Ananya Paul

Dimeric 1,3-Phenylene-bis(piperazinyl benzimidazole)s: Synthesis and Structure-Activity Investigations on their Binding with Human Telomeric G-Quadruplex DNA and Telomerase Inhibition Properties

Ligand-induced stabilization of G-quadruplex structures formed by the human telomeric DNA is an active area of research. The compounds which stabilize the G-quadruplexes often lead to telomerase inhibition. Herein we present the results of interaction of new monomeric and dimeric ligands having 1,3-phenylene-bis(piperazinyl benzimidazole) unit with G-quadruplex DNA (G4DNA) formed by human telomeric repeat d[(G(3)T(2)A)(3)G(3)]. These ligands efficiently stabilize the preformed G4DNA in the presence of 100 mM monovalent alkali metal ions. Also, the G4DNA formed in the presence of low concentrations of ligands in 100 mM K(+) adopts a highly stable parallel-stranded conformation. The G-quadruplexes formed in the presence of the dimeric compound are more stable than that induced by the corresponding monomeric counterpart. The dimeric ligands having oligo-oxyethylene spacers provide much higher stability to the preformed G4DNA and also exert significantly higher telomerase inhibition activity. Computational aspects have also been discussed.

Symmetrical bisbenzimidazoles with benzenediyl spacer: the role of the shape of the ligand on the stabilization and structural alterations in telomeric G-quadruplex DNA and telomerase inhibition

The extremities of chromosomes end in a G-rich single-stranded overhang that has been implicated in the onset of the replicate senescence. The repeated sequence forming a G-overhang is able to adopt a four-stranded DNA structure called G-quadruplex, which is a poor substrate for the enzyme telomerase. Small molecule based ligands that selectively stabilize the telomeric G-quadruplex DNA, induce telomere shortening eventually leading to cell death. Herein, we have investigated the G-quadruplex DNA interaction with two isomeric bisbenzimidazole-based compounds that differ in terms of shape (V-shaped angular vs linear). While the linear isomer induced some stabilization of the intramolecular G-quadruplex structure generated in the presence of Na(+), the other, having V-shaped central planar core, caused a dramatic structural alteration of the latter, above a threshold concentration. This transition was evident from the pronounced changes observed in the circular dichroism spectra and from the gel mobility shift assay involving the G-quadruplex DNA. Notably, this angular isomer could also induce the G-quadruplex formation in the absence of any added cation. The ligand-quadruplex complexes were investigated by computational molecular modeling, providing further information on structure-activity relationships. Finally, TRAP (telomerase repeat amplification protocol) experiments demonstrated that the angular isomer is selective toward the inhibition of telomerase activity.

Synthesis and Evaluation of a Novel Class of G-Quadruplex-Stabilizing Small Molecules Based on the 1,3-Phenylene-Bis(piperazinyl benzimidazole) System

Achieving stabilization of telomeric DNA in G-quadruplex conformation by various organic compounds has been an important goal for the medicinal chemists seeking to develop new anticancer agents. Several compounds are known to stabilize G-quadruplexes. However, relatively few are known to induce their formation and/or alter the topology of the preformed quadruplex DNA. Herein, four compounds having the 1,3-phenylene-bis(piperazinyl benzimidazole) unit as a basic skeleton have been synthesized, and their interactions with the 24-mer telomeric DNA sequences from Tetrahymena thermophilia d(T(2)G(4))(4) have been investigated using high-resolution techniques such as circular dichroism (CD) spectropolarimetry, CD melting, emission spectroscopy, and polyacrylamide gel electrophoresis. The data obtained, in the presence of one of three ions (Li(+), Na(+), or K(+)), indicate that all the new compounds have a high affinity for G-quadruplex DNA, and the strength of the binding with G-quadruplex depends on (i) phenyl ring substitution, (ii) the piperazinyl side chain, and (iii) the type of monovalent cation present in the buffer. Results further suggest that these compounds are able to abet the conversion of the intramolecular quadruplex into parallel stranded intermolecular G-quadruplex DNA. Notably, these compounds are also capable of inducing and stabilizing the parallel stranded quadruplex from randomly structured DNA in the absence of any stabilizing cation. The kinetics of the structural changes induced by these compounds could be followed by recording the changes in the CD signal as a function of time. The implications of the findings mentioned above are discussed in this paper.

