Yasumaru Hatanaka

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.

A novel biotinylated heterobifunctional cross-linking reagent bearing an aromatic diazirine.

The synthesis of a p-[(3-trifluoromethyl)diazirine-3-yl]benzoic acid derivative is described as a new carbene generating heterobifunctional cross-linking reagent. The cross-linker carries a biotin moiety in order to make use of avidin-biotin technology for specific manipulation of cross-linked components. To evaluate the ability of this reagent, the inter-subunit cross-linking of egg-white avidin tetramer was investigated. As a typical application of avidin-biotin technology for cross-linking experiments, a chemiluminescent detection method was examined to identify photobiotinylated components. A cross-linked dimeric product with an apparent molecular mass of 38 kDa was clearly visualized by the combined use of a horseradish peroxidase-streptavidin conjugate and a luminol-based chemiluminescent system.

CXCR4 Stimulates Macropinocytosis: Implications for Cellular Uptake of Arginine-Rich Cell-Penetrating Peptides and HIV

CXCR4 is a coreceptor of HIV-1 infection in host cells. Through a photocrosslinking study to identify receptors involved in internalization of oligoarginine cell-penetrating peptides (CPPs), we found that CXCR4 serves as a receptor that stimulates macropinocytic uptake of the arginine 12-mer peptide (R12) but not of the 8-mer. We also found that stimulating CXCR4 with its intrinsic ligands, stromal cell-derived factor 1α and HIV-1 envelope glycoprotein 120, induced macropinocytosis. R12 had activity to prevent viral infection for HIV-1(IIIB), a subtype of HIV-1 that uses CXCR4 as a coreceptor for entry into susceptible cells, whereas the addition of a macropinocytosis inhibitor, dimethylamiloride, resulted in enhancement of viral infection. The present study shows that CXCR4 triggers macropinocytosis, which may have implications for the cellular uptake of oligoarginine CPPs and internalization of HIV.

Synthesis of biotinylated bis(D-glucose) derivatives for glucose transporter photoaffinity labelling

New diazirine based bis-glucose derivatives for tagging glucose transporters have been synthesised. These included two biotinylated compounds linked either by an aminocaproate or by a cleavable dithiol link. These compounds have been derivatised via a key skeleton compound that can be easily used for introduction of additional tags. Studies on the erythrocyte glucose transporter (GLUT1) and the insulin-stimulated adipose cell transporter (GLUT4) have revealed the biotinylated photoreactive bis-glucose compounds are effective labelling reagents.

Characterization of cysteine residues of glutathione S-transferase P : Evidence for steric hindrance of substrate binding by a bulky adduct to cysteine 47

Glutathione S-transferase P (GST-P) lost the enzymatic activity by 7-fluoro-4-sulfamoyl-2, 1, 3-benzodiazole (ABD-F), a thiol-group chemical modifier, but did not by methylmethanethiol-sulfonate. Both ABD-F and methylmethanethiolsulfonate reacted with Cys47 and Cys101. These two cysteine residues were site-directedly mutated with serine residues. Only the Cys101Ser lost the enzymatic activity by the treatment of ABD-F. On carbon 13 NMR experiments, a NMR signal of S-[13C]CH3 adduct to Cys47 did not show any change by the addition of S-hexylglutathione. These facts revealed that Cys47 did not locate at the active site, and a bulky adduct to Cys47 hindered the binding of substrates to the active site.

Development of high-affinity ligands and photoaffinity labels for the D-fructose transporter GLUT5.

The GLUT5 transporter catalyses the specific uptake of D-fructose and can accept this hexose in its furanose and pyranose ring forms. The transporter does not accept fructose epimers and has very limited tolerance of bulky groups substituted at the 2-, 3-, 4- and 5-OH positions [Tatibouët, Yang, Morin and Holman (2000) Bioorg. Med. Chem. 8, 1825-1833]. To further explore whether bulky groups can be tolerated at the primary OH positions, a D-fructose analogue with an allylamine group substitution to replace the 1-OH group was synthesized and was found to be quite well tolerated ( K (i)=27.1 mM). However, this analogue occurs in multiple ring forms. By contrast, 2,5-anhydro-D-mannitol is a symmetrical molecule that occurs only in a furanose ring form in which C-1 and C-6 are equivalent. We have therefore synthesized new 2,5-anhydro-D-mannitol analogues (substituted at the equivalent of the 6-OH of D-fructose) and from studies in Chinese hamster ovary cells expressing GLUT5 cells report that (i) the allylamine derivative of 2,5-anhydro-D-mannitol is well tolerated ( K (i)=2.66 mM); (ii) introduction of a di-nitrophenyl-substituted secondary amine group enhances affinity ( K (i)=0.56 mM); (iii) introduction of amide-linked biotinylated photolabel moieties is possible without loss of affinity relative to 2,5-anhydro-D-mannitol but a small secondary amine spacer between the biotinylated photolabelling moiety and the fructofuranose ring increases affinity (fructose photolabel 2; K (i)=1.16 mM); (iv) introduction of a hydrophilic tartarate spacer between biotin and the diazirine photoreactive groups can be accomplished without reduction in affinity and (v) photoactivation of biotinylated fructose photolabels leads to specific biotin tagging of GLUT5. These data suggest that substitution of a secondary amine group (-NH) to replace the C-6 (or C-1) -OH of 2,5-anhydro-D-mannitol results in compounds of high affinity; the affinity is enhanced over 10-fold compared with D-fructose.

