Keizo Miyata

Novel small molecule nonpeptide aminopeptidase n inhibitors with a cyclic imide skeleton.

A novel series of small molecule nonpeptide aminopeptidase N (APN) inhibitors with a N-phenylphthalimide or N-phenylhomophthalimide skeleton were prepared. Evaluation of their protease inhibitory activities revealed that (i) some N-phenylphthalimide analogs are potent APN inhibitors, but they are also inhibitors of another protease, dipeptidylpeptidase IV (DPP-IV), and (ii) some N-phenylhomophthalimide analogs, including 2-(2,6-diethylphenyl)-1,2,3,4-tetrahydroisoquinoline-1,3-dione (PIQ-22), are potent and specific inhibitors of APN without DPP-IV-inhibitory activity. The structure-activity relationship studies of N-phenylphthalimides and N-phenylhomophthalimides are reviewed. PIQ-22 showed potent tumor-cell invasion-inhibitory activity.

Efficient delivery of antibody into living cells using a novel HVJ envelope vector system.

Introduction of antibodies which retain their function into cells using a simple and easy method would be very useful for study of the intracellular events in living cells. In this study, we developed a new method for intracellular delivery of antibody. It includes a combination of a novel IgG-capturing protein and hemagglutinating virus of Japan envelope (HVJ-E), an inactivated Sendai virus particle which can deliver a variety of molecules into mammalian cells via membrane-fusing activity. The IgG-capturing protein, which was molecularly designed to have two functions, was prepared as a fusion protein (ZZ-NP) of ZZ-dimer derived from an immunoglobulin-binding domain of protein A and nucleocapsid protein (NP), a part of the structural protein of HVJ. ZZ-NP was efficiently incorporated into the HVJ-E particle by treatment with detergent, and enhanced the incorporation of IgG. Moreover, fluorescence immunostaining revealed that the incorporated antibody was very efficiently introduced into living cells while retaining its function, i.e. anti-NPC (nuclear pore complex) monoclonal antibody was selectively located around cell nuclei. These findings suggest that this method is useful for intracellular delivery of antibody and for analysis of biological function of sub-cellular molecules in living cells.

Human tumor necrosis factor-α mutant RGD-V29 (F4614) shows potent antitumor activity and reduced toxicity against human tumor xenografted nude mice

The antitumor effects of human tumor necrosis factor-alpha (TNF) mutant RGD-V29 (code no. F4614), that includes the cell adhesive sequence (4)Arg-(5)Gly-(6)Asp and (29)Arg-->Val substitution, were evaluated. The therapeutic index, a measure of the extent of the therapeutically-effective range, using three constitutive administrations of RGD-V29 in Meth A-bearing mice was 4.8, whereas that of recombinant human TNF (rhTNF) ((1)SSS(4)RTPSDK...(29)RR...(155)L) was 2.8, clearly indicating that the effective RGD-V29 dose-range was extended. Furthermore, RGD-V29 showed potent antitumor activity against human lung cancer Mqnu-1 xenografted nude mice without severe gastrointestinal and other organ toxicities, even when administered at the maximal tolerated dose (MTD). In contrast, rhTNF induced severe toxicity at the MTD. Direct cytotoxicity of RGD-V29 against Mqnu-1 cells was similar to that of rhTNF. In addition, a cytotoxicity assay using a tumor-derived endothelial-like cell (tEC)/normal endothelial cell (nEC) system used to study TNF antitumor effects on tumor-associated endothelial cells, suggested that RGD-V29 showed preferential cytotoxicity toward tumor-associated endothelial cells compared with rhTNF. Thus, RGD-V29 appears to be a low-toxicity mutant of rhTNF that shows preferential activity towards tumors, and therefore merits further investigation in pre-clinical and clinical studies.

Preferential Activity of Wild‐type and Mutant Tumor Necrosis Factor‐α against Tumor‐derived Endothelial‐like Cells

Tumor‐derived endothelial‐like cells (tEC) were prepared by culturing human umbilical vein endothelial cells (HUVEC) in the presence of HT1080 human fibrosarcoma‐conditioned medium. tEC showed higher permeability and less cell‐adhesion activity than normal HUVEC (nEC). Tumor necrosis factor‐α (TNF) is known to have tumor‐vasculature disrupting activity. tEC showed higher cytotoxicity to recombinant human TNF (rhTNF) than nEC, and was not observed using HUVEC cultured with WI38 human diploid cell‐conditioned medium as a medium‐control. These results demonstrate that tEC acquire physiological properties of tumor‐associated vasculature, and may be a useful model system for the study of the mechanisms of TNF antitumor action. The TNF‐mutant RGD‐V29 (code No. F4614), which has an inserted 4Arg‐Gly‐Asp sequence and an 29Arg→Val replacement, was found to induce greater preferential destruction of tEC compared to rhTNF. When the preferential activities were evaluated in terms of 30% cytotoxicity (IC30) ratio (nEC/tEC), the ratio was 460 for RGD‐V29 compared to 4.2 for rhTNF. RGD‐V29 also exhibited cell‐adhesive function and bound preferentially to the p55 TNF‐receptor. Both these properties of RGD‐V29 contributed to the tEC selective cytotoxicity, indicating that the RGD ligands and selective p55 receptor binding on the cells, although uncharacterized, are involved in tEC targeting. Therefore, the TNF mutant RGD‐V29 may show greater selectivity toward tumor vasculature than wild‐type TNF.

Hemagglutinating Virus of Japan Envelope Vectors as High-Performance Vehicles for Delivery of Small RNAs

Hemagglutinating virus of Japan envelope (HVJ-E) vectors are particulate forms of the Sendai virus, and are characterized by maintained cell membrane fusion activities and completely inactivated genomes. HVJ-E vectors can be safely used as a non-viral transfection tools for laboratory research without the need for special protocols or equipment. HVJ-E particles are loaded with molecules such as DNA, proteins, antisense oligonucleotides, or small RNAs, to form HVJ-E vectors that carry these molecules into target cells by virtue of their membrane fusion activity. Interference by small RNAs such as small interfering RNA (siRNA) and microRNA (miRNA) is now established as an important biological strategy for gene silencing, and is becoming an essential method for analyzing biological processes. Various delivery reagents are currently available globally; however, delivery to non-adherent immune cells, particularly primary immune cells, remains extremely difficult. The simple and effective delivery capabilities of HVJ-E vectors overcome the above obstacle. Here we describe examples and demonstrate the utility and potential applications of HVJ-E vectors as high-performance vehicles for delivery of small RNAs.

Efficient Delivery of Antibody Into Living Cells Using Hemagglutinating Virus of Japan (HVJ) Envelope

This unit describes a novel method for antibody delivery into living cells using HVJ (hemagglutinating virus of Japan) envelope, an inactivated Sendai virus particle. Curr. Protoc. Immunol. 89:2.16.1‐2.16.12.

Use of the Hemagglutinating Virus of Japan (HVJ) Envelope as a Versatile Delivery System for Nucleic Acids and Proteins to Leukocytes In Vitro

The hemagglutinating virus of Japan (HVJ; also called the Sendai virus) envelope has been developed as a safe and efficient non‐viral vector. Because replication and transcription of genomic RNA is inactivated by β‐propiolactone treatment or UV‐irradiation, the HVJ envelope is extremely safe. This unit describes the method of transfection of siRNA and protein with the HVJ envelope. Curr. Protoc. Immunol. 89:10.17D.1‐10.17D.9.