Laurence Hurley

Abstract:

Gel electrophoresis analysis of CPI-I bisalkylation of a 21-mer duplex containing 5′-TAA²TTA¹-3′ (the palindromic preferred cross-linking sequence of the (+)-CC-1065 analog Bizelesin) shows same-strand (strand one) alkylation of first A¹ and then A² instead of the anticipated symmetrical A¹ alkylation of strands one and two. Two-dimensional NMR analyses (NOESY and COSY) confirm the head-to-tail minor groove orientation of the same-strand-bound drugs. CPI-I contrasts sharply with Bizelesin (two CPI-I units linked tail-to-tail by a ureadiyl "linker"), which symmetrically cross-links this sequence at A¹ (strands one and two), but only by first rearranging the duplex structure and consequently removing the duplex distortion stemming from monoadduct formation. CPI-I induces no such major DNA rearrangement prior to or during bisalkylation. Why does CPI-I react with the adenines of only a single strand? Two possible causes for the unexpected strand one A² alkylation are, first, retardation of strand two A' site's reactivity by focusing of monoadduct conformational distortion on this site and, second, elevation of A² reactivity above other competing adenine sites due to unusual monoadduct strand one A²T-step conformational properties. The relative importance of these two nonmutually exclusive factors was investigated using gel electrophoresis experiments: Time-course CPI-I bisalkylation studies were conducted on the AT-step sequence 5′-TAA²TTA1-3′ and an A-tract sequence, 5′-TAA²AAA¹-3′, to see if the former sequence's AT-step flexibility, high base-pair opening rate, and unwinding capability (traits not shared by the latter sequence) controlled selection of the second target site. The observed parallel AT-step and A-tract sequence A¹ and A² bisalkylation patterns suggest that AT-step properties play at best a secondary role (compared to 5′-end TA-step behavior) in directing the second alkylation reaction to the AT-step site. rMD (solvated) simulations of the AT-step and A-tract monoadducts display distortion that is focused on this 5′-end TA-step site. While two-dimensional ¹H NMR spectra of the final bisadduct reveal no significant TA-step conformational distortion, they demonstrate that conformational adjustment at the A² ligand attachment site diminishes head-to-tail steric clash of the two drugs. These results suggest that the CPI-I monoadduct propagates bending distortion (to the 5′-side) through five base pairs toward the TA-step junction site. In the AT-step and A-tract sequences, neither adenine straddling this TA-step junction site is alkylated by CPI-I, suggesting that the base pairs forming the junction site are distorted away from a suitable orientation or are unable to assume a conformation suitable for alkylation. Consequently, the second alkylation occurs at a site (AT-step) that requires a modest displacement of the second ligand away from the already attached drug. The results and analysis of the data included in this paper provide important lessons for the design of inter- and intrastrand DNA-DNA cross-linkers.

MYC is deregulated in most tumour types, but an effective means to selectively target its aberrant expression is not yet available. Supercoiling that is induced by transcription has been demonstrated to have dynamic effects on DNA in the MYC promoter element: it converts duplex DNA to non-duplex DNA structures, even at considerable distances from the transcriptional start site. These non-duplex DNA structures, which control both turning on and off of transcription and the rate of transcription firing, are amenable to small-molecule targeting. This dynamic system provides a unique opportunity for the treatment of tumours in which MYC is an important oncogene.

Abstract:

The quinobenzoxazines are a group of topoisomerase II catalytic inhibitors that have demonstrated promising anticancer activity in mice. They have been proposed to form an unprecedented 2:2 drug-Mg²⁺ self-assembly complex on DNA. We have exploited the photochemical decomposition of the quinobenzoxazines to gain further support and insights into the nature of 2:2 quinobenzoxazine-Mg²⁺ dimers and the 2:2 drug-Mg²⁺ complex on duplex DNA. The quinobenzoxazine A-62176 undergoes photodecomposition to highly fluorescent products. Methyl viologen (MV²⁺) accelerates this photoreaction almost 500-fold. The formation of 2:2 drug-Mg²⁺ dimers in solution is deduced from the Mg²⁺-dependent difference in the MV²⁺-facilitated photoreaction rates of racemic and scalemic A-62176. However, both racemic and scalemic A-62176 have identical MV²⁺-facilitated photoreaction rates in the presence of Mg²⁺ and the achiral fluoroquinolone norfloxacin, due to heterochemical norfloxacin/A-62176 dimer complex formation. DNA also accelerates the photochemical decomposition of A-62176 up to 80-fold. This DNA-acceleration requires Mg²⁺, duplex DNA, molecular oxygen, and intercalation of the drug into the DNA duplex. In the proposed model for drug-DNA complexation, only one drug molecule of each 2:2 drug-Mg²⁺ dimer intercalates into the DNA duplex, the other molecule binds externally to the DNA. Norfloxacin, which can only play the external binding role, was able to modulate the photochemical reaction of the quinobenzoxazines on DNA. Furthermore, it appears that the precise positioning of the intercalated molecule, which is modulated by the structure and stereochemistry of the externally bound molecule, plays an important role in determining the rate of photoreaction on DNA. The implications of the observed photochemical reaction of the quinobenzoxazines are described for human phototoxicity, photodynamic therapy, mechanism of action studies, and improved drug design for both topoisomerase and gyrase inhibitors.