Protein Profiling Probes Research Articles

Target Identification in Anti-Tuberculosis Drug Discovery.

Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis (TB), a disease that, although preventable and curable, remains a global epidemic due to the emergence of resistance and a latent form responsible for a long period of treatment. Drug discovery in TB is a challenging task due to the heterogeneity of the disease, the emergence of resistance, and uncomplete knowledge of the pathophysiology of the disease. The limited permeability of the cell wall and the presence of multiple efflux pumps remain a major barrier to achieve effective intracellular drug accumulation. While the complete genome sequence of Mtb has been determined and several potential protein targets have been validated, the lack of adequate models for in vitro and in vivo studies is a limiting factor in TB drug discovery programs. In current therapeutic regimens, less than 0.5% of bacterial proteins are targeted during the biosynthesis of the cell wall and the energetic metabolism of two of the most important processes exploited for TB chemotherapeutics. This review provides an overview on the current challenges in TB drug discovery and emerging Mtb druggable proteins, and explains how chemical probes for protein profiling enabled the identification of new targets and biomarkers, paving the way to disruptive therapeutic regimens and diagnostic tools.

Chemical Labeling of Protein 4'-Phosphopantetheinylation in Surfactin-Producing Nonribosomal Peptide Synthetases.

4'-Phosphopantetheinylation is an essential posttranslational modification of the primary and secondary metabolic pathways in prokaryotes and eukaryotes. Several peptide-based natural products are biosynthesized by large, multifunctional enzymes known as nonribosomal peptide synthetases (NRPSs), responsible for producing virulence factors and many pharmaceuticals. The thiolation (T) domain serves as a covalent tether for substrates and intermediates in nonribosomal peptide biosynthesis and must be posttranslationally modified with a 4'-phosphopantetheinyl group. To detect 4'-phosphopantetheinylation of NRPS in bacterial proteomes, we developed a 5'-(vinylsulfonylaminodeoxy)adenosine scaffold with a clickable functionality, enabling effective chemical labeling of 4'-phosphopantethylated NRPSs. In this chapter, we describe the design and synthesis of an activity-based protein profiling probe and summarize our work toward developing a series of protocols for the labeling and visualization of 4'-phosphopantetheinylation of endogenous NRPSs in complex proteomes.

Activity-Based Protein Profiling Probe for the Detection of Enzymes Catalyzing Polysorbate Degradation.

Polysorbates are nonionic surfactants that have been widely used in biotherapeutic formulations to prevent protein aggregation and denaturation. However, polysorbates are subject to degradation after prolonged storage if certain lipases are present in the biotherapeutic product. Because the degradation of polysorbates compromises the shelf life of biotherapeutics and leads to the formation of undesirable products such as protein aggregates and subvisible particles, it is important to identify the active enzymes that catalyze polysorbate hydrolysis. In this study, we developed a novel fluorophosphonate activity-based protein profiling (ABPP) probe (termed the REGN probe), which mimics the structure of polysorbate and targets lipases catalyzing polysorbate degradation. We demonstrated that the REGN probe could enrich certain lipases from Chinese hamster ovary (CHO) cell lysate by more than 100-fold compared with direct tryptic digestion. Furthermore, we found that the REGN probe had higher lipase enrichment efficiency than commercially available ABPP probes including fluorophosphonate-biotin (FP-biotin) and FP-desthiobiotin. Remarkably, the REGN probe can enrich several lipases that cannot be labeled by commercial probes, such as lysosomal acid lipase and cytosolic phospholipase A2. Additionally, we showed that lipases with abundances as low as 0.08 ppm in drug substances were detected by the REGN probe enrichment and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Collectively, we have developed a novel ABPP probe with higher enrichment efficiency and broader coverage for lipases compared with commercial probes, and this probe can be used to detect the trace level of lipases in biotherapeutic products and to facilitate their development and manufacturing.

Production of a Natural Dihydropteroate Synthase and Development of a Signal-Amplified Pseudo-Immunoassay for the Determination of Sulfonamides in Pork.

