Photoaffinity Labeling Method Research Articles

Interaction Studies between Carbonic Anhydrase and a Sulfonamide Inhibitor by Experimental and Theoretical Approaches

The most used approaches in structure-based drug design possess peculiar characteristics with advantages and limitations, and thus the management of complementary data from various techniques is of particular interest to synergistically achieve the development of effective enzyme inhibitors. In this Letter, we describe the application of experimental and computational techniques to study the interactions between human carbonic anhydrases and sulfonamide inhibitors. In particular, a series of affinity-labeled carbonic anhydrase inhibitors containing sulfonamido photoprobes was designed and synthesized, and one of these compounds, a benzophenone derivative, was chosen as a model photoprobe/inhibitor. A photoaffinity labeling method followed by mass spectrometry analysis was then applied to elucidate the inhibitor binding site, and a comparison with X-ray crystallography and molecular dynamics simulation data was carried out, highlighting that to have a comprehensive view of the protein/inhibitor complex stabilization all three kinds of experiments are necessary.

Target identification of baicalein derivative using DNA-programmed photoaffinity labeling

The natural product baicalein derivative baicalein-8-sulfonic acid (BaSO3H) showed significant inhibitory effects on hepatocarcinoma cells viabilities and colony formation, but its molecular target(s) and mechanism were still not clearly elucidated. Using a DNA-programmed photoaffinity labeling method, we identified 12 targets that specifically bound with BaSO3H. Among these, BaSO3H bound with c-Jun N-terminal kinase 2 (JNK2) at an affinity of 33.1 nM (Kd) to induce apoptosis and autophagy in hepatocarcinoma cells.

Target Protein Identification on Photocatalyst-Functionalized Magnetic Affinity Beads.

Although various affinity chromatography and photoaffinity labeling methods have been developed for target protein identification of bioactive molecules, it is often difficult to detect proteins that bind the ligand with weak transient affinity using these techniques. We have developed single electron transfer-mediated tyrosine labeling using ruthenium photocatalysts. Proximity labeling using 1-methyl-4-aryl-urazole (MAUra) labels proteins in close proximity to the photocatalyst with high efficiency and selectivity. Performing this labeling reaction on affinity beads makes it possible to label proteins that bind the ligand with weak transient affinity. In this article, novel protocols are described for target protein identification using photocatalyst proximity labeling on ruthenium photocatalyst-functionalized magnetic affinity beads. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of ruthenium photocatalyst Basic Protocol 2: Synthesis of azide- or desthiobiotin-conjugated labeling reagents Basic Protocol 3: Preparation of photocatalyst and ligand-functionalized affinity beads Basic Protocol 4: Target protein labeling in cell lysate Basic Protocol 5: Enrichment of labeled proteins with MAUra-DTB for LC-MS/MS analysis Basic Protocol 6: 2D-DIGE analysis of fluorescence-labeled proteins.

Abstract NT-119: TARGET DISCOVERY OF NATURAL PRODUCT INSPIRED PHYLLANTHUSMINS FOR TREATMENT OF HIGH GRADE SEROUS OVARIAN CANCER

Abstract BACKGROUND: High grade serous ovarian cancer (HGSOC) is a lethal gynecological malignancy with a need for new therapeutic agents. Many of the most widely used chemotherapeutic drugs are either derived from or are semi-synthetic derivatives of natural products. We developed potent synthetic analogs (PHYs) of the phyllanthusmin class inspired by prior natural product isolated from Phyllanthus poilanei Beille. MATERIALS & METHODS: HGSOC cell lines, OVCAR3 and OVCAR8, and non-tumorigenic controls, IOSE80 and FT33, were used in this study. Cytotoxicity assays included sulforhodamine B assay, and annexin X/PI staining and Western blotting for confirmation of apoptosis induction. A photo affinity labeling method was used to attach PHY analogs to solid phase support. Targets were isolated using a pulldown technique and mass spectrometry. CRISPR-Cas9 genome editing was used to knockout and confirm putative targets. RESULTS: The most potent analog, PHY34, has nanomolar potency in HGSOC cell lines in vitro and displayed cytotoxic activity through late-stage autophagy inhibition and activation of apoptosis. PHY34 was readily bioavailable through intraperitoneal administration in vivo where it significantly reduced HGSOC tumor burden. Targets were identified using photo affinity labeling-aided protein pulldown and mass spectrometry, and confirmed by generating knockout cell lines of targets. CONCLUSIONS: This class of compounds holds promise as a potential, novel chemotherapeutic approach and demonstrates the effectiveness of pleiotropically targeting autophagy and apoptosis as a viable strategy for combating high grade serous ovarian cancer. Citation Format: Alexandria N. Young, Steven Kurina, Andrew Huntsman, Rathnayake A. Rathnayake, Melissa Korkmaz, A. Douglas Kinghorn, Leslie Aldrich, Stephanie Cologna, James R. Fuchs, Joanna E. Burdette. TARGET DISCOVERY OF NATURAL PRODUCT INSPIRED PHYLLANTHUSMINS FOR TREATMENT OF HIGH GRADE SEROUS OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-119.

