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

Abstract

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 R_a and R_b arising from the nonequivalent PIX C6 and C9 2-aminoethyl side chains. In dsDNA formation of R_a was favored. In ssDNA, both products formed equally. Structures of reduced R_a and R_b 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 R_a less intercalated than R_b. The preferential formation of R_a vs R_b in dsDNA and deeper intercalation of R_b may be explained by the differential ability of the asymmetric PIX N2 nitrogen in R_a vs R_b to hydrogen bond with the exocyclic N⁴-amine of the cytosine displaced into the major groove. MD simulations in dsDNA with R_a and R_b 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 R_a and R_b conjugates. Binding energy analyses yielded values of −108.1 (±9.7) kcal/mol and −102.2 (±8.3) kcal/mol for the intercalated R_a and R_b 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.