The in vitro interaction of naphthyridinomycin with deoxyribonucleic acids.

The binding of naphthyridinomycin (NAP) to deoxyribonucleic acid was investigated using radioisotope labeled antibiotic. Dithiothreitol (DTT) enhances complex formation in a concentration dependent fashion but was found to be slightly inhibitory at concentrations above 10 mM. [C3H3]-NAP-DNA complexes, formed in the presence or absence of reducing reagents, were stable to Sephadex G-25 chromatography and precipitation with ethanol, indicating a strong bond formed between the drug and DNA. Time course studies showed that the difference between the binding of activated and non-activated antibiotic was a DTT-dependent burst. This was followed by a second phase of binding which was similar in both the activated and non-activated antibiotics. The activation of the antibiotic by DTT was a reversible reaction at pH 7.9. The activated form at pH 5.0 was extremely stable and did not revert to the unactivated form even after an 8-h incubation period. Antibiotic-DNA complex formation was pH independent between pH 5.0 and 7.0 for activated NAP. The non-activated antibiotic bound to DNA much better at pH 5.0 than at physiological pH values. Release of antibiotic from complexes (as followed by long term dialysis) formed in the presence of DTT and at pH 5.0 was biphasic, suggesting that the drug can bind to DNA in more than one way. A constant rate of antibiotic release was observed at pH 7.9 with or without DTT. At pH 2.0 and pH 12.0, greater than 95% of the antibiotic is released from the complexes. Most of the acid released antibiotic is NAP while most of the base released antibiotic had decomposed to a more polar compound. NAP binds well to calf thymus DNA, poly(dG) . poly(dC), and T4 DNA but shows significantly less affinity for poly(dA) . poly(dT), poly(dA . dT) . poly(dA . dT), poly(dG), poly(dC), poly(dI) . poly(dC) or poly(dG . dC) . poly(dG . dC). This specificity of NAP for DNA is similar to that observed for the pyrrolo(1,4)benzodiazepine antibiotics and saframycin A and S; all of which bind to double stranded DNA through their carbinolamine or masked carbinolamine functionalities. Two mechanisms which can explain the need for activation of NAP are also proposed.