Azido derivative of tricarboxylic acid for photoaffinity labeling

A new photoaffinity probe, 5-(1-hydroxy-4-azidophenylazo)-1,2,3-benzenetricarboxylic acid, was synthesized and characterized. This reagent can be potentially used in photoaffinity labeling of the mitochondrial tricarboxylate carrier, as well as of enzymes interacting with tricarboxylic acids. Inhibition and labeling of the mitochondrial tricarboxylate carrier is presented.     

Photoaffinity labeling of P2-purinergic and H1-histamine receptors in intact smooth muscle

This paper discusses the general applicability of photoaffinity labels as pharmacological receptor antagonists in functional studies of intact smooth muscle preparations. Guidelines are suggested that take into account the criteria for photoaffinity labeling studies as well as those for use with conventional antagonists.     

Tandem photoaffinity labeling–bioorthogonal conjugation in medicinal chemistry

Photoaffinity labeling has a longstanding history as a powerful biochemical technique. However, photoaffinity labeling has significantly evolved over the past decade principally due to its coupling with bioorthogonal/click chemistry reactions. This review aims to highlight tandem photoaffinity labeling–bioorthogonal conjugation as a chemical approach in medicinal chemistry and chemical biology. In particular, recent examples of using this strategy for affinity-based protein profiling (AfBPP), drug target identification, binding ensemble profiling, studying endogenous biological molecules, and imaging applications will be presented. Additionally, recent advances in the development of ‘all-in-one’ compact moieties possessing a photoreactive group and clickable handle will be discussed.     

Mechanism of interferon action. The interferon-induced phosphoprotein P1 possesses a double-stranded RNA-dependent ATP-binding site

Protein P1, the interferon-induced protein phosphorylated in the presence of dsRNA in human amnion U-cells, was covalently labeled with [alpha-32P]ATP following ultraviolet irradiation. The photoaffinity labeling of protein P1 was dependent upon double-stranded RNA. Antibody prepared against phosphorylated protein P1 immunoprecipitated the double-stranded RNA-dependent photoaffinity-labeled product. The extent of photoaffinity labeling was significantly decreased by the addition of unlabeled ATP, GTP, or AMP; adenosine had little effect on the photoaffinity labeling of protein P1. These results suggest that protein P1 possesses a site capable of binding an adenine nucleotide in a double-stranded RNA-dependent manner.     

Photoaffinity labeling probe for the substrate binding site of human phenol sulfotransferase (SULT1A1): 7-azido-4-methylcoumarin.

A novel fluorescent photoactive probe 7-azido-4-methylcoumarin (AzMC) has been characterized for use in photoaffinity labeling of the substrate binding site of human phenol sulfotransferase (SULT1A1 or P-PST-1). For the photoaffinity labeling experiments, SULT1A1 cDNA was expressed in Escherichia coli as a fusion protein to maltose binding protein (MBP) and purified to apparent homogeneity over an amylose column. The maltose moiety was removed by Factor Xa cleavage. Both MBSULT1A1 and SULT1A1 were efficiently photolabeled with AzMC. This labeling was concentration dependent. In the absence of light, AzMC competitively inhibited the sulfation of 4MU catalyzed by SULT1A1 (Ki = 0.47 +/- 0.05 mM). Moreover, enzyme activity toward 2-naphthol was inactivated in a time- and concentration-dependent manner. SULT1A1 inactivation by AzMC was protected by substrate but was not protected by cosubstrate. These results indicate that photoaffinity labeling with AzMC is highly suitable for the identification of the substrate binding site of SULT1A1. Further studies are aimed at identifying which amino acids modified by AzMC are localized in the binding site.     

Photoaffinity labeling combined with mass spectrometric approaches as a tool for structural proteomics

Protein chemistry, such as crosslinking and photoaffinity labeling, in combination with modern mass spectrometric techniques, can provide information regarding protein-protein interactions beyond that normally obtained from protein identification and characterization studies. While protein crosslinking can make tertiary and quaternary protein structure information available, photoaffinity labeling can be used to obtain structural data about ligand-protein interaction sites, such as oligonucleotide-protein, drug-protein and protein-protein interaction. In this article, we describe mass spectrometry-based photoaffinity labeling methodologies currently used and discuss their current limitations. We also discuss their potential as a common approach to structural proteomics for providing 3D information regarding the binding region, which ultimately will be used for molecular modeling and structure-based drug design.     

Photoaffinity labeling of dog pancreas microsomes with 8-azido-ATP inhibits association of nascent preprolactin with the signal sequence receptor complex.