A Binding Site Hotspot Map of the FKBP12-Rapamycin-FRB Ternary Complex by Photoaffinity Labeling and Mass Spectrometry-Based Proteomics.

Structural characterization of small molecule binding site hotspots within the global proteome is uniquely enabled by photoaffinity labeling (PAL) coupled with chemical enrichment and unbiased analysis by mass spectrometry (MS). MS-based binding site maps provide structural resolution of interaction sites in conjunction with identification of target proteins. However, binding site hotspot mapping has been confined to relatively simple small molecules to date; extension to more complex compounds would enable the structural definition of new binding modes in the proteome. Here, we extend PAL and MS methods to derive a binding site hotspot map for the immunosuppressant rapamycin, a complex macrocyclic natural product that forms a ternary complex with the proteins FKBP12 and FRB. Photo-rapamycin was developed as a diazirine-based PAL probe for rapamycin, and the FKBP12-photo-rapamycin-FRB ternary complex formed readily in vitro. Photoirradiation, digestion, and MS analysis of the ternary complex revealed a McLafferty rearrangement product of photo-rapamycin conjugated to specific surfaces on FKBP12 and FRB. Molecular modeling based on the binding site map revealed two distinct conformations of complex-bound photo-rapamycin, providing a 5.0 Å distance constraint between the conjugated residues and the diazirine carbon and a 9.0 Å labeling radius for the diazirine upon photoactivation. These measurements may be broadly useful in the interpretation of binding site measurements from PAL. Thus, in characterizing the ternary complex of photo-rapamycin by MS, we applied binding site hotspot mapping to a macrocyclic natural product and extracted precise structural measurements for interpretation of PAL products that may enable the discovery of new binding sites in the "undruggable" proteome.

Molecular Conjugation for the "Diazido Probe" Method

Target identification of screening hit compounds with unknown mechanisms of action obtained from chemical libraries by phenotypic assays has played an important role in the development of innovative drugs that work based on novel mechanisms. To improve the usability of the target identification based on the photoaffinity labeling method, we have studied the "diazido probe" method, wherein a photoreactive aromatic azido and relatively photostable aliphatic azido groups are sequentially used for photoreaction and introduction of a latent detectable tag via the Staudinger ligation or click reactions, and related synthetic methods that enable expeditious preparation of molecular probes. To facilitate the development of diazido probes, we established short synthetic routes to diazido building blocks with different connectable groups based on sequential iridium-catalyzed C-H borylation and copper-catalyzed azidation of 1,3-disubstituted benzenes, and subsequent diverse functional group transformations leaving the azido groups untouched. To improve the utility of click chemistry for efficient introduction of a latent detectable tag, we developed a transient protection method of cyclooctynes from cycloaddition with an azide by 1 : 1 complexation with a cationic copper(I) salt. Application of this protection method using a cationic copper salt to a cyclooctyne bearing a terminal alkyne allowed for the selective click conjugation with an azide at the terminal alkyne moiety, which rendered functionalized cyclooctyne derivatives easily synthesizable.

Water compatible photoarylation of amino acids and peptides.

