Yutaka Sadakane

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.

Isolation of acein-2, a novel angiotensin-I-converting enzyme inhibitory peptide derived from a tryptic hydrolysate of human plasma.

We previously described a novel angiotensin‐I‐converting enzyme (ACE) inhibitory peptide, designated Acein‐1, that was isolated from a tryptic hydrolysate of human plasma. We now report a second such inhibitory peptide, Acein‐2 obtained from the same hydrolysate. The peptide was purified by gel filtration and cation exchange chromatography followed by reversed‐phase gradient and isocratic high performance liquid chromatography. Acein‐2 was found to be a tripeptide, Leu‐Ile‐Tyr, which is thought to correspond to f(518–520) of human α2‐macroglobulin. The synthetic tripeptide showed a potent dose‐dependent inhibition of ACE, with an IC50 value of 0.82 μmol/l. Lineweaver–Burk plots suggested that Acein‐2 as well as the previously described Acein‐1 are non‐competitive inhibitors.

Membrane Incorporation, Channel Formation, and Disruption of Calcium Homeostasis by Alzheimer's β-Amyloid Protein.

Oligomerization, conformational changes, and the consequent neurodegeneration of Alzheimers β-amyloid protein (AβP) play crucial roles in the pathogenesis of Alzheimers disease (AD). Mounting evidence suggests that oligomeric AβPs cause the disruption of calcium homeostasis, eventually leading to neuronal death. We have demonstrated that oligomeric AβPs directly incorporate into neuronal membranes, form cation-sensitive ion channels (“amyloid channels”), and cause the disruption of calcium homeostasis via the amyloid channels. Other disease-related amyloidogenic proteins, such as prion protein in prion diseases or α-synuclein in dementia with Lewy bodies, exhibit similarities in the incorporation into membranes and the formation of calcium-permeable channels. Here, based on our experimental results and those of numerous other studies, we review the current understanding of the direct binding of AβP into membrane surfaces and the formation of calcium-permeable channels. The implication of composition of membrane lipids and the possible development of new drugs by influencing membrane properties and attenuating amyloid channels for the treatment and prevention of AD is also discussed.

Calcium dyshomeostasis and neurotoxicity of Alzheimer’s β-amyloid protein

Neurotoxicity of Alzheimer’s β-amyloid protein (AβP) is central to the pathogenesis of Alzheimer’s disease (AD). Recent approaches have emphasized the importance of AβP oligomerization, which causes synaptic degeneration and neuronal loss, finally leading to the pathogenesis of AD. Although the precise molecular mechanism of AβP neurotoxicity remains elusive, our and other numerous findings have demonstrated that AβP directly incorporated into neuronal membranes formed calcium-permeable ion channels (amyloid channels) and resulted in an abnormal elevation of the intracellular calcium levels. The formation of amyloid channels and the abnormal increase of intracellular Ca2+ have also been commonly observed in other neurodegenerative diseases, including conformational diseases such as prion disease or dementia with Lewy bodies. This article reviews the current understanding of the pathology of AD based on the hypothesis that the disruption of calcium homeostasis through amyloid channels may be the molecular basis of AβP neurotoxicity. The potential development of preventive agents is also discussed.

Acein-1, a novel angiotensin-I-converting enzyme inhibitory peptide isolated from tryptic hydrolysate of human plasma

A novel angiotensin‐I‐converting enzyme (ACE) inhibitory peptide, designated acein‐1, was isolated from the tryptic hydrolysate of human plasma. Gel filtration and cation exchange chromatography were performed to purify this peptide, followed by reversed‐phase gradient and isocratic high‐performance liquid chromatography. Acein‐1 was found to be a heptapeptide, Tyr‐Leu‐Tyr‐Glu‐Ile‐Ala‐Arg, corresponding to f(138–144) of human serum albumin. The synthetic heptapeptide, hexapeptide (Tyr‐Leu‐Tyr‐Glu‐Ile‐Ala, des‐7R acein‐1) and octapeptide (Tyr‐Leu‐Tyr‐Glu‐Ile‐Ala‐Arg‐Arg, acein‐1R) showed dose‐dependent inhibitions of ACE, and their IC50 values were 16 μmol/l, 500 μmol/l and 86 μmol/l, respectively. Acein‐1 might be a non‐competitive inhibitor, while acein‐1R may be an uncompetitive inhibitor, as shown by Lineweaver‐Burk plots.

