Fig. 3: TYR-catalyzed in-situ formed TYR degraders.

a Chemical structure of azido-modified VH032 ligands (VH032-Azi1, VH032-Azi2, VH032-Azi3); alkyne-modified TYR inhibitor (Alk-TIn), and click-catalyzed TYR degraders (DeTYR-1, DeTYR-2, DeTYR-3). b Western blot analysis of TYR and MITF in A375 and B16F10 cells treated with VH032, Alk-TIn, VH032 + Alk-TIn, VH032-Azi1 + Alk-TIn + SA (0.5 μM), VH032-Azi2 + Alk-TIn + SA (0.5 μM) and VH032-Azi3 + Alk-TIn + SA (0.5 μM) at the concentration of 0.1 μM for 24 h. (n  =  3 independent experiments). c Molecular dynamic simulation of TYR (wheat), Alk-TIn (yellow), VHL (cyan), and VH032-Azi3 (magenta). The distance was measured by the two linker atoms (circled in green). d Molecular dynamic simulation of DeTYR-3 (yellow) interacting with VHL (cyan) and TYR (wheat) proteins. e Co-immunoprecipitation of TYR and VHL from lysates of A375 and B16F10 cells treated with VH032-Azi3 (0.1 μM) + Alk-TIn (0.1 μM) + SA (0.5 μM) or epi-VH032-Azi3 (0.1 μM) + Alk-TIn (0.1 μM) + SA (0.5 μM) for 4 h. (n  =  3 independent experiments). TYR inhibition of blank control, kojic acid, TIn, Alk-TIn and VH032-Azi3 + Alk-TIn + SA (0.5 μM) at 0.1 μM in A375 (f) and B16F10 (g) cells, respectively. (n  =  3 biological replicates). (****P  <  0.0001). Data are presented as mean ± standard deviation (SD). Melanin content of blank control, kojic acid, TIn, Alk-TIn, and VH032-Azi3 + Alk-TIn + SA at 0.1 μM in A375 (h) (P = 0.0479) and B16F10 (i) (P = 0.0147) cells, respectively. (n  =  3 biological replicates). Data are presented as mean ± standard error (SEM). Statistical analysis was performed using ONE-WAY variance (ANOVA) by Dunnett’s multiple comparisons (*P < 0.05).