we report 4-oxo-β-lactams as a novel scaffold for inhibition of rhomboids. We show that they covalently react with the active site and that the covalent bond is sufficiently stable for detection of the covalent rhomboid-lactam complex. 4-Oxo-β-lactams may therefore find future use as both inhibitors and activity-based probes for rhomboid proteases.     

Fluorophosphonates on-Demand: A General and Simplified Approach toward Fluorophosphonate Synthesis

Herein, we report a general and simplified synthesis of fluorophosphonates directly from p-nitrophenylphosphonates. This FP on-demand reaction is mediated by a commercially available polymer-supported fluoride reagent that produces a variety (25 examples) of fluorophosphonates in high yields while only requiring reagent filtration for pure fluorophosphonate isolation. This reaction protocol facilitates the rapid profiling of serine hydrolases with diverse and novel sets of activated phosphonates with differential proteome reactivity. Moreover, slight modification of the procedure into a reaction-to-assay format has enabled additional screening efficiency.     

Fatty Acyl Sulfonyl Fluoride as an Activity-Based Probe for Profiling Fatty Acid-Associated Proteins in Living Cells

Fatty acids play fundamental structural, metabolic, functional, and signaling roles in all biological systems. Altered fatty acid levels and metabolism have been associated with many pathological conditions. Chemical probes have greatly facilitated biological studies on fatty acids. Herein, we report the development and characterization of an alkynyl-functionalized long-chain fatty acid-based sulfonyl fluoride probe for covalent labelling, enrichment, and identification of fatty acid-associated proteins in living cells. Our quantitative chemical proteomics show that this sulfonyl fluoride probe targets diverse classes of fatty acid-associated proteins including many metabolic serine hydrolases that are known to be involved in fatty acid metabolism and modification. We further validate that the probe covalently modifies the catalytically or functionally essential serine or tyrosine residues of its target proteins and enables evaluation of their inhibitors. The sulfonyl fluoride-based chemical probe thus represents a new tool for profiling the expression and activity of fatty acid-associated proteins in living cells.     

Activity-Based Protein Profiling of Bile Salt Hydrolysis in the Human Gut Microbiome with Beta-Lactam or Acrylamide-Based Probes

Microbial bile salt hydrolases (BSHs) found in the intestine catalyze the deconjugation of taurine- and glycine-linked bile salts produced in the liver. The resulting bile salts are biological detergents and are critical in aiding lipophilic nutrient digestion. Therefore, the activity of BSHs in the gut microbiome is directly linked to human metabolism and overall health. Bile salt metabolism has also been associated with disease phenotypes such as liver and colorectal cancer. In order to reshape the gut microbiome to optimize bile salt metabolism, tools to characterize and quantify these processes must exist to enable a much-improved understanding of how metabolism goes awry in the face of disease, and how it can be improved through an altered lifestyle and environment. Furthermore, it is necessary to attribute metabolic activity to specific members and BSHs within the microbiome. To this end, we have developed activity-based probes with two different reactive groups to target bile salt hydrolases. These probes bind similarly to the authentic bile salt substrates, and we demonstrate enzyme labeling of active bile salt hydrolases by using purified protein, cell lysates, and in human stool.     

Activity-based protein profiling: new developments and directions in functional proteomics

Proteomic screening has become increasingly insightful with the availability of novel analytical tools and technologies. Detailed analysis and integration of the profound datasets attained from comprehensive profiling studies are enabling researchers to dig deeper into the foundations of genomic and proteomic networks, towards a clearer understanding of the intricate cellular circuitries they manifest. The major difficulty often lies in correlating the patho/physiological state presented with the underlying biological mechanisms; therefore, identification of causal variants as therapeutic targets is extremely important. Herein, we will describe methods that address this challenge through activity-based protein profiling, which applies chemical probes to the comparison and monitoring of protein dynamics across complex proteomes. Over recent years such activity-based probes have been creatively augmented with applications in gel-based separations, microarrays and in vivo imaging. These developments offer a newfound ability to characterise and differentiate cells, tissues and proteomes through activity-dependent signatures; this has expanded the scope and impact of activity-based probes in biomedical research.     

