Development of a lysosomal activator for the treatment of neurodegenerative disease

Neurodegenerative diseases represent a severe and growing problem as the global population ages. For the vast majority of these conditions, there are no disease modifying treatments available. Drug development for these disorders is extremely challenging due to limited understanding of their pathogenesis, lack of suitable animal models, and reduction of potential chemical space for therapies due to the highly selective blood brain barrier. After noting a reduction in CNS Smad signaling with age and neurodegeneration, our lab set out to screen for a small molecule capable of activating this pathway in the brain. A hybrid phenotypic screen followed by structural perturbation and testing of the top hits yielded lead compound SRI-011381. SRI-011381 was chosen among the other hits due to its high permeability, low toxicity, and promising efficacy in vivo. SRI-011381 was found to have anti-inflammatory and neuroprotective activity in numerous mouse models of neuroinflammation and disease, with particular promise in a mouse model of Parkinson's Disease. Here, I describe my effort to characterize the metabolism of SRI-011381, prepare it for IND-submission to the FDA, and identify its biological target. To characterize the metabolism of SRI-011381 I utilized mass spectrometry to first identify the metabolites made by liver microsomes in vitro and then to confirm their presence and relative abundance in vivo. From their fragmentation spectra I deduced their probable structural identity, then commissioned the synthesis of these predicted compounds to confirm the structures of all major metabolites of SRI-011381. I then tested the metabolites in our in vivo models to determine if they were active. I also confirmed that no unique metabolites were generated by human microsomes, as required by the FDA. To prepare for IND-submission to the FDA, I worked to compile and summarize all of the toxicological and safety studies done with SRI-011381. Based on their results, I followed up on potential issues with veterinary specialists and prepared our safety package for the FDA. I also carried out a full battery of permeability, pharmacokinetic, and brain/plasma protein binding studies in collaboration with Takeda Pharmaceuticals. Using this data, I generated a human physiology-based pharmacokinetic model to determine the optimal dose for our first-in-human safety study, as well as an estimated efficacious dose. To uncover the mechanism and target of 381, I performed single-cell RNA sequencing on the brains of treated mice and performed a series of target identification experiments using various methods. I was eventually able to identify the biological target of 381 using a whole-genome CRISPRi screen. The screen pointed us to the lysosome, and specifically to the proton pump responsible for acidification -- vacuolar ATP-ase. Following the screen, I worked to validate a selection of the top hits with cell lines I generated, and to confirm lysosomal activation by SRI-011381. Finally, I confirmed functional lysosome activation with experiments showing increased rates of protein breakdown in lysosomes with SRI-011381 treatment

Keywords: Chemical Engineering.
Advisor: Swartz James R. 
Wyss-Coray Anton 
Bassik Michael