Stern blot results: In A), GFP-CDH13 fusion proteins (26 kDa+105 kDa = 131 kDa) were detected in HEK293 cells by an antibody against GFP (TA150041). Mock cells transfected with the empty GFP vector expressed only GFP (26 kDa). In B), GFP-CDH13 fusion proteins were detected by an antibody against CDH13 (AF3264). Two bands were detected by this antibody, one at approximately 131 kDa and another at 105 kDa. Mock cells transfected with the empty GFP vector did not express CDH13. In A), B) the protein loading control, A-tubulin (50 kDa), was detected by an antibody against a-tubulin (T9026). (TIF)Figure S2 Localization of GFP-CDH13 fusion proteins in living HEK293 cells. Images of living cells showed cytoplasmic localization of GFP-CDH13. In mock cells GFP was distributed all over the intracellular space. A) HEK293-GFP, B) HEK293-Wild Type CDH13, C) HEK293-GFP-A376T, D) HEK293-GFPG113R, E) HEK293-GFP-I585V, F) HEK293-GFP-L643R, G) HEK293-GFP-N39S, H) HEK293-GFP-R174W, I) HEK293GFP-V112I.(-)-Epigallocatechin Gallate (TIF) Figure S3 CDH13 stained HEK293 cells expressingGFP-wild type and variant CDH13 fusion proteins. Two distinct signals were observed in cells stained for cell surface CDH13:1. GFP-CDH13 (green) localized in the cytoplasm as it was observed in living cells and 2. CDH13 expressed on the cell membrane (red). Mock cells transfected with GFP did not express CDH13. A) HEK293-GFP, B) HEK293-Wild Type CDH13, C)CDH13 Coding Variants in ADHDHEK293-GFP-A376T, D) HEK293-GFP-G113R, E) HEK293GFP-I585V, F) HEK293-GFP-L643R, G) HEK293-GFP-N39S, H) HEK293-GFP-R174W, I) HEK293-GFP-V112I. (TIF)Author ContributionsConceived and designed the experiments: TM JH IW SJ. Performed the experiments: TM SJ PMK. Analyzed the data: TM SJ PMK IW JH. Contributed reagents/materials/analysis tools: JH PMK SJ. Wrote the paper: TM JH. Critical revision of the manuscript and final approval: SJ IW PMK JH.AcknowledgmentsWe thank Paal Henning Borge and Guri E Matre for help with sequencing and mutagenesis. We also thank Rune Kleppe for helpful discussions and Professor Ian F Pryme for a critical reading of the manuscript.
Letter pubs.acs.org/acsmedchemlettFused 3Hydroxy-3-trifluoromethylpyrazoles Inhibit Mutant Huntingtin ToxicitySalvatore La Rosa,*,,Tiziana Benicchi, Laura Bettinetti, Ilaria Ceccarelli, Enrica Diodato, Cesare Federico, Pasquale Fiengo, Davide Franceschini, Ozgun Gokce, Freddy Heitz,, Giulia Lazzeroni, Ruth Luthi-Carter,, Letizia Magnoni, Vincenzo Miragliotta,, Carla Scali, and Michela ValacchiSiena Biotech SpA, Strada del Petriccio e Belriguardo 35, 53100 Siena, Italy Brain Mind Institute, Ecole Polytechnique Federale de Lausanne (EPFL), Lausanne, SwitzerlandS * Supporting InformationABSTRACT: Here, we describe the selection and optimization of a chemical series active in both a full-length and a fragmentbased Huntington’s disease (HD) assay.Voriconazole Twenty-four thousand small molecules were screened in a phenotypic HD assay, identifying a series of compounds bearing a 3-hydroxy-3trifluoromethylpyrazole moiety as able to revert the toxicity induced by full-length mutant Htt by up to 50 .PMID:35850484 A chemical exploration around the series led to the identification of compound 4f, which demonstrated to be active in a Htt171- 82Q rat primary striatal neuron assay and a PC12-Exon-1 based assay. This compound was selected for testing in R6/2 mice, in which it was well-tolerated and showed a positive effect on body weight and a positive trend in preventing ventricular volume enlargment. These studies.