Photoaffinity Labeling Research Articles

Oxadiazolines as Tools for Photoaffinity Labeling

Oxadiazolines as Tools for Photoaffinity Labeling

End-to-End Throughput Chemical Proteomics for Photoaffinity Labeling Target Engagement and Deconvolution.

Photoaffinity labeling (PAL) methodologies have proven to be instrumental for the unbiased deconvolution of protein-ligand binding events in physiologically relevant systems. However, like other chemical proteomic workflows, they are limited in many ways by time-intensive sample manipulations and data acquisition techniques. Here, we describe an approach to address this challenge through the innovation of a carboxylate bead-based protein cleanup procedure to remove excess small-molecule contaminants and couple it to plate-based, proteomic sample processing as a semiautomated solution. The analysis of samples via label-free, data-independent acquisition (DIA) techniques led to significant improvements on a workflow time per sample basis over current standard practices. Experiments utilizing three established PAL ligands with known targets, (+)-JQ-1, lenalidomide, and dasatinib, demonstrated the utility of having the flexibility to design experiments with a myriad of variables. Data revealed that this workflow can enable the confident identification and rank ordering of known and putative targets with outstanding protein signal-to-background enrichment sensitivity. This unified end-to-end throughput strategy for processing and analyzing these complex samples could greatly facilitate efficient drug discovery efforts and open up new opportunities in the chemical proteomics field.

Oxadiazolines as Photoreleasable Labels for Drug Target Identification.

Photoaffinity labeling is a widely used technique for studying ligand-protein and protein-protein interactions. Traditional photoaffinity labels utilize nonspecific C-H bond insertion reactions mediated by a highly reactive intermediate. Despite being the most widely used photoaffinity labels, diazirines exhibit limited compatibility with downstream organic reactions and suffer from storage stability concerns. This study introduces oxadiazolines as innovative and complementary photoactivatable labels for addition to the toolbox and demonstrates their application in vitro and through in cellulo labeling experiments. Oxadiazolines can be easily synthesized from ketone moieties and cleaved with 302-330 nm light to cleanly liberate a diazo reactive moiety that can covalently modify nucleophilic amino acid residues. Notably, oxadiazolines are compatible with various organic reaction conditions and functional groups, allowing for the exploration of a large chemical space. Several known inhibitors featuring the oxadiazoline functionality were prepared without affecting their binding affinity. Furthermore, we confirmed the ability of oxadiazolines to form covalent bonds with proteins upon UV-irradiation, both in vitro and in cellulo, yielding comparable results to those of the matched diazirine compounds.

Three classes of propofol binding sites on GABAA receptors

Propofol is a widely used anesthetic and sedative that acts as a positive allosteric modulator (PAM) of gamma-aminobutyric acid type A (GABAA) receptors. Several potential propofol binding sites that may mediate this effect have been identified using propofol-analogue photoaffinity labeling. o-PD labels β-H267, a pore-lining residue, whereas AziPm labels residues β-M286, β-M227 and α-I239 in the two membrane-facing interfaces (β(+)/α(-) and α(+)/β(-)) between α and β subunits. This study used photoaffinity labeling of α1β3 GABAA receptors to reconcile the apparently conflicting results obtained with AziPm and o-PD labeling, focusing on whether β3-H267 identifies specific propofol binding site(s). The results show that propofol, but not AziPm protects β3-H267 from labeling by o-PD, whereas both propofol and o-PD protect against AziPm labeling of β3-M286, β3-M227 and α1I239. These data indicate that there are three distinct classes of propofol binding sites, with AziPm binding to two of the classes and o-PD to all three. Analysis of binding stoichiometry using native mass spectrometry in β3 homomeric receptors, demonstrated a minimum of five AziPm labeled residues and three o-PD labeled residues per pentamer, suggesting that there are two distinct propofol binding sites per β-subunit. The native MS data, coupled with photolabeling performed in the presence of zinc, indicate that the binding site(s) identified by o-PD are adjacent to, but not within the channel pore, since the pore at the 17’ H267 residue can accommodate only one propofol molecule. These data validate the existence of three classes of specific propofol binding sites on α1β3 GABAA receptors.

Profiling sorghum-microbe interactions with a specialized photoaffinity probe identifies key sorgoleone binders in Acinetobacter pittii.