Ligand-Directed Chemistry on Glycoside Hydrolases – A Proof of Concept Study

Selective covalent labelling of enzymes using small molecule probes has advanced the scopes of protein profiling. The covalent bond formation to a specific target is the key step of activity-based protein profiling (ABPP), a method which has become an indispensable tool for measuring enzyme activity in complex matrices. With respect to carbohydrate processing enzymes, strategies for ABPP so far involve labelling the active site of the enzyme, which results in permanent loss of activity. Here, we report in a proof of concept study the use of ligand-directed chemistry (LDC) for labelling glycoside hydrolases near – but not in – the active site. During the labelling process, the competitive inhibitor is cleaved from the probe, departs the active site and the enzyme maintains its catalytic activity. To this end, we designed a building block synthetic concept for small molecule probes containing iminosugar-based reversible inhibitors for labelling of two model β-glucosidases. The results indicate that the LDC approach can be adaptable for covalent proximity labelling of glycoside hydrolases.     

Activity-Based Protein Profiling (ABPP) of Oxidoreductases

Over the last two decades, activity-based protein profiling (ABPP) has been established as a tremendously useful proteomic tool for measuring the activity of proteins in their cellular context, annotating the function of uncharacterized proteins, and investigating the target profile of small-molecule inhibitors. Unlike hydrolases and other enzyme classes, which exhibit a characteristic nucleophilic residue, oxidoreductases have received much less attention in ABPP. In this minireview, the state of the art of ABPP of oxidoreductases is described and the scope and limitations of the existing approaches are discussed. It is noted that several ABPP probes have been described for various oxidases, but none so far for a reductase, which gives rise to opportunities for future research.     

Activity-Based Protein Profiling in Methicillin-Resistant Staphylococcus aureus Reveals the Broad Reactivity of a Carmofur-Derived Probe

Activity-based protein profiling is a powerful chemoproteomic technique to detect active enzymes and identify targets and off-targets of drugs. Here, we report the use of carmofur- and activity-based probes to identify biologically relevant enzymes in the bacterial pathogen Staphylococcus aureus. Carmofur is an anti-neoplastic prodrug of 5-fluorouracil and also has antimicrobial and anti-biofilm activity. Carmofur probes were originally designed to target human acid ceramidase, a member of the NTN hydrolase family with an active-site cysteine nucleophile. Here, we first profiled the targets of a fluorescent carmofur probe in live S. aureus under biofilm-promoting conditions and in liquid culture, before proceeding to target identification by liquid chromatography/mass spectrometry. Treatment with a carmofur-biotin probe led to enrichment of 20 enzymes from diverse families awaiting further characterization, including the NTN hydrolase-related IMP cyclohydrolase PurH. However, the probe preferentially labeled serine hydrolases, thus displaying a reactivity profile similar to that of carbamates. Our results suggest that the electrophilic N-carbamoyl-5-fluorouracil scaffold could potentially be optimized to achieve selectivity towards diverse enzyme families. The observed promiscuous reactivity profile suggests that the clinical use of carmofur presumably leads to inactivation of a number human and microbial enzymes, which could lead to side effects and/or contribute to therapeutic efficacy.     

Rapid Development of a Potent Photo‐triggered Inhibitor of the Serine Hydrolase RBBP9

The serine hydrolases constitute a large class of enzymes that play important roles in physiology. There is great interest in the development of potent and selective pharmacological inhibitors of these proteins. Traditional active‐site inhibitors often have limited selectivity within this superfamily and are tedious and expensive to discover. Using the serine hydrolase RBBP9 as a model target, we designed a rapid and relatively inexpensive route to highly selective peptoid‐based inhibitors that can be activated by visible light. This technology provides rapid access to photo‐activated tool compounds capable of selectively blocking the function of particular serine hydrolases.     

Synthetic Approaches of Epoxysuccinate Chemical Probes

Synthetic chemical probes are powerful tools for investigating biological processes. They are particularly useful for proteomic studies such as activity-based protein profiling (ABPP). These chemical methods initially used mimics of natural substrates. As the techniques gained prominence, more and more elaborate chemical probes with increased specificity towards given enzyme/protein families and amenability to various reaction conditions were used. Among the chemical probes, peptidyl-epoxysuccinates represent one of the first types of compounds used to investigate the activity of the cysteine protease papain-like family of enzymes. Structurally derived from the natural substrate, a wide body of inhibitors and activity- or affinity-based probes bearing the electrophilic oxirane unit for covalent labeling of active enzymes now exists. Herein, we review the literature regarding the synthetic approaches to epoxysuccinate-based chemical probes together with their reported applications, from biological chemistry and inhibition studies to supramolecular chemistry and the formation of protein arrays.     

