Identification of hepta-histidine as a candidate drug for Huntington's disease by in silico-in vitro- in vivo-integrated screens of chemical libraries.

Tomomi Imamura,Hidenori Homma,Kazunori Yamada,Kentaro Tomii,Teikichi Ikura,Ying Mao,Xigui Chen,Kazuhiko Tagawa,Juliana Bosso Taniguchi,Hitoshi Okazawa,Hideyuki Okano,Makoto Nakabayashi,Julia Kaye,Kyota Fujita,Nobutoshi Ito,Kazumi Motoki,Steven Finkbeiner,Takuya Tamura

doi:10.1038/srep33861

Abstract

We identified drug seeds for treating Huntington’s disease (HD) by combining in vitro single molecule fluorescence spectroscopy, in silico molecular docking simulations, and in vivo fly and mouse HD models to screen for inhibitors of abnormal interactions between mutant Htt and physiological Ku70, an essential DNA damage repair protein in neurons whose function is known to be impaired by mutant Htt. From 19,468 and 3,010,321 chemicals in actual and virtual libraries, fifty-six chemicals were selected from combined in vitro-in silico screens; six of these were further confirmed to have an in vivo effect on lifespan in a fly HD model, and two chemicals exerted an in vivo effect on the lifespan, body weight and motor function in a mouse HD model. Two oligopeptides, hepta-histidine (7H) and Angiotensin III, rescued the morphological abnormalities of primary neurons differentiated from iPS cells of human HD patients. For these selected drug seeds, we proposed a possible common structure. Unexpectedly, the selected chemicals enhanced rather than inhibited Htt aggregation, as indicated by dynamic light scattering analysis. Taken together, these integrated screens revealed a new pathway for the molecular targeted therapy of HD.

Highlights

We found that mutant Htt interacts with Ku70 and impairs its function in non-homologous end-joining (NHEJ), a type of DNA double-strand break repair (DDBR) that functions in non-dividing cells such as differentiated neurons[24]
For the first wet screening of our in-house chemical library (19,468 chemicals or peptides) at the TMDU Chemical Biology Screening Center, we employed single molecule fluorescence spectroscopy (MF20, Olympus) and examined the effect of chemicals or peptides from the library on the interaction between normal Ku70 protein (Ku70-HisTag expressed in E. coli by the pET28 vector) and mutant Htt protein (GST-HttExon1-110Q)
Prior to the start of the screening, we tested the efficiency of two methods, fluorescence correlation spectroscopy (FCS) and fluorescence intensity distribution analysis-polarization (FIDA-PO)

Summary

Introduction

The R6/2 transgenic mouse expressing Htt exon1-Q120 ± 523 has a long history of use in HD research, whether overexpression of a partial fragment of the mutant protein completely reflects human pathology has been debated. It is generally accepted that some features of the R6/2 mouse pathology mimic human HD We used this model to investigate the pathological function of Ku7024; protein-protein interaction screenings identified this protein as molecule that directly interacts with Htt[25]. Given that previous results have suggested the importance of Ku70 relative to various mediator molecules, we screened chemicals that could inhibit the interaction between Ku70 and mutant Htt. We combined an in vitro screen, in silico screen, Drosophila screen and mouse screen, and we validated the effect with human iPS cells obtained from HD patients. One chemical was difficult to synthesize on a large scale

Methods

Results

Conclusion