Activity-based Protein Profiling Research Articles

Activity-based Tools for Interrogating Host Biology During Infection.

Host cells sense and respond to pathogens by dynamically regulating cell signaling. The rapid modulation of signaling pathways is achieved by post-translational modifications (PTMs) that can alter protein structure, function, and/or binding interactions. By using chemical probes to broadly profile changes in enzyme function or side-chain reactivity, activity-based protein profiling (ABPP) can reveal PTMs that regulate host-microbe interactions. While ABPP has been widely utilized to uncover microbial mechanisms of pathogenesis, in this review, we focus on more recent applications of this technique to the discovery of host PTMs and enzymes that modulate signaling within infected cells. Collectively, these advances underscore the importance of ABPP as a tool for interrogating the host response to infection and identifying potential targets for host-directed therapies.

Serine Hydrolase Activity‐Based Probes for Use in Chemical Proteomics

AbstractSerine hydrolases (SHs) comprise a large superfamily of enzymes that play critical roles in many biological processes. Despite their importance, many SHs remain uncharacterized and the vast majority of SHs lack selective inhibitors. In response, activity‐based protein profiling (ABPP) and activity‐based probes (ABPs) have been leveraged to construct a more comprehensive picture of the SH proteome. Since the utility of ABPP is largely dictated by the reactivity profile of the ABPs deployed, novel scaffolds and chemotypes are needed to expand the breadth and selectivity of SH‐targeting ABPs. In this review, we highlight recent innovations in SH probe development, covering both established and emerging electrophilic warheads. We then discuss how strategic implementation of SH‐targeting ABPs has yielded selective, potent inhibitors and imaging agents with broad use. Finally, we present methods for ABP diversification and explore cutting‐edge applications in therapeutics development and discovery biology.

Inhibition of diacylglycerol lipase β modulates lipid and endocannabinoid levels in the ex vivo human placenta.

Lipids and fatty acids are key components in metabolic processes of the human placenta, thereby contributing to the development of the fetus. Placental dyslipidemia and aberrant activity of lipases have been linked to diverse pregnancy associated complications, such as preeclampsia and preterm birth. The serine hydrolases, diacylglycerol lipase α and β (DAGLα, DAGLβ) catalyze the degradation of diacylglycerols, leading to the formation of monoacylglycerols (MAG), including one main endocannabinoid 2-arachidonoylglycerol (2-AG). The major role of DAGL in the biosynthesis of 2-AG is evident from various studies in mice but has not been investigated in the human placenta. Here, we report the use of the small molecule inhibitor DH376, in combination with the ex vivo placental perfusion system, activity-based protein profiling (ABPP) and lipidomics, to determine the impact of acute DAGL inhibition on placental lipid networks. DAGLα and DAGLβ mRNA expression was detected by RT-qPCR and in situ hybridization in term placentas. Immunohistochemistry staining for CK7, CD163 and VWF was applied to localize DAGLβ transcripts to different cell types of the placenta. DAGLβ activity was determined by in- gel and MS-based activity-based protein profiling (ABPP) and validated by addition of the enzyme inhibitors LEI-105 and DH376. Enzyme kinetics were measured by EnzChek™ lipase substrate assay. Ex vivo placental perfusion experiments were performed +/- DH376 [1 µM] and changes in tissue lipid and fatty acid profiles were measured by LC-MS. Additionally, free fatty acid levels of the maternal and fetal circulations were determined. We demonstrate that mRNA expression of DAGLβ prevails in placental tissue, compared to DAGLα (p ≤ 0.0001) and that DAGLβ is mainly located to CK7 positive trophoblasts (p ≤ 0.0001). Although few DAGLα transcripts were identified, no active enzyme was detected applying in-gel or MS-based ABPP, which underlined that DAGLβ is the principal DAGL in the placenta. DAGLβ dependent substrate hydrolysis in placental membrane lysates was determined by the application of LEI-105 and DH376. Ex vivo pharmacological inhibition of DAGLβ by DH376 led to reduced MAG tissue levels (p ≤ 0.01), including 2-AG (p≤0.0001). We further provide an activity landscape of serine hydrolases, showing a broad spectrum of metabolically active enzymes in the human placenta. Our results emphasize the role of DAGLβ activity in the human placenta by determining the biosynthesis of 2-AG. Thus, this study highlights the special importance of intra-cellular lipases in lipid network regulation. Together, the activity of these specific enzymes may contribute to the lipid signaling at the maternal-fetal interface, with implications for function of the placenta in normal and compromised pregnancies.