Syndecan-4 Is a Receptor for Clathrin-Mediated Endocytosis of Arginine-Rich Cell-Penetrating Peptides

Arginine-rich cell-penetrating peptides (CPPs) such as Tat and oligoarginine peptides have been widely used as carriers for intracellular delivery of bioactive molecules. Despite accumulating evidence for involvement of endocytosis in the cellular uptake of arginine-rich CPPs, the primary cell-surface receptors for these peptide carriers that would initiate endocytic processes leading to intracellular delivery of bioactive cargoes have remained poorly understood. Our previous attempt to identify membrane receptors for octa-arginine (R8) peptide, one of the representative arginine-rich CPPs, using the photo-cross-linking probe bearing a photoreactive diazirine was not successful due to considerable amounts of cellular proteins nonspecifically bound to the affinity beads. To address this issue, here we developed a photo-cross-linking probe in which a cleavable linker of a diazobenzene moiety was employed to allow selective elution of cross-linked proteins by reducing agent-mediated cleavage. We demonstrated that introduction of the diazobenzene moiety into the photoaffinity probe enables efficient purification of cross-linked proteins with significant reduction of nonspecific binding proteins, leading to successful identification of 17 membrane-associated proteins that would interact with R8 peptide. RNAi-mediated knockdown experiments in combination with the pharmacological inhibitors revealed that, among the proteins identified, syndecan-4, one of the heparan sulfate proteoglycans, is an endogenous membrane-associated receptor for the cellular uptake of R8 peptide via clathrin-mediated endocytosis. This syndecan-4-dependent pathway was also involved in the intracellular delivery of bioactive proteins mediated by R8 peptide. These results reveal that syndecan-4 is a primary cell-surface target for R8 peptide that allows intracellular delivery of bioactive cargo molecules via clathrin-mediated endocytosis.

Helisterculins A and B, Two New (7.5′,8.2′)-Neolignans, and Helisorin, the First (6.4′,7.5′,8.2′)-Neolignan, from the Indonesian Medicinal PlantHelicteres isora

During a chemical study of Indonesian medicinal plants, we examined the constituents of fruits of Helicteres isora L. (Sterculiaceae), one of the famous Jamu medicines. From a water extract of the fruits, we isolated three new neolignans, helisterculins A (1) and B (2) and helisorin (3), and elucidated their structures by spectral analyses. Helisterculins A (1) and B (2) are (7.5′,8.2′)-neolignans with a bicyclo[2.2.2]octene C-framework, while helisorin (3) is a (6.4′,7.5′,8.2′)-neolignan with a very rare 4,4a,9,9a-tetrahydro-3,9-methano-3H-fluorene C-framework. The natural product with the latter C-framework has no literature precedent. The neolignans 1 – 3 showed weak inhibitory activity against reverse transcriptase from avian myeloblastosis virus.

Photolabeled sites with a tetrodotoxin derivative in the domain III and IV of the electroplax sodium channel

Forty three percent of the labeled sites, at least, in the electroplax sodium channel with a photoactivable tetrodotoxin derivative were identified by probing protease-digested labeled fragments with several sequence-directed antibodies. They are located in the loop between segments S5 and S6 of domain IV, as well as the region containing transmembrane segment S6 and adjacent extracellular and cytoplasmic sequences in domain III. No photolabeled fragments were detected in the corresponding region of domain I. These results suggest that C-11 of tetrodotoxin where the photoreactive moiety is attached orients to the region between S5 and S6 in domain III and IV. Probable orientation of the tetrodotoxin molecule in sodium channels is considered by taking together with the recent report of the site-directed mutagenesis.

Development and Leading-Edge Application of Innovative Photoaffinity Labeling

Photoaffinity labeling has become increasingly important with the development of powerful specific probes that are synthesized by installing a photo-activatable functional group (photophore) on the framework of biological ligands. The present review summarizes the development of diazirine-based photoaffinity labeling by focusing on its application to the structural elucidation of ligand-accepting sites within proteins.