In vitro activity: Rbin-1 is a potent and reversible triazinoindole-based inhibitors of eukaryotic ribosome biogenesis. Rbin-1 inhibits recombinant full-length Mdn1’s ATPase activity. Two of the active analogs (Rbin-1 and Rbin-2) inhibit the ATPase activity by 40% at 1 uM. In particular, an analog (Rbin-2) with a bromine substituent at postion-7 is 10-fold more active than Rbin-1 (GI50=14±1 nM (Rbin-2); 136±7 nM (Rbin-1), n=4, mean±SD).
Kinase Assay: Radioactive γ-P32-ATP is added to 600 mM MgATP (pH=7)
solutions at volume ratios of 1:1000-1:300, depending on the lifetime of
the radioactive reagent. The total volume of each reaction is 12 mL,
including 6 ml of protein from size exclusion chromatography fractions
(final concentration 0-50 nM for different fractions, peak fractions are
used for Rbin-1 and AMPPNP inhibition), 4 mL FPLC SEC buffer with 0.6
mM Na2SO4 and 2 mL MgATP (final concentration=100
mM). The reactions are then incubated at room temperature for 30 or 60
min before quenching with 12 mL 0.2 M EDTA. 1 mL from each reaction
mixture is spotted on to TLC PEI cellulose F plates. The TLC buffer
contained 0.15 M formic acid and 0.15 M lithium chloride. The TLC plates
are then imaged using the Typhoon Scanner 9400. ImageJ is used to
calculate the densitometric ratio of the spots corresponding to
radioactive free phosphate and ATP to determine the percent of ATP
hydrolyzed.
Cell Assay: Ribozinoindoles are potent chemical inhibitors of eukaryotic ribosome assembly. Rbin-1 targets Mdn1 or cellular processes that involve this protein. It efficiently inhibit the growth of yeast cells. | 
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