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. 2014 Aug 19;9(11):2471–2478. doi: 10.1021/cb500515r

Figure 2.

Figure 2

Characterization and comparison of SETD8 inhibition by SPS8I1–3. (a) Comparison of SPS8I1–3 as SETD8 inhibitors in vitro and in a cellular setting. The IC50 values of SPS8I1–3 were extracted from Figure 1b. The inhibition modes for SPS8I1–3 (SAM dependence, substrate dependence, slow onset/irreversible characters) were summarized according to the data in panels b–e. Values of kinact and Ki were extracted from Supplementary Figure S1. The EC50 values and cellular phenotypes of SPS8I1–3 are summarized according to the data in Figure 3. The off-target effects of SPS8I1–3 on other PMTs were based on the data in Figure 1c. The bottom line lists the reported non-PMT targets of SPS8I1–3.33−38 (b,c) Characterization of SAM-dependent inhibition of SETD8 by SPS8I1–3. Relative IC50 values were defined as the ratios of IC50 values for each inhibitor (the concentration range of 0.1–100 μM was examined) in the presence of the varied concentrations of the SAM cofactor or substrate in the presence of the lowest concentration of SAM (1 μM) or substrate (5 μM), respectively. These numbers were then plotted against the ratios of [SAM]/Km,SAM or [substrate]/Km,H4K20 (Km,SAM = 24 ± 1 μM and Km,H4K20 or Km,pep = 125 ± 3 μM, as will be reported elsewhere), respectively. The data were arbitrarily fit to 0–3 order exponential curves to show a general trend of IC50 versus the concentrations of SAM or H4K20 peptide. (d) Time-dependent, slow onset inactivation of SETD8 by SPS8I1–3. After incubating with the inhibitors, the relative methyltransferase activity of SETD8 was evaluated by measuring the initial rate and then plotted as the percentage of the loss of the activity versus the DMSO-treated control at different time intervals. (e) Irreversible character of SETD8 inhibition by SPS8I1–3. After preincubating SPS8I1–3 to inactivate SETD8, the mixtures of SETD8 and inhibitors were diluted by 200-fold to lower the concentrations of inhibitors below their IC50 values. The residual methyltransferase activity was monitored and presented as the percentage of the DMSO-treated control.