Design, Synthesis, and DNA Binding Properties of Photoisomerizable Azobenzene-Distamycin Conjugates : An Experimental and Computational Study

Here, we present the synthesis, photochemical, and DNA binding properties of three photoisomerizable azobenzene-distamycin conjugates in which two distamycin units were linked via electron-rich alkoxy or electron-withdrawing carboxamido moieties with the azobenzene core. Like parent distamycin A, these molecules also demonstrated AT-specific DNA binding. Duplex DNA binding abilities of these conjugates were found to depend upon the nature and length of the spacer, the location of protonatable residues, and the isomeric state of the conjugate. The changes in the duplex DNA binding efficiency of the individual conjugates in the dark and with their respective photoirradiated forms were examined by circular dichroism, thermal denaturation of DNA, and Hoechst displacement assay with poly[d(A-T).d(T-A)] DNA in 150 mM NaCl buffer. Computational structural analyses of the uncomplexed ligands using ab initio HF and MP2 theory and molecular docking studies involving the conjugates with duplex d[(GC(AT)10CG)]2 DNA were performed to rationalize the nature of binding of these conjugates.

Design and Synthesis of Heterocyclic Cations for Specific DNA Recognition: From AT-Rich to Mixed-Base-Pair DNA Sequences

The compounds synthesized in this research were designed with the goal of establishing a new paradigm for mixed-base-pair DNA sequence-specific recognition. The design scheme starts with a cell-permeable heterocyclic cation that binds to AT base pair sites in the DNA minor groove. Modifications were introduced in the original compound to include an H-bond accepting group to specifically recognize the G-NH that projects into the minor groove. Therefore, a series of heterocyclic cations substituted with an azabenzimidazole ring has been designed and synthesized for mixed-base-pair DNA recognition. The most successful compound, 12a, had an azabenzimidazole to recognize G and additional modifications for general minor groove interactions. It binds to the DNA site −AAAGTTT– more strongly than the −AAATTT– site without GC and indicates the design success. Structural modifications of 12a generally weakened binding. The interactions of the new compound with a variety of DNA sequences with and without GC base pairs were evaluated by thermal melting analysis, circular dichroism, fluorescence emission spectroscopy, surface plasmon resonance, and molecular modeling.

Binding of Gemini Bisbenzimidazole Drugs with Human Telomeric G-Quadruplex Dimers: Effect of the Spacer in the Design of Potent Telomerase Inhibitors

The study of anticancer agents that act via stabilization of telomeric G-quadruplex DNA (G4DNA) is important because such agents often inhibit telomerase activity. Several types of G4DNA binding ligands are known. In these studies, the target structures often involve a single G4 DNA unit formed by short DNA telomeric sequences. However, the 3′-terminal single-stranded human telomeric DNA can form higher-order structures by clustering consecutive quadruplex units (dimers or n-mers). Herein, we present new synthetic gemini (twin) bisbenzimidazole ligands, in which the oligo-oxyethylene spacers join the two bisbenzimidazole units for the recognition of both monomeric and dimeric G4DNA, derived from d(T2AG3)4 and d(T2AG3)8 human telomeric DNA, respectively. The spacer between the two bisbenzimidazoles in the geminis plays a critical role in the G4DNA stability. We report here (i) synthesis of new effective gemini anticancer agents that are selectively more toxic towards the cancer cells than the corresponding normal cells; (ii) formation and characterization of G4DNA dimers in solution as well as computational construction of the dimeric G4DNA structures. The gemini ligands direct the folding of the single-stranded DNA into an unusually stable parallel-stranded G4DNA when it was formed in presence of the ligands in KCl solution and the gemini ligands show spacer length dependent potent telomerase inhibition properties.

A short DNA sequence confers strong bleomycin binding to hairpin DNAs.