Transport of bovine preprolactin into dog pancreas microsomes involves a microsomal protein which is sensitive to photoaffinity labeling with azido-ATP and which is distinct from the ATP-binding protein, immunoglobulin heavy chain binding protein. Here we addressed the question of what stage of preprolactin transport is affected. Thus a nascent presecretory protein which is related to preprolactin, termed ppl-86mer, was employed. Here we show that the nascent preprolactin did not become associated with the alpha-subunit of the signal sequence receptor complex after photoaffinity labeling of microsomes with azido-ATP. Therefore, we conclude that the microsomal protein which is sensitive to photoaffinity labeling with azido-ATP acts prior to the signal sequence receptor complex.     

Photoaffinity labeling combined with mass spectrometric approaches as a tool for structural proteomics

Protein chemistry, such as crosslinking and photoaffinity labeling, in combination with modern mass spectrometric techniques, can provide information regarding protein–protein interactions beyond that normally obtained from protein identification and characterization studies. While protein crosslinking can make tertiary and quaternary protein structure information available, photoaffinity labeling can be used to obtain structural data about ligand–protein interaction sites, such as oligonucleotide–protein, drug–protein and protein–protein interaction. In this article, we describe mass spectrometry-based photoaffinity labeling methodologies currently used and discuss their current limitations. We also discuss their potential as a common approach to structural proteomics for providing 3D information regarding the binding region, which ultimately will be used for molecular modeling and structure-based drug design.     

Immuno-photoaffinity labeling of the D2-dopamine receptor

Azido-haloperidol was synthesized and applied as a photoaffinity ligand for the D2-dopamine receptor. In bovine striatal membranes, azido-haloperidol bound reversibly to the receptor (KD = 15 nM), and when exposed to light, it bound to the receptor irreversibly. This irreversible inactivation was prevented by the dopaminergic agonist N-propylnorapomorphine or the dopaminergic antagonists haloperidol and (+)-butaclamol. The photoaffinity labeled D2-receptor was probed with anti-haloperidol antibodies following gel electrophoresis and transfer to nitrocellulose. A major polypeptide of 94 kDa reacted with the anti-haloperidol antibodies. This polypeptide band was not observed when the photoaffinity labeling was performed in the presence of (+)-butaclamol or spiperone.     

Photoaffinity labeling of atrial natriuretic factor receptors of rat kidney cortex plasma membranes

Synthetic rat atrial natriuretic factor (ANF) was derivatized with the N-hydroxysuccinimide ester of [125I]iodoazidosalicylic acid to yield a radioactive photoaffinity probe. Incubation of purified plasma membranes from rat kidney cortex with this photoaffinity probe resulted in the specific labeling of a 140-kDa glycoprotein. The photoaffinity labeling of this protein was inhibited by ANF but not by reduced and alkylated ANF nor by other unrelated peptides. A 140-kDa band was also specifically labeled in liver plasma membranes but not in adipocyte plasma membranes. These observations suggest strongly that the 140-kDa glycoprotein is the ANF receptor.     

A new highly efficient photoreactive analogue of dCTP. Synthesis, characterization, and application in photoaffinity modification of DNA binding proteins

A new base-substituted analogue of dCTP, exo-N-{2-[N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]aminoethyl}-2'-deoxycytidine-5'-triphosphate (FAP-dCTP) has been synthesized and characterized. FAP-dCTP is an efficient substrate of mammalian DNA polymerase beta in the reaction of primer elongation displaying substrate properties as an analogue of dCTP and dTTP. FAP-dCTP was used for the photoaffinity modification of mammalian DNA polymerase beta. Two approaches to photoaffinity labeling were utilized. In one approach, photoreactive FAP-dCTP was first incorporated into radiolabeled primer-template, and photoreactive DNA was UV-irradiated in the presence of DNA polymerase beta, which resulted in the polymerase labeling by photoreactive primer. In an alternate approach, FAP-dCTP was first UV-cross-linked to the enzyme; subsequently, radiolabeled primer-template was added, and the enzyme-linked FAP-dCTP was incorporated into the 3'-end of radioactive primer. This "catalytic" modification pathway was shown to be less specific in recognition of FAP-dCTP as an analogue of dCTP than dTTP. FAP-dCTP was used as substrate of endogenous DNA polymerases of HeLa cell extract to synthesize photoreactive DNAs for photoaffinity modification of cell proteins. UV irradiation results in modification of DNA binding proteins of cell extract. The level of photoaffinity labeling of protein targets in the cell extract was strongly dependent on the efficiency of synthesis of photoreactive DNA.     

