Example 3
To support the mechanism of action by which phenylbenzamides directly block AQP4, we perform in vitro binding studies using purified AQP4b and Compound 4 radiolabeled with 3H. Using a Hummel-Dryer style assay, a gel filtration column is equilibratrated with buffer containing detergent, to maintain solubility of AQP4b, and 1 μM [3H]-Compound 4. AQP4b is diluted to 250 μM in this column buffer and incubated at RT for 30 min. The sample is then applied to the column, fractions collected and the presence of [3H]-Compound 4 detected by liquid scintillation counting. FIG. 3 shows the elution profile of [3H]-Compound 4 from the gel filtration column with the elution positions of tetrameric and monomeric AQP4b indicated. The rise in [3H]-Compound 4 from a baseline value of 1 μM represents binding to each of these proteins. Although no monomeric AQP4b can be readily detected in our highly purified AQP4b by conventional means, this assay reveals the presence of a small, albiet vanishing, amount of monomer. The relative affinities for Compound 4 are ˜100 μM and less than 1 μM for tetramer and monomer, respectively. This assay shows relatively weak binding of Compound 4 to solubilized AQP4b; nevertheless, it clearly demonstrates that this phenylbenzamide directly interacts with AQP4b.
