Research Institution: Walter and Eliza Hall Institute of Medical Research

Principle Investigator: A/Prof. Marnie Blewitt

Type: Australian – Nevada, Melbourne, AUSTRALIA

Project title: “Pre-clinical testing for FSHD CRISPR-inhibition therapy”

Status: Active


FSHD is caused by production of a protein called DUX4 in skeletal muscle, that should normally be switched off, so one potential treatment for FSHD is reinstating the off state at DUX4. The protein known as SMCHD1 is normally involved in switching off DUX4 and this role is known to be critical as reduced SMCHD1 function causes FSHD2 and results in more severe disease in FSHD1. Importantly, FSHD patients retain at least some functional SMCHD1, meaning there is potential to boost the function of the remaining normal SMCHD1 so that it can again turn off DUX4. Our previous work has shown that SMCHD1 function can indeed be enhanced and in this project we will screen a 120,000-chemical library of drug-like molecules in search of chemicals that can boost SMCHD1 function. Once we have identified such molecules, we will refine their design to produce a series of molecules that could be used to develop potent drugs that activate SMCHD1 and concomitantly repress DUX4, as potential therapeutics for FSHD. These molecules will form the basis of future commercial partnerships with pharmaceutical companies. We anticipate drugs that target SMCHD1 would be used in conjunction with drugs that directly target DUX4. This project will be run at The Walter and Eliza Hall Institute of Medical Research by Associate Professor Marnie Blewitt, Dr James Murphy and Associate Professor Chris Burns.



Update September 2019

Grant 39: High throughput chemical screens for activators of SMCHD1, as potential therapeutics for FSHD

The molecule SMCHD1 has been shown to play an important role in FSHD, where it keeps the specific DNA element that causes FSHD in check, by ensuring that it goes unnoticed in the cell i.e. it is switched off. Our project is to identify drug-like chemicals that boost SMCHD1’s activity, as potential therapeutics to treat FSHD. The hope is that by boosting SMCHD1 activity, the DNA element responsible for FSHD can be effectively switched off.

In Aim 1, we screened around 120,000 chemicals for their effect on SMCHD1’s activity (Aim 1, completed Feb 2018); however after validation and counter-screening, we did not isolate a chemical that has the capacity to enhance SMCHD1’s activity. This is not completely unexpected, as finding a chemical that makes a protein work harder is much more challenging than finding one that breaks that protein i.e. inhibits its activity. Without a hit, we were unable to progress to Aim 2.
We requested and had approved a variation to funding, introducing Variant Aim A and a new set of milestones (1-4). We proposed to use an alternative methodology, to screen chemical fragments via a higher throughput and more cost effective method. This is called a fragment screen, and rather than testing the effect these chemical fragments have on the enzyme activity, instead they test whether they bind to SMCHD1, and with what affinity. After fragments that bind are found, they are improved using the 3D structure of the protein to guide the design of chemical modifications. Finally, they would be tested for their effect on SMCHD1’s enzyme activity and engineered to become activators.

We have completed Milestone 1 of Variant Aim A, exactly the timing we anticipated (9 months).
As we planned, we outsourced the large-scale production of insect cells that overexpress the human SMCHD1 ATPase domain to the University of Queensland Protein Expression Facility. Back at WEHI, these insect cells were used to purify sufficient high-quality protein for the full high throughput chemical screen. We then transferred the protein to Monash Institute of Pharmaceutical Sciences for the Fragment screen. They performed a pilot screen to ensure that the protein was stable in the required conditions. They then performed the fragment screen, analyzing the 1142 chemical fragments for their capacity to bind SMCHD1’s ATPase domain. Each fragment is tested in a pool of 3-5 fragments. This screen has been successful and revealed 100 primary hits that are putatively bind SMCHD1’s ATPase domain. These hits are consistent with the number of hits predicted. They are now the subject of validation in Milestone 2.

The 100 putative hits from the screen are now being validated using three separate assays. A set of 10-20 validated binding fragments will then be progressed to Milestone 3. One important update to note is that in the past month, the 3D structure of SMCHD1’s ATPase domain has been reported by another group. This is excellent news as it will accelerate our progress and mean that we can work from this structure when optimizing the binding fragments in later milestones.

In summary, we have now performed a screen of 1142 chemical fragments and identified 100 putative SMCHD1 binding chemical fragments. These fragments will be validated in the coming 3 months, then optimized for their affinity to SMCHD1 over the subsequent 3 months. Taking advantage of the newly published 3D structure of SMCHD1’s ATPase domain, with appropriate medicinal chemistry staff we will be able to engineer binding fragments not only for their affinity, but also to try and create moieties that allow them to activate SMCHD1’s activity, as a potential treatment for FSHD.

Update July 2017

The molecule SMCHD1 has been shown to play an important role in FSHD, where it keeps the specific DNA element that causes FSHD in check, by ensuring that it goes unnoticed in the cell i.e. it is switched off. Our project is to identify drug-like chemicals that boost SMCHD1’s activity, as potential therapeutics to treat FSHD. To achieve this aim, we will screen more than 117,000 chemicals, then characterise those that enhance SMCHD1 function for how they achieve this enhanced activity and for their role in the context of living cells. In the first 6 months of the project, we have established all of the systems required to screen the large library of chemicals, and have performed a pilot screen of just over 4000 chemicals. From this pilot we have already identified 40 hits, that potentially activate SMCHD1. We are now keenly working on these molecules to validate their effect on SMCHD1, then study their interaction with SMCHD1. Alongside this preliminary work on the hits from our pilot screen, we will now screen the full library of around 113,000 chemicals, to find more such hits for future validation, with the clear aim of identifying, characterising and developing drug-like molecules that activate SMCHD1 as potential treatment for FSHD.