Photoactive Analogue Research Articles

Light-Controlled Anticancer Activity and Cellular Uptake of a Photoswitchable Cisplatin Analogue.

A photoactive analogue of cisplatin was synthesized with two arylazopyrazole ligands, able to undergo trans-cis/cis-trans photoisomerizations. The cis photoisomer showed a dark half-life of 9 days. The cytotoxicities of both photoisomers of the complex were determined in several cancer and normal cell lines and compared to that of cisplatin. The trans photoisomer of the complex was much more cytotoxic than both the cis photoisomer and cisplatin, and was more toxic for cancer (4T1) than for normal (NMuMG) murine breast cells. 4T1 cell death occurred through necrosis. Photoisomerization of the trans and cis photoisomers internalized by the 4T1 cells increased and decreased their viability, respectively. The cellular uptake of the trans photoisomer was stronger than that of both the cis photoisomer and cisplatin. Both photoisomers interacted with DNA faster than cisplatin. The trans photoisomer was bound stronger by bovine serum albumin and induced a greater decrease in cellular glutathione levels than the cis photoisomer.

Effect of Linker Distribution in the Photocatalytic Activity of Multivariate Mesoporous Crystals.

The use of Metal-Organic Frameworks as crystalline matrices for the synthesis of multiple component or multivariate solids by the combination of different linkers into a single material has emerged as a versatile route to tailor the properties of single-component phases or even access new functions. This approach is particularly relevant for Zr6-MOFs due to the synthetic flexibility of this inorganic node. However, the majority of materials are isolated as polycrystalline solids, which are not ideal to decipher the spatial arrangement of parent and exchanged linkers for the formation of homogeneous structures or heterogeneous domains across the solid. Here we use high-throughput methodologies to optimize the synthesis of single crystals of UiO-68 and UiO-68-TZDC, a photoactive analogue based on a tetrazine dicarboxylic derivative. The analysis of the single linker phases reveals the necessity of combining both linkers to produce multivariate frameworks that combine efficient light sensitization, chemical stability, and porosity, all relevant to photocatalysis. We use solvent-assisted linker exchange reactions to produce a family of UiO-68-TZDC% binary frameworks, which respect the integrity and morphology of the original crystals. Our results suggest that the concentration of TZDC in solution and the reaction time control the distribution of this linker in the sibling crystals for a uniform mixture or the formation of core-shell domains. We also demonstrate how the possibility of generating an asymmetric distribution of both linkers has a negligible effect on the electronic structure and optical band gap of the solids but controls their performance for drastic changes in the photocatalytic activity toward proton or methyl viologen reduction.

Thiophene-fused boracycles as photoactive analogues of diboraanthracenes.

The construction of thiophene-fused analogues of diboraanthracenes with different aryl substituents through boron-mercury exchange followed by the nucleophilic replacement of the chlorines of dichlorodiboradithiophene 2 with Grignard reagents is reported. These diboradithiophenes exhibited unusual photophysical and electrochemical properties. They all undergo photoisomerisation, which can be traced using photophysical and 1H NMR spectroscopy studies.

Propofol Inhibits SIRT2 Deacetylase through a Conformation-specific, Allosteric Site

meta-Azi-propofol (AziPm) is a photoactive analog of the general anesthetic propofol. We photolabeled a myelin-enriched fraction from rat brain with [(3)H]AziPm and identified the sirtuin deacetylase SIRT2 as a target of the anesthetic. AziPm photolabeled three SIRT2 residues (Tyr(139), Phe(190), and Met(206)) that are located in a single allosteric protein site, and propofol inhibited [(3)H]AziPm photolabeling of this site in myelin SIRT2. Structural modeling and in vitro experiments with recombinant human SIRT2 determined that propofol and [(3)H]AziPm only bind specifically and competitively to the enzyme when co-equilibrated with other substrates, which suggests that the anesthetic site is either created or stabilized in enzymatic conformations that are induced by substrate binding. In contrast to SIRT2, specific binding of [(3)H]AziPm or propofol to recombinant human SIRT1 was not observed. Residues that line the propofol binding site on SIRT2 contact the sirtuin co-substrate NAD(+) during enzymatic catalysis, and assays that measured SIRT2 deacetylation of acetylated α-tubulin revealed that propofol inhibits enzymatic function. We conclude that propofol inhibits the mammalian deacetylase SIRT2 through a conformation-specific, allosteric protein site that is unique from the previously described binding sites of other inhibitors. This suggests that propofol might influence cellular events that are regulated by protein acetylation state.

