Objective: To validate on-target engagement of disease-relevant ⍺-synuclein (a-syn) aggregates by WTX-607, WaveBreak’s small-molecule clinical candidate for Parkinson’s disease (PD) and Lewy body dementia (LBD), using ex vivo seeding and in situ binding assays to: a) demonstrate dose-dependent inhibition of a-syn aggregation in an amplification assay seeded with patient brain tissue; b) show binding of WTX-607 to disease-associated a-syn species by direct staining in patient brain sections.

Background: α-Synuclein (a-syn) aggregation and spreading represent key mechanisms for Parkinson’s disease (PD) and Dementia with Lewy Body (DLB) pathogenesis and progression. A role for templated-seeding is supported by the high sensitivity and specificity from cerebrospinal fluid (CSF) seed amplification assay (SAA) positivity in subjects with PD, DLB, or MSA.

WTX-607’s unique MOA directly targets a-syn nucleation mechanisms that are the initiating steps for multimer and aggregate formation. We determined the ability of WTX-607 to inhibit aggregation driven by patient-derived a-syn using a novel SAA that uses seeds extracted from neuropathologically-confirmed brain tissue.

Method: SAA was adapted to detect seeds extracted from formalin-fixed paraffin-embedded human brain tissue. Seeds were extracted from deparaffinized sections using a combination of buffers and high-power sonication. Resulting material was amplified using wildtype human a-syn monomer as substrate across a range of compound concentrations (0.1-10,000 nM). ThT fluorescence kinetics was analyzed to determine compound activity. Separately, we utilized fluorescently labeled WTX-607 to directly visualize binding with a-syn in situ in human tissue.

Results: Low nM concentrations of WTX-607 inhibited aggregation by ~90% in SAA reactions seeded with either PD, DLB, or MSA tissue-derived seeds (n=10 each). Tissues from synucleinopathy subjects exhibited higher levels of specific staining with labeled WTX-607 while no specific staining pattern was observed in samples from healthy control subjects. These data support direct engagement of WTX-607 with a-syn aggregates in human disease.

Conclusion: Collectively, these data suggest WTX-607 inhibits aggregation with selective and specific on-target engagement with a-syn aggregates in patient brain tissue.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/patient-derived-%ce%b1-synuclein-seed-amplification-as-a-tool-for-evaluating-small-molecule-aggregation-inhibitors-for-parkinsons-disease-and-lewy-body-dementia-therapeutics/