Koichi Nishigaki

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Organization: Saitama Univer , Japan

Department: Department of Functional Materials Science

Title: Professor(PhD)

Chemicals
References

Strong Inhibition of Beta-Amyloid Peptide Aggregation Realized by Two-Steps Evolved Peptides

Co-reporter:Sunita Ghimire Gautam;Masayuki Komatsu

Chemical Biology & Drug Design 2015 Volume 85( Issue 3) pp:356-368

Publication Date(Web):

DOI:10.1111/cbdd.12400

Several decades of cumulated research evidence have proven that aggregation of beta-amyloid 42 (Aβ42) is the main cause of neuronal death in the brains of patients with Alzheimer's disease. Therefore, inhibition of Aβ42 aggregation holds great promise for the prevention and treatment of Alzheimer's disease. To this end, we used a systematic in vitro evolution including a paired peptide library method. We identified two peptides with high binding affinity (with K_d in the nm range) for Aβ42. Functionally, these peptides strongly inhibited the aggregation of Aβ42 as shown by the thioflavin T assay and atomic force microscopy. Moreover, these peptides rescued PC12 cells from the cytotoxic effect of aggregated Aβ42 in vitro. Our results suggest that these novel peptides may be potential therapeutic seeds for the treatment of Alzheimer's disease.

A radioisotope-nondependent high-sensitivity method for measuring the activity of glioblastoma-related O6-methylguanine DNA methyltransferase

Co-reporter:Aya Hongo, Ran Gu, Miho Suzuki, Naoto Nemoto, Koichi Nishigaki

Analytical Biochemistry 2015 480() pp: 82-84

Publication Date(Web):1 July 2015

DOI:10.1016/j.ab.2014.08.012

O6-Methylguanine DNA methyltransferase (MGMT) cancels the anticancer effect of temozolomide (drug for glioblastoma), which introduces methylation to DNA. Therefore, developing an MGMT inhibitor is a promising strategy for the treatment of this cancer. For this purpose, a sensitive detection method that does not depend on the conventional radioisotope (RI) method was developed. This was realized by a fluorescence-based method that measured the amount of cleavable restriction sites demethylated by the action of MGMT; this method was enhanced by introducing a polymerase chain reaction (PCR) amplification step. As an assay of enzyme activity, 20-fold higher sensitivity (subnanomolar) was attained compared with our and others’ fluorescence-based approaches.

Selection-by-function: efficient enrichment of cathepsin E inhibitors from a DNA library

Co-reporter:Mohammed Naimuddin;Yasunori Kinoshita;Masato Ito;Kenji Yamamoto;Koichirou Kitamura;Yoko Honda-Takahashi;Marina Murakami;Kazunori Hanada;Yuzuru Husimi

Journal of Molecular Recognition 2007 Volume 20(Issue 1) pp:58-68

Publication Date(Web):18 DEC 2006

DOI:10.1002/jmr.812

A method for efficient enrichment of protease inhibitors out of a DNA library was developed by introducing SF-link technology. A two-step selection strategy was designed consisting of the initial enrichment of aptamers based on binding function while the second enrichment step was based on the inhibitory activity to a protease, cathepsin E (CE). The latter was constructed by covalently linking of a biotinylated peptide substrate to each of the ssDNA molecule contained in the preliminarily selected DNA library, generating ‘SF-link’. Gradual enrichment of inhibitory DNAs was attained in the course of selection. One molecule, SFR-6-3, showed an IC₅₀ of around 30 nM, a K_d of around 15 nM and high selectivity for CE. Sequence and structure analysis revealed a C-rich sequence without any guanine and possibly an i-motif structure, which must be novel to be found in in vitro-selected aptamers. SF-link technology, which is novel as the screening technology, provided a remarkable enrichment of specific protease inhibitors and has a potential to be further developed. Copyright © 2006 John Wiley & Sons, Ltd.

Development of Systemic in vitro Evolution and Its Application to Generation of Peptide-Aptamer-Based Inhibitors of Cathepsin E

Co-reporter:Koichiro Kitamura, Chuya Yoshida, Yasunori Kinoshita, Tomoko Kadowaki, ... Koichi Nishigaki

Journal of Molecular Biology (17 April 2009) Volume 387(Issue 5) pp:1186-1198

Publication Date(Web):17 April 2009

DOI:10.1016/j.jmb.2008.12.028

Proteases are involved in various biological functions. Thus, inhibition of their activities is scientifically interesting and medically important. However, there is no systematic method established to date to generate endopeptidase inhibitory peptides. Here, we report a general system to identify endopeptidase inhibitory peptides based on the use of in vitro evolution. Using this system, we generated peptides that inhibit cathepsin E (CE) specifically at a submicromolar IC50. This system generates protease inhibitor peptides utilizing techniques of cDNA display, selection-by-function, Y-ligation-based block shuffling, and others. We further demonstrated the importance and effectiveness of a secondary library for obtaining small-sized and active peptides. CE inhibitory peptides generated by this method were characterized by a small size (8 to 12 aa) and quite different sequences, suggesting that they bind to different sites on CE. Typical CE inhibitory peptide aptamers obtained here (Pi101; SCGG IIII SCIA) have half an inhibition activity (Ki; 5 nM) of pepstatin A (potent CE inhibitor) without inhibiting cathepsin D (structurally similar to CE). The general applicability of this system suggests that it may be useful to identify inhibitory peptides for various kinds of proteases and that it may therefore contribute to protein science and drug discovery. The peptide binding to a protein is discussed in comparison with the antibody binding to an antigen.