Characterization of Regioisomeric Conjugates Arising from Pixantrone at AP Sites in DNA

Alkylation at N7 of guanine leads to formation of abasic (AP) sites in DNA. These equilibrate between α and β anomers and form conjugates with 6,9-bis[(2-aminoethyl)amino]benzo[g]isoquinoline-5,10-dione (Pixantrone, PIX). NMR revealed that these conjugates formed as regioisomeric Schiff base products Ra and Rb arising from the nonequivalent PIX C6 and C9 2-aminoethyl side chains. In dsDNA formation of Ra was favored. In ssDNA, both products formed equally. Structures of reduced Ra and Rb conjugates from molecular dynamics calculations restrained by 1H NOESY data in 5'-d(C1A2G3A4C5T6X7A8G9A10C11T12)-3':5'-d(A13G14T15C16T17C18A19G20T21C22T23G24)-3', X = reduced PIX-AP site conjugate, showed that both regioisomers intercalated on the 3' side of base pair T6/A19, displacing C18 into the major groove, with Ra less intercalated than Rb. The preferential formation of Ra vs Rb in dsDNA and deeper intercalation of Rb may be explained by the differential ability of the asymmetric PIX N2 nitrogen in Ra vs Rb to hydrogen bond with the exocyclic N4-amine of the cytosine displaced into the major groove. MD simulations in dsDNA with Ra and Rb reduced PIX AP site conjugates revealed that for both, the isoquinoline first reoriented within the minor groove. The complementary cytosine rotated toward the major groove, allowing intercalation of the Ra and Rb conjugates. Binding energy analyses yielded values of -108.1 (±9.7) kcal/mol and -102.2 (±8.3) kcal/mol for the intercalated Ra and Rb conjugates, respectively. A crystal structure in the Dickerson-Drew Dodecamer at 2.5 Å resolution revealed looped-out PIX stacking with PIX from a neighboring duplex.