Photo-affinity and Metabolic Labeling Probes Based on the Opioid Alkaloids.

The opioids are powerful analgesics yet possess contingencies that can lead to opioid-use disorder. Chemical probes derived from the opioid alkaloids can provide deeper insight into the molecular interactions in a cellular context. Here, we designed and developed photo-click morphine (PCM-2) as a photo-affinity probe based on morphine and dialkynyl-acetyl morphine (DAAM) as a metabolic acetate reporter based on heroin. Application of these probes to SH-SY5Y, HEK293T, and U2OS cells revealed that PCM-2 and DAAM primarily localize to the lysosome amongst other locations inside the cell by confocal microscopy and chemical proteomics. Interaction site identification by mass spectrometry revealed the mitochondrial phosphate carrier protein, solute carrier family 25 member 3, SLC25A3, and histone H2B as acylation targets of DAAM. These data illustrate the utility of chemical probes to measure localization and protein interactions in a cellular context and will inform the design of probes based on the opioids in the future.

Chemical tools for the opioids.

The opioids are potent and widely used pain management medicines despite also possessing severe liabilities that have fueled the opioid crisis. The pharmacological properties of the opioids primarily derive from agonism or antagonism of the opioid receptors, but additional effects may arise from specific compounds, opioid receptors, or independent targets. The study of the opioids, their receptors, and the development of remediation strategies has benefitted from derivatization of the opioids as chemical tools. While these studies have primarily focused on the opioids in the context of the opioid receptors, these chemical tools may also play a role in delineating mechanisms that are independent of the opioid receptors. In this review, we describe recent advances in the development and applications of opioid derivatives as chemical tools and highlight opportunities for the future.

γ-Secretase inhibitors and modulators: Mechanistic insights into the function and regulation of γ-Secretase.

Over two decades, γ-secretase has been the target for extensive therapeutic development due to its pivotal role in pathogenesis of Alzheimer's disease and cancer. However, it has proven to be a challenging task owing to its large set of substrates and our limited understanding of the enzyme's structural and mechanistic features. The scientific community is taking bigger strides towards solving this puzzle with recent advancement in techniques like cryogenic electron microscopy (cryo-EM) and photo-affinity labelling (PAL). This review highlights the significance of the PAL technique with multiple examples of photo-probes developed from γ-secretase inhibitors and modulators. The binding of these probes into active and/or allosteric sites of the enzyme has provided crucial information on the γ-secretase complex and improved our mechanistic understanding of this protease. Combining the knowledge of function and regulation of γ-secretase will be a decisive factor in developing novel γ-secretase modulators and biological therapeutics.

Effect of Alkynyl Group on Reactivity in Photoaffinity Labeling with 2-Thienyl-Substituted α-Ketoamide.

Minimalist photo-reactive probes, which consist of a photo-reactive group and a tag for detection of target proteins, are useful tools in chemical biology. Although several diazirine-based and aryl azide-based minimalist probes are available, no keto-based minimalist probe has yet been reported. Here we describe minimalist probes based on a 2-thienyl-substituted α-ketoamide bearing an alkyne group on the thiophene ring. The 3-alkyne probe showed the highest photo-affinity labeling efficiency.

Target identification of anti-diabetic and anti-obesity flavonoid derivative (Fla-CN).

Fla-CN is a flavonoid derivative with anti-diabetic and anti-obesity effects; however, its biological targets are still unknown. In this study, we developed bifunctional affinity-based probes to identify the direct targets of Fla-CN. When using probe 3, we observed the co-location of probe 3 and mitochondria in both HepG2 and 3T3-L1 cells. The putative target proteomes were obtained using activity-based protein profiling (ABPP) and photo-affinity labelling. Pyruvate carboxylase, mitochondrial malate dehydrogenase, mitochondrial complex I, and F1FO-ATPase were validated as the direct targets of Fla-CN by surface plasmon resonance (SPR) and biochemical assays. It was elucidated that the Tyr651, Gln870 and Lys912 were the key amino acid residues near the binding site of pyruvate carboxylase with Fla-CN. The direct interaction of Fla-CN and the above four targets allowed elucidation of its complicated molecular mechanism, including the activation of adenosine 5-monophosphate (AMP)-activated protein kinase (AMPK), and the inhibition of gluconeogenesis. Further investigation for activation of AMPK in normal and insulin resistance (IR) HepG2 cells, indicated that Fla-CN could target insulin resistance tissues.

Probes for Photoaffinity Labelling of Kinases.