Improvements,Variations and Biomedical Applications of the Michaelis–Arbuzov Reaction

Compounds bearing the phosphorus–carbon (P–C) bond have important pharmacological, biochemical, and toxicological properties. Historically, the most notable reaction for the formation of the P–C bond is the Michaelis–Arbuzov reaction, first described in 1898. The classical Michaelis–Arbuzov reaction entails a reaction between an alkyl halide and a trialkyl phosphite to yield a dialkylalkylphosphonate. Nonetheless, deviations from the classical mechanisms and new modifications have appeared that allowed the expansion of the library of reactants and consequently the chemical space of the yielded products. These involve the use of Lewis acid catalysts, green methods, ultrasound, microwave, photochemically-assisted reactions, aryne-based reactions, etc. Here, a detailed presentation of the Michaelis–Arbuzov reaction and its developments and applications in the synthesis of biomedically important agents is provided. Certain examples of such applications include the development of alkylphosphonofluoridates as serine hydrolase inhibitors and activity-based probes, and the P–C containing antiviral and anticancer agents.     

First Identification of a Large Set of Serine Hydrolases by Activity-Based Protein Profiling in Dibutyl Phthalate-Exposed Zebrafish Larvae

Despite the involvement of several serine hydrolases (SHs) in the metabolism of xenobiotics such as dibutyl phthalate (DBP), no study has focused on mapping this enzyme class in zebrafish, a model organism frequently used in ecotoxicology. Here, we survey and identify active SHs in zebrafish larvae and search for biological markers of SH type after exposure to DBP. Zebrafish were exposed to 0, 5, and 100 µg/L DBP from 4 to 120 h post-fertilization. A significant decrease in vitellogenin expression level of about 2-fold compared to the control was found in larvae exposed to 100 µg/L DBP for 120 h. The first comprehensive profiling of active SHs in zebrafish proteome was achieved with an activity-based protein profiling (ABPP) approach. Among 49 SHs identified with high confidence, one was the carboxypeptidase ctsa overexpressed in larvae exposed to 100 µg/L DBP for 120 h. To the best of our knowledge, this is the first time that a carboxypeptidase has been identified as deregulated following exposure to DBP. The overall results indicate that targeted proteomics approaches, such as ABPP, can, therefore, be an asset for understanding the mechanism of action related to xenobiotics in ecotoxicology.     

Serine Hydrolases in Lipid Homeostasis of the Placenta-Targets for Placental Function?

The metabolic state of pregnant women and their unborn children changes throughout pregnancy and adapts to the specific needs of each gestational week. These adaptions are accomplished by the actions of enzymes, which regulate the occurrence of their endogenous substrates and products in all three compartments: mother, placenta and the unborn. These enzymes determine bioactive lipid signaling, supply, and storage through the generation or degradation of lipids and fatty acids, respectively. This review focuses on the role of lipid-metabolizing serine hydrolases during normal pregnancy and in pregnancy-associated pathologies, such as preeclampsia, gestational diabetes mellitus, or preterm birth. The biochemical properties of each class of lipid hydrolases are presented, with special emphasis on their role in placental function or dysfunction. While, during a normal pregnancy, an appropriate tonus of bioactive lipids prevails, dysregulation and aberrant signaling occur in diseased states. A better understanding of the dynamics of serine hydrolases across gestation and their involvement in placental lipid homeostasis under physiological and pathophysiological conditions will help to identify new targets for placental function in the future.     

Leaf Apoplast of Field-Grown Potato Analyzed by Quantitative Proteomics and Activity-Based Protein Profiling

Multiple biotic and abiotic stresses challenge plants growing in agricultural fields. Most molecular studies have aimed to understand plant responses to challenges under controlled conditions. However, studies on field-grown plants are scarce, limiting application of the findings in agricultural conditions. In this study, we investigated the composition of apoplastic proteomes of potato cultivar Bintje grown under field conditions, i.e., two field sites in June–August across two years and fungicide treated and untreated, using quantitative proteomics, as well as its activity using activity-based protein profiling (ABPP). Samples were clustered and some proteins showed significant intensity and activity differences, based on their field site and sampling time (June–August), indicating differential regulation of certain proteins in response to environmental or developmental factors. Peroxidases, class II chitinases, pectinesterases, and osmotins were among the proteins more abundant later in the growing season (July–August) as compared to early in the season (June). We did not detect significant differences between fungicide Shirlan treated and untreated field samples in two growing seasons. Using ABPP, we showed differential activity of serine hydrolases and β-glycosidases under greenhouse and field conditions and across a growing season. Furthermore, the activity of serine hydrolases and β-glycosidases, including proteins related to biotic stress tolerance, decreased as the season progressed. The generated proteomics data would facilitate further studies aiming at understanding mechanisms of molecular plant physiology in agricultural fields and help applying effective strategies to mitigate biotic and abiotic stresses.     