Interactions between plants and soil microbial communities that benefit plant growth and enhance nutrient acquisition are driven by the selective release of metabolites from plant roots, or root exudation. To investigate these plant-microbe interactions, we developed a photoaffinity probe based on sorgoleone (sorgoleone diazirine alkyne for photoaffinity labeling, SoDA-PAL), a hydrophobic secondary metabolite and allelochemical produced in Sorghum bicolor root exudates. We applied SoDA-PAL to the identification of sorgoleone-binding proteins in Acinetobacter pittii SO1, a potential plant growth-promoting microbe isolated from sorghum rhizosphere soil. Competitive photoaffinity labeling of A. pittii whole cell lysates with SoDA-PAL identified 137 statistically enriched proteins, including putative transporters, transcriptional regulators, and a subset of proteins with predicted enzymatic functions. We performed computational protein modeling and docking with sorgoleone to prioritize candidates for experimental validation and then confirmed binding of sorgoleone to four of these proteins in vitro: the α/β fold hydrolase SrgB (OH685_09420), a fumarylacetoacetase (OH685_02300), a lysophospholipase (OH685_14215), and an unannotated hypothetical protein (OH685_18625). Our application of this specialized sorgoleone-based probe coupled with structural bioinformatics streamlines the identification of microbial proteins involved in metabolite recognition, metabolism, and toxicity, widening our understanding of the range of cellular pathways that can be affected by a plant secondary metabolite.IMPORTANCEHere, we demonstrate that a photoaffinity-based chemical probe modeled after sorgoleone, an important secondary metabolite released by sorghum roots, can be used to identify microbial proteins that directly interact with sorgoleone. We applied this probe to the sorghum-associated bacterium Acinetobacter pittii and showed that probe labeling is dose-dependent and sensitive to competition with purified sorgoleone. Coupling the probe with proteomics and computational analysis facilitated the identification of putative sorgoleone binders, including a protein implicated in a conserved pathway essential for sorgoleone catabolism. We anticipate that discoveries seeded by this workflow will expand our understanding of the molecular mechanisms by which specific metabolites in root exudates shape the sorghum rhizosphere microbiome.

Chemoproteomic Strategy Identifies PfUCHL3 as the Target of Halofuginone.

The human malaria parasite Plasmodium falciparum (P. falciparum) continues to pose a significant public health challenge, leading to millions of fatalities globally. Halofuginone (HF) has shown a significant anti-P. falciparum effect, suggesting its potential as a therapeutic agent for malaria treatment. In this study, we synthesized a photoaffinity labeling probe of HF to identify its direct target in P. falciparum. Our results reveal that ubiquitin carboxyl-terminal hydrolase 3 (PfUCHL3) acts as a crucial target protein of HF, which modulates parasite growth in the intraerythrocytic cycle. In particular, we discovered that HF potentially forms hydrogen bonds with the Leu10, Glu11, and Arg217 sites of PfUCHL3, thereby inducing an allosteric effect by promoting the embedding of the helix 6' region on the protein surface. Furthermore, HF disrupts the expression of multiple functional proteins mediated by PfUCHL3, specifically those that play crucial roles in amino acid biosynthesis and metabolism in P. falciparum. Taken together, this study highlights PfUCHL3 as a previously undisclosed druggable target of HF, which contributes to the development of novel anti-malarial agents in the future.

A natural small molecule alleviates liver fibrosis by targeting apolipoprotein L2.

Liver fibrosis is an urgent clinical problem without effective therapies. Here we conducted a high-content screening on a natural Euphorbiaceae diterpenoid library to identify a potent anti-liver fibrosis lead, 12-deoxyphorbol 13-palmitate (DP). Leveraging a photo-affinity labeling approach, apolipoprotein L2 (APOL2), an endoplasmic reticulum (ER)-rich protein, was identified as the direct target of DP. Mechanistically, APOL2 is induced in activated hepatic stellate cells upon transforming growth factor-β1 (TGF-β1) stimulation, which then binds to sarcoplasmic/ER calcium ATPase 2 (SERCA2) to trigger ER stress and elevate its downstream protein kinase R-like ER kinase (PERK)-hairy and enhancer of split 1 (HES1) axis, ultimately promoting liver fibrosis. As a result, targeting APOL2 by DP or ablation of APOL2 significantly impairs APOL2-SERCA2-PERK-HES1 signaling and mitigates fibrosis progression. Our findings not only define APOL2 as a novel therapeutic target for liver fibrosis but also highlight DP as a promising lead for treatment of this symptom.

Site-Selective Functionalization of Molecularly Imprinted Nanoparticles to Recognize Lysine-Rich Peptides.