In this study, a type of magnetic photoaffinity-labeled activity-based protein profiling probe for sulfonamide drugs was first synthesized for the purpose of capturing the natural dihydropteroate synthase of Escherichia coli by using simple incubation and magnetic separation. After characterization of its identity with LC-ESI-MS/MS, this enzyme was used as a recognition reagent to develop a direct competitive pseudo-ELISA for the determination of the residues of 40 sulfonamides in pork. Because of the use of streptavidin-horseradish peroxidase and biotinylated horseradish peroxidase as a signal-amplified system, the limits of detection for the 40 drugs were in the range of 0.001-0.016 ng/mL. Compared to the steps in a conventional assay formation, the operation steps were the same, but the sensitivities increased 32-88-fold. Furthermore, the assay performances were better than the previously reported immunoassays performances for sulfonamides. Therefore, this method could be used as a practical tool for multiscreening the trace levels of sulfonamides residues in food samples.

Flipping the Switch: Innovations in Inducible Probes for Protein Profiling.

Over the past two decades, activity-based probes have enabled a range of discoveries, including the characterization of new enzymes and drug targets. However, their suitability in some labeling experiments can be limited by nonspecific reactivity, poor membrane permeability, or high toxicity. One method for overcoming these issues is through the development of “inducible” activity-based probes. These probes are added to samples in an unreactive state and require in situ transformation to their active form before labeling can occur. In this Review, we discuss a variety of approaches to inducible activity-based probe design, different means of probe activation, and the advancements that have resulted from these applications. Additionally, we highlight recent developments which may provide opportunities for future inducible activity-based probe innovations.

Synthesis of photoaffinity labeled activity-based protein profiling probe and production of natural TetR protein for immunoassay of tetracyclines in milk

In this study, a photoaffinity labeled activity-based protein profiling probe for tetracycline drugs was first synthesized that was used to capture a type of natural TetR protein from Escherichia coli. The TetR simultaneously recognized 10 tetracycline drugs. Then it was used as recognition reagent to develop a direct competitive chemiluminescence immunoassay for detection of the 10 drugs in milk. The IC50 values for these drugs were in the range of 0.084–0.32 ng/mL, and the limits of detection were in the range of 2.0–9.0 pg/mL. Their recoveries from the standards fortified blank milk samples were in the range of 68.4%-91.3%. After comparison, the natural TetR protein and the developed method showed better performances than the previous TetR proteins and the related immunoassays. Therefore, this method could be used as a practical tool for routine screening the residues of tetracycline drugs in milk. Furthermore, this type of probe could also be used to produce the receptors and develop different immunoassays for other classes of drugs.

Activity-Based Hydrazine Probes for Protein Profiling of Electrophilic Functionality in Therapeutic Targets.

Most known probes for activity-based protein profiling (ABPP) use electrophilic groups that tag a single type of nucleophilic amino acid to identify cases in which its hyper-reactivity underpins function. Much important biochemistry derives from electrophilic enzyme cofactors, transient intermediates, and labile regulatory modifications, but ABPP probes for such species are underdeveloped. Here, we describe a versatile class of probes for this less charted hemisphere of the proteome. The use of an electron-rich hydrazine as the common chemical modifier enables covalent targeting of multiple, pharmacologically important classes of enzymes bearing diverse organic and inorganic cofactors. Probe attachment occurs by both polar and radicaloid mechanisms, can be blocked by molecules that occupy the active sites, and depends on the proper poise of the active site for turnover. These traits will enable the probes to be used to identify specific inhibitors of individual members of these multiple enzyme classes, making them uniquely versatile among known ABPP probes.

A Class of Valuable (Pro-)Activity-Based Protein Profiling Probes: Application to the Redox-Active Antiplasmodial Agent, Plasmodione.

Plasmodione (PD) is a potent antimalarial redox-active drug acting at low nM range concentrations on different malaria parasite stages. In this study, in order to determine the precise PD protein interactome in parasites, we developed a class of (pro-)activity-based protein profiling probes (ABPP) as precursors of photoreactive benzophenone-like probes based on the skeleton of PD metabolites (PDO) generated in a cascade of redox reactions. Under UV-photoirradiation, we clearly demonstrate that benzylic oxidation of 3-benzylmenadione 11 produces the 3-benzoylmenadione probe 7, allowing investigation of the proof-of-concept of the ABPP strategy with 3-benzoylmenadiones 7–10. The synthesized 3-benzoylmenadiones, probe 7 with an alkyne group or probe 9 with -NO2 in para position of the benzoyl chain, were found to be the most efficient photoreactive and clickable probes. In the presence of various H-donor partners, the UV-irradiation of the photoreactive ABPP probes generates different adducts, the expected “benzophenone-like” adducts (pathway 1) in addition to “benzoxanthone” adducts (via two other pathways, 2 and 3). Using both human and Plasmodium falciparum glutathione reductases, three protein ligand binding sites were identified following photolabeling with probes 7 or 9. The photoreduction of 3-benzoylmenadiones (PDO and probe 9) promoting the formation of both the corresponding benzoxanthone and the derived enone could be replaced by the glutathione reductase-catalyzed reduction step. In particular, the electrophilic character of the benzoxanthone was evidenced by its ability to alkylate heme, as a relevant event supporting the antimalarial mode of action of PD. This work provides a proof-of-principle that (pro-)ABPP probes can generate benzophenone-like metabolites enabling optimized activity-based protein profiling conditions that will be instrumental to analyze the interactome of early lead antiplasmodial 3-benzylmenadiones displaying an original and innovative mode of action.

