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 marine natural product ecteinascidin 743 (Et 743) is currently in phase II clinical trials. We have undertaken parallel structural and modeling studies of an Et 743-(N2-guanine) 12-mer DNA adduct and an adduct involving the structurally related Et 736 of the same sequence in order to ascertain the structural basis for the ecteinascidin-DNA sequence selectivity. In contrast to the C-subunit differences found in Et 736 and Et 743, they have identical A-B-subunit scaffolds, which are the principal sites of interaction with DNA bases. These identical scaffolds generate parallel networks of drug- DNA hydrogen bonds that associate the drugs with the three base pairs at the recognition site. We propose that these parallel hydrogen bonding networks stabilize the Et 736 and Et 743 A- and B-subunit prealkylation binding complex with the three base pairs and are the major factors governing sequence recognition and reactivity. The possibility that a unique hydrogen- bonding network directs the course of sequence recognition was examined by first characterizing the hydrogen-bonding substituents using ¹H NMR properties of the exchangeable protons attached to the hydrogen-bond donor and other protons near the proposed acceptor. Using these experimental findings as indicators of hydrogen bonding, Et 736-12-mer duplex adduct models (binding and covalent forms) containing the favored sequences 5'-AGC and 5'-CGG were examined by molecular dynamics (MD) in order to evaluate the stability of the hydrogen bonds in the resulting conformations. The MD- generated models of these favored sequences display optimal donor/acceptor positions for maximizing the number of drug-DNA hydrogen bonds prior to covalent reaction. The results of MD analysis of the carbinolamine (binding) forms of the sequences 5'-GGG (moderately reactive) and 5'-AGT (poorly reactive) suggested reasons for their diminished hydrogen-bonding capability. These experimental and modeling results provide the structural basis for the following sequence specificity rules: For the target sequence 5'-XGY, the favored base to the 3'-side, Y, is either G or C. When Y is G, then a pyrimidine base (T or C) is favored for X. When Y is C, a purine (A or G) is favored for X.