DNA recognition by lexitropsins,minor groove binding agents

Consideration is given to alternative approaches to the development of DNA sequences selective binding agents because of their potential applications in diagnosis and treatment of cancer as well as in molecular biology. The concept of lexitropsins, or information-reading molecules, is introduced within the antigene strategy as an alternative to, and complementary with, the antigene approach for cellular intervention and gene control The chemical, physical and paharmacological factors involved in the design of effective lexitropsins are discussed and illustrated with experimental results. Among the factors contributing to the molecular recognition processes are: the presence and disposition of hydrogen bond accepting and donating groups, ligand shape, chirality, stereochemistry, flexibility and charge. For longer ligands, such as are required to target unique sequences in biological systems (14–16 base pairs), the critical feature is the phasing or spatial corresponding between repeat units in the ligand and receptor. The recently discovered 2:1 lexitropsin-DNA binding motif provides a further refinement in molecular recognition in permitting discrimination between GC and CG base pairs. The application of these factors in the design and synthesis of novel agents which exhibits anticancer, antiviral and antitretroviral properties, and inhibition of critical cellular enzymes including topoisomerases is discussed. The emerging evidence of a relationship between sequence selectivity of the new agents and the biological responses they invoke is also described.     

DNA minor groove binding of cross-linked lexitropsins: experimental conditions required to observe the covalently linked WPPW (groove wall-peptide-peptide-groove wall) motif.

A theoretical analysis of binding interactions between covalently cross-linked lexitropsins and DNA is undertaken, in which a novel cyclic symmetric 2:2 dimeric lexitropsin-DNA-binding model is proposed. Applicability of commonly used techniques including NMR, quantitative footprinting, CD, and ethidium fluorometry to differentiate the covalently linked WPPW (groove Wall-Peptide-Peptide-groove Wall) from a 2:2 cross-linked lexitropsin-DNA duplex structure is examined.     

Mechanistic studies on DNA damage by minor groove binding copper-phenanthroline conjugates

Copper–phenanthroline complexes oxidatively damage and cleave nucleic acids. Copper bis-phenanthroline and copper complexes of mono- and bis-phenanthroline conjugates are used as research tools for studying nucleic acid structure and binding interactions. The mechanism of DNA oxidation and cleavage by these complexes was examined using two copper–phenanthroline conjugates of the sequence-specific binding molecule, distamycin. The complexes contained either one or two phenanthroline units that were bonded to the DNA-binding domain through a linker via the 3-position of the copper ligand. A duplex containing independently generated 2-deoxyribonolactone facilitated kinetic analysis of DNA cleavage. Oxidation rate constants were highly dependent upon the ligand environment but rate constants describing elimination of the alkali-labile 2-deoxyribonolactone intermediate were not. Rate constants describing DNA cleavage induced by each molecule were 11–54 times larger than the respective oxidation rate constants. The experiments indicate that DNA cleavage resulting from β-elimination of 2-deoxyribonolactone by copper–phenanthroline complexes is a general mechanism utilized by this family of molecules. In addition, the experiments confirm that DNA damage mediated by mono- and bis-phenanthroline copper complexes proceeds through distinct species, albeit with similar outcomes.     

DAPI binding to the DNA minor groove: a continuum solvent analysis

A continuum solvent model based on the generalized Born (GB) or finite-difference Poisson-Boltzmann (FDPB) approaches has been employed to compare the binding of 4'-6-diamidine-2-phenyl indole (DAPI) to the minor groove of various DNA sequences. Qualitative agreement between the results of GB and FDPB approaches as well as between calculated and experimentally observed trends regarding the sequence specificity of DAPI binding to B-DNA was obtained. Calculated binding energies were decomposed into various contributions to solvation and DNA-ligand interaction. DNA conformational adaptation was found to make a favorable contribution to the calculated total interaction energy but did not change the DAPI binding affinity ranking of different DNA sequences. The calculations indicate that closed complex formation is mainly driven by nonpolar contributions and was found to be disfavored electrostatically due to a desolvation penalty that outbalances the attractive Coulomb interaction. The calculated penalty was larger for DAPI binding to GC-rich sequences compared with AT-rich target sequences and generally larger for the FDPB vs the GB continuum model. A radial interaction profile for DAPI at different distances from the DNA minor groove revealed an electrostatic energy minimum a few Angstroms farther away from the closed binding geometry. The calculated electrostatic interaction up to this distance is attractive and it may stabilize a nonspecific binding arrangement.     

A new protein domain for binding to DNA through the minor groove.