Cover Feature: Differently Tagged Probes for Protein Profiling of Mitochondria (ChemBioChem 9/2019)

Cover Feature: Differently Tagged Probes for Protein Profiling of Mitochondria (ChemBioChem 9/2019)

Differently Tagged Probes for Protein Profiling of Mitochondria.

The mitochondrion is one of the most important organelles in the eukaryotic cell. Characterization of the mitochondrial proteome is a prerequisite for understanding its cellular functions at the molecular level. Here we report a proteomics method based on mitochondrion-targeting groups and click chemistry. In our strategy, three different mitochondrion-targeting moieties were each augmented with a clickable handle and a cysteine-reactive group. Fluorescence-based bioimaging and fractionation experiments clearly showed that most signals arising from the labels were localized in the mitochondria of cells, as a result of covalent attachment between probe and target proteins. The three probes had distinct profiling characteristics. Furthermore, we successfully identified more than two hundred mitochondrial proteins. The results showed that different mitochondrion-targeting groups targeted distinct proteins with partial overlap. Most of the labeled proteins were localized in the mitochondrial matrix and inner mitochondrial membrane. Our results provide a tool for chemoproteomic analysis of mitochondrion-related proteins.

Dissection, Optimization, and Structural Analysis of a Covalent Irreversible DDAH1 Inhibitor.

Inhibitors of the human enzyme dimethylarginine dimethylaminohydrolase-1 (DDAH1) can control endogenous nitric oxide production. A time-dependent covalent inactivator of DDAH1, N5-(1-imino-2-chloroethyl)-l-ornithine ( KI = 1.3 μM, kinact = 0.34 min-1), was conceptually dissected into two fragments and each characterized separately: l-norvaline ( Ki = 470 μM) and 2-chloroacetamidine ( KI = 310 μM, kinact = 4.0 min-1). This analysis suggested that the two fragments were not linked in a manner that allows either to reach full affinity or reactivity, prompting the synthesis and characterization of three analogues: two that mimic the dimethylation status of the substrate, N5-(1-imino-2-chloroisopropyl)-l-ornithine ( kinact /KI = 208 M-1 s-1) and N5-(1-imino-2-chlorisopropyl)-l-lysine ( kinact /KI = 440 M-1 s-1), and one that lengthens the linker beyond that found in the substrate, N5-(1-imino-2-chloroethyl)-l-lysine (Cl-NIL, KI = 0.19 μM, kinact = 0.22 min-1). Cl-NIL is one of the most potent inhibitors reported for DDAH1, inactivates with a second order rate constant (1.9 × 104 M-1 s-1) larger than the catalytic efficiency of DDAH1 for its endogenous substrate (1.6 × 102 M-1 s-1), and has a partition ratio of 1 with a >100 000-fold selectivity for DDAH1 over arginase. An activity-based protein-profiling probe is used to show inhibition of DDAH1 within cultured HEK293T cells (IC50 = 10 μM) with cytotoxicity appearing only at higher concentrations (ED50 = 118 μM). A 1.91 Å resolution X-ray crystal structure reveals specific interactions made with DDAH1 upon covalent inactivation by Cl-NIL. Dissecting a covalent inactivator and analysis of its constituent fragments proved useful for the design and optimization of this potent and effective DDAH1 inhibitor.

Rational Tuning of Fluorobenzene Probes for Cysteine-Selective Protein Modification.

