(A) RGB-286638 chemical structure (left panel). Chemical name: 1-(3-{4-[4-(2-Methoxyethyl)-piperazin-1-ylmethyl]-phenyl}-4-oxo-1,4-dihydroindeno[1,2-c]pyrazol-5-yl)-3-morpholin-4-yl-urea dihydrochloride. Molecular formula: C29H37N7O4 2HCl. Molecular weight: 618.52.
(B) Dose- and time-dependent cytotoxicity of RGB-286638 in MM cell lines. Expressing wild-type p53 (MM.1S, MM.1R, H929) or mutant-p53 (U266, OPM1, RPMI) cells were incubated with RGB-286638 (0–100nM), and viability was determined at 24 and 48 h using MTT assay. EC50 ranged between 20–70nM at 48h.
(C) RGB-286638-mediated cytotoxicity in primary tumor cells from MM patients. Myeloma cells isolated from BM via CD138+ positive selection were cultured with RGB-286638 (0–100nM), and viability was assessed by MTT at 48h. RGB-286638 demonstrated comparable cytotoxicity to MM cell lines. Data represents means (+/−SD) of triplicate cultures.
(D) RGB-286638 treatment results in transcriptional CDK inhibition. The indicated cell lines were exposed to RGB-286638 (0–100nM) 1, 4, and 8 h treatment. Whole lysates were subjected to western blotting and membranes probed for phosphorylated RNAPII at S2/S5 and Rb at S807/811/S780 sites using phosphospecific antibodies.