On October 31, researchers at Fudan University, including Profs. Boxun Lu (School of Life Sciences), Yiyan Fei (School of Informatics Science and Engineering), and Yu Ding (School of Life Sciences) published online under the title “Allele-selective Lowering of Mutant HTT Protein by HTT-LC3 Linker Compounds” in Nature and showed their study about an innovative method of drug discovery: using the autophagosome-tethering compounds (ATTEC) to degrade pathogenic proteins and treat the disease. The team carried out a smartly-designed screening featuring small-molecule microarray and front-edge optical technologies, and managed to identify four small molecule compounds that specifically reduced the protein that caused Huntington's disease, bringing hope to the disease-progression-modifying treatment of this disease and similar diseases. Lu Boxun, Fei Yiyan and Ding Yu are the corresponding authors of the paper. Li Zhaoyang, Wang Cen, Wang Ziying and Zhu Chenggang, PhD. students at Fudan University, are the first authors. The research was jointly funded by National Natural Science Foundation of China and the Ministry of Science and Technology of China.

Dr. Fei’s group has developed a label-free high-throughput optical platform for biomolecular interactions which has been applied in various systems including proteins, small molecules, viruses and bacteria. The optical platform consists of biochip and oblique-incidence reflectivity difference (OI-RD) technique. Based on detection of changes on thickness or refractive index, OI-RD is able to measure biomolecular interactions without labels. With 2D scanning, OI-RD is able to image biochip with area about 8~10 cm2 so that it can measure up to 10,000 biomolecular interactions at the same time. The optical platform provides an efficient method to probe biomolecular interactions in label-free and high throughput mode. 

With joint efforts, the team found four small molecules that can specifically reduce the protein that caused Huntington’s disease. Huntington's disease (HD) is one of the four major neurodegenerative diseases that have been most extensively studied. Since the biochemical activity of the mutant huntingtin protein (mHTT) that causes the disease is uncharacterized, the conventional drug discovery approach relied on inhibitors that block the bioactivity of the pathogenic proteins is not applicable. Protein degradation is a promising way for HD. Autophagy is an intracellular protein degradation machinery and will degrade all proteins engulfed into autophagosomes, including the normal wild-type huntingtin protein that plays a role in neuroprotection and other important proteins, if enhanced non-specifically. This would be possibly self-defeating. How can we identify compounds that only degrades mHTT but not wild-type HTT? The team envisioned a “small molecule glue” functioning as Autophagosome Tethering Compound (ATTEC), which could tether LC3 and mHTT together so that mHTT is engulfed into autophagosomes for degradation. Meanwhile, the ATTEC does not interact with the wild-type HTT protein, leaving it unaffected.

In fact, such ATTECs were highly challenging to identify. Only one out of about two thousand compounds has the desired properties. It is like a needle in a haystack. Thus finding it had been a major obstacle of this project for a long time. The platform of Dr. Fei’s group provided an efficient and economic tool to look for ATTECs. The research team stamped nearly four thousand small-molecule compounds onto a chip and had the target protein flow through the chip. If it binds to a specific compound immobilized onto the chip, the molecular layer at the position will thicken, generating a tiny change that can be detected by OI-RD. Using this cutting-edge screening approach, the team found two small molecules that could bind to both LC3 and mHTT proteins, but not to wild-type HTT. After studying a panel of small molecule compounds with similar structures, a total of four ATTECs that bind LC3 and mutant HTT were identified and validated.

(a)OI-RD image of small molecule microarray; (b) Two small molecules that can bind to LC3 and mHTT but not to HTT.

The team found that these four compounds significantly reduced mHTT levels in HD mouse neurons, HD patient cells, and HD Drosophila models at ~10 to 100 nanomolar concentrations, with little effect on wild-type HTT levels. Excitingly, at least two out of these four compounds are able to cross the blood-brain barrier, and a small dose of intraperitoneal injection would significantly reduce mHTT levels in the cortex and striatum of HD mice, without affecting wild-type HTT levels. They also significantly improved disease-related phenotypes, providing an entry point for the development of oral or injectable drugs for HD. The team further explored the intrinsic mechanisms by which these small molecule compounds could distinguish between mutant and wild-type HTT proteins, which were almost identical except in the glutamine repeat (polyQ) length. It turned out that these compounds were bound to excessively long polyQ stretches that only appeared in mHTT. Based on this, the team realized that the application of these small molecule compounds may reach far beyond the potential treatment of Huntington's disease. Nine human diseases are so called polyQ diseases, because they are caused by specific mutant proteins containing excessively long polyQ. Among them, spinocerebellar ataxia type III (SCA3) is the most common polyQ diseases in the Chinese population. A world class expert in the neurodegenerative disease field and a member of National Academy of Sciences and winner of Breakthrough Prize in Life Sciences, Huda Zoghbi, wrote a commentary article in Nature for this research: “exciting discovery”, “this therapeutic strategy might be useful not only for Huntington’s disease, but also for other diseases involving expanded polyglutamine tracts”.

Based on the high throughput optical platform, Dr. Haipeng Liu of Shanghai Pulmonary Hospital and Dr. Fei collaborated also published their important collaboration work in Nature (Nuclear cGAS suppresses DNA repair and promotes tumorigenesis). The application of this optical platform in the fields of biomedicine and other fields will be greatly enhanced.