Fig. 1: Discovery of potent ligands against proteasome subunit PSMD2.
From: Targeted degradation via direct 26S proteasome recruitment
a, Phage and mRNA display approaches yield macrocycle ligands with high affinity to PSMD2. ClAc-F, N-chloroacetyl l-phenylalanine; Sar, sarcosine (N-methyl glycine); ϕ, phage display. b, Top macrocycle ligands bind the 26S proteasome with nanomolar affinity. An AlphaScreen assay using a biotinylated analog of macrocycle MC2 and an antibody to PSMD1 shows dose-dependent 26S proteasome binding for three of the four ligands; curves represent the mean ± s.d. from three independent experiments. SA, streptavidin. c, Alanine and lysine scanning of MC2 and MC1 using in vitro translation and ELISA reveals that an HRYxGW/F motif is critical for PSMD2 binding. Bars represent the mean ± s.d. of three independent experiments; WT, wild type. d, AP–MS and TMT–MS with MC1biotin from HEK293 lysates shows enrichment of proteasome subunits for the active l-enantiomer over the inactive d-enantiomer control in a binary comparison volcano plot. Data represent three biological replicates per condition (l-CIDE, d-CIDE and DMSO control). The horizontal dotted line shows a P value of 0.05. The vertical dotted lines show log2 (fold change) of –1 or 1. For details of the data and statistical analyses, see the Methods. e, Macrocycle MC2 dramatically stabilizes PSMD2, increasing its Tm by 9.2 °C, as observed by differential scanning fluorimetry.