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Cyclopropenone, Cyclopropeniminium Ion, and Cyclopropenethione as Novel Electrophilic Warheads for Potential Target Discovery of Triple-Negative Breast Cancer.

Because very few targets are currently available for drug development, triple-negative breast cancer (TNBC) has been defined as one of the most difficult diseases for chemotherapy. Herein, we describe a suite of novel electrophilic warheads, which we have used in chemical proteomics studies in a search for potential targets for TNBC. Binding site analysis revealed that these warheads can modify not only highly nucleophilic residues, including cysteine and lysine, but also weakly nucleophilic residues. Cys12 of Kirsten rat sarcoma (KRASG12C) was successfully labeled by cyclopropenone and the cyclopropeniminium ions. Moderate inhibitory activity against TNBC cells was achieved with these novel electrophile-based probes. Activity-based protein profiling reveals that these electrophiles can covalently label a series of essential protein targets, including ALDH2, LRPPRC, and FABP5 from MDA-MB-231 cells. Further functional validation experiments demonstrated that FABP5 might be a potential target for TNBC.

Accelerating inhibitor discovery for deubiquitinating enzymes

Deubiquitinating enzymes (DUBs) are an emerging drug target class of ~100 proteases that cleave ubiquitin from protein substrates to regulate many cellular processes. A lack of selective chemical probes impedes pharmacologic interrogation of this important gene family. DUBs engage their cognate ligands through a myriad of interactions. We embrace this structural complexity to tailor a chemical diversification strategy for a DUB-focused covalent library. Pairing our library with activity-based protein profiling as a high-density primary screen, we identify selective hits against 23 endogenous DUBs spanning four subfamilies. Optimization of an azetidine hit yields a probe for the understudied DUB VCPIP1 with nanomolar potency and in-family selectivity. Our success in identifying good chemical starting points as well as structure-activity relationships across the gene family from a modest but purpose-build library challenges current paradigms that emphasize ultrahigh throughput in vitro or virtual screens against an ever-increasing scope of chemical space.

Ligand‐Directed Chemistry for Protein Labeling for Affinity‐Based Protein Analysis

AbstractStructural and functional analyses of proteins‐of‐interest (POI) in multimolecular crowding conditions (mMCC) such as live cells and tissues are regarded as inevitable challenges for in depth understanding of the real shapes of POIs in their existing natural environments. Activity‐based protein profiling (ABPP) is a definitely powerful tool capable of analyzing a proteome possessing a particular activity under mMCC. While ABPP usually targets a proteome of interest, study of a particular protein in mMCC is also valuable. Although activity‐based probes (ABPs) are often used for this aim, most of conventional ABPs cause the loss of original activities, and therefore are not perfectly suitable for functional analysis of labeled proteins. Ligand‐directed chemistry (LDchem) developed by our group is an alternative approach of ABPs, that can modify a surface of POI rather than its active site using a cleavable electrophile in a traceless manner. LDchem thus enables the POI labeling with a synthetic fluorophore with no or minimal effects on the original functions of POIs even in mMCC. In this review, we briefly describe a principle of LDchem for native protein labeling and summarize its recent chemical biology applications such as the imaging‐based biological analysis of POI functions and construction of POI‐based biosensors.

Understanding and Targeting the Endocannabinoid System with Activity‐Based Protein Profiling

AbstractThe endocannabinoid system (ECS) is a widespread modulatory system composed of cannabinoid receptors, endogenous signaling lipids termed endocannabinoids and the enzymes for the biosynthesis and degradation of these endocannabinoids. It plays a crucial role in diverse (patho)physiological functions, such as neurotransmission, neural development, synaptic plasticity, mood, food intake, and inflammation. Enzymes involved in the biosynthesis (e. g. NAPE‐PLD, DAGLs) and degradation (e. g. FAAH, MAGL) of endocannabinoids have attracted attention from both academia and industry due to their therapeutic potential. The discovery of selective inhibitors for these enzymes is important for functional assignment and biomedical applications. Activity‐based protein profiling (ABPP) has emerged as a powerful technique, which to a large extend accelerated the development of selective inhibitors for ECS enzymes. This review summarizes the representative activity‐based probes (ABPs) and small molecular inhibitors developed in the past two decades for ECS enzymes and will discuss the biological discoveries attributed by the application of these small molecules.

