Laurence Hurley

PMID: 17172304;PMCID: PMC1861781;Abstract:

Regulation of the structural equilibrium of G-quadruplex-forming sequences located in the promoter regions of oncogenes by the binding of small molecules has shown potential as a new avenue for cancer chemotherapy. In this study, microcalorimetry (isothermal titration calorimetry and differential scanning calorimetry), electronic spectroscopy (ultraviolet-visible and circular dichroism), and molecular modeling were used to probe the complex interactions between a cationic porphryin mesotetra (N-methyl-4-pyridyl) porphine (TMPyP4) and the c-MYC PU 27-mer quadruplex. The stoichiometry at saturation is 4:1 mol of TMPyP4/c-MYC PU 27-mer G-quadruplex as determined by isothermal titration calorimetry, circular dichroism, and ultraviolet-visible spectroscopy. The four independent TMPyP4 binding sites fall into one of two modes. The two binding modes are different with respect to affinity, enthalpy change, and entropy change for formation of the 1:1 and 2:1, or 3:1 and 4:1 complexes. Binding of TMPyP4, at or near physiologic ionic strength ([K⁺] = 0.13 M), is described by a "two-independent-sites model." The two highest-affinity sites exhibit a K1 of 1.6 x 10⁷ M^-1 and the two lowest-affinity sites exhibit a K2 of 4.2 x 10⁵ M ^-1. Dissection of the free-energy change into the enthalpy- and entropy-change contributions for the two modes is consistent with both "intercalative" and "exterior" binding mechanisms. An additional complexity is that there may be as many as six possible conformational quadruplex isomers based on the sequence. Differential scanning calorimetry experiments demonstrated two distinct melting events (Tm1 = 74.7°C and Tm2 = 91.2°C) resulting from a mixture of at least two conformers for the c-MYC PU 27-mer in solution. © 2007 by the Biophysical Society.

Abstract:

TDP (TATA binding protein), the primary transcription factor tliat recruits subsequent transcription machinery, binds io the TATA box through the extensive minor groove contacts and locally bends ihe DNA. This protein-induced distortion transiently creates an unv-ound site on the downstream side of the TATA box. and is favorably seized by piuramyrins, a group of novel threading intercalating arititumor antibiotics. To understand the detail of the dynamics of Ihe TATA box upon TBP binding, we have investigated the TUP-1 ATA box complexes using D Nase I arid piuramyrins. D Nase I foot print ing ex periinpnts revealed overdigested pattern rather than protection on both the if half A tract of the TATA box and the downstream flanking sequences at low i oncen t rations of protein, implying the unusual déformai ion of t he minor groove. A downstream ba.se-pair step in the protein-DNA complex showed the enhanced modification by a.11 pluramycins. However, pluramycins that have distinct, sugar substituants alkylated different guanines in the same base-pair step (CG : GC), indicating t hat the sugar substituents modulated orientations of ihe drugs. Taken together, it is proposed that the asymmetric recognition of the TBP-TATA complex originates trom the preferred distortion on the 3′ half A tract of the TATA box and that the propagated distortion results in the un winding of the specific downstream base-pair step. Those results also suggest the potential of pluramycins as molecular probes that detect uniquely deformed DNA duplexes through the specifii positioning of sugar substituents.

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.