Protein kinases, one of the largest enzyme superfamilies, regulate many physiological and pathological processes. They are drug targets for multiple human diseases, including various cancer types. Probes for the photoaffinity labelling of kinases are important research tools for the study of members of this enzyme superfamily. In this review, we discuss the design principles of these probes, which are mainly derived from inhibitors targeting the ATP pocket. Overall, insights from crystal structures guide the placement of photoreactive groups and detection tags. This has resulted in a wide variety of probes, of which we provide a comprehensive overview. We also discuss several areas of application of these probes, including the identification of targets and off-targets of kinase inhibitors, mapping of their binding sites, the development of inhibitor screening assays, the imaging of kinases, and identification of protein binding partners.

Targeting of parvulin interactors by diazirine mediated cross-linking discloses a cellular role of human Par14/17 in actin polymerization.

The peptidyl-prolyl cis/trans isomerases (PPIases) Parvulin 14 (Par14) and Parvulin 17 (Par17) result from alternative transcription initiation of the PIN4 gene. Whereas Par14 is present in all metazoan, Par17 is only expressed in Hominidae. Par14 resides mainly within the cellular nucleus, while Par17 is translocated into mitochondria. Using photo-affinity labeling, cross-linking and mass spectrometry (MS) we identified binding partners for both enzymes from HeLa lysates and disentangled their cellular roles. Par14 is involved in biogenesis of ribonucleoprotein (RNP)-complexes, RNA processing and DNA repair. Its elongated isoform Par17 participates in protein transport/translocation and in cytoskeleton organization. Nuclear magnetic resonance (NMR) spectroscopy reveals that Par17 binds to ß-actin with its N-terminal region, while both parvulins initiate actin polymerization depending on their PPIase activity as monitored by fluorescence spectroscopy. The knockdown (KD) of Par17 in HCT116 cells results in a defect in cell motility and migration.

Photo Cross-Linking Probes Containing ϵ-N-Thioacyllysine and ϵ-N-Acyl-(δ-aza)lysine Residues.

Posttranslational modifications (PTMs) are important in the regulation of protein function, trafficking, localization, and marking for degradation. This work describes the development of peptide activity/affinity-based probes for the discovery of proteins that recognize novel acyl-based PTMs on lysine residues in the proteome. The probes contain surrogates of ϵ-N-acyllysine by introduction of either hydrazide or thioamide functionalities to circumvent hydrolysis of the modification during the experiments. In addition to the modified PTMs, the developed chemotypes were analyzed with respect to the effect of peptide sequence. The photo cross-linking conditions and subsequent functionalization of the covalent adducts were systematically optimized by applying fluorophore labeling and gel electrophoresis (in-gel fluorescence measurements). Finally, selected probes, containing the ϵ-N-glutaryllysine and ϵ-N-myristoyllysine analogues, were successfully applied for the enrichment of native, endogenous proteins from cell lysate, recapitulating the expected interactions of SIRT5 and SIRT2, respectively. Interestingly, the latter mentioned was able to pull down two different splice variants of SIRT2, which has not been achieved with a covalent probe before. Based on this elaborate proof-of-concept study, we expect that the technology will have broad future applications for pairing of novel PTMs with the proteins that target them in the cell.

Recent advances in the discovery and development of glyoxalase I inhibitors.

Glyoxalase I (GLO1) is a homodimeric Zn2+-metalloenzyme that catalyses the transformation of methylglyoxal (MG) to d-lacate through the intermediate S-d-lactoylglutathione. Growing evidence indicates that GLO1 has been identified as a potential target for the treatment cancer and other diseases. Various inhibitors of GLO1 have been discovered or developed over the past several decades including natural or natural product-based inhibitors, GSH-based inhibitors, non-GSH-based inhibitors, etc. The aim of this review is to summarize recent achievements of concerning discovery, design strategies, as well as pharmacological aspects of GLO1 inhibitors with the target of promoting their development toward clinical application.

From Phenotypic Hit to Chemical Probe: Chemical Biology Approaches to Elucidate Small Molecule Action in Complex Biological Systems.

Biologically active small molecules have a central role in drug development, and as chemical probes and tool compounds to perturb and elucidate biological processes. Small molecules can be rationally designed for a given target, or a library of molecules can be screened against a target or phenotype of interest. Especially in the case of phenotypic screening approaches, a major challenge is to translate the compound-induced phenotype into a well-defined cellular target and mode of action of the hit compound. There is no "one size fits all" approach, and recent years have seen an increase in available target deconvolution strategies, rooted in organic chemistry, proteomics, and genetics. This review provides an overview of advances in target identification and mechanism of action studies, describes the strengths and weaknesses of the different approaches, and illustrates the need for chemical biologists to integrate and expand the existing tools to increase the probability of evolving screen hits to robust chemical probes.

Direct and indirect approaches to identify drug modes of action.