Unraveling Complex MicroRNA Signaling Pathways with Activity‐Based Protein Profiling to Guide Therapeutic Discovery**

AbstractmicroRNAs (miRNAs) are important cell factors that can play essential roles, such as regulating transcription factors in embryonic development. Traditionally, microarrays or high‐throughput sequencing of crosslinked protein‐RNA complexes have been applied to deduce direct targets, but these techniques fail to account for biomolecular interactions with functional consequences. Activity‐based protein profiling (ABPP) represents a promising alternative to conventional methods and was applied to the discovery of functional targets of human miRNAs. ABPP identified downregulation of MGLL and LIPC, in response to miR‐185 and miR‐27b, respectively. Hepatitis C virus (HCV) infection was found to be modulated by these indirect downstream effectors. These findings highlight the importance of identifying functional targets in developing novel ways to mimic or inhibit miRNA pathways. This review will focus on miRNA regulation and how ABPP can be used to study these pathways.

Characterizing metabolic drivers of Clostridioides difficile infection with activity-based hydrazine probes.

Many enzymes require post-translational modifications or cofactor machinery for primary function. As these catalytically essential moieties are highly regulated, they act as dual sensors and chemical handles for context-dependent metabolic activity. Clostridioides difficile is a major nosocomial pathogen that infects the colon. Energy generating metabolism, particularly through amino acid Stickland fermentation, is central to colonization and persistence of this pathogen during infection. Here using activity-based protein profiling (ABPP), we revealed Stickland enzyme activity is a biomarker for C. difficile infection (CDI) and annotated two such cofactor-dependent Stickland reductases. We structurally characterized the cysteine-derived pyruvoyl cofactors of D-proline and glycine reductase in C. difficile cultures and showed through cofactor monitoring that their activity is regulated by their respective amino acid substrates. Proline reductase was consistently active in toxigenic C. difficile, confirming the enzyme to be a major metabolic driver of CDI. Further, activity-based hydrazine probes were shown to be active site-directed inhibitors of proline reductase. As such, this enzyme activity, via its druggable cofactor modality, is a promising therapeutic target that could allow for the repopulation of bacteria that compete with C. difficile for proline and therefore restore colonization resistance against C. difficile in the gut.

(Glycan Binding) Activity-Based Protein Profiling in Cells Enabled by Mass Spectrometry-Based Proteomics.

The presence of glycan modifications at the cell surface and other locales positions them as key regulators of cell recognition and function. However, due to the complexity of glycosylation, the annotation of which proteins bear glycan modifications, which glycan patterns are present, and which proteins are capable of binding glycans is incomplete. Inspired by activity-based protein profiling to enrich for proteins in cells based on select characteristics, these endeavors have been greatly advanced by the development of appropriate glycan-binding and glycan-based probes. Here, we provide context for these three problems and describe how the capability of molecules to interact with glycans has enabled the assignment of proteins with specific glycan modifications or of proteins that bind glycans. Furthermore, we discuss how the integration of these probes with high resolution mass spectrometry-based technologies has greatly advanced glycoscience.

Fluorogenic Granzyme A Substrates Enable Real-Time Imaging of Adaptive Immune Cell Activity.

Cytotoxic immune cells, including T lymphocytes (CTLs) and natural killer (NK) cells, are essential components of the host response against tumors. CTLs and NK cells secrete granzyme A (GzmA) upon recognition of cancer cells; however, there are very few tools that can detect physiological levels of active GzmA with high spatiotemporal resolution. Herein, we report the rational design of the near-infrared fluorogenic substrates for human GzmA and mouse GzmA. These activity-based probes display very high catalytic efficiency and selectivity over other granzymes, as shown in tissue lysates from wild-type and GzmA knock-out mice. Furthermore, we demonstrate that the probes can image how adaptive immune cells respond to antigen-driven recognition of cancer cells in real time.

Fluorogenic Granzyme A Substrates Enable Real-Time Imaging of Adaptive Immune Cell Activity.

Cytotoxic immune cells, including T lymphocytes (CTLs) and natural killer (NK) cells, are essential components of the host response against tumors. CTLs and NK cells secrete granzyme A (GzmA) upon recognition of cancer cells; however, there are very few tools that can detect physiological levels of active GzmA with high spatiotemporal resolution. Herein, we report the rational design of the near-infrared fluorogenic substrates for human GzmA and mouse GzmA. These activity-based probes display very high catalytic efficiency and selectivity over other granzymes, as shown in tissue lysates from wild-type and GzmA knock-out mice. Furthermore, we demonstrate that the probes can image how adaptive immune cells respond to antigen-driven recognition of cancer cells in real time.

Combinatorial assembly and design of enzymes.

