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Browsing by Author "Srivastava, Ankit"
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Item Discovery of potent inhibitors of α-synuclein aggregation using structure-based iterative learning(Springer Nature, 2024) Horne, Robert I.; Andrzejewska, Ewa A.; Alam, Parvez; Brotzakis, Z. Faidon; Srivastava, Ankit; Aubert, Alice; Nowinska, Magdalena; Gregory, Rebecca C.; Staats, Roxine; Possenti, Andrea; Chia, Sean; Sormanni, Pietro; Ghetti, Bernardino; Caughey, Byron; Knowles, Tuomas P. J.; Vendruscolo, Michele; Pathology and Laboratory Medicine, School of MedicineMachine learning methods hold the promise to reduce the costs and the failure rates of conventional drug discovery pipelines. This issue is especially pressing for neurodegenerative diseases, where the development of disease-modifying drugs has been particularly challenging. To address this problem, we describe here a machine learning approach to identify small molecule inhibitors of α-synuclein aggregation, a process implicated in Parkinson's disease and other synucleinopathies. Because the proliferation of α-synuclein aggregates takes place through autocatalytic secondary nucleation, we aim to identify compounds that bind the catalytic sites on the surface of the aggregates. To achieve this goal, we use structure-based machine learning in an iterative manner to first identify and then progressively optimize secondary nucleation inhibitors. Our results demonstrate that this approach leads to the facile identification of compounds two orders of magnitude more potent than previously reported ones.Item Enhanced quantitation of pathological α-synuclein in patient biospecimens by RT-QuIC seed amplification assays(Public Library of Science, 2024-09-20) Srivastava, Ankit; Wang, Qinlu; Orrù, Christina D.; Fernandez, Manel; Compta, Yaroslau; Ghetti, Bernardino; Zanusso, Gianluigi; Zou, Wen-Quan; Caughey, Byron; Beauchemin, Catherine A. A.; Pathology and Laboratory Medicine, School of MedicineDisease associated pathological aggregates of alpha-synuclein (αSynD) exhibit prion-like spreading in synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Seed amplification assays (SAAs) such as real-time quaking-induced conversion (RT-QuIC) have shown high diagnostic sensitivity and specificity for detecting proteopathic αSynD seeds in a variety of biospecimens from PD and DLB patients. However, the extent to which relative proteopathic seed concentrations are useful as indices of a patient's disease stage or prognosis remains unresolved. One feature of current SAAs that complicates attempts to correlate SAA results with patients' clinical and other laboratory findings is their quantitative imprecision, which has typically been limited to discriminating large differences (e.g. 5-10 fold) in seed concentration. We used end-point dilution (ED) RT-QuIC assays to determine αSynD seed concentrations in patient biospecimens and tested the influence of various assay variables such as serial dilution factor, replicate number and data processing methods. The use of 2-fold versus 10-fold dilution factors and 12 versus 4 replicate reactions per dilution reduced ED-RT-QuIC assay error by as much as 70%. This enhanced assay format discriminated as little as 2-fold differences in αSynD seed concentration besides detecting ~2-16-fold seed reductions caused by inactivation treatments. In some scenarios, analysis of the data using Poisson and midSIN algorithms provided more consistent and statistically significant discrimination of different seed concentrations. We applied our improved assay strategies to multiple diagnostically relevant PD and DLB antemortem patient biospecimens, including cerebrospinal fluid, skin, and brushings of the olfactory mucosa. Using ED αSyn RT-QuIC as a model SAA, we show how to markedly improve the inter-assay reproducibility and quantitative accuracy. Enhanced quantitative SAA accuracy should facilitate assessments of pathological seeding activities as biomarkers in proteinopathy diagnostics and prognostics, as well as in patient cohort selection and assessments of pharmacodynamics and target engagement in drug trials.