Short Lexitropsin that Recognizes the DNA Minor Groove at 5‘-ACTAGT-3‘:  Understanding the Role of Isopropyl-thiazole

Abstract

Isopropyl-thiazole ((iPr)Th) represents a new addition to the building blocks of nucleic acid minor groove-binding molecules. The DNA decamer duplex d(CGACTAGTCG)(2) is bound by a short lexitropsin of sequence formyl-PyPy(iPr)Th-Dp (where Py represents N-methyl pyrrole, (iPr)Th represents thiazole with an isopropyl group attached, and Dp represents dimethylaminopropyl). NMR data indicate ligand binding in the minor groove of DNA to the sequence 5'-ACT(5)AG(7)T-3' at a 2:1 ratio of ligand to DNA duplex. Ligand binding, assisted by the enhanced hydrophobicity of the (iPr)Th group, occurs in a head-to-tail fashion, the formyl headgroups being located toward the 5'-ends of the DNA sequence. Sequence reading is augmented through hydrogen bond formation between the exocyclic amine protons of G(7) and the (iPr)Th nitrogen, which lies on the minor groove floor. The B(I)/B(II) DNA backbone equilibrium is altered at the T(5) 3'-phosphate position to accommodate a B(II) configuration. The ligands bind in a staggered mode with respect to one another creating a six base pair DNA reading frame. The introduction of a new DNA sequence-reading element into the recognition jigsaw, combined with an extended reading frame for a small lexitropsin with enhanced hydrophobicity, holds great promise in the development of new, potentially commercially viable drug lead candidates for gene targeting.

Ranking Ligand Affinity for the DNA Minor Groove by Experiment and Simulation

“…Analysis of the 1 H NMR data for the complex between 1 and 5 0 -CGACTAGTCG-3 0 ( Figure 1a) had already established that minor groove binding occurs with a staggered 2:1, head-totail, side-by-side binding motif 16,17 at the indicated (underlined) reading frame. 11 The same characteristic NMR resonance pattern also occurs for the binding of 1 to both (Figure 1b,c, respectively), all of which produce outstanding quality twodimensional (2D) nuclear Overhauser effect spectroscopy (NOESY) NMR data sets. In stark contrast, the binding of 1 to 5 0 -d(CGCCTAGGCG)-3 0 can at best be described as "poor", being characterized by broad NMR resonances (Figure 1d) and ill-defined cross-peaks in 2D NOESY NMR spectra.…”

Recognition of the DNA Minor Groove by Thiazotropsin Analogues

“…[14] Further structural and energetic investigations by NMR spectroscopy and ITC for the same ligand with other DNA sequences revealed additional key features that were responsible for stable complex formation and factors (particularly steric) that readily destabilise complex formation. [17] In an extension to these studies, we report here structures of the DNA complexes formed by both thiazotropsin B and AIK-18/51, thus allowing comparisons between all three complexes to be made.…”

“…Their free solution behaviour and their respective DNA complexes have been studied to establish how structural and functional alterations to the ligand affect the formation and stability of their DNA complexes. DNase I footprinting, [13] ITC [20] and NMR spectroscopy [14] studies previously showed thiazotropsin A to bind to the hexanucleotide 5'-ACTAGT-3'. Replacing one of the N-methylpyrrole groups of thiazotropsin A with N-methylimidazole, to form thiazotropsin B, changes the preferred binding sequence to 5'-(A/T)CGCG(T/A)-3'.…”

“…[10] Similar to many classical examples of lexitropsin/ DNA complexes, [11,12] our studies show this class of ligand (which contains the isopropyl-thiazole building block for enhanced lipophilicity) to bind as antiparallel, face-to-face dimers in the DNA minor groove. [13][14][15][16][17] These ligands and their later generation analogues aggregate in free aqueous solution. The extent of aggregation depends on ligand structure.…”

Dimerization Energetics of DNA Minor Groove Binders

“…In this work, we carried out the energy analysis of the molecular binding between seven MGB ligands, the complex structures of which were studied relatively well [10][11][12][13][14][15][16], and DNA following the dimer mechanism. This makes it possible to carry out the energy calculations for dimer structures corresponding to nuclear magnetic resonance (NMR) or X-ray diffraction (XRD) analysis data and directly "cut out" from experimentally determined dimer-DNA structures.…”