Activity-based Protein Profiling Method Research Articles

Investigating pH Effects on Enzymes Catalyzing Polysorbate Degradation by Activity-Based Protein Profiling

Host cell proteins (HCPs) are process-related impurities that can negatively impact the quality of biotherapeutics. Some HCPs possess enzymatic activity and can affect the active pharmaceutical ingredient (API) or excipients such as polysorbates (PS). PSs are a class of non-ionic surfactants commonly used as excipients in biotherapeutics to enhance the stability of APIs. The enzyme activity of certain HCPs can result in the degradation of PSs, leading to particle formation and decreased shelf life of biotherapeutics. Identifying and characterizing these HCPs is therefore crucial. This study employed the Activity-Based Protein Profiling (ABPP) technique to investigate the effect of pH on the activity of HCPs that have the potential to degrade polysorbates. Two probes were utilized: the commercially available fluorophosphonate (FP)-Desthiobiotin probe and a probe based on the antiobesity drug, Orlistat. Over 50 HCPs were identified, showing a strong dependence on pH-milieu regarding their enzyme activity. These findings underscore the importance of accounting for pH variations in the ABPP method and other investigations of HCP activity. Notably, the Orlistat-based probe (OBP) enabled us to investigate the enzymatic activity of a wider range of HCPs, emphasizing the advantage of using more than one probe for ABPP. Finally, this study led to the discovery of previously unreported active enzymes, including three HCPs from the carboxylesterase enzyme family.

Activity‐Based Protein Profiling – Celebrating the Groundbreaking Contributions of Benjamin Cravatt

Activity‐Based Protein Profiling – Celebrating the Groundbreaking Contributions of Benjamin Cravatt

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.

An Activity-Based Oxaziridine Platform for Identifying and Developing Covalent Ligands for Functional Allosteric Methionine Sites: Redox-Dependent Inhibition of Cyclin-Dependent Kinase 4.

Activity-based protein profiling (ABPP) is a versatile strategy for identifying and characterizing functional protein sites and compounds for therapeutic development. However, the vast majority of ABPP methods for covalent drug discovery target highly nucleophilic amino acids such as cysteine or lysine. Here, we report a methionine-directed ABPP platform using Redox-Activated Chemical Tagging (ReACT), which leverages a biomimetic oxidative ligation strategy for selective methionine modification. Application of ReACT to oncoprotein cyclin-dependent kinase 4 (CDK4) as a representative high-value drug target identified three new ligandable methionine sites. We then synthesized a methionine-targeting covalent ligand library bearing a diverse array of heterocyclic, heteroatom, and stereochemically rich substituents. ABPP screening of this focused library identified 1oxF11 as a covalent modifier of CDK4 at an allosteric M169 site. This compound inhibited kinase activity in a dose-dependent manner on purified protein and in breast cancer cells. Further investigation of 1oxF11 found prominent cation-π and H-bonding interactions stabilizing the binding of this fragment at the M169 site. Quantitative mass-spectrometry studies validated 1oxF11 ligation of CDK4 in breast cancer cell lysates. Further biochemical analyses revealed cross-talk between M169 oxidation and T172 phosphorylation, where M169 oxidation prevented phosphorylation of the activating T172 site on CDK4 and blocked cell cycle progression. By identifying a new mechanism for allosteric methionine redox regulation on CDK4 and developing a unique modality for its therapeutic intervention, this work showcases a generalizable platform that provides a starting point for engaging in broader chemoproteomics and protein ligand discovery efforts to find and target previously undruggable methionine sites.

Discovery of Leishmania Druggable Serine Proteases by Activity-Based Protein Profiling.