Sites and functional consequence of VDAC–alkylphenol anesthetic interactions

General anesthetics have previously been shown to bind mitochondrial VDAC. Here, using a photoactive analog of the anesthetic propofol, we determined that alkylphenol anesthetics bind to Gly56 and Val184 on rat VDAC1. By reconstituting rat VDAC into planar bilayers, we determined that propofol potentiates VDAC gating with asymmetry at the voltage polarities; in contrast, propofol does not affect the conductance of open VDAC. Additional experiments showed that propofol also does not affect gramicidin A properties that are sensitive to lipid bilayer mechanics. Together, this suggests propofol affects VDAC function through direct protein binding, likely at the lipid-exposed channel surface, and that gating can be modulated by ligand binding to the distal ends of VDAC β-strands where Gly56 and Val184 are located.

Mechanisms Revealed Through General Anesthetic Photolabeling

General anesthetic photolabels are used to reveal molecular targets and molecular binding sites of anesthetic ligands. After identification, the relevance of anesthetic substrates or binding sites can be tested in biological systems. Halothane and photoactive analogs of isoflurane, propofol, etomidate, neurosteroids, anthracene, and long chain alcohols have been used in anesthetic photolabeling experiments. Interrogated protein targets include the nicotinic acetylcholine receptor, GABAA receptor, tubulin, leukocyte function-associated antigen-1, and protein kinase C. In this review, we summarize insights revealed by photolabeling these targets, as well as general features of anesthetics, such as their propensity to partition to mitochondria and bind voltage-dependent anion channels. The theory of anesthetic photolabel design and the experimental application of photoactive ligands are also discussed.

Direct Modulation of Microtubule Stability Contributes to Anthracene General Anesthesia

Recently, we identified 1-aminoanthracene as a fluorescent general anesthetic. To investigate the mechanism of action, a photoactive analogue, 1-azidoanthracene, was synthesized. Administration of 1-azidoanthracene to albino stage 40-47 tadpoles was found to immobilize animals upon near-UV irradiation of the forebrain region. The immobilization was often reversible, but it was characterized by a longer duration consistent with covalent attachment of the ligand to functionally important targets. IEF/SDS-PAGE examination of irradiated tadpole brain homogenate revealed labeled protein, identified by mass spectrometry as β-tubulin. In vitro assays with aminoanthracene-cross-linked tubulin indicated inhibition of microtubule polymerization, similar to colchicine. Tandem mass spectrometry confirmed anthracene binding near the colchicine site. Stage 40-47 tadpoles were also incubated 1 h with microtubule stabilizing agents, epothilone D or discodermolide, followed by dosing with 1-aminoanthracene. The effective concentration of 1-aminoanthracene required to immobilize the tadpoles was significantly increased in the presence of either microtubule stabilizing agent. Epothilone D similarly mitigated the effects of a clinical neurosteroid general anesthetic, allopregnanolone, believed to occupy the colchicine site in tubulin. We conclude that neuronal microtubules are "on-pathway" targets for anthracene general anesthetics and may also represent functional targets for some neurosteroid general anesthetics.

Photoactive Analogs of Farnesyl Diphosphate and Related Isoprenoids: Design and Applications in Studies of Medicinally Important Isoprenoid- Utilizing Enzymes

Farnesyl diphosphate (FPP) is an important metabolic intermediate in the biosynthesis of a variety of molecules including sesquiterpenes and the side chains of a number of cofactors. FPP is also the source of isoprenoid side chains found attached to proteins. Enzymes that employ FPP as a substrate are of interest because they are involved in the semisynthesis of drugs as well as targets for drug design. Photoactive analogs of FPP have been useful for identifying enzymes that use this molecule as a substrate. A variety of photocrosslinking groups have been employed to prepare FPP analogs for use in such experiments including aryl azides, diazotrifluoropropionates and benzophenones. In this review, the design of these probes is described along with an examination of how they have been used in crosslinking experiments.