Sequence-selective binding of peptides has been a long-standing goal of chemists. As one of the most abundant amino acids in proteins, lysine plays an important role in protein functions as well as in antimicrobial and cell-penetrating peptides. Herein, we report molecularly imprinted nanoparticles (NPs) with high sequence selectivity for lysine-rich peptides. The NPs are prepared from molecular imprinting of cross-linkable surfactant micelles and postmodification of the imprinted pockets by photoaffinity labeling. The method allows carboxylic acids to be installed precisely near the lysine amino side chains, greatly enhancing the binding strengths of lysine-rich peptides. Small variations in the peptide sequence can be distinguished, and the binding affinity correlates positively with the number of lysine groups in model tripeptides. The method applies to complex lysine-rich biological peptides, achieving hundreds of nanomolar binding affinities and excellent binding specificities.

Photoactivatable mRNA 5' Cap Analogs for RNA-Protein Crosslinking.

Chemical modification of messenger RNA (mRNA) has paved the way for advancing mRNA-based therapeutics. The intricate process of mRNA translation in eukaryotes is orchestrated by numerous proteins involved in complex interaction networks. Many of them bind specifically to a unique structure at the mRNA 5'-end, called 5'-cap. Depending on the 5'-terminal sequence and its methylation pattern, different proteins may be involved in the translation initiation and regulation, but a deeper understanding of these mechanisms requires specialized molecular tools to identify natural binders of mRNA 5'-end variants. Here, a series of 8 new synthetic 5'-cap analogs that allow the preparation of RNA molecules with photoreactive tags using a standard in vitro transcription reaction are reported. Two photoreactive tags and four different modification sites are selected to minimize potential interference with cap-protein contacts and to provide complementary properties regarding crosslinking chemistry and molecular interactions. The tailored modification strategy allows for the generation of specific crosslinks with model cap-binding proteins, such as eIF4E and Dcp2. The usefulness of the photoreactive cap analogs is also demonstrated for identifying the cap-binding subunit in a multi-protein complex, which makes them perfect candidates for further development of photoaffinity labeling probes to study more complex mRNA-related processes.

Pan-serotype dengue virus inhibitor JNJ-A07 targets NS4A-2K-NS4B interaction with NS2B/NS3 and blocks replication organelle formation

Dengue fever represents a significant medical and socio-economic burden in (sub)tropical regions, yet antivirals for treatment or prophylaxis are lacking. JNJ-A07 was described as highly active against the different genotypes within each serotype of the disease-causing dengue virus (DENV). Based on clustering of resistance mutations it has been assumed to target DENV non-structural protein 4B (NS4B). Using a photoaffinity labeling compound with high structural similarity to JNJ-A07, here we demonstrate binding to NS4B and its precursor NS4A-2K-NS4B. Consistently, we report recruitment of the compound to intracellular sites enriched for these proteins. We further specify the mechanism-of-action of JNJ-A07, which has virtually no effect on viral polyprotein cleavage, but targets the interaction between the NS2B/NS3 protease/helicase complex and the NS4A-2K-NS4B cleavage intermediate. This interaction is functionally linked to de novo formation of vesicle packets (VPs), the sites of DENV RNA replication. JNJ-A07 blocks VPs biogenesis with little effect on established ones. A similar mechanism-of-action was found for another NS4B inhibitor, NITD-688. In summary, we unravel the antiviral mechanism of these NS4B-targeting molecules and show how DENV employs a short-lived cleavage intermediate to carry out an early step of the viral life cycle.

Mitochondrial respiratory complex I can be inhibited via bypassing the ubiquinone-accessing tunnel.

Mitochondrial NADH-ubiquinone oxidoreductase (complex I) couples electron transfer from NADH to ubiquinone with proton translocation in its membrane part. Structural studies have identified a long (~ 30 Å), narrow, tunnel-like cavity within the enzyme, through which ubiquinone may access a deep reaction site. Although various inhibitors are considered to block the ubiquinone reduction by occupying the tunnel's interior, this view is still debatable. We synthesized a phosphatidylcholine-quinazoline hybrid compound (PC-Qz1), in which a quinazoline-type toxophore was attached to the sn-2 acyl chain to prevent it from entering the tunnel. However, PC-Qz1 inhibited complex I and suppressed photoaffinity labeling by another quinazoline derivative, [125I]AzQ. This study provides further experimental evidence that is difficult to reconcile with the canonical ubiquinone-accessing tunnel model.