Leishmaniasis are a group of diseases caused by parasitic protozoa of the genus Leishmania. Current treatments are limited by difficult administration, high cost, poor efficacy, toxicity, and growing resistance. New agents, with new mechanisms of action, are urgently needed to treat the disease. Although extensively studied in other organisms, serine proteases (SPs) have not been widely explored as antileishmanial drug targets. Herein, we report for the first time an activity-based protein profiling (ABPP) strategy to investigate new therapeutic targets within the SPs of the Leishmania parasites. Active-site directed fluorophosphonate probes (rhodamine and biotin-conjugated) were used for the detection and identification of active Leishmania serine hydrolases (SHs). Significant differences were observed in the SHs expression levels throughout the Leishmania life cycle and between different Leishmania species. Using iTRAQ-labelling-based quantitative proteomic mass spectrometry, we identified two targetable SPs in Leishmania mexicana: carboxypeptidase LmxM.18.0450 and prolyl oligopeptidase LmxM.36.6750. Druggability was ascertained by selective inhibition using the commercial serine protease inhibitors chymostatin, lactacystin and ZPP, which represent templates for future anti-leishmanial drug discovery programs. Collectively, the use of ABPP method complements existing genetic methods for target identification and validation in Leishmania.

MicroRNA-27b regulates hepatic lipase enzyme LIPC and reduces triglyceride degradation during hepatitis C virus infection

miRNAs are short, noncoding RNAs that negatively and specifically regulate protein expression, the cumulative effects of which can result in broad changes to cell systems and architecture. The miRNA miR-27b is known to regulate lipid regulatory pathways in the human liver and is also induced by the hepatitis C virus (HCV). However, the functional targets of miR-27b are not well established. Herein, an activity-based protein profiling method using a serine hydrolase probe, coupled with stable isotope labeling and mass spectrometry identified direct and indirect targets of miR-27b. The hepatic lipase C (LIPC) stood out as both highly dependent on miR-27b and as a major modulator of lipid pathway misregulation. Modulation of miR-27b using both exogenous miRNA mimics and inhibitors demonstrated that transcription factors Jun, PPARα, and HNF4α, all of which also influence LIPC levels and activity, are regulated by miR-27b. LIPC was furthermore shown to affect the progress of the life cycle of HCV and to decrease levels of intracellular triglycerides, upon which HCV is known to depend. In summary, this work has demonstrated that miR-27b mediates HCV infection by downregulating LIPC, thereby reducing triglyceride degradation, which in turn increases cellular lipid levels.

Detecting and identifying glycoside hydrolases using cyclophellitol-derived activity-based probes.

The ability to detect active enzymes in a complex mixture of folded proteins (e.g., secretome, cell lysate) generally relies on observations of catalytic ability, necessitating the development of an activity assay that is compatible with the sample and selective for the enzyme(s) of interest. Deconvolution of the contributions of different enzymes to an observed catalytic ability further necessitates an often-challenging protein separation. The advent of broadly reactive activity-based probes (ABPs) for retaining glycoside hydrolases (GHs) has enabled an alternative, often complementary, assay for active GHs. Using activity-based protein profiling (ABPP) techniques, many retaining glycoside hydrolases can be separated, detected, and identified with high sensitivity and selectivity. This chapter outlines ABPP methods for the detection and identification of retaining glycoside hydrolases from microbial sources, including protein sample preparation from bacterial lysates and fungal secretomes, enzyme labeling and detection via fluorescence, and enzyme identification using affinity-based enrichment coupled to peptide sequencing following isobaric labeling.

Chemical Proteomic Analysis of Serine Hydrolase Activity in Niemann-Pick Type C Mouse Brain.

The endocannabinoid system (ECS) is considered to be an endogenous protective system in various neurodegenerative diseases. Niemann-Pick type C (NPC) is a neurodegenerative disease in which the role of the ECS has not been studied yet. Most of the endocannabinoid enzymes are serine hydrolases, which can be studied using activity-based protein profiling (ABPP). Here, we report the serine hydrolase activity in brain proteomes of a NPC mouse model as measured by ABPP. Two ABPP methods are used: a gel-based method and a chemical proteomics method. The activities of the following endocannabinoid enzymes were quantified: diacylglycerol lipase (DAGL) α, α/β-hydrolase domain-containing protein 4, α/β-hydrolase domain-containing protein 6, α/β-hydrolase domain-containing protein 12, fatty acid amide hydrolase, and monoacylglycerol lipase. Using the gel-based method, two bands were observed for DAGL α. Only the upper band corresponding to this enzyme was significantly decreased in the NPC mouse model. Chemical proteomics showed that three lysosomal serine hydrolase activities (retinoid-inducible serine carboxypeptidase, cathepsin A, and palmitoyl-protein thioesterase 1) were increased in Niemann-Pick C1 protein knockout mouse brain compared to wild-type brain, whereas no difference in endocannabinoid hydrolase activity was observed. We conclude that these targets might be interesting therapeutic targets for future validation studies.