The design of structurally diverse enzymes is constrained by long-range interactions that are necessary for accurate folding. We introduce an atomistic and machine learning strategy for the combinatorial assembly and design of enzymes (CADENZ) to design fragments that combine with one another to generate diverse, low-energy structures with stable catalytic constellations. We applied CADENZ to endoxylanases and used activity-based protein profiling to recover thousands of structurally diverse enzymes. Functional designs exhibit high active-site preorganization and more stable and compact packing outside the active site. Implementing these lessons into CADENZ led to a 10-fold improved hit rate and more than 10,000 recovered enzymes. This design-test-learn loop can be applied, in principle, to any modular protein family, yielding huge diversity and general lessons on protein design principles.

Synthesis and structure-activity relationships of N - (3 - (1H-imidazol-2-yl) phenyl) - 3-phenylpropionamide derivatives as a novel class of covalent inhibitors of p97/VCP ATPase

Noncovalent inhibitors of p97 have entered clinical studies. Compared with noncovalent inhibitors, covalent inhibitors have unique advantages in maintaining inhibitory effect and improving the resistance of the target. We previously employed the activity-based protein profiling to definitely identify p97 as the protein target of FL-18 that has a unique scaffold of benpropargylamide coupled with an imidazole. In this study, we report a thorough structure-activity-relationship study involving the new scaffold. A total of three rounds of optimization led to the discovery of the most potent covalent inhibitor of p97 to date. A chemical proteomics study indicated that the newly-synthesized compounds still targeted the C522 residue of p97 and retained selectivity among the complicated whole proteome. This study provides a suite of new covalent inhibitors of p97 to assist in its biological study and drug discovery.

Chemically diverse activity-based probes with unexpected inhibitory mechanisms targeting trypsin-like serine proteases.

Activity-based probes (ABP) are molecules that bind covalently to the active form of an enzyme family, making them an attractive tool for target and biomarker identification and drug discovery. The present study describes the synthesis and biochemical characterization of novel activity-based probes targeting trypsin-like serine proteases. We developed an extensive library of activity-based probes with "clickable" affinity tags and a diaryl phosphonate warhead. A wide diversity was achieved by including natural amino acid analogs as well as basic polar residues as side chains. A detailed enzymatic characterization was performed in a panel of trypsin-like serine proteases. Their inhibitory potencies and kinetic profile were examined, and their IC50 values, mechanism of inhibition, and kinetic constants were determined. The activity-based probes with a benzyl guanidine side chain showed the highest inhibitory effects in the panel. Surprisingly, some of the high-affinity probes presented a reversible inhibitory mechanism. On the other hand, probes with different side chains exhibited the expected irreversible mechanism. For the first time, we demonstrate that not only irreversible probes but also reversible probes can tightly label recombinant proteases and proteases released from human mast cells. Even under denaturing SDS-PAGE conditions, reversible slow-tight-binding probes can label proteases due to the formation of high-affinity complexes and slow dissociation rates. This unexpected finding will transform the view on the required irreversible nature of activity-based probes. The diversity of this library of activity-based probes combined with a detailed enzyme kinetic characterization will advance their applications in proteomic studies and drug discovery.

Universal Strategy to Develop Fluorogenic Probes for Lysine Deacylase/Demethylase Activity and Application in Discriminating Demethylation States.

Dynamically controlling the post-translational modification of the ε-amino groups of lysine residues is critical for regulating many cellular events. Increasing studies have revealed that many important diseases, including cancer and neurological disorders, are associated with the malfunction of lysine deacylases and demethylases. Developing fluorescent probes that are capable of detecting lysine deacylase and demethylase activity is highly useful for interrogating their roles in epigenetic regulation and diseases. Due to the distinct substrate recognition of these epigenetic eraser enzymes, designing a universal strategy for detecting their activity poses substantial difficulty. Moreover, designing activity-based probes for differentiating their demethylation states is even more challenging and still remains largely unexplored. Herein, we report a universal strategy to construct probes that can detect the enzymatic activity of epigenetic "erasers" through NBD-based long-distance intramolecular reactions. The probes can be easily prepared by installing the O-NBD group at the C-terminal residue of specific peptide substrates by click chemistry. Based on this strategy, detecting the activity of lysine deacetylase, desuccinylase, or demethylase with superior sensitivity and selectivity has been successfully achieved through single-step probe development. Furthermore, the demethylase probe based on this strategy is capable of distinguishing different demethylation states by both absorption and fluorescence lifetime readout. We envision that these newly developed probes will provide powerful tools to facilitate drug discovery in epigenetics in the future.