Phenotypic assays are becoming increasingly more common among drug discovery practices, expanding drug target diversity as lead compounds identified through such screens are not limited to known targets. While increasing diversity is beneficial to the drug discovery process and the fight against disease, the unknown modes of action of new lead compounds can hamper drug discovery as, in most cases, the process of lead compound optimization is made difficult due to the unknown nature of the target; blindly changing substituents can prove fruitless due to the inexhaustible number of potential combinations, and it is therefore desirable to rapidly identify the targets of lead compounds developed through phenotypic screening. In addition, leads identified through target-based screening often have off-target effects that contribute towards drug toxicity, and by identifying those secondary targets, the drugs can be improved. However, the identification of a leads mode of action is far from trivial and now represents a major bottleneck in the drug discovery pipeline. This review looks at some of the recent developments in the identification of drug modes of action, focusing on phenotype-based methods using metabolomics, proteomics, transcriptomics, and genomics to detect changes in phenotype in response to the presence of the drug, and affinity-based methods using modified/unmodified drug as bait to capture and identify targets. © 2017 IUBMB Life, 70(1):9-22, 2018.

Detection of protein targets with a single binding epitope using DNA-templated photo-crosslinking and strand displacement.

DNA-based probes are powerful analytical tools for protein detection and analysis. Target-induced DNA assembly is a widely used strategy to transduce target-ligand binding to detectable signals. However, most of the existing methods based on DNA assembly require two or more binding sites on the target protein. Here we report a novel detection method suitable for protein targets with just a single binding site. This method is based on target-induced probe assembly, DNA-templated photo-crosslinking, and DNA-mediated toehold strand displacement to form a tri-probe complex that is specific for target protein.

A photo-cleavable biotin affinity tag for the facile release of a photo-crosslinked carbohydrate-binding protein.

The use of photo-crosslinking glycoprobes represents a powerful strategy for the covalent capture of labile protein complexes and allows detailed characterization of carbohydrate-mediated interactions. The selective release of target proteins from solid support is a key step in functional proteomics. We envisaged that light activation can be exploited for releasing labeled protein in a dual photo-affinity probe-based strategy. To investigate this possibility, we designed a trifunctional, galactose-based, multivalent glycoprobe for affinity labeling of carbohydrate-binding proteins. The resulting covalent protein-probe adduct is attached to a photo-cleavable biotin affinity tag; the biotin moiety enables specific presentation of the conjugate on streptavidin-coated beads, and the photolabile linker allows the release of the labeled proteins. This dual probe promotes both the labeling and the facile cleavage of the target protein complexes from the solid surfaces and the remainder of the cell lysate in a completely unaltered form, thus eliminating many of the common pitfalls associated with traditional affinity-based purification methods.

Development of a bioluminescent immunoassay based on Fc-specific conjugated antibody-nanoluciferase immunoreagents for determining aflatoxin B1.

Aflatoxin B1 (AFB1) is a potent carcinogen, and is among the most hazardous mycotoxins in agricultural products. Therefore, the development of sensitive and convenient detection methods for AFB1 is significant for food safety against mycotoxins. Herein, a bioluminescent enzyme immunoassay (BLEIA) was developed for ultrasensitive detection of AFB1, based on the novel Fc-specific antibody-nanoluciferase (Ab-Nluc) conjugates which were fabricated using an IgG-binding protein-assisted photo-conjugation strategy. In indirect competitive immunoassay format, the proposed BLEIA exhibited the detection limit of 0.0232 ng mL-1, which was 37.4-fold lower than that obtained using the classical enzyme-linked immunosorbent assay (ELISA) based on Ab-horseradish peroxidase (Ab-HRP) chemical conjugates (0.868 ng mL-1). Meanwhile, the BLEIA exhibited high accuracy and precision. Thus, the proposed Fc-specific Ab-Nluc conjugates-based BLEIA provides an ultrasensitive and reliable method for detecting toxins and has potential for use in food safety monitoring.

Proteomics-Based Transporter Identification by the PICK Method: Involvement of TM7SF3 and LHFPL6 in Proton-Coupled Organic Cation Antiport at the Blood-Brain Barrier.

A proton-coupled organic cation (H+/OC) antiporter working at the blood-brain barrier (BBB) in humans and rodents is thought to be a promising candidate for the efficient delivery of cationic drugs to the brain. Therefore, it is important to identify the molecular entity that exhibits this activity. Here, for this purpose, we established the Proteomics-based Identification of transporter by Crosslinking substrate in Keyhole (PICK) method, which combines photo-affinity labeling with comprehensive proteomics analysis using SWATH-MS. Using preselected criteria, the PICK method generated sixteen candidate proteins. From these, knockdown screening in hCMEC/D3 cells, an in vitro BBB model, identified two proteins, TM7SF3 and LHFPL6, as candidates for the H+/OC antiporter. We synthesized a novel H+/OC antiporter substrate for functional analysis of TM7SF3 and LHFPL6 in hCMEC/D3 cells and HEK293 cells. The results suggested that both TM7SF3 and LHFPL6 are components of the H+/OC antiporter.