Opportunities for Lipid-Based Probes in the Field of Immunology.

Lipids perform a wide range of functions inside the cell, ranging from structural building block of membranes and energy storage to cell signaling. The mode of action of many signaling lipids has remained elusive due to their low abundance, high lipophilicity, and inherent instability. Various chemical biology approaches, such as photoaffinity or activity-based protein profiling methods, have been employed to shed light on the biological role of lipids and the lipid-protein interaction profile. In this review, we will summarize the recent developments in the field of chemical probes to study lipid biology, especially in immunology, and indicate potential avenues for future research.

Deciphering T Cell Immunometabolism with Activity-Based Protein Profiling.

As a major sentinel of adaptive immunity, T cells seek and destroy diseased cells using antigen recognition to achieve molecular specificity. Strategies to block checkpoint inhibition of T cell activity and thus reawaken the patient's antitumor immune responses are rapidly becoming standard of care for treatment of diverse cancers. Adoptive transfer of patient T cells genetically engineered with tumor-targeting capabilities is redefining the field of personalized medicines. The diverse opportunities for exploiting T cell biology in the clinic have prompted new efforts to expand the scope of targets amenable to immuno-oncology. Given the complex spatiotemporal regulation of T cell function and fate, new technologies capable of global molecular profiling in vivo are needed to guide selection of appropriate T cell targets and subsets. In this chapter, we describe the use of activity-based protein profiling (ABPP) to illuminate different aspects of T cell metabolism and signaling as fertile starting points for investigation. We highlight the merits of ABPP methods to enable target, inhibitor, and biochemical pathway discovery of T cells in the burgeoning field of immuno-oncology.

Activity-Based Protein Profiling in Bacteria.

Understanding the molecular mechanisms of bacterial pathogenesis and virulence is of great importance from both an academic and clinical perspective, especially in view of an alarming increase in bacterial resistance to existing antibiotics and antibacterial agents. Use of small molecules to dissect the basis of these dynamic processes is a very attractive approach due to their ability for rapid spatiotemporal control of specific biochemical functions. Activity-based protein profiling (ABPP), employing small molecule probes to interrogate enzyme activities in complex proteomes, has emerged as a powerful tool to study bacterial pathogenesis. In this chapter, we present a set of ABPP methods to identify and analyze enzymes essential for growth, metabolism and virulence of different pathogens including S. aureus and L. monocytogenes using natural product-inspired activity-based probes.

Chemical Proteomics of Host-Pathogen Interaction

In less than two decades, activity-based protein profiling (ABPP) has expanded to become the de facto tool for the study of small molecule-protein interactions in a proteomic environment. In this issue, Na etal. (2015) present another ABPP method, which they called reactive probe-based chemical proteomics, to study host-pathogen interaction and subsequently identify the protein PheA as a potential key effector during the pathogen infection process.

Proteomics meets microbiology: technical advances in the global mapping of protein expression and function

The availability of complete genome sequences for a large number of pathogenic organisms has opened the door for large-scale proteomic studies to dissect both protein expression/regulation and function. This review highlights key proteomic methods including two-dimensional gel electrophoresis, reference mapping, protein expression profiling and recent advances in gel-free separation techniques that have made a significant impact on the resolution of complex proteomes. In addition, we highlight recent developments in the field of chemical proteomics, a branch of proteomics aimed at functionally profiling a proteome. These techniques include the development of activity-based probes and activity-based protein profiling methods as well as the use of synthetic small molecule libraries to screen for pharmacological tools to perturb basic biological processes. This review will focus on the applications of these technologies to the field of microbiology.