Photoaffinity labeling approaches to elucidate lipid–protein interactions

Lipid–protein interactions serve as the basis for many of the diverse roles of lipids. However, these noncovalent binding events are often weak, transient, or dependent upon environmental cues. Photoaffinity labeling can preserve these interactions under native conditions, enabling their biochemical profiling. Typically, photoaffinity labeling probes contain a diazirine photocrosslinker and a click chemistry handle for enrichment and downstream analysis. In this review, we summarize recent advances in the understanding the mechanisms of diazirine photocrosslinking, and we provide an overview of recent applications of photoaffinity labeling to reveal the interactions of diverse types of lipids with specific members of the proteome.     

Mechanism of photoaffinity labeling of P2-purinergic receptors by arylazido aminopropionyl atp in isolated guinea-pig vas deferens

Following photolysis in the presence of the isolated guinea-pig vas deferens, arylazido aminopropionyl ATP (ANAPP₃), a photoaffinity label analog of ATP, produces an irreversible and specific pharmacological antagonism of contractile responses to adenine nucleotides. Experiments were performed to determine whether the antagonism follows the photolysis-dependent formation of nitrene intermediates at occupied receptors (true photoaffinity labeling) or if the reactive intermediate is photogenerated in solution prior to diffusion to the receptor and formation of covalent bonds (pseudo-photoaffinity labeling). When present during photolysis, para-aminobenzoic acid (PABA; 10 mM), a scavenger for nitrenes generated in solution, did not prevent the antagonism of ATP-induced responses by ANAPP₃. However, the absorption spectrum of ANAPP₃ photolyzed in the presence of PABA was different from that obtained when PABA was not present. This evidence for the formation of additional photolysis products suggests that ANAPP₃ had inserted into PABA during photolysis. Thus, covalent bonds arise from true photoaffinity labeling of the receptor. An analysis of the pharmacological effects of PABA indicated that responses to ATP, KCl and acetylcholine were unaffected either in the presence of, or after a 23 min incubation, with 10 mM PABA. In contrast, PABA produced a substantial, but reversible, antagonism of histamine- and norepinephrine-induced contractions. Irradiation of tissues in the presence of 10 mM PABA produced a slight potentiation of responses to ATP. Thus, information on the mechanisms of photoaffinity labeling may be obtained from functional studies on intact tissues. However, the pharmacological effects of agents used to define these mechanisms should be evaluated as well.     

Photoaffinity labeling of the multidrug resistance protein 2 (ABCC2/cMOAT) with a photoreactive analog of LTC4

Several studies have shown that the multidrug resistant protein MRP2 mediates the transport of chemotherapeutic drugs and normal cell metabolites, including Leukotriene C (LTC₄); however direct binding of the LTC₄ to MRP2 has not been demonstrated. In this study, a photoreactive analog of LTC₄ (IAALTC₄) was used to demonstrate its direct binding to MRP2. Our results show specific photoaffinity labeling of MRP2 with IAALTC₄ in plasma membranes from MDCKII^MRP2 cells. The photoaffinity labeling signal of MRP2 with IAALTC₄ was much lower than that of MRP1, consistent with previous studies whereby the measured K_m values of MRP1 and MRP2 for LTC₄ were 1 μM and 0.1 μM LTC₄, respectively. Competition of IAALTC₄ photoaffinity labeling to MRP2 with MK571, a well characterized inhibitor of MRP2 function, showed ~75% reduction in binding in the presence of 50 μM excess MK571. Interestingly, unmodified LTC₄ enhanced the photoaffinity labeling of IAALTC₄ to MRP2, whereas excess GSH and Quercetin had no significant effect. Mild tryptic digestion of photoaffinity labeled MRP2 revealed several photoaffinity labeled peptides that localized the IAALTC₄ binding to a 15 kDa amino acid sequence that contains transmembrane 16 and 17. Together these results provide the first demonstration of direct LTC₄ binding to MRP2.     