Fluorobenzene probes for protein profiling through selective cysteine labeling have been developed by rational reactivity tuning. Tuning was achieved by selecting an electron-withdrawing para substituent in combination with variation of the number of fluorine substituents. Optimized probes chemoselectively arylated cysteine residues in proteins under aqueous conditions. Probes linked to azide, biotin, or a fluorophore were applicable to labeling of eGFP and albumin. Selective inhibition of cysteine proteases was also demonstrated with the probes. Additionally, probes were tuned for site-selective labeling of cysteine residues and for activity-based protein profiling in cell lysates.

Rational Tuning of Fluorobenzene Probes for Cysteine‐Selective Protein Modification

AbstractFluorobenzene probes for protein profiling through selective cysteine labeling have been developed by rational reactivity tuning. Tuning was achieved by selecting an electron‐withdrawing para substituent in combination with variation of the number of fluorine substituents. Optimized probes chemoselectively arylated cysteine residues in proteins under aqueous conditions. Probes linked to azide, biotin, or a fluorophore were applicable to labeling of eGFP and albumin. Selective inhibition of cysteine proteases was also demonstrated with the probes. Additionally, probes were tuned for site‐selective labeling of cysteine residues and for activity‐based protein profiling in cell lysates.

Activity-Based Phosphatidylinositol Kinase Probes Detect Changes to Protein-Protein Interactions During Hepatitis C Virus Replication.

Protein-protein interactions are integral to host-virus interactions and can contribute significantly to enzyme regulation by changing the localization of both host and viral enzymes within the cell, inducing conformational change relevant to enzyme activity or recruiting other additional proteins to form functional complexes. Identifying the interactors of active enzymes using an activity-based protein profiling probe has allowed us to characterize both normal enzyme activation mechanisms and the manner by which these mechanisms are hijacked and altered by the hepatitis C virus (HCV). Here, we report use of a novel activity-based probe, PIKBPyne, which labels phosphatidylinositol kinases (PIKs) in an activity-based manner, to investigate HCV-dependent changes in protein-protein interactions for PI4KB. Herein, we report the synthesis of new variations on PIKBPyne, compare their ability to label the interacting partners of PI4KB, and demonstrate the utility of our approach in characterizing virus-mediated changes to host function.

The Development of Benzimidazole-Based Clickable Probes for the Efficient Labeling of Cellular Protein Arginine Deiminases (PADs).

Citrullination is the post-translational hydrolysis of peptidyl-arginines to form peptidyl-citrulline, a reaction that is catalyzed by the protein arginine deiminases (PADs), a family of calcium-regulated enzymes. Aberrantly increased protein citrullination is associated with a slew of autoimmune diseases (e.g., rheumatoid arthritis (RA), multiple sclerosis, lupus, and ulcerative colitis) and certain cancers. Given the clear link between increased PAD activity and human disease, the PADs are therapeutically relevant targets. Herein, we report the development of next generation cell permeable and "clickable" probes (BB-Cl-Yne and BB-F-Yne) for covalent labeling of the PADs both in vitro and in cell-based systems. Using advanced chemoproteomic technologies, we also report the off targets of both BB-Cl-Yne and BB-F-Yne. The probes are highly specific for the PADs, with relatively few off targets, especially BB-F-Yne, suggesting the preferential use of the fluoroacetamidine warhead in next generation irreversible PAD inhibitors. Notably, these compounds can be used in a variety of modalities, including the identification of off targets of the parent compounds and as activity-based protein profiling probes in target engagement assays to demonstrate the efficacy of PAD inhibitors.

Artemisinin activity-based probes identify multiple molecular targets within the asexual stage of the malaria parasites Plasmodium falciparum 3D7

The artemisinin (ART)-based antimalarials have contributed significantly to reducing global malaria deaths over the past decade, but we still do not know how they kill parasites. To gain greater insight into the potential mechanisms of ART drug action, we developed a suite of ART activity-based protein profiling probes to identify parasite protein drug targets in situ. Probes were designed to retain biological activity and alkylate the molecular target(s) of Plasmodium falciparum 3D7 parasites in situ. Proteins tagged with the ART probe can then be isolated using click chemistry before identification by liquid chromatography-MS/MS. Using these probes, we define an ART proteome that shows alkylated targets in the glycolytic, hemoglobin degradation, antioxidant defense, and protein synthesis pathways, processes essential for parasite survival. This work reveals the pleiotropic nature of the biological functions targeted by this important class of antimalarial drugs.

