Potent inhibitors of toxic alpha-synuclein identified via cellular time-resolved FRET biosensors

Abstract

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.

Neuroprotective Effects of a Novel Demeclocycline Derivative Lacking Antibiotic Activity: From a Hit to a Promising Lead Compound

“…High-throughput screening systems (HTS) based on the inhibition of α-Syn toxic species provide starting chemical matters for drug discovery processes, and different selection platforms are constantly providing small molecules as hit compounds for PD [18] [28,53] These molecules must be studied in the subsequent steps of drug design pipelines in order to obtain leaders for preclinical and clinical trials. Recently, Braun et al (2021) identified DMC and the bi-benzimidazole Ro 90-7501 as compounds that prevented α-Syn aggregation at nanomolar concentrations and protected SH-SY5Y cells from αSyn-induced death [18]. Of these, DMC was shown to be the most potent, was effective at a nanomolar range, and could also inhibit α-Syn Ser219 phosphorylation in a murine primary neuron assay [18].…”

“…The ability of DMC to interfere with α-Syn amyloid-like aggregation has been previously reported [18]; however, its antibiotic activity would hinder its repositioning as a neuroprotector. To study if DDMC retained the antiaggregant property shown by its precursor DMC, we monitored the ability of both TCs to inhibit α-Syn self-assembly.…”

“…DMC is clinically used to treat susceptible bacterial infections and can be systemically administrated due to its low toxicity in human cells, even neurons [34]. In order to uncover if this was also the case for DDMC, we first evaluated its impact on cell viability by measuring MTT turnover [35] in SH-SY5Y cell cultures, using similar concentrations as those shown in Braun et al for DMC [18]. The results showed no statistical differences between both compounds, tested at 10 and 50 µM, with respect to the control conditions (Figure 1c).…”

“…Recently, using a high-throughput drug discovery biosensor platform, demeclocycline (DMC) was selected from a 1280-compound small-molecule library (LOPAC) as a hit compound with enough potential to prevent neurodegeneration induced by the aggregation of α-Syn [18]. Interestingly, among 21 hit compounds identified by the biosensor, DMC showed the highest inhibition of αSyn-induced toxicity in the low nanomolar range (EC50 = 65 nM) and was tenfold more potent than previously described small molecules.…”

Challenges in Discovering Drugs That Target the Protein–Protein Interactions of Disordered Proteins

“…Much effort has been focused on stabilizing SYN monomers or inhibiting oligomer formation in order to prevent aggregation [49][50][51]. For example, apomorphine, a nonselective dopamine receptor agonist utilized in PD therapy, inhibited SYN fibrillation; however, it resulted in large, toxic oligomeric species [52].…”