Synthesis of a fluorescent photoaffinity probe of OSW-1 by site-selective acylation of an inactive congener and biological evaluation.

A novel fluorescent photoaffinity probe of OSW-1 was prepared in two steps from a naturally occurring inactive congener by a sequential site-selective acylation strategy using Me2SnCl2. It displayed highly potent anticancer activity and a similar intracellular localization property to that of a fluorescently-tagged OSW-1, thereby demonstrating its potential utility in live cell studies.

Photoaffinity labeling of the anionic sites in Caco-2 cells mediating saturable transport of hydrophilic cations ranitidine and famotidine.

The H(2) antagonists, ranitidine and famotidine, exhibit saturable absorptive transport across Caco-2 cell monolayers and human intestine via a yet unidentified mechanism. A photoreactive derivative of famotidine has been synthesized and evaluated as a photoaffinity probe for the putative transporter protein(s). The probe irreversibly inhibited ranitidine transport across Caco-2 cell monolayers and irreversibly increased the transepithelial electrical resistance (TEER) after UV activation. Photoaffinity labeling was protected by a molar excess of famotidine.

Design, synthesis, and evaluation of 5'-S-aminoethyl-N(6)- azidobenzyl-5'-thioadenosine biotin conjugate: a bifunctional photoaffinity probe for the es nucleoside transporter.

A bifunctional biotinylated photoaffinity label for the nitrobenzylmercaptopurine riboside (NBMPR)-sensitive (es) nucleoside transporter (ENT1) has been synthesized and evaluated. This new probe,5'-S-aminoethyladenosine-N(6)-azidobenzyl-5'-thioadenosine biotin conjugate (SAEATA-14-biotin), exhibited high-affinity binding to the es transporter in K562 cells as determined by flow cytometry, with a K(i) of 2.69 nM in competition against 5-(SAENTA)-x8-fluorescein. It also exhibited covalent linking to the es transporter in BeWo cell membranes upon UV irradiation. This new bifunctional probe is a potential tool for determining the amino acid residues involved in ligand binding at the NBMPR-binding site of the ENT1 nucleoside transporter, as well as for the purification of the transporter.

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.

Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain of mouse P-glycoprotein: structure-activity relationships for flavonoid binding.

Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain (NBD2) of mouse P-glycoprotein were investigated by using two recombinantly expressed soluble proteins of different lengths and photoactive ATP analogues, 8-azidoadenosine triphosphate (8N(3)-ATP) and 2',3',4'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP). The two proteins, Thr(1044)-Thr(1224) (NBD2(short)) and Lys(1025)-Ser(1276) (NBD2(long)), both incorporated the four consensus sequences of ABC (ATP-binding cassette) transporters, Walker A and B motifs, the Q-loop, and the ABC signature, while differing in N-terminal and C-terminal extensions. Radioactive photolabeling of both proteins was characterized by hyperbolic dependence on nucleotide concentration and high-affinity binding with K(0.5)(8N(3)-ATP) = 36-37 microM and K(0.5)(TNP-8N(3)-ATP) = 0.8-2.6 microM and was maximal at acidic pH. Photolabeling was strongly inhibited by TNP-ATP (K(D) = 0.1-5 microM) and ATP (K(D) = 0.5-2.7 mM). Since flavonoids display bifunctional interactions at the ATP-binding site and a vicinal steroid-interacting hydrophobic sequence [Conseil, G., Baubichon-Cortay, H., Dayan, G., Jault, J.-M., Barron, D., and Di Pietro, A. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9831-9836], a series of 30 flavonoids from different classes were investigated for structure-activity relationships toward binding to the ATP site, monitored by protection against photolabeling. The 3-OH and aromaticity of conjugated rings A and C appeared important, whereas opening of ring C abolished the binding in all but one case. It can be concluded that the benzopyrone portion of the flavonoids binds at the adenyl site and the phenyl ring B at the ribosyl site. The Walker A and B motifs, intervening sequences, and small segments on both sides are sufficient to constitute the ATP site.

Evidence for a central apolipoprotein A-I domain loosely bound to lipids in discoidal lipoproteins that is capable of penetrating the bilayer of phospholipid vesicles.

Previous evidence indicated that discoidal reconstituted high density lipoproteins (rHDL) of apolipoprotein A-I (apoA-I) can interact with lipid membranes (Tricerri, M. A., Córsico, B., Toledo, J. D., Garda, H. A., and Brenner, R. R. (1998) Biochim. Biophys. Acta 1391, 67-78). With the aim of studying this interaction, photoactivable reagents and protein cleavage with CNBr and hydroxylamine were used. The generic hydrophobic reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine gave information on the apoA-I regions in contact with the lipid phase in the rHDL discs. Two protein regions loosely bound to lipids were detected: a C-terminal domain and a central one located between residues 87 and 112. They consist of class Y amphipathic alpha-helices that have a different distribution of the charged residues in their polar faces by comparison with class A helices, which predominate in the rest of the apoA-I molecule. The phospholipid analog 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoro-methyl-3-H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, which does not undergo significant exchange between membranes and lipoproteins, was used to identify the apoA-I domain directly involved in the interaction of rHDL discs with membranes. By incubating either rHDL or lipid-free apoA-I with lipid vesicles containing 125I-TID-PC, only the 87-112 apoA-I segment becomes labeled after photoactivation. These results indicate that the central domain formed by two type Y helices swings away from lipid contact in the discoidal lipoproteins and is able to insert into membrane bilayers, a process that may be of great importance for the mechanism of cholesterol exchange between high density lipoproteins and cell membranes.

Selective inhibition of MDR1 P-glycoprotein-mediated transport by the acridone carboxamide derivative GG918.

The acridone carboxamide derivative GG918 (N-{4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]-pheny l}-9,10dihydro-5-methoxy-9-oxo-4-acridine carboxamide) is a potent inhibitor of MDR1 P-glycoprotein-mediated multidrug resistance. Direct measurements of ATP-dependent MDR1 P-glycoprotein-mediated transport in plasma membrane vesicles from human and rat hepatocyte canalicular membranes indicated 50% inhibition at GG918 concentrations between 8 nM and 80 nM using N-pentyl-[3H]quinidinium, ['4C]doxorubicin and [3H]daunorubicin as substrates. The inhibition constant K for GG918 was 35 nM in rat hepatocyte canalicular membrane vesicles with [3H]daunorubicin as the substrate. Photoaffinity labelling of canalicular and recombinant rat Mdr1b P-glycoprotein by [3H]azidopine was suppressed by 10 muM and 40 muM GG918. The high selectivity of GG918-induced inhibition was demonstrated in canalicular membrane vesicles and by analysis of the hepatobiliary elimination in rats using [3H]daunorubicin, [3H]taurocholate and [3H]cysteinyl leukotrienes as substrates for three distinct ATP-dependent export pumps. Almost complete inhibition of [3H]daunorubicin transport was observed at GG918 concentrations that did not affect the other hepatocyte canalicular export pumps. The high potency and selectivity of GG918 for the inhibition of human MDR1 and rat Mdr1b P-glycoprotein may serve to interfere with this type of multidrug resistance and provides a tool for studies on the function of these ATP-dependent transport proteins.

Affinity labelling of the tick-borne encephalitis virus RNA replicase proteins by 4-N-exo-base-substituted photoreactive CTP analogs.

4-N-exo-base-substituted photoreactive analogs of CTP were designed and synthesized. Two flavivirus proteins NS5 and NS3 are shown to be labelled after RNA synthesis in the presence of the analogs, irradiation by UV-light (313 nm) and subsequent [alpha-32P]NTP incorporation.