Engineering a region of bulk tolerance in the 5-HT2A receptor

Abstract Anew binding model for ketanserin was constructed; this model and preliminary mutagenesis data suggested that a highly conserved phenylalanine (F340) introduces a potential region of bulk-intolerance into the binding pocket. We tested this hypothesis by evaluating the binding affinities of several novel analogues of the 5-HT 2A antagonist ketanserin at both native and mutant receptors. We found that replacing the bulky F340 with a less bulky leucine (F340L) dramatically enhances (37 ± 16-fold; P 3 – 5 ) which are characterized by having bulky substituents in the benzylic position. By contrast, four other ketanserin analogues ( 2 , 6 – 8 ) which lacked these substituents at the benzylic position show minimally altered binding affinities at the F340L mutant receptor (1.3 ± 0.3-fold; P > 0.05 vs. native receptor). This structural modification of the receptor allowed for the increased affinity of low-affinity, bulky ketanserin analogues.