Age-related changes of alpha-crystallin aggregate in human lens

Summary.Lens alpha-crystallin, composed of two subunits alpha A- and alpha B-crystallin, forms large aggregates in the lens of the eye. The present study investigated the aggregate of human lens alpha-crystallin from elderly and young donors. Recombinant alpha A- and alpha B-crystallins in molar ratios of alpha A to alpha B at 1:1, corresponding to the aged sample, were also studied in detail. We found by ultra-centrifugation analysis that the alpha-crystallin aggregate from elderly donors was large and heterogeneous with an average sedimentation coefficient of 30 S and a range of 20–60 S at 37 °C. This was higher compared to the young samples that had an average sedimentation coefficient of 17 S. The sedimentation coefficients of recombinant alpha A- and alpha B-crystallins were approximately 12 S and 15 S, respectively. Even when recombinant alpha-crystallins were mixed in molar ratios equivalent to those found in vivo, similar S values as the native aged alpha-crystallin aggregates were not obtained.Changes in the self-association of alpha-crystallin aggregate were correlated to changes in chaperone activity. Alpha-crystallin from young donors, and recombinant alpha A- and alpha B-crystallin and their mixtures showed chaperone activity, which was markedly lost in samples from the aged alpha-crystallin aggregates.

Synthesis of Diazirinyl Photoprobe Carrying a Novel Cleavable Biotin

Photoaffinity labeling is a powerful tool for the identification of receptor proteins and their binding sites. The major drawback of this method, however, is the reliability of the labeling and the purity of labeled peptides after purification from a large number of unlabeled fragments. The recent development of diazirine-based biotinylated probes provides an efficient solution to this problem. In these probes, the diazirine photophore contributes to the formation of a stable cross-link that greatly improves the efficiency. The application of an N-acetylglucosamine photoprobe carrying biotinylated diazirine provided the first information regarding acceptor-site peptides of b-1,4-galactosyltransferase. We have also developed a novel method for the one-step introduction of a biotinylated diazirine photophore into unprotected carbohydrate ligands. Since biotin–avidin binding is essentially irreversible (Kd = 10 15 m), several approaches have been investigated to achieve efficient recovery of biotinylated products from an immobilized avidin matrix. Although the use of monomeric avidin contributes to the isolation of biotinylated products because of the lower affinity to biotin (Kd = 10 8 m), it requires a high

Quantification of the isomerization of Asp residue in recombinant human αA-crystallin by reversed-phase HPLC

A method for determining the isomerization of Asp residues in proteins is described and demonstrated by quantifying the isomerization of Asp(151) in recombinant human alphaA-crystallin. First, four types of dodecapeptide fragment ((146)IQTGLD(151)ATHAER(157)) in which the Asp residue was either L-Asp, D-Asp, L-isoAsp or D-isoAsp were synthesized, and RP-HPLC conditions were established for their separation. Next, the Asp(151)-containing peptide fragments isolated from the tryptic hydrolysate of recombinant alphaA-crystallin were analyzed under these conditions. New peaks, the retention times of which were the same as those of peptides containing D-Asp, L-isoAsp and D-isoAsp, were generated when alphaA-crystallin was incubated for 140 days at 37 degrees C. An amino acid composition, amino acid sequence, and enantiomeric analysis revealed that two peaks with retention times identical to those of peptides containing L-isoAsp and D-isoAsp represented dodecapeptide fragments containing L-isoAsp(151) and D-isoAsp(151), respectively. RP-HPLC analysis under other condition suggested that the peak with retention time identical to that of peptide containing D-Asp represented dodecapeptide fragments containing D-Asp(151). The present method does not require acid hydrolysis, which generates further isomerization products as artifacts, and thus make possible the sensitive quantification of each type of Asp isomer individually at a specific site in a protein. In our analysis of the Asp(151) residue in human alphaA-crystallin, the degree of isomerization from L-Asp to D-Asp can be determined to a level as low as 0.3%.

Protein domain of chicken α1-acid glycoprotein is responsible for chiral recognition

Abstract Ovoglycoprotein from chicken egg whites (OGCHI) has been used as a chiral selector to separate drug enantiomers. However, neither the amino acid sequence of OGCHI nor the responsible part for the chiral recognition (protein domain or sugar moiety) has yet to be determined. First, we isolated a cDNA clone encoding OGCHI, and clarified the amino acid sequence of OGCHI, which consists of 203 amino acids including a predictable signal peptide of 20 amino acids. The mature OGCHI shows 31–32% identities to rabbit and human α1-acid glycoproteins (α1-AGPs). Thus, OGCHI should be the chicken α1-AGP. Second, the recombinant chicken α1-AGP was prepared by the Escherichia coli expression system, and its chiral recognition ability was confirmed by capillary electrophoresis. Since proteins expressed in E. coli are not modified by any sugar moieties, this result shows that the protein domain of the chicken α1-AGP is responsible for the chiral recognition.

Synthesis of tag introducible (3-trifluoromethyl)phenyldiazirine based photoreactive phenylalanine.

An efficient synthesis of tag introducible (3-trifluoromethyl)phenyldiazirine based phenylalanine derivatives is described. Alkylation of a chiral glycine equivalent with a spacer containing (3-trifluoromethyl)phenyldiazirinyl bromides enables us to create photoreactive L-phenylalanine derivatives. After introduction of biotin at the spacer, the biotinylated and photoreactive amino acid was applied for L-amino acid oxidase and incorporated into a substrate binding site. These compounds will be powerful tools not only for photoaffinity labeling to elucidate properties of bioactive peptides but also as trifunctional photophors to introduce a ligand skeleton.