Protein p6 of the Bacillus subtilis phage phi 29 binds with low sequence specificity to DNA through the minor groove, forming a multimeric nucleoprotein complex that activates the initiation of phi 29 DNA replication. Deletion analysis suggested that the N-terminal part of protein p6, predicted to form an amphipathic alpha-helix, is involved in DNA binding. We have constructed site-directed mutants at the polar side of the putative alpha-helix. DNA binding and activation of initiation of phi 29 DNA replication were impaired in most of the mutant proteins obtained. A 19 amino acid peptide comprising the N-terminus of protein p6 interacted with a DNA fragment containing high-affinity signals for protein p6 binding with approximately 50-fold higher affinity than the peptide corresponding to an inactive mutant. Both wild-type peptide and protein p6 recognized the same sequences in this DNA fragment. This result, together with distamycin competition experiments, suggested that the wild-type peptide also binds to DNA through the minor groove. In addition, CD spectra of the wild-type peptide showed an increase in the alpha-helical content when bound to DNA. All these results indicate that an alpha-helical structure located in the N-terminal region of protein p6 is involved in DNA binding through the minor groove.     

DNA sequence preferences of several AT-selective minor groove binding ligands.

We have examined the interaction of distamycin, netropsin, Hoechst 33258 and berenil, which are AT-selective minor groove-binding ligands, with synthetic DNA fragments containing different arrangements of AT base pairs by DNase I footprinting. For fragments which contain multiple blocks of (A/T)4 quantitative DNase I footprinting reveals that AATT and AAAA are much better binding sites than TTAA and TATA. Hoechst 33258 shows that greatest discrimination between these sites with a 50-fold difference in affinity between AATT and TATA. Alone amongst these ligands, Hoechst 33258 binds to AATT better than AAAA. These differences in binding to the various AT-tracts are interpreted in terms of variations in DNA minor groove width and suggest that TpA steps within an AT-tract decrease the affinity of these ligands. The behaviour of each site also depends on the flanking sequences; adjacent pyrimidine-purine steps cause a decrease in affinity. The precise ranking order for the various binding sites is not the same for each ligand.     

Mode of reversible binding of neocarzinostatin chromophore to DNA: evidence for binding via the minor groove

Two general approaches have been taken to understand the mechanism of the reversible binding of the nonprotein chromophore of neocarzinostatin to DNA: (1) measurement of the relative affinity of the chromophore for various DNAs that have one or both grooves blocked by bulky groups and (2) studies on the influence of adenine-thymine residue-specific, minor groove binding agents such as the antibiotics netropsin and distamycin on the chromophore-DNA interaction. Experiments using synthetic DNAs containing halogen group (Br, I) substituents in the major groove or natural DNAs with glucosyl moieties projecting into the major groove show that obstruction of the major groove does not decrease the binding stoichiometry or the binding constant for the DNA-chromophore interaction. Chemical methylation of bases in both grooves of calf thymus DNA, resulting in 13% methylation of N-7 of guanine in the major groove and 7% methylation of N-3 of adenine in the minor groove, decreases the binding affinity and increases the size of the binding site for neocarzinostatin chromophore. Similar results were obtained whether binding parameters were determined directly by spectroscopic measurements or indirectly by measuring the ability of the DNA to protect the chromophore against degradation. On the other hand, netropsin and distamycin compete with neocarzinostatin chromophore for binding to the minor groove of DNA, as shown by their decrease in the ability of poly(dA-dT) to protect the chromophore against degradation and their reduction in chromophore-induced DNA damage as measured by thymine release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modeling DNA deformations induced by minor groove binding proteins

Molecular modeling is used to demonstrate that the major structural deformations of DNA caused by four different minor groove binding proteins, TBP, SRY, LEF-1, and PurR, can all be mimicked by stretching the double helix between two 3'-phosphate groups flanking the binding region. This deformation reproduces the widening of the minor groove and the overall bending and unwinding of DNA caused by protein binding. It also reproduces the principal kinks associated with partially intercalated amino acid side chains, observed with such interactions. In addition, when protein binding involves a local transition to an A-like conformation, phosphate neutralization, via the formation of protein-DNA salt bridges, appears to favor the resulting deformation.     

The width of the minor groove affects the binding of the bisquaternary heterocycle SN-6999 to duplex DNA.

