We have developed a high-throughput drug discovery platform, measuring fluorescence resonance energy transfer (FRET) with fluorescent alpha-synuclein (αSN) biosensors, to detect spontaneous pre-fibrillar oligomers in living cells. Our two αSN FRET biosensors provide complementary insight into αSN oligomerization and conformation in order to improve the success of drug discovery campaigns for the treatment of Parkinson’s disease. We measure FRET by fluorescence lifetime, rather than traditional fluorescence intensity, providing a structural readout with greater resolution and precision. This facilitates identification of compounds that cause subtle but significant conformational changes in the ensemble of oligomeric states that are easily missed using intensity-based FRET. We screened a 1280-compound small-molecule library and identified 21 compounds that changed the lifetime by >5 SD. Two of these compounds have nanomolar potency in protecting SH-SY5Y cells from αSN-induced death, providing a nearly tenfold improvement over known inhibitors. We tested the efficacy of several compounds in a primary mouse neuron assay of αSN pathology (phosphorylation of mouse αSN pre-formed fibrils) and show rescue of pathology for two of them. These hits were further characterized with biophysical and biochemical assays to explore potential mechanisms of action. In vitro αSN oligomerization, single-molecule FRET, and protein-observed fluorine NMR experiments demonstrate that these compounds modulate αSN oligomers but not monomers. Subsequent aggregation assays further show that these compounds also deter or block αSN fibril assembly.

Alfalfa, like other legumes, establishes a symbiotic relationship with the soil bacteria, Sinorhizobium meliloti , which results in the formation of the root nodules. Nodules contain the bacteria enclosed in a membrane‐bound vesicle, the symbiosome where it fixes atmospheric N 2 and converts it into ammonia using the bacterial enzyme, nitrogenase. The ammonia released into the cytoplasm from the symbiosome is assimilated into glutamine (Gln) using carbon skeletons produced by the metabolism of sucrose (Suc), which is imported into the nodules from the leaves. The key enzyme involved in the synthesis of Suc in the leaves is sucrose phosphate synthase ( SPS ) and glutamine synthetase ( GS ) is the enzyme with a role in ammonia assimilation in the root nodules. Alfalfa plants, overexpressing SPS or GS , or both showed increased growth and an increase in nodule function. The endogenous genes for the key enzymes in C/N metabolism showed increased expression in the nodules of both sets of transformants. Furthermore, the endogenous SPS and GS genes were also induced in the leaves and nodules of the transformants, irrespective of the transgene, suggesting that the two classes of plants share a common signaling pathway regulating C/N metabolism in the nodules. This study reaffirms the utility of the nodulated legume plant to study C/N interaction and the cross talk between the source and sink for C and N.

Aggregation of endogenous a-synuclein is the hallmark of Parkinson's disease pathology. Under normal conditions, a-synuclein plays an important role in neurological function. Under pathologic conditions, misfolded a-synuclein forms cytotoxic oligomers and fibrils. There has been significant effort to identify small molecule inhibitors of a-synuclein fibrillization. However, recent evidence suggests the kinetically unstable oligomeric species, and not fibrils, are the source of a-synuclein cytotoxicity. We interrogated the selfassociation of the oligomeric species under aggregation prone conditions using FRET. To achieve this, we developed a set of cell-free FRET based biosensors that monitor oligomer-oligomer interactions and fibril formation through fluorescence lifetime. Using fluorescence lifetime provides a 30-fold increase in sensitivity over more traditional FRET measurements. We have achieved an optimal FRET of 14.99% for oligomeric interactions and a seeded induced change in FRET of 15.34%. We are currently optimizing these biosensors for scale up to facilitate high throughput screening. Hits will be validated for FRET dose response and biophysical changes in aggregation kinetics via thioflavin T assays and AFM characterization.