Advances in applications of activity-based chemical probes in the characterization of amino acid reactivities

The discovery of novel drug targets enhances the development of novel drugs, and the discovery of novel target proteins depends on highly accurate high-throughput methods of analyzing drug-protein interactions. Protein expression levels, spatial localization, and structural differences directly affect pharmacodynamics. To date, >20000 proteins have been discovered in the human proteome by the genome and proteome projects via gene and protein sequencing. Understanding the biological functions of proteins is critical in identifying and regulating biological processes, with most remaining unidentified. Until recently, >85% of proteins were considered undruggable, mainly because of the lack of binding pockets and active sites targeted by small molecules. Therefore, characterization of the reactive sites of amino acids based on proteomic hierarchy is the key to novel drug design. Recently, with the rapid development of mass spectrometry (MS), the study of drug-target protein interactions based on proteomics technology has been considerably promoted. Activity-based protein profiling (ABPP) is an active chemical probe-based method of detecting functional enzymes and drug targets in complex samples. Compared with classical proteomics strategies, ABPP is based mainly on protein activity. It has been successfully utilized to characterize the activities of numerous protease families with crucial biological functions, such as serine hydrolases, protein kinases, glycosidases, and metalloenzymes. It has also been used to identify key enzymes that are closely related to diseases and develop covalent inhibitors for use in disease treatment. The technology used in proteome analysis ranges from gel electrophoresis to high-throughput MS due to the progress of MS technology. ABPP strategies combined with chemical probe labeling and quantitative MS enable the characterization of amino acid activity, which may enhance the discovery of novel drug targets and the development of lead compounds. Amino acid residues play critical roles in protein structures and functions, and covalent drugs targeting these amino acids are effective in treating numerous diseases. There are 20 main types of natural amino acids, with different reactivities, in the proteins in the human body. In addition, the proteins and amino acids are affected by the spatial microenvironment, leading to significant differences in their spatial reactivities. The key in evaluating the reactivities of amino acids via ABPP is to select those with high reactivities. The core of the ABPP strategy is the use of chemical probes to label amino acid sites that exhibit higher activities in certain environments. The activity-based probe (ABP) at the core of ABPP consists of three components: reactive, reporter groups and a linker. The reactive group is the basis of the ABP and anchors the drug target via strong forces, such as covalent bonds. The reaction exhibits a high specificity and conversion rate and should display a good biocompatibility. Activity probes based on different amino acid residues have been developed, and the screening of amino acid activity combined with isotope labeling is a new focus of research. Currently, different types of ABPs have been developed to target amino acids and characterize amino acid reactivity, such as cysteine labeled with an electrophilic iodoacetamide probe and lysine labeled with activated esters. ABPP facilitates the discovery of potentially therapeutic protein targets, the screening of lead compounds, and the identification of drug targets, thus aiding the design of novel drugs. This review focuses on the development of ABPP methods and the progress in the screening of amino acid reactivity using ABPs, which should be promising methods for use in designing targeted drugs with covalent interactions.

Affinity- and activity-based probes synthesized from structurally diverse hops-derived xanthohumol flavonoids reveal highly varied protein profiling in Escherichia coli.

Xanthohumol, the principle prenylflavonoid found in hops (Humulus lupulus) and a reported anti-inflammatory agent, has great potential for pharmaceutical interventions related to inflammatory disorders in the gut. A suite of probes was prepared from xanthohumol and its structural isomer isoxanthohumol to enable profiling of both protein affinity binding and catalytic enzyme reactivity. The regiochemistry of the reactive group on the probes was altered to reveal how probe structure dictates protein labeling, and which probes best emulate the natural flavonoids. Affinity- and activity-based probes were applied to Escherichia coli, and protein labeling was measured by chemoproteomics. Structurally dependent activity-based probe protein labeling demonstrates how subtle alterations in flavonoid structure and probe reactive groups can result in considerably different protein interactions. This work lays the groundwork to expand upon unexplored cellular activities related to xanthohumol interactions, metabolism, and anti-inflammatory mechanisms.

Neutrophil serine proteases

The identification and characterization of the four active neutrophil serine proteases (NSPs) have provided a better understanding of their roles in various physiological and pathological processes. The availability of appropriate tools such as substrates, inhibitors, and activity-based probes (ABPs) for studying their activity and functions in cells has become increasingly important. In this paper, the authors provide a comprehensive overview of the current state of knowledge on the tools available for studying NSPs. The substrates, inhibitors, and ABPs that have been developed to date are described, including their strengths and limitations. The authors also discuss the potential implications of these tools for future research on NSPs, including their potential use in the development of new therapeutics for various diseases. Overall, this paper highlights the importance of understanding the activity and functions of NSPs and provides valuable information on the tools available for studying these proteases.