Modulation of DNA-polyamide interaction by ß-alanine substitutions: a study of positional effects on binding affinity, kinetics and thermodynamics.

Hairpin polyamides (PAs) are an important class of sequence-specific DNA minor groove binders, and frequently employ a flexible motif, ß-alanine (ß), to reduce the molecular rigidity to maintain the DNA recognition register. To better understand the diverse effects that ß can have on DNA-PA binding affinity, selectivity, and especially kinetics, which have rarely been reported, we have initiated a detailed study for an eight-heterocyclic hairpin PA and its ß derivatives with their cognate and mutant sequences. With these derivatives, all internal pyrroles of the parent PA are systematically substituted with single or double ßs. A set of complementary experiments have been conducted to evaluate the molecular interactions in detail: UV-melting, biosensor-surface plasmon resonance, circular dichroism and isothermal titration calorimetry. The ß substitutions generally weaken the binding affinities of these PAs with cognate DNA, and have large and diverse influences on PA binding kinetics in a position- and number-dependent manner. The DNA base mutations have also shown positional effects on the binding of a single PA. Besides the ß substitutions, the monocationic Dp group [3-(dimethylamino)propylamine] in parent PA has been modified into a dicationic Ta group (3,3'-diamino-N-methyldipropylamine) to minimize the frequently observed PA aggregation with ITC experiments. The results clearly show that the Ta modification not only maintains the DNA binding mode and affinity of PA, but also significantly reduces PA aggregation and allows the complete thermodynamic signature of eight-ring hairpin PA to be determined for the first time. This combined set of results significantly extends our understanding of the energetic basis of specific DNA recognition by PAs.

DNA Binding Polyamides and the Importance of DNA Recognition in their use as Gene-Specific and Antiviral Agents.

There is a long history for the bioorganic and biomedical use of N-methyl-pyrrole-derived polyamides (PAs) that are higher homologs of natural products such as distamycin A and netropsin. This work has been pursued by many groups, with the Dervan and Sugiyama groups responsible for many breakthroughs. We have studied PAs since about 1999, partly in industry and partly in academia. Early in this program, we reported methods to control cellular uptake of polyamides in cancer cell lines and other cells likely to have multidrug resistance efflux pumps induced. We went on to discover antiviral polyamides active against HPV31, where SAR showed that a minimum binding size of about 10 bp of DNA was necessary for activity. Subsequently we discovered polyamides active against two additional high-risk HPVs, HPV16 and 18, a subset of which showed broad spectrum activity against HPV16, 18 and 31. Aspects of our results presented here are incompatible with reported DNA recognition rules. For example, molecules with the same cognate DNA recognition properties varied from active to inactive against HPVs. We have since pursued the mechanism of action of antiviral polyamides, and polyamides in general, with collaborators at NanoVir, the University of Missouri-St. Louis, and Georgia State University. We describe dramatic consequences of ß-alanine positioning even in relatively small, 8-ring polyamides; these results contrast sharply with prior reports. This paper was originally presented by JKB as a Keynote Lecture in the 2nd International Conference on Medicinal Chemistry and Computer Aided Drug Design Conference in Las Vegas, NV, October 2013.