In vitro activity: RGB-286638 is an indenopyrazole-derived CDK inhibitor (CDKI) with Ki-nanomolar activity against transcriptional CDKs. RGB-286638 inhibits several tyrosine and serine/threonine non-CDK enzymes, i.e. GSK-3β, TAK1, AMPK, Jak2, MEK1. The dose- and time-dependent effect of treatment with RGB-286638 (12.5-100nM) is investigated on the growth of human p53-wt (MM.1S, MM.1R, and H929) and p53-mutant (U266, OPM1, and RPMI) MM cells by MTT assay, assessing viability at 24 and 48 hours. The half-maximally effective concentrations (EC50) range between 20 and 70 nM at 48 hours. Dose-dependent differences in growth among p53-wt and -mutant cells are observed after 50nM treatment, with p53-wt MM.1S, MM.1R and H929 being slightly more sensitive to RGB-286638 treatment at 48h.

Kinase Assay: Nuclear Extraction Kit was used to isolated the nuclear proteins from MM.1S cells treated with 50 nM RGB-286638 for 1, 4, and 8 h. Nuclear protein are aliquoted and added to the 96-well plate coated with specific double-stranded DNA sequence containing the p53 response element for overnight incubation. p53 in the nuclear extract is detected by addition of a specific primary antibody directed against p53. A secondary antibody conjugated to HRP is added to provide a sensitive colorimetric readout at 450 nm. All experiments are performed in triplicates

Cell Assay: Colorimetric assays are used to evaluate drug activity at increasing concentrations of RGB-286638 (0-100 nM). Expressing wild-type p53 (MM.1S, MM.1R, H929) or mutant-p53 (U266, OPM1, RPMI) cells from 24- or 48-h cultures are pulsed with 10μL of 5mg/mL MTT to each well, followed by incubation at 37°C for 4h, and addition of 100 μL isopropanol containing 0.04 HCl. Absorbance readings at a wavelength of 570nm (with correction using readings at 630nm) are taken on a spectrophotometer. All experiments are performed in triplicates.