Find our wrap-up report below!
Last quarter we embarked on an exciting 12-week drug discovery challenge to discover new, efficient and safe treatments for the gruelling neurodegenerative disease, Amyotrophic Lateral Sclerosis (ALS). After continuous hard work which was documented in our weekly blogs, we now proudly look back at the main achievements.
Arctoris’ part of the challenge was the in vitro validation and prioritisation of computationally derived compounds as potential drug candidates for ALS treatment. The compounds were shortlisted by GenieUs Genomics’ advanced machine learning and molecular dynamic modelling tools in Australia and validated in complex cell models at Arctoris’ robotics-powered automated labs in Oxford, UK.
In a nutshell, the challenge had two key parts:
Firstly, showcasing that the selected compounds exhibit a neuroprotective effect by reducing eIF2α phosphorylation in motor neurons.
Demonstrating that the compounds exhibit an anti-inflammatory effect, by inhibiting the impact of activated (inflammatory) glial cells that can induce and exacerbate a distinct ALS phenotype on motor neurons.
Preparing for the challenge itself, GenieUs Genomics designed and shortlisted 4 compound inhibitors for both parts. And at Arctoris, automated protocols were developed to grow and differentiate motor neurons and astrocytes with the highest robustness and reproducibility.
What did we achieve?
Part 1: eIF2α Phosphorylation in Motor Neurons
Increased eIF2α phosphorylation is a known pathological hallmark of several major neurodegenerative diseases (Kim H. J., Nat; Genet. 2014; Bond S., J Neuropathol Exp Neurol. 2020), including ALS, and so we set out to test the effects of 4 eIF2α phosphorylation inhibitors in modulating this pathological biomarker.
First of all, we showed that we could induce an ALS-like state by treating healthy motor neurons with a known eIF2α activator and directly quantifying eIF2α phosphorylation via homogeneous time-resolved fluorescence. As expected, treated cells showed higher eIF2α phosphorylation than the healthy motor neurons (similar to what is observed in ALS cells) (week 2). The images below show immunofluorescence (IF) imaging of our motor neurons. We did this to ensure they underwent complete differentiation prior to experimental work.
IF images of motor neurons 10 days post-seeding with different types of staining. Blue stains nuclei, red stains beta III tubulin - one of two structural components that form the neuronal microtubule network, green stains choline acetyltransferase - an enzyme involved in synthesising acetylcholine, the neurotransmitter that regulates signal transduction at the neuromuscular joint. Scale bar = 200 um (zoom out) and 50 um (zoom in).
Next, we treated the healthy and ALS-like motor neurons with increasing amounts of the 4 compound inhibitors and measured their effect on cell viability and eIF2α phosphorylation (weeks 7 and 8). Our data indicate that 3 of the 4 compound inhibitors were well tolerated over the dose range and had a positive impact in reducing EIF2α phosphorylation, in particular in ALS-like motor neurons (i.e. those treated with the eIF2α activator). One of the compound inhibitors showed neurotoxicity at the highest doses, narrowing the possible therapeutic range. Cell viability graphs for the 4 compound inhibitors are shown below. Following these findings, we tested whether there were synergistic effects between 2 of the 4 compound inhibitors, however, the results were inconclusive at this stage (week 11).
Cell viability graphs of each compound inhibitor. K-control line = a known compound inhibitor.
Overall these results are promising: 3 of the 4 tested compound inhibitors point in the right direction and are potentially worth pursuing to understand if this translates into neuroprotective effects. Further investigations using increased dosage and treatment times and recapitulating similar effects in patient-derived ALS motor neurons will be beneficial.
Part 2: Inhibition of Glial Cell Activation
Secretion of inflammatory cytokines is expected to lead to the loss of motor neurons (Komine O. and Yamanaka K., Nagoya J Med Sci. 2015). We set out to test the effect of 4 compounds in counteracting cytokines released from activated astrocytes. BX559 is hypothesised to activate astrocytes and lead to cytokine release, and therefore a potentially useful tool for this challenge. Our goal is to evaluate if the 4 BX559 inhibitors can reduce the toxic effects of cytokines on healthy motor neurons when the media containing the cytokines is transferred to these cells.
First, we needed to find out the optimal dose and time for BX559 to elicit an inflammatory response in astrocytes. To this end, we measured (via flow cytometry) the amount of cytokines (TNFa, IL-1B, IL-8 and IL-6) released at different drug concentrations over time (weeks 4 and 6). Unfortunately, there was no clear dose-dependent response over time, so we continued the tests with the most promising BX559 condition. We co-cultured astrocytes with BX559 and varying doses of the BX559 inhibitors and then transferred the cell media onto healthy motor neurons to gauge its effects on cell viability (week 10).
Unfortunately, the results were not favourable. None of the compounds showed an effect on motor neuron viability. A potential reason for the lack of response lies in the low degree of inflammation-induced on astrocytes upon BX559 addition, thus reducing any subsequent effect we could have seen on motor neurons. We tested for this potential explanation and saw that when astrocytes were treated with a control known to potently activate an inflammatory response we did detect a significant increase in the amount of inflammatory cytokines released, indicating that the astrocytes themselves were behaving as expected, but BX559 might not have (week 10).
For more conclusive evidence, further experiments will be needed to identify potential issues with BX559 treatment and delivery, optimise its dosing, and confirm that a proper immune response is induced on astrocytes.
Thanks to the combined strengths of machine learning approaches and advanced automation, we made extremely promising progress on a very tough disease in a record time of just 12 weeks. All of our planned experiments were executed on time, yielding answers to some of our key questions. We confirmed viable drug targets and drugs worth following up on laying the foundations for the next stages of work.
It was an intense 12 weeks for everyone involved in the study, but it was certainly worth it, and we hope this can be taken as an example of how a concerted effort between committed organisations leveraging enabling technologies can lead to rapid progress in an area of large unmet clinical need.
About GenieUs Genomics
GenieUs is a precision health company using breakthrough data analytics and machine learning to solve the mystery of neurodegenerative diseases, some of the most misunderstood conditions on the planet. Founded in 2017, its analytics solution uses cutting-edge algorithms and the deepest integration of multi omic datasets to map and sub-categorise neurodegenerative diseases for more personalised treatment. The GenieUs Deep Integrated Genomics Analysis Platform (DiGAP) uses some of the most advanced and highly tuned Artificial Intelligence available.
Arctoris is a tech-enabled biopharma platform company founded and headquartered in Oxford, UK with its US operations based in Boston and its Asia-Pacific operations based in Singapore. Arctoris combines robotics and data science with a world-class team for small molecule and biologics discovery. Ulysses®, the unique technology platform developed by Arctoris, enables the company and its partners to conduct their R&D – from target to hit, lead, and candidate – significantly faster, and with considerably improved data quality and depth.
Bringing together the expertise of seasoned biotech and pharma veterans with its proprietary technologies, Arctoris leads to higher success rates and an accelerated progression of programs towards the clinic. Arctoris pursues an internal pipeline of assets in oncology and neuro and also collaborates with select biotech and pharma partners in the US, Europe, and Asia-Pacific.