Identifying the Propofol Binding Site(S) in Heterologously Expressed Human Alpha1 Beta3 Gaba-A Receptors using a Photoreactive Propofol Analog

Propofol, a widely used intravenous general anesthetic, potentiates GABA-A receptor (GABAAR) function at anesthetic concentrations, and “knock-in” mice expressing a single mutation at position 15 in the β3 subunit M2 helix (βM2-15) have reduced sensitivity to many of the anesthetic effects of propofol. However, the location of propofol's binding site(s) remains to be determined. Here we use a photoactive analog of propofol, [3H]m-azipropofol(2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol), an aryl diazirine with broad amino acid side chain reactivity, to identify propofol binding site(s) in affinity-purified, expressed human α1β3 GABAARs in detergent/asolectin solution. m-Azipropofol acts as a tadpole anesthetic with potency (EC50 = 3μM) similar to propofol, and it acts as a low efficacy GABAAR potentiator (Hall et al. 2010, J Med. Chem. 53, 5667). Irradiation of GABAAR for 30 min at 365nm resulted in [3H]m-azipropofol photoincorporation into both the α1 and β3 subunits that was inhibitable by propofol and non-radioactive m-azipropofol with IC50s of 28±3 μM and 6.7±1 μM, respectively. Individual amino acids photolabeled by [3H]m-azipropofol were identified by protein microsequencing of subunit fragments generated by enzymatic or chemical digestion. In β3, there was propofol-inhibitable photolabeling of β3Met-286, an amino acid that is photolabeled by [3H]azietomidate in the etomidate binding site at the β3M3-α1M1 interface (Li et al. 2006, J Neurosci. 26, 11599, Chiara et al. 2012, Biochem. 51, 836). In α1, [3H]m-azipropofol photolabeled α1Ile-239 within α1M1, located one helical turn below α1Met-236 that is photolabeled by [3H]azietomidate. Consistent with propofol and m-azipropofol binding to this etomidate site, they completely inhibit [3H]azietomidate photolabeling with IC50s of 46±3 μM and 23±3 μM, respectively. Both β3Met-286 and α1Met-236 are in close proximity to βM2-15, which docking calculations suggest may also be part of this propofol binding site.

Synthesis of a-factor peptide from Saccharomyces cerevisiae and photoactive analogues via Fmoc solid phase methodology

a-Factor from Saccharomyces cerevisiae is a farnesylated dodecapeptide involved in mating. The molecule binds to a G-protein coupled receptor and hence serves as a simple system for studying the interactions between prenylated molecules and their cognate receptors. Here, we describe the preparation of a-factor and two photoactive analogues via Fmoc solid-phase peptide synthesis using hydrazinobenzoyl AM NovaGel™ resin; the structure of the synthetic a-factor was confirmed by MS-MS analysis and NMR; the structures of the analogues were confirmed by MS-MS analysis. Using a yeast growth arrest assay, the analogues were found to have activity comparable to a-factor itself.

Synthesis, Properties, and Applications of Diazotrifluropropanoyl‐Containing Photoactive Analogs of Farnesyl Diphosphate Containing Modified Linkages for Enhanced Stability

Photoactive analogs of farnesyl diphosphate (FPP) are useful probes in studies of enzymes that employ this molecule as a substrate. Here, we describe the preparation and properties of two new FPP analogs that contain diazotrifluoropropanoyl photophores linked to geranyl diphosphate via amide or ester linkages. The amide-linked analog (3) was synthesized in 32P-labeled form from geraniol in seven steps. Experiments with Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase) showed that 3 is an alternative substrate for the enzyme. Photolysis experiments with [(32)P]3 demonstrate that this compound labels the beta-subunits of both farnesyltransferase and geranylgeranyltransferase (types 1 and 2). However, the amide-linked probe 3 undergoes a rearrangement to a photochemically unreactive isomeric triazolone upon long term storage making it inconvenient to use. To address this stability issue, the ester-linked analog 4 was prepared in six steps from geraniol. Computational analysis and X-ray crystallographic studies suggest that 4 binds to protein farnesyl transferase (PFTase) in a similar fashion as FPP. Compound 4 is also an alternative substrate for PFTase, and a 32P-labeled form selectively photocrosslinks the beta-subunit of ScPFTase as well as E. coli farnesyldiphosphate synthase and a germacrene-producing sesquiterpene synthase from Nostoc sp. strain PCC7120 (a cyanobacterial source). Finally, nearly exclusive labeling of ScPFTase in crude E. coli extract was observed, suggesting that [32P]4 manifests significant selectivity and should hence be useful for identifying novel FPP-utilizing enzymes in crude protein preparations.