Design and synthesis of an activity-based protein profiling probe derived from cinnamic hydroxamic acid

In our continued effort to discover new anti-hepatitis C virus (HCV) agents, we validated the anti-replicon activity of compound 1, a potent and selective anti-HCV hydroxamic acid recently reported by us. Generally favorable physicochemical and in vitro absorption, distribution, metabolism, and excretion (ADME) properties exhibited by 1 made it an ideal parent compound from which activity-based protein profiling (ABPP) probe 3 was designed and synthesized. Evaluation of probe 3 revealed that it possessed necessary anti-HCV activity and selectivity. Therefore, we have successfully obtained compound 3 as a suitable ABPP probe to identify potential molecular targets of compound 1. Probe 3 and its improved analogs are expected to join a growing list of ABPP probes that have made important contributions to not only the studies of biochemical and cellular functions but also discovery of selective inhibitors of protein targets.

Photoaffinity Probes for Identification of Carbohydrate‐Binding Proteins

AbstractCarbohydrate‐protein interactions mediate cellular signals, which are crucial in a diverse array of biological and pathological processes. Despite their importance, many carbohydrate‐protein interactions remain unknown due to inherent difficulties in studying them. With the aim to provide the first step in elucidating the biological roles of carbohydrates, photoaffinity labeling has been used as a promising chemical strategy for the detection and identification of carbohydrate‐binding proteins in their native environment. Recent efforts in the design of carbohydrate‐based photoaffinity probes include the use of selective photoreactive groups, development of biotinylated photoreactive groups and linkers, multivalent carbohydrate ligands, affinity‐based protein profiling probes, and photoreactive monosaccharide precursors for metabolic glycan labeling. These methods offer new tools to address challenges associated in capturing the often elusive carbohydrate‐protein interactions.

Design, Synthesis, and Optimization of Novel Epoxide Incorporating Peptidomimetics as Selective Calpain Inhibitors

Hyperactivation of the calcium-dependent cysteine protease calpain 1 (Cal1) is implicated as a primary or secondary pathological event in a wide range of illnesses and in neurodegenerative states, including Alzheimer's disease (AD). E-64 is an epoxide-containing natural product identified as a potent nonselective, calpain inhibitor, with demonstrated efficacy in animal models of AD. By use of E-64 as a lead, three successive generations of calpain inhibitors were developed using computationally assisted design to increase selectivity for Cal1. First generation analogues were potent inhibitors, effecting covalent modification of recombinant Cal1 catalytic domain (Cal1cat), demonstrated using LC-MS/MS. Refinement yielded second generation inhibitors with improved selectivity. Further library expansion and ligand refinement gave three Cal1 inhibitors, one of which was designed as an activity-based protein profiling probe. These were determined to be irreversible and selective inhibitors by kinetics studies comparing full length Cal1 with the general cysteine protease papain.

Activity-Based Protein Profiling of the Escherichia coli GlpG Rhomboid Protein Delineates the Catalytic Core

Rhomboid proteins comprise the largest class of intramembrane protease known, being conserved from bacteria to humans. The functional status of these proteases is typically assessed through direct or indirect detection of peptide cleavage products. Although these assays can report on the ability of a rhomboid to catalyze peptide bond cleavage, differences in measured hydrolysis rates can reflect changes in the structure and activity of catalytic residues, as well as the ability of the substrate to access the active site. Here we show that a highly reactive and sterically unencumbered fluorophosphonate activity-based protein profiling probe can be used to report on the catalytic integrity of active site residues in the Escherichia coli GlpG protein. We used results obtained with this probe on GlpG in proteomic samples, in combination with a conventional assay of proteolytic function on purified samples, to identify residues that are located on the cytoplasmic side of the lipid bilayer that are required for maximal proteolytic activity. Regions tested include the 90-residue aqueous-exposed N-terminus that encompasses a globular structure that we have determined by solution nuclear magnetic resonance, along with residues on the cytoplasmic side of the transmembrane domain core. While in most cases mutation or elimination of these residues did not significantly alter the catalytic status of the GlpG active site, the lipid-facing residue Arg227 was found to be important for maintaining a catalytically competent active site. In addition, we found a functionally critical region outside the transmembrane domain (TMD) core that is required for maximal protease activity. This region encompasses an additional 8-10 residues on the N-terminal side of the TMD core that precedes the first transmembrane segment and was not previously known to play a role in rhomboid function. These findings highlight the utility of the activity-based protein profiling approach for the characterization of rhomboid function.