Figure 2. Identification of kinetin riboside by drug library screening for inhibitors of CCND2 promoter trans-activation in an NIH3T3 cell reporter system.

(A) Compounds were screened for inhibition of CCND2 promoter trans-activation at 16 hours in a 3T3 reporter model in which the MAF–trans-activating factor was coexpressed with the CCND2 promoter–driving LUC expression. The results of screening the Spectrum library is shown as a dot plot comparing each compound’s assay position (x axis) with its effect on MAF-driven CCND2 promoter trans-activation (measured by LUC) relative to effects on 3T3 viability (measured by MTS; y axis). Compounds below the dotted line were defined as putative hits. LOPAC and Prestwick drug libraries were also screened but are not shown. (B) Repeat testing of kinetin riboside (kinetin R), dexamethasone, and vehicle against reporter cells expressing LUC driven by a control RSV promoter or the CCND2 promoter, with and without MAF coexpression, showing that LUC suppression by these drugs is mediated specifically by the CCND2 promoter. Repression of CCND2 promoter activity by kinetin riboside is not restricted to trans-activation induced by MAF. (C) Suppression of CCND2-driven LUC protein levels in 3T3 following kinetin riboside (10 μM) treatment. Results of separate experiments are plotted (triangles and circles); each point is the mean of duplicates ± SEM. The overall curve of best fit is depicted as a solid line. Despite an estimated LUC half-life of 3 hours, kinetin riboside induces approximately 80% suppression by 9 hours, suggesting that suppression of the CCND2 promoter begins within 1–4 hours. (D) Chemical structure of kinetin riboside.