human amyloid β1  (R&D Systems)

Journal: bioRxiv

Article Title: Small-molecule degraders reduce Aβ production through CAPRIN1-mediated lysosomal degradation of APP in Alzheimer’s iPSC-derived neurons

doi: 10.1101/2023.12.29.573648

Figure Lengend Snippet: The compound 0043 reduces Aβ42 and APP in AD-iPSC-Ns. (A) ELISA of Aβ42 levels in culture media of iPSC-Ns under two Dox inducing protocols (means ± SD; ***p<0.001, ****p<0.0001, ns=non-significance). (B) ELISA of Aβ42 in culture media of sAD2.3-Ns treated with each compound (10 µM) for 4 days. (C) Cell viability assay of sAD2.3-Ns treated with a series of doses of indicated compounds for 4 days (means ± SD; n=3). (D) ELISA of Aβ42 levels in culture media of each of the panel of eight iPSC-Ns treated with indicated compounds (10 µM) for 4 days (means ± SD; ****p<0.0001; n=4). (E) Contrast microscopes of APP SWE mutant AG27606-Ns (the scale = 100 µm). (F) ELISA of Aβ42 levels in the cultures of APP SWE mutant and APP WT AG27606-Ns (means ± SD; ****p<0.0001; n=4). (G) ELISA of Aβ42 levels in the cultures of APP SWE AG27606-Ns treated with 0043 (10 µM) for 4 days (means ± SD; ****p<0.0001; n=4). (H) ELISA of Aβ42 in the cultures of sAD2.3-Ns treated with a series of doses of 0043 ( top ) for 5 days or one dose of 0043 (10 µM) for indicated days ( bottom ; means ± SD; *p<0.05; ***p<0.001; n=3). (I) ELISA of Aβ42 in the cultures of sAD2.3-Ns and AG27606-Ns treated with a series of dilutions of 0043 for 5 days with the IC 50 values indicated. (J) Western blots of indicated proteins in sAD2.3-Ns treated with 0043 (10 µM) or DMSO for 3 days. (K) Western blots of sAD2.3-Ns and AG27606-Ns with the indicated doses of 0043 for 3 days.

Article Snippet: Secreted Aβ42 peptides were quantified using the Human Amyloid β1-42 human ELISA kit (Thermo Fisher) according to the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Viability Assay, Mutagenesis, Western Blot

Journal: bioRxiv

Article Title: Small-molecule degraders reduce Aβ production through CAPRIN1-mediated lysosomal degradation of APP in Alzheimer’s iPSC-derived neurons

doi: 10.1101/2023.12.29.573648

Figure Lengend Snippet: The compound 0043 directly binds CAPRIN1 and APP. (A) NMR spectrum of 200 µM 0043 in green ( top ) and 1D-WaterLOGSY spectra recorded of 200 µM 0043 where red and black spectrum corresponds to 0043 in the presence and absence of 20 µM CAPRIN1, respectively. (B) Representative backbone amide peaks in the 1 H- 15 N-HSQC spectrum of CAPRIN1 alone (blue) and in the presence of 0043 (red). The direction of peak shifts is shown by the black arrows. (C,D) Thermofluor shift assay of rhCAPRIN1 Full (C) and rhAPP Full (D) in the presence of indicated doses of 0043. (E,F) Western blots of CETSA (E) and levels of indicated proteins at a temperature point relative to that 37°C (F) in AG27606-Ns in the absence and presence of 0043 (10 µM) with increased temperature. (G) Western blots for APP and CAPRIN1 in sgCAPRIN1 and sgRNA control HEK-APP695 WT clones treated or untreated with 0043 (10µM). (H). ELISA of Aβ42 levels in culture media of sgCAPRIN1 and sgControl HEK-APP695 WT clones after treated with 0043 (10 µM) for 3 days (means ± SD; ****P < 0.0001, ns: non-significance, n=3).

Article Snippet: Secreted Aβ42 peptides were quantified using the Human Amyloid β1-42 human ELISA kit (Thermo Fisher) according to the manufacturer’s protocol.

Techniques: Shift Assay, Western Blot, Control, Clone Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Small-molecule degraders reduce Aβ production through CAPRIN1-mediated lysosomal degradation of APP in Alzheimer’s iPSC-derived neurons

doi: 10.1101/2023.12.29.573648

Figure Lengend Snippet: Identification of the lead 0152 from chemical modification of the hit 0043. (A) The chemical structures of the hit compound 0043 and its derivative lead 0152. (B) The PK assessment of 0043 and 0152 by analyzing mouse plasma at different time points after oral administration of the compounds (n= 3). (C) The PK values of 0043 and 0152 determined by analyzing the compounds in mouse plasma samples after administrated orally in indicated doses and solution formulations. (D) NMR spectrum of 0152 in green ( top ) and 1D-WaterLOGSY spectra recorded of 0152 where red and black spectrum corresponds to 0152 in the presence or absence of 20 µM CAPRIN1. (E) Western blots of biotin-0152/streptavidin-coated bead-pull downs of AG27606-Ns lysates in the presence of various doses of 0152 and HB007. (F,G) ELISA of Aβ42 in culture media of AG27606-Ns (f) and UKBi011A-Ns (g) after treated with 0043 and 0152 (10 µM) for 4 days (means ± SD; **p<0.01, ****p<0.0001; n=4). (H,I) ELISA of Aβ42 in culture media of AG27606-Ns (H) and UKBi011A-Ns (I) after treated with various doses of 0043 and 0152 for 5 days with the IC 50 values calculated. (J) Western blot of APP in AG27606-Ns after treated with 0043 (10 µM) and 0152 (10 µM) for 5 days. (K) Confocal microscopes ( left ) of AG27606-Ns after treated with 0043 (10 µM) and 0152 (10 µM) for 24 hours and stained as indicated (green, APP antibodies; red: CAPRIN1 antibodies; and blue: HOECHST (scale bar = 20 µm) and the correlation coefficient values ( right ) of APP and CAPRIN1 analyzed using the Image J program (means ± SD; *p<0.05, ***p<0.001).

Article Snippet: Secreted Aβ42 peptides were quantified using the Human Amyloid β1-42 human ELISA kit (Thermo Fisher) according to the manufacturer’s protocol.

Techniques: Modification, Clinical Proteomics, Western Blot, Enzyme-linked Immunosorbent Assay, Staining