Five micrograms of MeOH-solubilized flu antigen–p7 protein was dried by evaporation, then resolubilized overnight at room temperature in 20 mM sodium phosphate buffer (pH 7.0) containing 100 mM lyso-myristoylphosphatidylglycerol (LMPG) (monomeric) or 100 mM 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC) (oligomeric),31 incorporating 4 mM rimantadine-HCl (Sigma) or 4 mM N-nonyl deoxynojirimycin (NN-DNJ) (Toronto Biochemicals); 2× native polyacrylamide gel electrophoresis (PAGE) loading dye (150 mM Tris-Cl (pH 7.0), 30% glycerol, 0.05% bromophenol

blue) was added and samples were separated on a 4-20% TGX gel (Biorad) prior to staining with Coomassie Brilliant Blue. We have modeled the heptameric GT1b J4 isolate p7 complex31 with lumenal His17.35 We extended these studies to include a low-pH, open form wherein His17 protonation selleck kinase inhibitor caused p7 protomers to rotate, inducing channel opening (Fig. 1A). This is consistent with p7 opening

being stimulated at low pH,33 as well as cellular proton conductance.19 We also generated a GT2a JFH-1 PLX4032 model (Fig. 1B) with similar structural characteristics to the J4 channel, despite significant sequence diversity. Autodock 4.0 was used to model binding sites (residue interactions <4 Å) on J4 and JFH-1 channels for amantadine (Ama), rimantadine (Rim), and NN-DNJ. Adamantanes bound to a peripheral, membrane-exposed

region of the channel complex (Fig. 1B, left panel), preventing channel opening. The location of this pocket agreed with NMR studies of p7-amantadine interactions36 and overlapped with J4 L(50-55)A, a mutation shown to alter amantadine sensitivity in vitro.31NN-DNJ did not interact with channel complexes, instead docking to p7 monomers at the protomer interface (Fig. 1B, right panel), thus potentially disrupting oligomerization. Accordingly, active nonyl-IS derivatives were predicted to bind this site with >10-fold higher affinity than inactive butyl-derivatives15 (data not shown). Although relatively well conserved PIK3C2G in other genotypes (Fig. 1C), variation at these positions may alter compound binding, providing a basis for genotype-dependent sensitivity.21 J4 and JFH-1 adamantane binding sites contained L20, which mutated to F20 in GT1b patients unresponsive to IFN/Rib/Ama.29 Comparison of predicted binding affinities (Autodock) revealed that Rim bound to wild-type channels with higher affinity compared with Ama, explaining its increased potency.19, 21 Ama-resistant JFH-1 p7 provided a threshold value for effective drug binding (Kd>7.41 μM). L20F increased predicted Kd values for both Ama and Rim above 7.41 μM (Fig. 2A), with one exception.