Identification of hormone-interacting amino acid residues within the steroid-binding domain of the glucocorticoid receptor in relation to other steroid hormone receptors

Purified rat liver glucocorticoid receptor was covalently charged with [3H]glucocorticoid by photoaffinity labeling (UV irradiation of [3H]triamcinolone acetonide-glucocorticoid receptor) or affinity labeling (incubation with [3H]dexamethasone mesylate). After labeling, separate samples of the denatured receptor were cleaved with trypsin (directly or after prior succinylation), chymotrypsin, and cyanogen bromide. Labeled residues in the peptides obtained were identified by radiosequence analysis. The peaks of radioactivity corresponded to Met-622 and Cys-754 after photoaffinity labeling with [3H]triamcinolone acetonide and Cys-656 after affinity labeling with [3H]dexamethasone mesylate. The labeled residues are all positioned within hydrophobic segments of the steroid-binding domain. The patterns of hydropathy and secondary structure for the glucocorticoid receptor are highly similar to those for the progestin receptor and similar but less so to those for the estrogen receptor and to those for c-erb A.     

Photoaffinity labeling of functionally different lysine-binding sites in human plasminogen and plasmin

Photoaffinity labeling of human plasmin using 4-azidobenzoylglycyl-L-lysine inhibits clot lysis activity, while the activity toward the active-site titrant, p-nitrophenyl-p'-guanidinobenzoate, or alpha-casein are maintained. Photoaffinity labeling of native Glu-plasminogen with the same reagent causes incorporation of approximately 1.5 mol label per mol plasminogen. This labeled plasminogen can be activated to plasmin by either urokinase or streptokinase. The resulting plasmin has full clot lysis activity and can be subsequently photoaffinity labeled with a loss of clot lysis activity. The rate of activation of labeled plasminogen by urokinase is increased relative to that of native plasminogen. epsilon-Aminocaproic acid blocks incorporation of photoaffinity label into both plasminogen and plasmin, indicating that the labeling is specific to the lysine-binding sites. The labels are located in the kringle 1+2+3 fragment in either photoaffinity-labeled plasminogen or plasmin. These results indicate that the specific lysine-binding site blocked in plasmin acts in concert with the active-site in binding and using fibrin as a substrate. This clot lysis regulating site is not available for labeling in plasminogen, but is exposed or changed upon activation to plasmin. The different lysine-binding sites labeled in plasminogen may regulate the conformation of the molecule as evidence by an enhanced rate of activation to plasmin.     

Photoaffinity labeling and mass spectrometry identify ribosomal protein S3 as a potential target for hybrid polar cytodifferentiation agents.

The ability of a novel class of hybrid polar compounds (HPCs) to induce differentiation and consequent cessation of proliferation of transformed cells has led to their development as potential chemotherapeutic agents in the treatment of cancer. Suberoylanilide hydroxamic acid (SAHA) is a prototype of a family of hydroxamic acid based compounds (SAHA-like HPCs) that can, at micromolar concentrations, induce a variety of transformed cell lines to differentiate. The mechanism of action of the HPCs is not entirely understood. Searching for a cellular target of the SAHA-like HPCs, we synthesized a photoaffinity labeling reagent structurally based on SAHA, and probed for SAHA-binding proteins in murine erythroleukemia (MEL) cells. Photoaffinity labeling in cell free extracts identified a 32-kDa protein (p32) that was specifically labeled by the photoaffinity reagent. Cell fractionation assays localized p32 to the P100 fraction. p32 was partially purified and identified by mass spectrometry as the 40 S ribosomal protein S3. Expression of epitope-tagged S3 in bacterial lysates followed by photoaffinity labeling confirmed its specific labeling. Identification of a cytodifferentiation agent target may shed light on the mechanism by which the SAHA-like HPCs exert their antitumor effects.     

Characterization of a photosensitive glucose derivative. A photoaffinity reagent for the erythrocyte hexose transporter

The photosensitive reagent 6-N-(4-azido-2-hydroxy-3,5-diiodobenzoyl)-D-glucosamine has been assessed as a potential photoaffinity label for the hexose transporter. Under zero-trans conditions, transport experiments performed in the dark reveal that the reagent inhibits the uptake of D-glucose in resealed human erythrocyte ghosts. Increasing the concentration of glucose in the transport medium has a protective effect, reducing the inhibition. Kinetic analysis indicates that the probe acts as a competitive inhibitor with high affinity for the erythrocyte hexose transporter (Ki between 0.07 and 0.2 microM). Exposure to a 280 nm filtered high intensity mercury-vapor lamp results in a rapid and efficient photolysis. At low concentrations of the probe, specific labeling of membrane preparations was observed. Autoradiograms of 10% SDS gels revealed the specific labeling of bands 4.51 and 6. This labeling was concentration-dependent and protected by D-glucose (not the L-isomer) and phloretin in the medium. When subjected to multiple exposures of low concentration of the photoaffinity reagent, apparent saturation was achieved.