Kazuki Saito

Novel non-peptidic and small-sized BACE1 inhibitors

Recently, we reported substrate-based beta-secretase (BACE1) inhibitors with a hydroxymethylcarbonyl (HMC) isostere as a substrate transition-state mimic. These inhibitors showed potent BACE1 inhibitory activities (approximately 1.2 nM IC(50)). In order to improve in vivo enzymatic stability and permeability across the blood-brain barrier, these penta-peptidic inhibitors would need to be further optimized. On the other hand, non-peptidic inhibitors possessing isophthalic residue at the P(2) position were reported from other research groups. We selected isophthalic-type aromatic residues at the P(2) position and an HMC isostere at the P(1) position as lead compounds. On the basis of the design approach focused on the conformer of docked inhibitor in BACE1, we found novel non-peptidic and small-sized BACE1 inhibitors possessing a 2,6-pyridinedicarboxylic, chelidamic or chelidonic residue at the P(2) position.

BACE1 inhibitors: optimization by replacing the P1' residue with non-acidic moiety.

Recently, we reported potent BACE1 inhibitors KMI-429, -684, and -574 possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. These inhibitors showed potent inhibitory activities in enzymatic and cell assays, especially, KMI-429 was confirmed to significantly inhibit Abeta production in vivo. However, acidic moieties at the P(4) and P(1) positions of KMI-compounds were thought to be unfavorable for membrane permeability across the blood-brain barrier. Herein, we replaced acidic moieties at the P(4) position with other hydrogen bond acceptor groups, and these inhibitors exhibited improved BACE1 inhibitory activities in cultured cells. In this study, we replaced the acidic moieties at the P(1) position with non-acidic and low molecular sized moieties.

Intact-cell-based surface plasmon resonance measurements for ligand affinity evaluation of a membrane receptor

Toward future applications to the discovery of drugs against membrane receptors on pathological cells, an intact-cell-based surface plasmon resonance (SPR) methodology has been developed. The injection of a suspension of epidermal carcinoma A431 cells (5×10(7)cells/ml), as an analyte, generated clear SPR responses to epidermal growth factor (EGF) immobilized on the sensor chip. Because the responses were competitively reduced by the free ligand EGF, added to the analyte cell suspension, they certainly reflect the specific interaction of the immobilized EGF with the extracellular region of its receptor, which is highly expressed on the surface of the A431 cells.

Inhibitory effect of a dimerization-arm-mimetic peptide on EGF receptor activation

A cyclic decapeptide was chemically synthesized that mimics the loop structure of a beta-hairpin arm of the EGF receptor, which is highly involved in receptor dimerization upon activation by ligand binding. This peptide was revealed to reduce dimer formation of the receptor in a detergent-solubilized extract of epidermoid carcinoma A431 cells and to inhibit receptor autophosphorylation at less than 10 microM in the intact cells.

Verification of protein disulfide bond arrangement by in-gel tryptic digestion under entirely neutral pH conditions

To develop a concise proteomic procedure to verify the protein disulfide bond arrangement, non‐reductive trypsin digestion of neuregulin 1‐β1 (176–246), a model disulfide‐containing protein, was assessed by a proteolytic 18O‐labeling analysis. As a result, the commonly used in‐gel tryptic digestion method has been improved for use entirely under neutral pH conditions. With this procedure, the disulfide arrangement of proteins could represent a clinical index candidate in pathological proteomic studies.

Concise machinery for monitoring ubiquitination activities using novel artificial RING fingers: Concise Machinery Using Artificial RING Fingers

Protein ubiquitination is involved in many cellular processes, such as protein degradation, DNA repair, and signal transduction pathways. Ubiquitin‐conjugating (E2) enzymes of the ubiquitination pathway are associated with various cancers, such as leukemia, lung cancer, and gastric cancer. However, to date, detection of E2 activities is not practicable for capturing the pathological conditions of cancers due to complications related to the enzymatic cascade reaction. To overcome this hurdle, we have recently investigated a novel strategy for measuring E2 activities. Artificial RING fingers (ARFs) were developed to conveniently detect E2 activities during the ubiquitination reaction. ARFs were created by grafting the active sites of ubiquitin‐ligating (E3) enzymes onto amino acid sequences with 38 residues. The grafting design downsized E3s to small molecules (ARFs). Such an ARF is a multifunctional molecule that possesses specific E2‐binding capabilities and ubiquitinates itself without a substrate. In this review, we discuss the major findings from recent investigations on a new molecular design for ARFs and their simplified detection system for E2 activities. The use of the ARF allowed us to monitor E2 activities using acute promyelocytic leukemia (APL)‐derived cells following treatment with the anticancer drug bortezomib. The molecular design of ARFs is extremely simple and convenient, and thus, may be a powerful tool for protein engineering. The ARF methodology may reveal a new screening method of E2s that will contribute to diagnostic techniques for cancers.

