The AKT modulator A-443654 reduces α-synuclein expression and normalizes ER stress and autophagy

• DOI: 10.1016/j.jbc.2021.101191
• Publication Date: 2021-09-11T23:13:26Z
• Authors: Mandi Gandelman; Warunee Dansithong; Stephen C. Kales; Sharan Paul; Gentrie Maag; Erika Aoyama; Alexey Zakharov; Ganesha Rai; Thomas Dexheimer; Brooke M. Whitehill; Hongmao Sun; Ajit Jadhav; Anton Simeonov; Mark J. Henderson; Duong P. Huynh; Stefan M. Pulst; Daniel R. Scoles
• Keywords: Synucleinopathies; LRRK2; HEK 293 cells

Accumulation of α-synuclein is a main underlying pathological feature Parkinson's disease and α-synucleinopathies, for which lowering expression the gene (SNCA) potential therapeutic avenue. Using cell-based luciferase reporter SNCA we performed quantitative high-throughput screen 155,885 compounds identified A-443654, an inhibitor multiple functional kinase AKT, as potent SNCA. HEK-293 cells with CAG repeat expanded ATXN2 (ATXN2-Q58 cells) have increased levels α-synuclein. We found that A-443654 normalized both mRNA monomers oligomers in ATXN2-Q58 cells. also fibroblasts iPSC-derived dopaminergic neurons from patient carrying triplication gene. Analysis autophagy endoplasmic reticulum stress markers showed successfully prevented toxicity restored cell function cells, normalizing mTOR, LC3-II, p62, STAU1, BiP, CHOP. decreased DCLK1, lysosomal degradation. Our study identifies AKT inhibition strategy reducing treating pathology. 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Quantitative screening: titration-based efficiently biological activities large chemical libraries.Proc. 103: 11473-11478Crossref (586) primary included viability measurement, Gly-Phe-7-amino-4-trifluoromethylcoumarin (GF-AFC), eliminated consideration if cytotoxic. number active passing cytotoxicity (cytotox) triage 1996 (1.3%). screened, doses, Z′ factors provided Table 1. After silico analysis (clustering, removal frequent hitter nuisance chemotypes), 842 selected follow-up retested at (luciferase GF-AFC confirmation) CMV-luc expressing flag general repressors. Following this triage, 33 tested enzyme-linked immunosorbent (ELISA) measure endogenous protein 3XSNCA fibroblasts. Triaging validation counter-screens orthogonal assays shown 2. Five confirmed secondary ELISA NCGC00470280, NCGC00419055, NCGC00448911, NCGC00345809, NCGC00347278 (A-443654) (Table 3). NCGC00345809 having least favorable cytotox parameters Among remaining displayed profile assays, ELISA, Western blot results considered study. structure Figure 1A. General observed counter-screen (IC50 8.91 μM) 1B).Table 1Libraries, numbers, factors, activitiesLibraryCompoundsDosesZ' (SNCA-luc)Active (SNCA-luc)Passing triageNPCaThe NPC library only three so curve classes −1.1, −1.2, −2.1, −2.2 could computed. There 12 (all negative classes).281630.421210Epigenetics Focused Library33940.5164NPACT515770.59299Genesis96,26040.619371562PTL300040.621411MIPE 4.01978110.6515547LOPAC128040.601815Sytravon45,05540.632719333Sum all libraries155,885----48901996The 155,885.a classes). Open table tab 2Triage (SNCA-luc, CMV-luc, Cytotox) (ELISA)Triage stepCriterionaCriteria advancing compounds: (1) Negative class screen. (2) Confirmed class, >30% (3) ΔAUC (single metric potency efficacy) >40 comparing versus assays. 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