Small Molecule Interactome Mapping by Photo-Affinity Labeling (SIM-PAL) to Identify Binding Sites of Small Molecules on a Proteome-Wide Scale.

Identification and characterization of small molecule-protein interactions is critical to understanding the mechanism of action of bioactive small molecules. Photo-affinity labeling (PAL) enables the capture of noncovalent interactions for enrichment and unbiased analysis by mass spectrometry (MS). Quantitative proteomics of the enriched proteome reveals potential interactions, and MS characterization of binding sites provides validation and structural insight into the interactions. Here, we describe the identification of the protein targets and binding sites of a small molecule using small molecule interactome mapping by PAL (SIM-PAL). Cells are exposed to a diazirine-alkyne-functionalized small molecule, and binding interactions are covalently captured upon UV irradiation. An isotopically coded, acid-cleavable biotin azide handle is attached to the conjugated proteins using copper-catalyzed azide-alkyne cycloaddition. Biotin-labeled proteins are enriched for on-bead digestion and quantitative proteomics. Acid cleavage of the handle releases the bead-bound conjugated peptides for MS analysis and isotope-directed assignment of the binding site. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Generation of a small molecule-conjugated protein sample following treatment of live cells Alternate Protocol: Generation of a small molecule-conjugated protein sample following treatment of cell lysate Basic Protocol 2: Copper-catalyzed azide-alkyne cycloaddition functionalization and enrichment of labeled peptides Support Protocol 1: Synthesis of acid-cleavable, isotopically coded biotin picolyl azide handle Support Protocol 2: Monitoring enrichment by immunoblotting Basic Protocol 3: Mass spectrometry analysis to identify interacting proteins and conjugation sites.

Chemical Proteomics for Target Discovery of Head-to-Tail Cyclized Mini-Proteins.

Target deconvolution is one of the most challenging tasks in drug discovery, but a key step in drug development. In contrast to small molecules, there is a lack of validated and robust methodologies for target elucidation of peptides. In particular, it is difficult to apply these methods to cyclic and cysteine-stabilized peptides since they exhibit reduced amenability to chemical modification and affinity capture; however, such ribosomally synthesized and post-translationally modified peptide natural products are rich sources of promising drug candidates. For example, plant-derived circular peptides called cyclotides have recently attracted much attention due to their immunosuppressive effects and oral activity in the treatment of multiple sclerosis in mice, but their molecular target has hitherto not been reported. In this study, a chemical proteomics approach using photo-affinity crosslinking was developed to determine a target for the circular peptide [T20K]kalata B1. Using this prototypic nature-derived peptide enabled the identification of a possible functional modulation of 14-3-3 proteins. This biochemical interaction was validated via competition pull down assays as well as a cellular reporter assay indicating an effect on 14-3-3-dependent transcriptional activity. As proof of concept, the presented approach may be applicable for target elucidation of various cyclic peptides and mini-proteins, in particular cyclotides, which represent a promising class of molecules in drug discovery and development.

Combining Amine-Reactive Cross-Linkers and Photo-Reactive Amino Acids for 3D-Structure Analysis of Proteins and Protein Complexes.

During the last 15 years, the combination of chemical cross-linking and high-resolution mass spectrometry (MS) has matured into an alternative approach for analyzing 3D-structures of proteins and protein complexes. Using the distance constraints imposed by the cross-links, models of the protein or protein complex under investigation can be created. The majority of cross-linking studies are currently conducted with homobifunctional amine-reactive cross-linkers. We extend this "traditional" cross-linking/MS strategy by adding complementary photo-cross-linking data. For this, the diazirine-containing unnatural amino acids photo-leucine and photo-methionine are incorporated into the proteins and cross-link formation is induced by UV-A irradiation. The advantage of the photo-cross-linking strategy is that it is not restricted to lysine residues and that hydrophobic regions in proteins can be targeted, which is advantageous for investigating membrane proteins. We consider the strategy of combining cross-linkers with orthogonal reactivities and distances to be ideally suited for maximizing the amount of structural information that can be gained from a cross-linking experiment.

Target Identification by Diazirine Photo-Cross-linking and Click Chemistry.

Target identification of biologically active small-molecules is often the rate-determining step in forward chemical genetics. Photo-affinity labeling (PAL) represents a useful biochemical strategy for target identification in complex protein mixtures. This unit describes the use of alkyl diazirine-based photo-affinity probes and Cu(I)-catalyzed click chemistry to covalently label and visualize the targets of biologically active small-molecules. A general method for affinity purification of probe-modified proteins, useful for identification of protein targets, is also described.