Laurence Hurley

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.

PMID: 1782349;Abstract:

Analysis of the anomalous migration in electrophoretic mobilities of (+)-CC-1065-modified oligomers following ligation reveals that (+)-CC-1065 induces DNA bending and winding of the helix. (+)-CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. This drug selectively bonds covalently to N3 of adenine and lies in the minor groove of DNA, reacting in a highly sequence-selective manner. Structurally, (+)-CC-1065 consists of three subunits: two identical pyrroloindole units (subunits B and C) and a third subunit containing the DNA-reactive cyclopropane ring (subunit A). While the bonding reaction is the main determinant of DNA sequence selectivity of (+)-CC-1065, binding interactions between the inside edge substituents of the B and C subunits and the floor of the minor groove of DNA can modulate or fine tune this sequence selectivity [Hurley, L. H., Lee, C.-S., McGovren, J. P., Mitchell, M. A., Warpehoski, M. A., Kelly, R. C., & Aristoff, P. A. (1988) Biochemistry 27, 3886-3892]. The A subunit of (+)-CC-1065 is responsible for the bending of DNA, and close van der Waals contacts between the inside edge of (+)-CC-1065 and the floor of the minor groove of DNA cause winding equivalent to about 1 base pair per alkylation site and stiffening of DNA. The magnitude of DNA bending induced by (+)-CC-1065 and related compounds is about 14-19°, which is equivalent to that produced by an adenine-thymine tract of about 5-6 base pairs in length. Experiments using oligomers containing both an adenine tract and a unique (+)-CC-1065 bonding site approximately one helix turn apart demonstrate that the directionality of drug-induced bending is in toward the minor groove and the locus of bending is about 2-3 base pairs to the 5′-side of the covalently modified adenine. A circularization efficiency assay shows that the optimum size of circles produced by (+)-CC-1065 and related drugs is between 168 and 180 base pairs. These results are discussed in relation to the molecular basis of the DNA sequence selectivity of (+)-CC-1065, and the (+)-CC-1065-induced DNA bending is compared with the intrinsic bending associated with adenine tracts. Since (+)-CC-1065 induces effects on local DNA structure that appear similar to those produced naturally by adenine tracts and certain DNA binding proteins, the relevance of this phenomenon to biological effects of (+)-CC-1065 and related drugs is considered. © 1991 American Chemical Society.

Abstract:

The sequence specificity of bizelesin, an interstrand DNA-DNA cross-linker related to the monoalkylating compound (+)-CC-1065, was studied using restriction enzyme fragments. Bizelesin, like (+)-CC-1065, forms monoalkylation adducts through N3 of adenine but can also form DNA-DNA cross-links six base pairs apart on opposite strands. Compared to many other minor groove cross-linking compounds, bizelesin is very efficient at cross-linking DNA. There is a higher than expected proportion of cross-linked adducts based upon the relative number of cross-linked vs monoalkylated adducts. This is rationalized based upon the relative thermodynamic stability of the cross-linked vs monoalkylated species. Where bizelesin monoalkylation occurs, the sequence specificity is significantly higher than those of (+)-CC-1065 and other monoalkylating (+)-CPI analogs. The bizelesin GC tolerance at cross-linking sites is twice as high as for the monoalkylation sites. This increased GC tolerance can be largely explained by the covalent immobilization of the second alkylation arm at sequences that are not normally reactive toward CPI monoalkylation compounds but are made reactive due to a proximity effect. This same rationale can be used to explain the reactivity of the second alkylation arm of bizelesin with guanine, cytosine, and thymine on some sequence. There are some sequences that appear to be unusual in their reactivity with bizelesin in that bizelesin formed cross-linking spanning seven base pairs, and bizelesin forms monoakylation adducts on guanine. In these cases, it is proposed that bizelesin may trap out rare conformational forms during the second alkylation step, or bizelesin may alkylate unusual sites due to the strong precovalent affinity of bizelesin for those sites.

PMID: 20657837;PMCID: PMC2906509;Abstract:

The GC-rich nuclease hypersensitivity element III1 (NHE III1) of the c-MYC promoter largely controls the transcriptional activity of the c-MYC oncogene. The C-rich strand in this region can form I-motif DNA secondary structures. We determined the folding pattern of the major I-motif formed in the NHE III1, which can be formed at near-neutral pH. While we find that the I-motif formed in the four 3′ consecutive runs of cytosines appears to be the most favored, our results demonstrate that the C-rich strand of the c-MYC NHE III1 exhibits a high degree of dynamic equilibration. Using a trisubstituted oligomer of this region, we determined the formation of two equilibrating loop isomers, one of which contains a flipped-out cytosine. Our results indicate that the intercalative cytosine⁺-cytosine base pairs are not always necessary for an intramolecular Imotif. The dynamic character of the c-MYC I-motif is intrinsic to the NHE III¹ sequence and appears to provide stability to the c-MYC I-motif. © 2010 Dai et al.