Fig. 7: CN-0928 downregulates PCBP2 protein by INTS1.

a The workflow of chemical proteomics for target identification of CN-0928 includes three groups: In the CN-0928 group (middle), SH-SY5Y cell lysate was incubated with CN-0928-conjugated beads to enrich putative CN-0928 target proteins. In the competition group (lower), the lysate was preincubated with excessive free CN-0928 and then incubated with CN-0928-conjugated beads. In the blank control (upper), beads without CN-0928 were used to identify nonspecifically bound proteins. Each experiment was performed in duplicate, and the proteins pulled down were analyzed by LC-MS/MS. b, c Representative Western blots (left) and quantification (right) of BACE1 and PCBP2 in SH-SY5Y cells with INTS1 (b) or mH2A1 (c) knockdown, followed by treatment with either 1 μM CN-0928 or DMSO (Ctrl). d Relative mRNA levels of PCBP2 and BACE1 in SH-SY5Y cells transiently transfected with siINTS1, measured in the absence (Ctrl) or presence of 1 µM CN-0928. e Molecular docking results of CN-0928 to INTS1. The panoramic view shows the spatial position of CN-0928 in the structure of INTS1. The hydrogen bond is presented in the local interaction image. f Per-residue decomposition analysis of the binding free energy. In the CN-0928-INTS1 binary complex, CN-0928 forms hydrogen bonds with ARG-1404 during molecular dynamics simulations. Violin width is proportional to the kernel-density estimate of the distribution; the central line denotes the median and the thin lines indicate the 25th and 75th percentiles (n = 1000 analyzed frames). g CN-0928-biotin pulldown of flag-tagged wild type (INTS1-WT-flag) and ARG-1404 mutated INTS1 (R1404A-flag, R1404K-flag, and R1404L-flag) proteins. Cell lysates were mixed without and drug (blank), or with CN-0928-biotin (CN-biotin). Data: mean ± s.e.m (b–d). Significance was determined by one-way ANOVA with Bonferroni’s test (b–d). ns nonsignificant. In vitro: n  =  3 biological replicates (b–d, g). Source data are provided as a Source Data file.