3,6-DMAD hydrochloride functions as an inhibitor for the IRE1α-XBP1 pathway within the unfolded protein response mechanism.

The compound 3,6-DMAD effectively impedes IRE1α oligomerization as well as its endoribonuclease (RNase) activity in vitro.

3,6-DMAD (10 mg/kg, i.p., once every 12 h, total thrice) significantly inhibits in vivo XBP1-luciferase activity assessed 3.5 days after the initial treatment. 3,6-DMAD-treatment significantly inhibits tumor xenograft growth.

IRE1α oligomerization assay is set up using the nuclease reaction buffer, 2 μM recombinant hIRE1α, 2 mM ADP, and 60 μM of 3,6-DMAD. All reactions are performed in 20 μL with 10% DMSO to account for the vehicle and incubated for 15 min at 30 °C to allow for oligomerization. The optical density of the sample is measured at 500 nm using a NanoDrop 2000.

2×10^4 cells per well are plated into 96-well plates and treatment started 0-12 hours after plating. RPMI 8226 and MM1.R human MM cells are treated with 0-6 μM 3,6-DMAD. After 24 hours of treatment, XTT reagent (ATCC) is added to the wells then cells are incubated for 2 hours and absorbance measured at both 475 nM and 660 nM using a plate reader.

5×10^6 RPMI 8226 cells are implanted subcutaneously into the flanks of 4-6 weeks' old NOD Scid mice. Drug treatment started when the sizes of the tumors reached 150 mm3. Four tumor-bearing mice per group are treated with 10 mg/kg 3,6-DMAD or vehicle (saline) intraperitoneally once every 2 days.