A novel photoarylation of amino acids and peptides is described, which tolerates the presence of water. Irradiation of Boc-protected amino acids in the presence of N-protected 2-azidobenzimidazoles leads to selective arylation of carboxy termini or side chains. The new reaction also works for peptides. Irradiation of the nonapeptide H-SPSYVYHQF-OH also resulted in selective arylation of the tyrosine side chains, as indicated by ESI-MS/MS fragmentation. Chemo- and regioselectivity could add the title reaction to the repertoire of photoaffinity labeling methods.

Multivalent photoaffinity probe for labeling small molecule binding proteins.

Characterization of small molecule (SM)-protein interaction is of high importance in biomedical research such as target identification and proteomic profiling. Photo-cross-linking is a powerful and straightforward strategy to covalently capture SM's binding proteins. The DNA-based photoaffinity labeling method is able to capture SM's protein targets with high specificity but suffers low cross-linking efficiency, which limits its utility for low abundance and low affinity proteins. After screening a variety of cross-linkers, by utilizing the multivalency effect, the cross-linking efficiency was improved by nearly 7-fold without compromising probe specificity. The generality and performance of multivalent photoaffinity probes have been validated with a variety of SM-protein pairs in the complexity of cell lysates.

Photoaffinity Labeling of Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Targets in Mammalian Cells*♦

Nicotinic acid adenine dinucleotide phosphate (NAADP) is an agonist-generated second messenger that releases Ca(2+) from intracellular acidic Ca(2+) stores. Recent evidence has identified the two-pore channels (TPCs) within the endolysosomal system as NAADP-regulated Ca(2+) channels that release organellar Ca(2+) in response to NAADP. However, little is known about the mechanism coupling NAADP binding to calcium release. To identify the NAADP binding site, we employed a photoaffinity labeling method using a radioactive photoprobe based on 5-azido-NAADP ([(32)P-5N(3)]NAADP) that exhibits high affinity binding to NAADP receptors. In several systems that are widely used for studying NAADP-evoked Ca(2+) signaling, including sea urchin eggs, human cell lines (HEK293, SKBR3), and mouse pancreas, 5N(3)-NAADP selectively labeled low molecular weight sites that exhibited the diagnostic pharmacology of NAADP-sensitive Ca(2+) release. Surprisingly, we were unable to demonstrate labeling of endogenous, or overexpressed, TPCs. Furthermore, labeling of high affinity NAADP binding sites was preserved in pancreatic samples from TPC1 and TPC2 knock-out mice. These photolabeling data suggest that an accessory component within a larger TPC complex is responsible for binding NAADP that is unique from the core channel itself. This observation necessitates critical evaluation of current models of NAADP-triggered activation of the TPC family.

20-Iodo-14,15-epoxyeicosa-8(Z)-enoyl-3-azidophenylsulfonamide: Photoaffinity Labeling of a 14,15-Epoxyeicosatrienoic Acid Receptor

Endothelium-derived epoxyeicosatrienoic acids (EETs) relax vascular smooth muscle by activating potassium channels and causing membrane hyperpolarization. Recent evidence suggests that EETs act via a membrane binding site or receptor. To further characterize this binding site or receptor, we synthesized 20-iodo-14,15-epoxyeicosa-8(Z)-enoyl-3-azidophenylsulfonamide (20-I-14,15-EE8ZE-APSA), an EET analogue with a photoactive azido group. 20-I-14,15-EE8ZE-APSA and 14,15-EET displaced 20-(125)I-14,15-epoxyeicosa-5(Z)-enoic acid binding to U937 cell membranes with K(i) values of 3.60 and 2.73 nM, respectively. The EET analogue relaxed preconstricted bovine coronary arteries with an ED(50) comparable to that of 14,15-EET. Using electrophoresis, 20-(125)I-14,15-EE8ZE-APSA labeled a single 47 kDa band in U937 cell membranes, smooth muscle and endothelial cells, and bovine coronary arteries. In U937 cell membranes, the 47 kDa radiolabeling was inhibited in a concentration-dependent manner by 8,9-EET, 11,12-EET, and 14,15-EET (IC(50) values of 444, 11.7, and 8.28 nM, respectively). The structurally unrelated EET ligands miconazole, MS-PPOH, and ketoconazole also inhibited the 47 kDa labeling. In contrast, radiolabeling was not inhibited by 8,9-dihydroxyeicosatrienoic acid, 5-oxoeicosatetraenoic acid, a biologically inactive thiirane analogue of 14,15-EET, the opioid antagonist naloxone, the thromboxane mimetic U46619, or the cannabinoid antagonist AM251. Radiolabeling was not detected in membranes from HEK293T cells expressing 79 orphan receptors. These studies indicate that vascular smooth muscle, endothelial cells, and U937 cell membranes contain a high-affinity EET binding protein that may represent an EET receptor. This EET photoaffinity labeling method with a high signal-to-noise ratio may lead to new insights into the expression and regulation of the EET receptor.