Solution structure of the PHD finger from the human KIAA1045 protein: PHD Structure of the KIAA1045 Protein

Cross‐brace structural motifs are required as a scaffold to design artificial RING fingers (ARFs) that function as ubiquitin ligase (E3) in ubiquitination and have specific ubiquitin‐conjugating enzyme (E2)‐binding capabilities. The Simple Modular Architecture Research Tool database predicted the amino acid sequence 131–190 (KIAA1045ZF) of the human KIAA1045 protein as an unidentified structural region. Herein, the stoichiometry of zinc ions estimated spectrophotometrically by the metallochromic indicator revealed that the KIAA1045ZF motif binds to two zinc atoms. The structure of the KIAA1045ZF motif bound to the zinc atoms was elucidated at the atomic level by nuclear magnetic resonance. The actual structure of the KIAA1045ZF motif adopts a C4HC3‐type PHD fold belonging to the cross‐brace structural family. Therefore, the utilization of the KIAA1045ZF motif as a scaffold may lead to the creation of a novel ARF.

Unique auto-ubiquitination activities of artificial RING fingers in cancer cells: Auto-Ubiquitination of Artificial RING Fingers in Cancer Cells

Ubiquitin‐conjugating (E2) enzymes in protein ubiquitination are associated with various diseases. An artificial RING finger (ARF) is a useful tool, and E2 activities are conveniently estimated based on ARF reactivities. To extend the use of ARF in cells, we constructed a TAT‐ARF using a cell‐penetrating trans‐activator protein (TAT) peptide. An in vitro ubiquitination assay without substrates showed auto‐ubiquitination of TAT‐ARF via its TAT region. TAT‐ARF was translocated into MCF7 breast cancer cells, and then TAT‐ARF ubiquitinated itself via its ARF. Experiments using confocal laser‐scanning microscopy revealed that FAM‐labeled TAT‐ARF was readily internalized in cells and it remained encapsulated in vesicles. The Cell Counting Kit‐8 assay indicated that the TAT‐ARF uptake occurred without cytotoxicity in MCF7 cells at concentrations below 5.0 μM. By taking advantage of TAT‐ARF, we, for the first time, succeeded in detecting E2 activities in cells. Thus, the present work opens up new avenues in the investigation of protein ubiquitination.

Unique RING finger structure from the human HRD1 protein: Unique RING finger from human HRD1

Artificial RING fingers (ARFs) are created by transplanting active sites of RING fingers onto cross‐brace structures. Human hydroxymethylglutaryl‐coenzyme A reductase degradation protein 1 (HRD1) is involved in the degradation of the endoplasmic reticulum (ER) proteins. HRD1 possesses the RING finger domain (HRD1_RING) that functions as a ubiquitin‐ligating (E3) enzyme. Herein, we determined the solution structure of HRD1_RING using nuclear magnetic resonance (NMR). Moreover, using a metallochromic indicator, we determined the stoichiometry of zinc ions spectrophotometrically and found that HRD1_RING binds to two zinc atoms. The Simple Modular Architecture Research Tool database predicted the structure of HRD1_RING as a typical RING finger. However, it was found that the actual structure of HRD1_RING adopts an atypical RING‐H2 type RING fold. This structural analysis unveiled the position and range of the active site of HRD1_RING that contribute to its specific ubiquitin‐conjugating enzyme (E2)‐binding capability.

Quantitative evaluation of refolding conditions for a disulfide‐bond‐containing protein using a concise 18O‐labeling technique

A concise method was developed for quantifying native disulfide‐bond formation in proteins using isotopically labeled internal standards, which were easily prepared with proteolytic 18O‐labeling. As the method has much higher throughput to estimate the amounts of fragments possessing native disulfide arrangements by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) than the conventional high performance liquid chromatography (HPLC) analyses, it allows many different experimental conditions to be assessed in a short time. The method was applied to refolding experiments of a recombinant neuregulin 1‐β1 EGF‐like motif (NRG1‐β1), and the optimum conditions for preparing native NRG1‐β1 were obtained by quantitative comparisons. Protein disulfide isomerase (PDI) was most effective at the reduced/oxidized glutathione ratio of 2:1 for refolding the denatured sample NRG1‐β1 with the native disulfide bonds.