A versatile photoactivatable probe designed to label the diphosphate binding site of farnesyl diphosphate utilizing enzymes

Farnesyl diphosphate (FPP) is a substrate for a diverse number of enzymes found in nature. Photoactive analogues of isoprenoid diphosphates containing either benzophenone, diazotrifluoropropionate or azide groups have been useful for studying both the enzymes that synthesize FPP as well as those that employ FPP as a substrate. Here we describe the synthesis and properties of a new class of FPP analogues that links an unmodified farnesyl group to a diphosphate mimic containing a photoactive benzophenone moiety; thus, importantly, these compounds are photoactive FPP analogues that contain no modifications of the isoprenoid portion of the molecule that may interfere with substrate binding in the active site of an FPP utilizing enzyme. Two isomeric compounds containing meta- and para-substituted benzophenones were prepared. These two analogues inhibit Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase) with IC 50 values of 5.8 ( meta isomer) and 3.0 μM ( para isomer); the more potent analogue, the para isomer, was shown to be a competitive inhibitor of ScPFTase with respect to FPP with a K I of 0.46 μM. Radiolabeled forms of both analogues selectively labeled the β-subunit of ScPFTase. The para isomer was also shown to label Escherichia coli farnesyl diphosphate synthase and Drosophila melanogaster farnesyl diphosphate synthase. Finally, the para isomer was shown to be an alternative substrate for a sesquiterpene synthase from Nostoc sp. strain PCC7120, a cyanobacterial source; the compound also labeled the purified enzyme upon photolysis. Taken together, these results using a number of enzymes demonstrate that this new class of probes should be useful for a plethora of studies of FPP-utilizing enzymes.

Photoincorporation of azialcohol to the C1B domain of PKCδ is buffer dependent

Protein kinase C (PKC) is a signal transducing protein that has been implicated in binding alcohol and anesthetics. The alcohol and anesthetic binding of protein kinase C delta C1B domain has been determined previously by photolabeling and mass spectrometry [J. Das, G.H. Addona, W.S. Sandberg, S.S. Husain, T. Stehle, K.W. Miller, Identiffcation of a general anesthetic binding site in the diacylglycerol-binding domain of protein kinase C delta, J. Biol. Chem. 279 (2004) 37964-37972]. Here we studied photoincorporation of 3-azioctanol, a photoactive analog of octanol into PKC delta C1B in two buffer systems containing tris and hepes. The extent of photoincorporation was higher in hepes compared to tris as determined by high performance liquid chromatography and mass spectrometric analysis. The results are explained on the basis of the presence of number of primary hydroxyl and amino groups in tris and hepes molecules that could affect the binding of alcohol molecules to protein. This observation will be useful in selecting buffer system for biochemical studies on PKC.

Novel Ouabain Receptors in Bovine Adrenal Cortex: Evidence for Ouabain Transport

Novel high affinity binding sites for ouabain (NOR) are present in bovine adrenal cortex (Ward SC, Hypertension 2002; 39[pt. 2]:536). To distinguish NOR from classical sodium‐potassium pumps (Na pumps), we used fresh adrenal cortical membranes. Scatchard analysis showed that K+ (20mM) reduced the apparent Kd for ouabain binding to Na pumps from 4.6 ± 0.2 to 240 ± 7.2 nM with no change in Bmax (10.1 ± 0.1 vs. 10.3 ± 0.2 pmoles/mg). K+ increased the Kd for ouabain binding to NOR and increased the Bmax (0.15 ± 0.02 vs. 0.26 ± 0.06 pmoles/mg). Moreover, the effect of Na+ was more dramatic; Na+ inhibited ouabain binding to Na pumps with no change in Bmax, while Na+ increased the apparent Kd for ouabain binding to NOR and increased Bmax > 25‐fold. Ouabain binding to the Na pump in stored membranes remained constant for up to 50 days whereas NOR related binding declined with a t1/2 of 6.8 days. The association of ouabain with membranes was 0.538 pmoles/mg when [Na+]o » [Na+]i, and was 0.152 pmoles/mg when [Na+]o « [Na+]i, and was sensitive to permeable anions. NOR binding was sensitive to osmolarity. Photoactive analogs of ouabain labeled a 35 kDa polypeptide. We conclude that NOR mediates the secondary active sodium‐dependent transport of 3H‐ouabain into sealed membrane vesicles and that this process is electrogenic. NOR may mediate the secretion of cardiac glycosides by the adrenal cortex in vivo. Supported by HL078870 and HL075584.

Synthesis of Photoactive Analogues of a Cystine Knot Trypsin Inhibitor Protein

We describe the preparation of a recently described photoactive amino acid analogue (photoMethionine) by two novel synthetic routes, one of which is flexible and enantiospecific, and the site-specific chemical incorporation of photoMethionine into a defined and functionally active protein using a combination of solid-phase peptide synthesis and modern chemical ligation methodology. Site-specific labeling of proteins with this amino acid analogue through chemical synthesis provides valuable probes for photoaffinity cross-linking studies.