アジド基の特性を利用したｎｏｎ‐ＲＩ光親和性標識法の開発

We have designed and synthesized an azido-functionalized photoaffinity probe, [125I] GIF-0082, based on the structural modification of dantrolene. This compound specifically suppresses physiological Ca2+ release from sarcoplasmic reticulum (SR) of skeletal muscle without affecting Ca2+-induced Ca2+ release (CICR). Learning from the experience of handling the radioisotope (RI) labeled probe, we have developed a novel method of RI-free photoaffinity labeling in order to realize direct analysis of photolabeled proteins. This method utilizes a compact bifunctional probe consist of a photoreactive group and an aliphatic azido group that survives photolysis. By using this probe, a target protein is photo-cross-linked (Step 1), and then a detectable group is introduced by Staudinger-Bertozzi ligation using an alkyl azido moiety (Step 2). The utility of the method has been demonstrated by specific labeling of the catalytic portion of human HMG-CoA reductase using a diazido-functionalized cerivastatin derivative, photovastatin CAA 1, and a triarylphosphine derivative, GIF-0373, equipped with a fluorescein structure. The protocol has been also applied to the photolabeling of target proteins for a diazido-functionalized dantrolene derivative, GIF-0430.

アクチン脱重合分子の合成と活性

Aplyronine A 1 is a potent antitumor macrolide isolated from the sea hare Aplysia kurodai. Aplyronine A interacts with actin, one of the major proteins in cytoskeleton. We achieved total synthesis of aplyronine A and its 18 analogs and investigated structure-activity relationships. Analysis of the interaction of aplyronine analogs with actin by a photoaffinity labeling method and X-ray crystal structure analysis of actin complex with aplyronine A have been carried out. These results revealed that aplyronine A binds to actin at the hydrophobic cleft between subdomains 1 and 3 of actin. The binding mode of the macrolactone part of aplyronine A was different from previously reported macrolides. In addition, the trimethylserine moiety of aplyronine A, the essential functional group for cytotoxicity against HeLa S3 cells, was sticking out from a surface of actin-aplyro-nine A complex.

Cross‐linking chemistry and biology: development of multifunctional photoaffinity probes

An efficient method of photoaffinity labeling has been developed based on rationally designed multifunctional photoprobes. Photoaffinity techniques have been used to elucidate the protein structure at the interface of biomolecules by the photochemical labeling of interacting sites. However, the identification of labeled sites within target proteins is often difficult. Novel biotinyl bioprobes bearing a diazirine photophore have contributed significantly to the rapid elucidation of ligand binding sites within proteins, thereby extending conventional photoaffinity methods. This article discusses the synthesis and applications of various photoprobes bearing a biotin, including strategies using cleavable linkages between photophores. The combination of photoaffinity methods with chip technology is also described as a novel entry to rapid affinity-based screening of inhibitors. This review focuses on a rapid and reliable photoaffinity method utilizing diazirine-based multifunctional photoprobes with numerous potential applications in functional proteomics of biomolecular interactions.

Photoaffinity labeling in drug discovery and developments: chemical gateway for entering proteomic frontier.

