Promising Outcomes of Original Grant:
Our Rapid Response Innovation Award (RRIA) enabled us to develop a conformational change assay for monomeric alpha-synuclein. We identified positive and negative controls for conformational change and measured the characteristics of the assay. Our original hypothesis was that we could detect ligand-induced conformational change of alpha-synuclein by second-harmonic generation (SHG) if it was labeled site-specifically. This hypothesis was confirmed by our work for the RRIA grant.
Objectives for Supplemental Investigation:
Having developed a conformational change assay for alpha-synuclein, we will screen several small molecule libraries to identify compounds which stabilize the monomeric protein. Whatever the mechanism of PD pathogenesis (e.g., aggregation), the premise of our approach is to identify small molecules that stabilize monomeric alpha-synuclein in one of the various conformations it adopts.
Our hypothesis, given all the experimental evidence on alpha-synuclein, is that targeting specific conformations of the alpha-synuclein monomer – the protein in its most fundamental form – will inhibit or halt neurodegeneration. Once we have identified hits, we will verify them as true hits by testing them in a counterscreen for aggregators. We will then use a screening-by-catalog computational method to identify commercially available structural analogs of the hits from various sources, including the NINDS library of approved CNS drugs. We will also measure the ability of the compounds to inhibit aggregation of alpha-synuclein in vitro.
Importance of This Research for the Development of a New PD Therapy
Compounds we identify in our screen will be rigorously tested in vitro for their ability to modulate the conformation of monomeric alpha-synuclein. We intend to take the most promising ones forward in a drug development program that includes synthesis to optimize their potency and testing in animal models to validate both the compounds and our approach to develop therapeutics for PD. Compounds that stabilize monomeric alpha-synuclein in a particular conformation could be effective therapeutics and, as pharmacological tools, provide important insight into PD pathogenesis.
The goal of our original funded grant was to use our alpha-synuclein assay to identify compounds that prevent conformational change to the fully open form of the monomeric protein. Spermine binding induces a fully extended alpha-synuclein conformation and this leads to a great increase in its aggregation. We successfully applied our assay to identify a number of compounds that prevent this conformational change and appear to stabilize the monomeric protein in its compact conformation.