A complex between d(GGGAAAAACGG).d(CCGTTTTTCCC) and the minor groove binding drug SN-6999 has been studied by 1H nuclear magnetic resonance spectroscopy. The drug is found to bind in the d(A)5 tract, but with interactions extending one residue in the 3'-direction along each strand. Doubling of resonances in the complex indicates slow to intermediate exchange between two binding modes. An orientational preference (7:3) is found, the first such example in an SN-6999 complex. Furthermore, the upper limit of the lifetime for the major species is longer than was found for SN-6999 with other DNA duplexes. The preferred orientation of SN-6999 has the pyridinium ring near the 5'-end of the (+) strand; the minor binding mode has the reverse orientation. The orientational preference and slower exchange rate relative to other SN-6999 complexes is attributed to increased stabilization from van der Waals interactions due to better shape complementarity between the DNA duplex and ligand. The comparison of these results with studies of SN-6999 complexed to other DNA duplexes reveals the sensitivity of the binding properties to the delicate interplay between the molecular structure of the ligand and the specific characteristics of the DNA minor groove.     

Hydration changes accompanying the binding of minor groove ligands with DNA

4',6-diamidino-2-phenylindole (DAPI), netropsin, and pentamidine are minor groove binders that have terminal -C(NH2)2+ groups. The hydration changes that accompany their binding to the minor groove of the (AATT)2 sequence have been studied using the osmotic stress technique with fluorescence spectroscopy. The affinity of DAPI for the binding site decreases with the increasing osmolality of the solution, resulting in acquisition of 35+/-1 waters upon binding. A competition fluorescence assay was utilized to measure the binding constants and hydration changes of the other two ligands, using the DNA-DAPI complex as the fluorescence reporter. Upon their association to the (AATT)2 binding site, netropsin and pentamidine acquire 26+/-3 and 34+/-2 additional waters of hydration, respectively. The hydration changes are discussed in the context of the terminal functional groups of the ligands and conformational changes in the DNA.     

Polyhydroxylated azepanes as new motifs for DNA minor groove binding agents.

The synthesis of 1,3-bis-[3,4,5,6-tetrahydroxyazepane-N-p-phenoxy] and 1,3-bis-[3,4,5,6-tetrahydroxyazepane-N-p-benzyloxy] propanes is reported. These compounds have been prepared to investigate the potential of incorporating iminosugars as useful recognition elements in DNA minor groove binding agents. The compounds were shown to have very moderate binding affinities for DNA in thermal denaturation and ethidium bromide displacement assays when compared with propamidine. They were also found to possess some in vitro anticancer activity that did not correlate with their DNA binding affinity.     

Out-of-shape DNA minor groove binders: induced fit interactions of heterocyclic dications with the DNA minor groove

DB921 and DB911 are benzimidazole-biphenyl isomers with terminal charged amidines. DB911 has a central meta-substituted phenyl that gives it a shape similar to those of known minor groove binding compounds. DB921 has a central para-substituted phenyl with a linear conformation that lacks the appropriate radius of curvature to match the groove shape. It is thus expected that DB911, but not DB921, should be an effective minor groove binder, but we find that DB921 not only binds in the groove but also has an unusually high binding constant in SPR experiments (2.9 x 10(8) M(-)(1), vs 2.1 x 10(7) M(-)(1) for DB911). ITC thermodynamic analysis with an AATT sequence shows that the stronger binding of DB921 is due to a more favorable binding enthalpy relative to that of DB911. CD results support minor groove binding for both compounds but do not provide an explanation for the binding of DB921. X-ray crystallographic analysis of DB921 bound to AATT shows that an induced fit structural change in DB921 reduces the twist of the biphenyl to complement the groove, and places the functional groups in position to interact with bases at the floor of the groove. The phenylamidine of DB921 forms indirect contacts with the bases through a bound water. The DB921-water pair forms a curved binding module that matches the shape of the minor groove and provides a number of strong interactions that are not possible with DB911. This result suggests that traditional views of compound curvature required for minor groove complex formation should be reevaluated.     

FTIR study of specific binding interactions between DNA minor groove binding ligands and polynucleotides.

The use of FTIR spectroscopy is made to study the interactions between polynucleotides and two series of minor groove binding compounds. The latter were developed and described previously as part of an ongoing program of rational design of modified ligands based on naturally occurring pyrrole amidine antibiotic netropsin, and varying the structure of bisbenzimidazole chromosomal stain Hoechst 33258. Characteristic IR absorptions due to the vibrations of thymidine and cytosine keto groups in polynucleotides containing AT and GC base pairs respectively are used to monitor their interaction with the added ligands. Although the two thiazole based lexitropsins based on netropsin structure differ in the relative orientation of nitrogen and sulfur atoms with respect to the concave edge of the molecules, they interact exclusively with the thymidine C2 = O carbonyl groups in the minor groove of the alternating AT polymer as evidenced by specific changes in the IR spectra. In the second series of compounds based on Hoechst 33258, the structure obtained by replacing the two benzimidazoles in the parent compound by a combination of pyridoimidazole and benzoxazole, exhibits changes in the carbonyl frequency region of poly dG.poly dC which is attributed to the ligand interaction at the minor groove of GC base pairs. In contrast, Hoechst 33258 itself interacts only with poly dA.poly dT. Weak or no interaction exists between the ligands and any of the polynucleotides at the levels of the phosphate groups or the deoxyribose units.     