One of the major events occurring at biological interfaces is the specific recognition of bioactive ligands by their receptor proteins. The elucidation of interacting partners is an immediate entrance into the discovery of medicinal leads. The method of photoaffinity labeling enables the direct probing of target protein through a covalent bond introduced between a ligand and its specific receptor. Thus, the photoaffinity labeling is applied in two stages of drug discovery and development processes. First, the method is useful for the screening of early leads. If the binding site analysis of target protein is important for defining a particular pharmacophore, the photoaffinity labeling will give the structural information at the contact point of drugs with receptors. Second, emerging new technologies, combinatorial chemistry, recombinant DNA techniques, and high-throughput analysis, are extending the potential of photoaffinity labeling to become a rapid and more sensitive means for the identification of drug-receptor pairs as well as the elucidation of molecular recognition mechanism at drug-receptor interfaces. This review focuses on several recent impacts of photoaffinity labeling as a useful tool for drug discovery and developments.

Photoactivatable Reagents Based on Aryl(trifluoromethyl)diazirines: Synthesis and Application for Studying Nucleic Acid–Protein Interactions

Recent data on the synthesis of photoactivatable derivatives of nucleic acids and proteins on the basis of aryl(trifluoromethyl)diazirines—analogs of nucleosides, nucleotides, oligonucleotides, as well as amino acids and peptides—are reviewed. The synthesis of bi- and polyfunctional photoactivatable reagents, including those containing a cleavable function, designed for postsynthetic modification of biopolymers is described. Data are given on the use of the photoactivatable derivatives for studying nucleic acid–protein interactions by the method of photoaffinity labeling. Special consideration is paid to the results obtained by the authors" team in cooperation with other researchers as well as graduate students of the Chemistry of Natural Products Chair, Chemical Faculty, Moscow State University and destined to solve various scientific tasks in the domain of nucleic acid–protein recognition with the use of photoaffinity crosslinking.

Characterization of the Taxol Binding Site on the Microtubule: IDENTIFICATION OF Arg282 IN β-TUBULIN AS THE SITE OF PHOTOINCORPORATION OF A 7-BENZOPHENONE ANALOGUE OF TAXOL

Photoaffinity labeling methods have allowed a definition of the sites of interaction between Taxol and its cellular target, the microtubule, specifically beta-tubulin. Our previous studies have indicated that [(3)H]3'-(p-azidobenzamido)Taxol photolabels the N-terminal 31 amino acids of beta-tubulin (Rao, S., Krauss, N. E., Heerding, J. M., Swindell, C. S., Ringel, I., Orr, G. A., and Horwitz, S. B. (1994) J. Biol. Chem. 269, 3132-3134) and [(3)H]2-(m-azidobenzoyl)Taxol photolabels a peptide containing amino acid residues 217-233 of beta-tubulin (Rao, S., Orr, G. A., Chaudhary, A. G., Kingston, D. G. I., and Horwitz, S. B. (1995) J. Biol. Chem. 270, 20235-20238). The site of photoincorporation of a third photoaffinity analogue of Taxol, [(3)H]7-(benzoyldihydrocinnamoyl) Taxol, has been determined. This analogue stabilizes microtubules polymerized in the presence of GTP, but in contrast to Taxol, does not by itself enhance the polymerization of tubulin to its polymer form. CNBr digestion of [(3)H]7-(benzoyldihydrocinnamoyl)Taxol-labeled tubulin, with further arginine-specific cleavage by clostripain resulted in the isolation of a peptide containing amino acid residues 277-293. Amino acid sequence analysis indicated that the photoaffinity analogue cross-links to Arg(282) in beta-tubulin. Advances made by electron crystallography in understanding the structure of the tubulin dimer have allowed us to visualize the three sites of photoincorporation by molecular modeling. There is good agreement between the binding site of Taxol in beta-tubulin as determined by photoaffinity labeling and electron crystallography.

