Effects of head group substitution on large antiviral polyamide-DNA binding kinetics

Hairpin polyamides are pyrrole-based polymers that bind in the minor groove of DNA with remarkable affinity and specificity. While the natural product netropsin that inspired PA design features a terminal guanidinium group, few studies have examined how the head groups affect PA-DNA binding behavior and biological activity. Here the effects of two headgroup substitutions on DNA binding and antiviral behavior are explored with two large (14- and 20-ring) hairpin polyamides. To complete two sets of three polyamides, the syntheses of two, 20-ring hairpin polyamides with N-terminal cationic substitutions (guanidinium (Guan) and tetramethyl guanidine (TMG)) are reported. Using a fluorescence binding assay, some small differences in binding affinity toward a small DNA hairpin are observed for the PA14 series, with head-group substitution weakening low-nM binding by at least several-fold. DNA binding kinetics for head group-substituted PA14 exhibit biphasic traces. The head groups have no effect on a diffusion-controlled binding event, but result in a newly observed, concentration-independent slower phase. In contrast, head group substitutions significantly weaken DNA binding for PA20, and in the case of PA20-Guan, by 1000-fold. Head group substitution results in a faster first binding event for PA20, and a newly detected, second, slower binding event. Head group substitutions do not significantly alter dissociation decay constants for the PA14 series, with little effect across the PA20 series. Perturbations in pM to low nM PA-DNA binding affinities have little to no impact on μM IC₅₀ and IC₉₀ values. However there is a positive correlation between long residence times and antiviral behavior.