The thermodynamics of polyamide-DNA recognition: hairpin polyamide binding in the minor groove of duplex DNA

Crescent-shaped synthetic ligands containing aromatic amino acids have been designed for specific recognition of predetermined DNA sequences in the minor groove of DNA. Simple rules have been developed that relate the side-by-side pairings of Imidazole (Im) and Pyrrole (Py) amino acids to their predicted target DNA sequences. We report here thermodynamic characterization of the DNA-binding properties of the six-ring hairpin polyamide, ImImPy-gamma-PyPyPy-beta-Dp (where gamma = gamma-aminobutyric acid, beta = beta-alanine, and Dp = dimethylaminopropylamide). Our data reveal that, at 20 degrees C, this ligand binds with a relatively modest 1.8-fold preference for the designated match site, 5'-TGGTA-3', over the single base pair mismatch site, 5'-TGTTA-3'. By contrast, we find that the ligand exhibits a 102-fold greater affinity for its designated match site relative to the double base pair mismatch site, 5'-TATTA-3'. These results demonstrate that the energetic cost of binding to a double mismatch site is not necessarily equal to twice the energetic cost of binding to a single mismatch site. Our calorimetrically measured binding enthalpies and calculated entropy data at 20 degrees C reveal the ligand sequence specificity to be enthalpic in origin. We have compared the DNA-binding properties of ImImPy-gamma-PyPyPy-beta-Dp with the hairpin polyamide, ImPyPy-gamma-PyPyPy-beta-Dp (an Im --> Py "mutant"). Our data reveal that both ligands exhibit high affinities for their designated match sites, consistent with the Dervan pairing rules. Our data also reveal that, relative to their corresponding single mismatch sites, ImImPy-gamma-PyPyPy-beta-Dp is less selective than ImPyPy-gamma-PyPyPy-beta-Dp for its designated match site. This result suggests, at least in this case, that enhanced binding affinity can be accompanied by some loss in sequence specificity. Such systematic comparative studies allow us to begin to establish the thermodynamic database required for the rational design of synthetic polyamides with predictable DNA-binding affinities and specificities.     

New complex of post-activated neocarzinostatin chromophore with DNA: bulge DNA binding from the minor groove

Neocarzinostatin (NCS-chrom), a natural enediyne antitumor antibiotic, undergoes either thiol-dependent or thiol-independent activation, resulting in distinctly different DNA cleavage patterns. Structures of two different post-activated NCS-chrom complexes with DNA have been reported, revealing strikingly different binding modes that can be directly related to the specificity of DNA chain cleavage caused by NCS-chrom. The third structure described herein is based on recent studies demonstrating that glutathione (GSH) activated NCS-chrom efficiently cleaves DNA at specific single-base sites in sequences containing a putative single-base bulge. In this structure, the GSH post-activated NCS-chrom (NCSi-glu) binds to a decamer DNA, d(GCCAGAGAGC), from the minor groove. This binding triggers a conformational switch in DNA from a loose duplex in the free form to a single-strand, tightly folded hairpin containing a bulge adenosine embedded between a three base pair stem. The naphthoate aromatic moiety of NCSi-glu intercalates into a GG step flanked by the bulge site, and its substituent groups, the 2-N-methylfucosamine carbohydrate ring and the tetrahydroindacene, form a complementary minor groove binding surface, mostly interacting with the GCC strand in the duplex stem of DNA. The bulge site is stabilized by the interactions involving NCSi-glu naphthoate and GSH tripeptide. The positioning of NCSi-glu is such that only single-chain cleavage via hydrogen abstraction at the 5'-position of the third base C (which is opposite to the putative bulge base) in GCC is possible, explaining the observed single-base cleavage specificity. The reported structure of the NCSi-glu-bulge DNA complex reveals a third binding mode of the antibiotic and represents a new family of minor groove bulge DNA recognition structures. We predict analogue structures of NCSi-R (R = glu or other substituent groups) may be versatile probes for detecting the existence of various structures of nucleic acids. The NMR structure of this complex, in combination with the previously reported NCSi-gb-bulge DNA complex, offers models for specific recognition of DNA bulges of various sizes through binding to either the minor or the major groove and for single-chain cleavage of bulge DNA sequences.