In vitro activity: DTP3 physically interacts with MKK7, both in isolation and within the complex with GADD45β, and dissociates the GADD45β/MKK7 complex via an allosteric mechanism. DTP3 selectively kills cells and induces apoptosis in MM cells with functional MKK7 and elevated GADD45β expression without toxicity to normal cells. In addition, DTP3 displays synergistic activity with bortezomib in two different MM cell lines, exhibiting a combination index of 0.21 in U266 cells and of 0.56 in KMS-12 cells.

Kinase Assay: The stoichiometry and KD value of the DTP3/MKK7 interaction are determined by tryptophan fluorescence quenching analysis, after fitting the fluorescence data with a nonlinear regression algorithm.

Cell Assay: [3H]Thymidine incorporation assays are performed using standard protocols. Briefly, cell lines (CD138+ cells) are seeded into wells of 96-well plates at a concentration of 1.0x104 cells/well and then left untreated or treated daily with the indicated concentrations of peptides and maintained in complete RPMI-1640 medium at 37°C in 5% CO2, splitting them with medium as necessary. At 24, 72 or 144 hr, cells are incubated for an additional 16 hr with 0.037 MBq/well of [3H]thymidine, and then harvested onto glass fibre filter mats using a 96-well plate automated Tomtec cell harvester and analyzed by liquid scintillation spectroscopy with a LKB Wallac Trilux Microbeta 3-counter. Values are expressed as the percentage of the counts per minute (cpm) measured in the treated cultures relative to the cpm measured in the respective untreated cultures. The IC50 values are calculated using either 5 or 7 concentrations of compound and are defined as the mean concentration of compound inducing 50% inhibition of [3H]thymidine uptake relative to the uptake measured in untreated cells. Trypan blue exclusion assays are performed. Briefly, cells from lentivirus-infected cell lines are seeded into wells of 48-well plates in complete medium at a concentration of 2.0x105 cells/well, and then cultured at 37°C in 5% CO2, splitting them as necessary during the assays. Cell viability is monitored over a period of up to 8 days by cell counting using trypan blue, and the numbers of live infected cells in the cultures are extrapolated, where appropriate, from the cell counts by accounting for the percentages of eGFP+ cells, using flow cytometry. Values are expressed as the percentage of live infected cells present in the cultures at the times indicated relative to the number of live infected cells present in the same cultures at day 0.

Cancer Cell. 2014 Oct 13;26(4):495-508.