Sanglifehrin A mitigates multiorgan fibrosis by targeting the collagen chaperone cyclophilin B.

Pathological deposition and crosslinking of collagen type I by activated myofibroblasts drives progressive tissue fibrosis. Therapies that inhibit collagen synthesis have potential as antifibrotic agents. We identify the collagen chaperone cyclophilin B as a major cellular target of the natural product sanglifehrin A (SfA) using photoaffinity labeling and chemical proteomics. Mechanistically, SfA inhibits and induces the secretion of cyclophilin B from the endoplasmic reticulum (ER) and prevents TGF-β1-activated myofibroblasts from synthesizing and secreting collagen type I in vitro, without inducing ER stress or affecting collagen type I mRNA transcription, myofibroblast migration, contractility, or TGF-β1 signaling. In vivo, SfA induced cyclophilin B secretion in preclinical models of fibrosis, thereby inhibiting collagen synthesis from fibrotic fibroblasts and mitigating the development of lung and skin fibrosis in mice. Ex vivo, SfA induces cyclophilin B secretion and inhibits collagen type I secretion from fibrotic human lung fibroblasts and samples from patients with idiopathic pulmonary fibrosis (IPF). Taken together, we provide chemical, molecular, functional, and translational evidence for demonstrating direct antifibrotic activities of SfA in preclinical and human ex vivo fibrotic models. Our results identify the cellular target of SfA, the collagen chaperone cyclophilin B, as a mechanistic target for the treatment of organ fibrosis.

Identification of proteins involved in intracellular ubiquinone trafficking in Saccharomyces cerevisiae using artificial ubiquinone probe

Ubiquinone (UQ) is an essential player in the respiratory electron transfer system. In Saccharomyces cerevisiae strains lacking the ability to synthesize UQ6, exogenously supplied UQs can be taken up and delivered to mitochondria through an unknown mechanism, restoring the growth of UQ6-deficient yeast in non-fermentable medium. Since elucidating the mechanism responsible may markedly contribute to therapeutic strategies for patients with UQ deficiency, many attempts have been made to identify the machinery involved in UQ trafficking in the yeast model. However, definite experimental evidence of the direct interaction of UQ with a specific protein(s) has not yet been demonstrated. To gain insight into intracellular UQ trafficking via a chemistry-based strategy, we synthesized a hydrophobic UQ probe (pUQ5), which has a photoreactive diazirine group attached to a five-unit isoprenyl chain and a terminal alkyne to visualize and/or capture the labeled proteins via click chemistry. pUQ5 successfully restored the growth of UQ6-deficient S. cerevisiae (Δcoq2) on a non-fermentable carbon source, indicating that this UQ was taken up and delivered to mitochondria, and served as a UQ substrate of respiratory enzymes. Through photoaffinity labeling of the mitochondria isolated from Δcoq2 yeast cells cultured in the presence of pUQ5, we identified many labeled proteins, including voltage-dependent anion channel 1 (VDAC1) and cytochrome c oxidase subunit 3 (Cox3). The physiological relevance of UQ binding to these proteins is discussed.

Identification and Validation of PKR as a Direct Target for the Novel Sulfonamide-Substituted Tetrahydroquinoline Nonselective Inhibitor of the NLRP3 Inflammasome.

The NLRP3 inflammasome is a critical component of the innate immune system. The persistent abnormal activation of the NLRP3 inflammasome is implicated in numerous human diseases. Herein, sulfonamide-substituted tetrahydroquinoline derivative S-9 was identified as the most promising NLRP3 inhibitor, without obvious cytotoxicity. In vitro, S-9 inhibited the priming and activation stages of the NLRP3 inflammasome. Incidentally, we also observed that S-9 had inhibitory effects on the NLRC4 and AIM2 inflammasomes. To elucidate the multiple anti-inflammatory activities of S-9, photoaffinity probe P-2, which contained a photoaffinity label and a functional handle, was developed for target identification by chemical proteomics. We identified PKR as a novel target of S-9 in addition to NLRP3 by target fishing. Furthermore, S-9 exhibited a significant anti-neuroinflammatory effect in vivo. In summary, our findings show that S-9 is a promising lead compound targeting both PKR and NLRP3 that could emerge as a molecular tool for treating inflammasome-related diseases.

Proteomic Profiling of Antimalarial Plasmodione Using 3-Benz(o)ylmenadione Affinity-Based Probes.

