University of Plymouth
PEARL https://pearl.plymouth.ac.uk
Faculty of Health: Medicine, Dentistry and Human Sciences Peninsula Medical School
2019-10-30
Allele-selective lowering of mutant HTT
protein by HTTLC3 linker compounds
Li, Z
http://hdl.handle.net/10026.1/15145
10.1038/s41586-019-1722-1
Nature
Springer Science and Business Media LLC
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Allele-selective Lowering of Mutant HTT Protein by HTT-LC3 Linker Compounds 1
2
Zhaoyang Li
1#
, Cen Wang
1#
, Ziying Wang
1#
, Chenggang Zhu
2#
, Jie Li
3
, Tian Sha
1
, 3
Lixiang Ma
4
, Chao Gao
5
, Yi Yang
6
, Yimin Sun
1
, Jian Wang
1
, Xiaoli Sun
1
, Chenqi Lu
1
, 4
Marian Difiglia
7
, Yanai Mei
1
, Chen Ding
1$
, Shouqing Luo
6$
, Yongjun Dang
8
, Yu Ding
1*
, 5
Yiyan Fei
2*
, Boxun Lu
1*
6
7
1
Neurology Department at Huashan Hospital, State Key Laboratory of Medical 8
Neurobiology and MOE Frontiers Center for Brain Science, Institutes of Brain Science, 9
School of Life Sciences, Fudan University, Shanghai, China. 10
2
Department of Optical Science and Engineering, Shanghai Engineering Research 11
Center of Ultra-Precision Optical Manufacturing, Key Laboratory of Micro and Nano 12
Photonic Structures (Ministry of Education), Fudan University, Shanghai, China. 13
3
National Facility for Protein Science in Shanghai, Large-scale Preparation System, 14
Shanghai, China. 15
4
Department of Anatomy, Histology and Embryology, Shanghai Medical College, 16
Fudan University, Shanghai, China. 17
5
Institutes of Biomedical Sciences, Fudan University, Shanghai, China. 18
6
Peninsula Schools of Medicine and Dentistry, Institute of Translational and Stratified 19
Medicine, University of Plymouth, Plymouth, UK. 20
7
Laboratory of Cellular Neurobiology, Department of Neurology, Massachusetts 21
General Hospital, Charlestown, Massachusetts, United States of America 22
8
Key Laboratory of Metabolism and Molecular Medicine, Shanghai Medical College, 23
Fudan University, Shanghai, China.
24
# These authors contribute equally; 25
*
Corresponding authors: 26
Boxun Lu (lead contact): luboxun@fudan.edu.cn 27
Yiyan Fei: fyy@fudan.edu.cn 28
Yu Ding: yuding@fudan.edu.cn 29
$
Co-senior authors 30
- 2 -
Summary 31
Accumulation of mutant proteins is the major cause for many diseases 32
(proteopathies), and lowering the level of these proteins is highly desired for treatment. 33
We hypothesized that compounds interacting with both the autophagosome protein 34
LC3
1
and the disease-causing protein may target the latter for autophagic clearance. 35
We tested this hypothesis in the context of lowering mutant HTT protein (mHTT), 36
which contains an expanded polyglutamine (polyQ) tract and causes Huntington’s 37
disease (HD), an incurable neurodegenerative disorder
2
. Through 38
small-molecule-microarray based screening, we identified four compounds interacting 39
with both LC3 and mHTT, but not the wild-type HTT protein (wtHTT). Some of these 40
compounds targeted mHTT to autophagosomes, reduced mHTT levels in an 41
allele-selective manner, and rescued HD-relevant phenotypes in cells and in vivo in 42
the fly and mouse HD models. We further revealed that these compounds interacted 43
with the expanded polyQ stretch and could lower the level of mutant ATXN3, another 44
disease-causing protein with expanded polyQ
3
. Our study provides candidate 45
compounds for lowering mHTT and potentially other disease-causing proteins with 46
polyQ expansion, demonstrating the concept of lowering disease-causing proteins by 47
autophagosome-tethering compounds (ATTEC). 48
- 3 -
Background 49
An emerging approach for disease treatment is to lower the levels of 50
disease-causing proteins, especially those with unknown activities. Biological tools 51
such as RNAi or CRISPR may achieve this goal
4-6
, but their clinical delivery is 52
challenging. Enhancing proteasomal degradation of target proteins by “PROTAC” is a 53
promising emerging approach
7
, but proteasomes alone are inefficient in degrading 54
certain large proteins or aggregates
8
. Another independent protein degradation 55
pathway is macroautophagy (referred to as autophagy hereafter), which is a bulk 56
degradation system that engulfs proteins into autophagosomes for subsequent 57
lysosomal degradation
9
. Autophagy is present in all eukaryotic cells, and thus 58
harnessing the power of autophagy to degrade certain target proteins may open new 59
windows for drug discovery. Here we investigate this possibility in the context of 60
lowering mHTT, which contains an expanded polyglutamine (polyQ) stretch (≥36Q) 61
and causes HD, an incurable monogenetic neurodegenerative disorder
2
. 62
mHTT could be degraded by autophagy, during which protein substrates are 63
engulfed into double-membrane autophagosomes associated with lipidated LC3
1
. We 64
thus hypothesized that linker compounds interacting with both mHTT and LC3 may 65
tether them together to enhance the recruitment of mHTT into autophagosomes, 66
facilitating mHTT degradation. In addition, mHTT-LC3 linker compounds that do not 67
interact with wtHTT may promote allele-selective degradation of mHTT. Since no 68
mHTT/LC3-interacting compounds have been reported, we performed 69
small-molecule-microarray-based screening for desired compounds, and utilized 70
- 4 -
wtHTT for the counter-screen to identify allele-selective candidates. 71
Results 72
Identification of mHTT-LC3 linker compounds 73
We stamped 3375 compounds (Fig. 1a) in duplicates into a microarray on 74
isocyanate-functionalized glass slides via the nucleophile-isocyanate reaction, which 75
forms covalent bonds between the compounds and the glass slides
10,11
. We then 76
purified the human LC3B protein
1
(Extended Data Fig. 1a-b & Supplementary Table 1), 77
a pathogenic mHTT exon1 fragment
12
with expanded polyQ (mHTTexon1-Q72), and a 78
control wtHTT exon1 fragment (HTTexon1-Q25) (Extended Data Fig. 1c-d) for the 79
screen. We fused a maltose-binding-protein (MBP) tag to both HTT exon1 proteins to 80
increase their solubility required for later experiments. 81
To identify LC3B- and mHTT- interacting compounds, we had these purified 82
proteins flow through the SMMs, and detected the compound-protein interaction using 83
an optical biosensor, the scanning oblique-incidence reflectivity difference (OI-RD) 84
microscope. OI-RD is a widely used interaction measurement technology
13-15
, whose 85
working principle has been validated and reported previously
16-18
. We then performed 86
experiments with HTTexon1-Q25 or buffer alone to exclude non-specific signals, and 87
identified two compounds that interact with both LC3B and mHTTexon1-Q72, but not 88
HTTexon1-Q25: 10O5 (GW5074, 89
3-3-[(3,5-Dibromo-4-hydroxyphenyl)methylidene]-5-iodo-1H-indol-2-one) and 8F20 90
(ispinesib, 91
N-(3-aminopropyl)-N-[(1R)-1-[7-chloro-4-oxo-3-(phenylmethyl)-2-quinazolinyl]-2-meth92