Sequence specificity, enantiospecificity and polyelectrolyte effect in the binding to DNA of a 6-(2-pyridyl)phenanthridine chiral photonuclease.
In order to establish the basis for the rational design of a novel family of intercalating chiral photonuclease drugs aimed at photochemotherapy, namely N, N'-dialkylated 6-(2-pyridinium)phenanthridinium (pyp) dications, a detailed investigation of the DNA binding of the dq2pyp member (where dq2 stands for -CH2CH2-), was conducted. The study addresses the sequence- and enantiospecificity, as well as polyelectrolyte effects in the drug-DNA interaction. Binding isotherms with synthetic polynucleotides, forcefield calculations, affinity chromatography in a DNA-cellulose stationary phase and salt-dependent equilibrium and kinetic studies with DNA were used. dq2pyp shows a strong preference for alternating GC over AT base pairs; binding to homopolymeric DNA is weak (< 3 x 10(4) M-1). Affinity chromatography shows enantiospecific binding of dq2pyp to DNA. The polyelectrolyte contribution to the binding free energy are shown to be relatively important (-4.8 kcal/nmol out of an overall value of -7.2 kcal/mmol at 10.2 mM Na+). The slope of the logkd (dissociation rate constant) vs. log[Na+] plot (0.7) agrees with the values predicted from counterion condensation theory for a dicationic intercalator, giving further support to such a DNA binding mode for dq2pyp. The relatively high kinetic dissociation constants (logkd = 0.70log[Na+] + 3.79) in comparison with those of propidium (two orders of magnitude larger at any Na+ concentration) seems to originate from the absence of amino groups in dq2pyp. The kinetic association constants (logka = -1.06log[Na+] + 5.53) are twice these of propidium, probably due to the less restrictive positioning of dq2pyp at the intercalation site. The kinetic studies support a mechanism of intercalation in which the drug forms a pre-equilibrium outside the complex followed by the intercalation of the drug. Molecular modelling is used throughout to rationalize all the experimental data, as well as to propose new candidates with improved DNA affinity and residence time.
Colmenarejo
G
G
Departamento de Bioquímica y Biologia Molecular I, Universidad Complutense de Madrid, Spain.
Montero
F
F
Orellana
G
G
D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D004247:DNA / Q000187:drug effects / Q000378:metabolism*; D015195:Drug Design; D004573:Electrolytes / Q000378:metabolism / Q000494:pharmacology; D004734:Energy Metabolism; D007364:Intercalating Agents / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D010617:Phenanthridines / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D017319:Photosensitizing Agents / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011119:Polynucleotides / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014755:Viologens / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology
D004573:Electrolytes; D007364:Intercalating Agents; C094137:N,N'-ethylene-6-(2-pyridinium)phenanthridinium; D010617:Phenanthridines; D017319:Photosensitizing Agents; D011119:Polynucleotides; D014755:Viologens; C014740:phenanthridinium; D004247:DNA