The main aim of this project was to investigate our drug compound in selected secondary indications through in vitro cell based assays. We performed these tests in all three selected pathologies: spinobulbar muscular atrophy (SBMA), Huntington disease (HD) and Parkinson disease (PD). These tests were either carried out by CRO (for SBMA and PD) or internally (for HD).
Our approach focuses on degrading protein aggregates in these different diseases. Therefore, we identified CRO that were able to study aggregation in SBMA (WuXi AppTech) and Parkinson (Neuro Sys) with the use of relevant cellular models. As for HD, since no CRO was offering assays that allowed to properly study aggregation in these diseases, we developed the assay by combining data and approaches from the literature.
SBMA:
We were able to use WuXi AppTech knowledge in terms of cell line generation and automated image quantification to develop a SBMA aggregation model. Briefly, the GFP bearing mutated form of the androgen receptor (AR, mutated in SBMA patients) was stably expressed in cells then treated with the AR agonist R1881 that triggers AR aggregation. AR was then analyzed either by western blot (expression) or by microscopy (aggregation). By comparing cells treated solely with vehicle and cells treated with ATS-033, we were able to show that our compound was able to decrease both expression and aggregation of AR in this model, one of the main goals of the drugs being developed currently in the field of SBMA.
PD:
We identified the French CRO Neuro Sys has one of the specialists in the field of Parkinson disease, and particularly on alpha-synuclein, the protein that aggregates in PD. We conducted two assays with Neuro Sys, both alpha-synuclein aggregation models. The first one relies on the use of MPP+, a toxin that triggers alpha-synuclein aggregation, and the other one relies on the addition to the cells of preformed-fibrils of alpha-synuclein that end up aggregating. Both models are done in primary dopaminergic neurons that are the affected cell type in the brain of PD patients. These assays showed that ATS-033 was able to reduce aggregation of alpha-synuclein, and that was accompanied in both models by a reduction of neuronal death, validating PD as an indication of interest for our compound.
HD:
Finaly, we developed a test internally to be able to study the aggregation of the mutated protein in HD: huntingtin (HTT). We transiently expressed both the wild type and the mutated form of HTT in cells and were able to show that, as expected, the mutated form led to an increase in aggregation (increased number of aggregates per cell, and increase number of cells with aggregates). We then looked at whether ATS-033 was able to reduce aggregation in this context, and our preliminary results suggest that indeed, ATS-033 is able to reduce mutated HTT aggregation.
Overall, we were able to show that ATS-033 could reduce protein aggregation in the three pathologies that we studied, therefore strengthening our pipeline. The next steps will be to further validating ATS-033 as a drug candidate for these diseases by moving to in vivo animal models.