Understanding the mechanisms of drug action in malarial parasites is crucial for the development of new drugs to combat infection and to counteract drug resistance. Proteomics is a widely used approach to study host-pathogen systems and to identify drug protein targets. Plasmodione is an antiplasmodial early-lead drug exerting potent activities against young asexual and sexual blood stages in vitro with low toxicity to host cells. To elucidate its molecular mechanisms, an affinity-based protein profiling (AfBPP) approach was applied to yeast and P. falciparum proteomes. New (pro-) AfBPP probes based on the 3-benz(o)yl-6-fluoro-menadione scaffold were synthesized. With optimized conditions of both photoaffinity labeling and click reaction steps, the AfBPP protocol was then applied to a yeast proteome, yielding 11 putative drug-protein targets. Among these, we found four proteins associated with oxidoreductase activities, the hypothesized type of targets for plasmodione and its metabolites, and other proteins associated with the mitochondria. In Plasmodium parasites, the MS analysis revealed 44 potential plasmodione targets that need to be validated in further studies. Finally, the localization of a 3-benzyl-6-fluoromenadione AfBPP probe was studied in the subcellular structures of the parasite at the trophozoite stage.

Chemoproteomic Profiling of Signaling Metabolite Fructose-1,6-Bisphosphate Interacting Proteins in Living Cells.

Fructose-1,6-bisphosphate (FBP), a cellular endogenous sugar metabolite in the glycolytic pathway, has recently been reported to act as a signaling molecule to regulate various cellular events through the engagement of important proteins. Though tremendous progress has been made in identifying specific FBP-protein interactions, the comprehensive identification of FBP-interacting proteins and their regulatory mechanisms remains largely unexplored. Here, we describe a concise synthetic approach for the scalable preparation of a photoaffinity FBP probe that enables the quantitative chemoproteomic profiling of FBP-protein interactions based on photoaffinity labeling (PAL) directly in living cells. Using such a protocol, we captured known FBP targets including PKM2 and MDH2. Furthermore, among unknown FBP-interacting proteins, we identified a mitochondrial metabolic enzyme aldehyde dehydrogenase 2 (ALDH2), against which FBP showed inhibitory activity and resulted in cellular ROS upregulation accompanied by mitochondrial fragmentation. Our findings disclosed a new mode of glucose signaling mediating by the FBP-ALDH2-ROS axis.

Photoaffinity labelling with small molecules

Photoaffinity labelling with small molecules

Photoaffinity labelling with small molecules

Photoaffinity labelling with small molecules

Integrated Proteomics Characterization of NLRP3 Inflammasome Inhibitor MCC950 in Monocytic Cell Line Confirms Direct MCC950 Engagement with Endogenous NLRP3.

Inhibition of the NLRP3 inflammasome is a promising strategy for the development of new treatments for inflammatory diseases. MCC950 is a potent and selective small-molecule inhibitor of the NLRP3 pathway and has been validated in numerous species and disease models. Although the capacity of MCC950 to block NLRP3 signaling is well-established, it is still critical to identify the mechanism of action and molecular targets of MCC950 to inform and derisk drug development. Quantitative proteomics performed in disease-relevant systems provides a powerful method to study both direct and indirect pharmacological responses to small molecules to elucidate the mechanism of action and confirm target engagement. A comprehensive target deconvolution campaign requires the use of complementary chemical biology techniques. Here we applied two orthogonal chemical biology techniques: compressed Cellular Thermal Shift Assay (CETSA) and photoaffinity labeling chemoproteomics, performed under biologically relevant conditions with LPS-primed THP-1 cells, thereby deconvoluting, for the first time, the molecular targets of MCC950 using chemical biology techniques. In-cell chemoproteomics with inlysate CETSA confirmed the suspected mechanism as the disruption of inflammasome formation via NLRP3. Further cCETSA (c indicates compressed) in live cells mapped the stabilization of NLRP3 inflammasome pathway proteins, highlighting modulation of the targeted pathway. This is the first evidence of direct MCC950 engagement with endogenous NLRP3 in a human macrophage cellular system using discovery proteomics chemical biology techniques, providing critical information for inflammasome studies.

Abstract 1166 A photoaffinity labeling strategy to elucidate interactions and physiological functions of N-acyl-phosphatidylethanolamine, a unique triply acylated phospholipid

Abstract 1166 A photoaffinity labeling strategy to elucidate interactions and physiological functions of N-acyl-phosphatidylethanolamine, a unique triply acylated phospholipid