Proteolytic Mapping of the Thymidine/Thymidylate Binding Site of Herpes Simplex Virus Type 1 Thymidine Kinase: A General Photoaffinity Labeling Method for Identifying Active-Site Peptides

The herpes simplex virus type 1 thymidine kinase (HSV-1 TK) is an important pharmacological target of antiviral nucleoside drugs and it uniquely possesses both a thymidine kinase and a thymidylate kinase activity. The structural relationship between these two activities is addressed in this study using a combination of active-site directed photoaffinity analogs, proteases, and tricine–SDS–polyacrylamide gel electrophoresis. For analysis of the thymidylate binding site, the thymidylate analog [32P]5-azido-dUMP was specifically photocrosslinked to the active site of HSV-1 TK. Because the amino acid sequence of HSV-1 TK is known, endoprotease Lys-C, V8 protease, trypsin, or chymotrypsin was used to generate a proteolytic map of photoincorporated peptides by separation on high-resolution tricine–SDS–polyacrylamide gels. Analysis of the resulting peptides indicated that the photoprobe was localized to one region comprising amino acids Ile112-Tyr132. Photolabeling of this region indicates that the thymine base of thymidine and TMP bind at one shared site in HSV-1 TK. In addition, the results reported in this study demonstrate that photolabeling with azidonucleotides can be used to identify photolabeled peptides by proteolytic mapping. This technique bypasses the problems of peptide purification and sequencing and yields rapid results when the primary amino acid structure of the protein of interest is known.

5445937 Detection of alzheimer's disease and other diseases using an improved photoaffinity labeling method : Haley Boyd Nicholasville, KY, United States assigned to The University of Kentucky Research Foundation

5445937 Detection of alzheimer's disease and other diseases using an improved photoaffinity labeling method : Haley Boyd Nicholasville, KY, United States assigned to The University of Kentucky Research Foundation

Characterization of the Taxol Binding Site on the Microtubule: 2-(m-AZIDOBENZOYL)TAXOL PHOTOLABELS A PEPTIDE (AMINO ACIDS 217-231) of β-TUBULIN

Photoaffinity labeling methods are being used to define the molecular contacts between taxol and its target protein, tubulin. Our laboratory has demonstrated previously that [3H]3'-(p-azidobenzamido)taxol photolabels the N-terminal 31 amino acids of beta-tubulin (Rao, S., Krauss, N.E., Heerding, J.M., Swindell, C.S., Ringel, I., Orr, G.A., and Horwitz, S.B. (1994) J. Biol. Chem. 269, 3132-3134). The interaction of a second photoaffinity analogue of taxol, [3H]2-(m-azidobenzoyl)taxol, with tubulin has been investigated. This analogue specifically photolabels beta-tubulin and the photolabeling is completed by both taxol and unlabeled 2-(m-azidobenzoyl)-taxol indicating a common binding domain. To identify the site(s) of photoincorporation, [3H]2-(m-azidobenzoyl)taxol-photolabeled beta-tubulin was subjected to sequential cyanogen bromide and tryptic digestions. Radiolabeled peptides were purified by reverse phase high performance liquid chromatography, and amino acid sequencing studies identified a peptide containing amino acid residues 217-231 of beta-tubulin as the major photolabeled domain.

Direct Identification of a Polyamine Binding Domain on the Regulatory Subunit of the Protein Kinase Casein Kinase 2 by Photoaffinity Labeling

Phosphorylation of many protein substrates by the protein kinase casein kinase 2 (CK2) is stimulated severalfold in the presence of polyamines such as spermine. Previous experiments have shown that CK2 is a polyamine binding protein and that the regulatory beta subunit is required for this binding activity. To delineate the spermine binding site of CK2, we have applied a photoaffinity labeling method using a tritiated photoactivable analog of spermine, [3H]sperminediazonium. The photoaffinity labeled beta subunit was cleaved with cyanogen bromide, and two labeled peptides were separated by high performance liquid chromatography. The major one was the peptide T72EQAAEM78 and the minor one was a 22-amino acid peptide comprising residues Ile98 to Met119. Thr72 and His108 were identified as the labeled amino acids of the Thr72-Met78 and Ile98-Met119 peptides, respectively. In the same manner, we succeeded in determining the residue Leu220 as an alpha subunit residue covalently bound to the probe. The photoaffinity labeling method described here enabled the first elucidation, by direct microsequencing, of a polyamine binding site on CK2 for which we propose a provisional structural model. These observations suggest a possible mechanism for CK2 activation by polyamines at the molecular level.