Photoactive Analogues of the Haloether Anesthetics Provide High-Resolution Features from Low-Affinity Interactions

The difficulty in obtaining binding target and site information for low-affinity drugs, like the inhaled anesthetics, has limited identification of their molecular effectors. Because such information can be provided by photoactive analogues, we designed, synthesized, and characterized a novel diazirnyl haloether that closely mimics isoflurane, the most widely used clinical general anesthetic. This compound, H-diaziflurane, is a nontoxic, potent anesthetic that potentiates GABA-gated ion channels in primary cultures of hippocampal neurons. Calorimetric and structural characterizations show that H-diaziflurane binds a model anesthetic host protein with similar energetics as isoflurane and forms photoadducts with residues lining the isoflurane binding site. H-diaziflurane will be immediately useful for identifying targets and sites important for the molecular pharmacology of the inhaled haloether anesthetics.

A photochemical snapshot of CD22 binding

Identification of endogenous glycan-binding ligands for cell-surface receptors has been difficult. Incorporation of a photoactive sialic acid analog into B-cell surface glycoproteins suggests that CD22 molecules may cluster by binding carbohydrate antigens on neighboring CD22 molecules.

GSH Inhibits Trypsinization of the C-terminal Half of Human MRP1

MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance to tumor cells. The accumulated evidence has proved that GSH interacts with MRP1 and stimulates drug transport. However, the mechanism of GSH-dependent drug transport by MRP1 remains unclear. In this study, we used limited tryptic digestion of MRP1 in isolated membrane vesicles, in the presence and absence of GSH, to investigate the influence of GSH on MRP1 conformation. We found that GSH inhibited the generation of an approximately 35-kDa C-terminal tryptic fragment (including a C-terminal His tag) termed C2 from MRP1. This effect of GSH was not because of direct inhibition of trypsin activity, and agosterol A enhanced the inhibitory effect of GSH. The main cleavage site in MRP1 for the generation of the C2 fragment by trypsin resided between TMD2 and NBD2 of MRP1. Limited tryptic digestion of membrane vesicles expressing various truncated and co-expressed MRP1 fragments in the presence and absence of GSH revealed that GSH inhibited the production of the C2 fragment only in the presence of the L(0) region of MRP1. Thus the L(0) region is required for the inhibition of trypsinization of the C-terminal half of MRP1 by GSH. These findings, together with previous reports, suggest that GSH induces a conformational change at a site within the MRP1 that is indispensable for the interaction of MRP1 with its substrates.

Synthesis of high specific activity 35S‐labelled N‐methanesulfonyl farnesylcysteine and a photoactive analog

AbstractPrenylated cysteine analogs, which mimic the prenylated cysteine residue of prenylated GTP‐binding proteins (G‐proteins), have been used in a variety of contexts for the study of prenylated G‐protein behavior. In earlier work in this area, we prepared the photoactive analog [35S]4 and showed that it labelled RhoGDI upon photolysis; those results were consistent with the idea that GDI contains an isoprenoid binding site. Here, we describe the preparation of [35S]N‐methanesulfonyl labelled analogs (1a and 2a) of N‐acetyl farnesylcysteine and its methyl ester together with an improved synthetic procedure for photoactive analogs 3 and 4; specific activities of ∼1100 Ci/mmol were achieved. Compounds 1a and 2a in unlabelled form were used as competitors in photolysis reactions to show that the methanesulfonamido group is a reasonable acetamide substitution. Additional experiments show that the photoactive ester [35S]3 can cross‐link GDI in both purified form and crude bacterial extract. However, the extent of cross‐linking obtained with the ester ([35S]3) is significantly less than that observed with the free acid ([35S]4) despite the fact that the esterified form probably more closely reflects the structure of the C‐terminus of a prenylated protein; using the GDI·Cdc42 co‐crystal structure, the structural basis for these results is discussed. Copyright © 2002 John Wiley & Sons, Ltd.

3D1015 ハロゲン置換アナログ発色団を持つPhotoactive Yellow Proteinの分光学的解析(18.光生物(A)視覚,一般講演,日本生物物理学会第40回年会)

3D1015 ハロゲン置換アナログ発色団を持つPhotoactive Yellow Proteinの分光学的解析(18.光生物(A)視覚,一般講演,日本生物物理学会第40回年会)