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. Author manuscript; available in PMC: 2013 Apr 20.
Published in final edited form as: Chem Biol. 2012 Apr 20;19(4):518–528. doi: 10.1016/j.chembiol.2012.03.007

Figure 2. In vitro characterization of thiadiazolidinedione inhibitors against viral oncoproteins.

Figure 2

(A) Ability of inhibitors to prevent LxCxE containing viral oncoproteins from disrupting E2F/pRb complexes. Ten-fold dilutions of inhibitor (compound 478166 is shown) were added to GST-pRbABC/6xHis-HPV1AE7CR2-3 or GST-pRbABC/6xHis-Ad5E1ACR2-3. (B) Ability of inhibitors to disrupt complexes between pRb and LxCxE containing viral oncoproteins. Compound 478166 was used for the experiment shown. (C) Binding of inhibitors to pRb as measured by isothermal titration calorimetry. The curve fit for pRb binding to compound 478081 reveals 1:1 binding with a KD of 165nM, and dH of -1237 cal/mol. (D) HPV-E7 binding to pRb in the presence of increasing concentrations of inhibitor. The ELISA-based assay was used to determine the mechanism of binding of the small molecules to pRb. Five-fold dilutions of inhibitor were added to pRb and the amount of E7 that was able to bind to pRb was determined. The calculated apparent KD values for pRB-E7 in the presence of 0.025, 0.25, 0.5 and 5.0 µM of inhibitor 478165 (KD for pRb of 104 nM) were 140 ± 22, 313 ± 21, 304 ± 76 and 764 ± 72 nM, respectively. See also Figure S3.