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. 2023 Jan 18;11(2):253. doi: 10.3390/biomedicines11020253

Figure 2.

Figure 2

AHK2 compound binds FKBP12 and functions as a RyR/FKBP12 stabilizer. (a) Target engagement of AHK2 was evaluated in HEK293 cells using a luminescence protein complementation assay. FKBP12/FRB dimerization was induced with rapamycin, while AHK2 did not induce FKBP12/FRB dimerization (left panel). AHK2 inhibits formation of FKBP12-Rapamycin-FRB interaction with an IC50 value of 2.98 mM (right panel). Graph shows data from a representative experiment repeated three times. Data are expressed as mean + SEM of n = 3 replicates. (b) AHK2 rescues FKBP12/RyR interaction in human myotubes under nitro-oxidative stress. In situ proximity ligation assay (red) shows that reduced FKBP12/RyR interaction in myotubes challenged with peroxynitrite donor SIN1 (3-morpholino-sydoniminium chloride) is partially rescued by AHK2 with an increase of 89%. Myotubes are counterstained with myosin heavy chain (MyHC, green). Statistical significance was determined using one-way ANOVA followed by Dunnett’s test. n = 11–15 images from 3–4 independent experiments. Scale bar: 50 μm.