Peptidyl Acyloxymethyl Ketones as Activity-Based Probes for the Main Protease of SARS-CoV-2**

The global pandemic caused by SARS-CoV-2 calls for the fast development of antiviral drugs against this particular coronavirus. Chemical tools to facilitate inhibitor discovery as well as detection of target engagement by hit or lead compounds from high-throughput screens are therefore in urgent need. We here report novel, selective activity-based probes that enable detection of the SARS-CoV-2 main protease. The probes are based on acyloxymethyl ketone reactive electrophiles combined with a peptide sequence including unnatural amino acids that targets the nonprimed site of the main protease substrate binding cleft. They are the first activity-based probes for the main protease of coronaviruses and display target labeling within a human proteome without background. We expect that these reagents will be useful in the drug-development pipeline, not only for the current SARS-CoV-2, but also for other coronaviruses.     

Inhibitor and protein microarrays for activity-based recognition of lipolytic enzymes

Protein and small-molecule microarrays are useful tools for high-throughput analysis of DNA-protein, protein-protein, and protein-small molecule interactions. Here we report on novel microarrays for activity screening of lipases and esterases based on phosphonic acid ester inhibitors. These compounds are activity recognition probes (ARPs) and bind to active serine hydrolases in a stoichiometric and irreversible manner. Protein microarrays were generated by spotting six different lipolytic enzymes onto hydrogel-coated glass slides. The activity of immobilized enzymes was determined after treatment with fluorescently labeled ARPs. Alternatively, biotinylated ARPs were bound to streptavidin slides in order to identify their affinity for enzymes in solution. Both systems, the protein- and ARP microarrays proved to be useful and versatile tools for the rapid identification and characterization of novel and known lipolytic enzymes.     

Chemical Probes and Activity-Based Protein Profiling for Cancer Research

Chemical probes can be used to understand the complex biological nature of diseases. Due to the diversity of cancer types and dynamic regulatory pathways involved in the disease, there is a need to identify signaling pathways and associated proteins or enzymes that are traceable or detectable in tests for cancer diagnosis and treatment. Currently, fluorogenic chemical probes are widely used to detect cancer-associated proteins and their binding partners. These probes are also applicable in photodynamic therapy to determine drug efficacy and monitor regulating factors. In this review, we discuss the synthesis of chemical probes for different cancer types from 2016 to the present time and their application in monitoring the activity of transferases, hydrolases, deacetylases, oxidoreductases, and immune cells. Moreover, we elaborate on their potential roles in photodynamic therapy.     

Two-Step Bioorthogonal Activity-Based Protein Profiling of Individual Human Proteasome Catalytic Sites

Bioorthogonal chemistry allows the selective modification of biomolecules in complex biological samples. One application of this methodology is in two-step activity-based protein profiling (ABPP), a methodology that is particularly attractive where direct ABPP using fluorescent or biotinylated probes is ineffective. Herein we describe a set of norbornene-modified, mechanism-based proteasome inhibitors aimed to be selective for each of the six catalytic sites of human constitutive proteasomes and immunoproteasomes. The probes developed for β1i, β2i, β5c, and β5i proved to be useful two-step ABPs that effectively label their developed proteasome subunits in both Raji cell extracts and living Raji cells through inverse-electron-demand Diels–Alder (iEDDA) ligation. The compound developed for β1c proved incapable of penetrating the cell membrane, but effectively labels β1c in vitro. The compound developed for β2c proved not selective, but its azide-containing analogue LU-002c proved effective in labeling of β2c via azide–alkyne click ligation chemistry both in vitro and in situ. In total, our results contribute to the growing list of proteasome activity tools to include five subunit-selective activity-based proteasome probes, four of which report on proteasome activities in living cells.