Bleomycins A5 and B2 were used to study the structural features in hairpin DNAs conducive to strong BLM–DNA interaction. Two members of a 10-hairpin DNA library previously found to bind most tightly to these BLMs were subsequently noted to share the sequence 5′-ACGC (complementary strand sequence 5′-GCGT). Each underwent double-strand cleavage at five sites within, or near, an eight base pair region of the DNA duplex which had been randomized to create the original library. A new hairpin DNA library was selected based on affinity for immobilized Fe(III)·BLM A5. Two of the 30 newly identified DNAs also contained the sequence 5′-ACGC/5′-GCGT. These DNAs bound to the Fe(II)·BLMs more tightly than any DNA characterized previously. Surface plasmon resonance confirmed tight Fe(III)·BLM B2 binding and gave an excellent fit for a 1:1 binding model, implying the absence of significant secondary binding sites. Fe(II)·BLM A5 was used to assess sites of double-strand DNA cleavage. Both hairpin DNAs underwent double-strand cleavage at five sites within or near the original randomized eight base region. For DNA 12, four of the five double-strand cleavages involved independent single-strand cleavage reactions; DNA 13 underwent double-strand DNA cleavage by independent single-strand cleavages at all five sites. DNA 14, which bound Fe·BLM poorly, was converted to a strong binder (DNA 15) by insertion of the sequence 5′-ACGC/5′-GCGT. These findings reinforce the idea that tighter DNA binding by Fe·BLM leads to increased double-strand cleavage by a novel mechanism and identify a specific DNA motif conducive to strong BLM binding and cleavage.

The unusual monomer recognition of guanine-containing mixed sequence DNA by a dithiophene heterocyclic diamidine.

DB1255 is a symmetrical diamidinophenyl-dithiophene that exhibits cellular activity by binding to DNA and inhibiting binding of ERG, an ETS family transcription factor that is commonly overexpressed or translocated in leukemia and prostate cancer [Nhili, R., Peixoto, P., Depauw, S., Flajollet, S., Dezitter, X., Munde, M. M., Ismail, M. A., Kumar, A., Farahat, A. A., Stephens, C. E., Duterque-Coquillaud, M., Wilson, W. D., Boykin, D. W., and David-Cordonnier, M. H. (2013) Nucleic Acids Res. 41, 125–138]. Because transcription factor inhibition is complex but is an attractive area for anticancer and antiparasitic drug development, we have evaluated the DNA interactions of additional derivatives of DB1255 to gain an improved understanding of the biophysical chemistry of complex function and inhibition. DNase I footprinting, biosensor surface plasmon resonance, and circular dichroism experiments show that DB1255 has an unusual and strong monomer binding mode in minor groove sites that contain a single GC base pair flanked by AT base pairs, for example, 5′-ATGAT-3′. Closely related derivatives, such as compounds with the thiophene replaced with furan or selenophane, bind very weakly to GC-containing sequences and do not have biological activity. DB1255 is selective for the ATGAT site; however, a similar sequence, 5′-ATGAC-3′, binds DB1255 more weakly and does not produce a footprint. Molecular docking studies show that the two thiophene sulfur atoms form strong, bifurcated hydrogen bond-type interactions with the G-N-H sequence that extends into the minor groove while the amidines form hydrogen bonds to the flanking AT base pairs. The central dithiophene unit of DB1255 thus forms an excellent, but unexpected, single-GC base pair recognition module in a monomer minor groove complex.

Mixed up minor groove binders: Convincing A·T specific compounds to recognize a G·C base pair.

DNA minor-groove-binding compounds have limited biological applications, in part due to problems with sequence specificity that cause off-target effects. A model to enhance specificity has been developed with the goal of preparing compounds that bind to two AT sites separated by G·C base pairs. Compounds of interest were probed using thermal melting, circular dichroism, mass spectrometry, biosensor-SPR, and molecular modeling methods. A new minor groove binder that can strongly and specifically recognize a single G·C base pair with flanking AT sequences has been prepared. This multi-site DNA recognition mode offers novel design principles to recognize entirely new DNA motifs.

Understanding mixed sequence DNA recognition by novel designed compounds: the kinetic and thermodynamic behavior of azabenzimidazole diamidines.

Sequence-specific recognition of DNA by small organic molecules offers a potentially effective approach for the external regulation of gene expression and is an important goal in cell biochemistry. Rational design of compounds from established modules can potentially yield compounds that bind strongly and selectively with specific DNA sequences. An initial approach is to start with common A·T bp recognition molecules and build in G·C recognition units. Here we report on the DNA interaction of a synthetic compound that specifically binds to a G·C bp in the minor groove of DNA by using an azabenzimidazole moiety. The detailed interactions were evaluated with biosensor-surface plasmon resonance (SPR), isothermal calorimetric (ITC), and mass spectrometry (ESI-MS) methods. The compound, DB2277, binds with single G·C bp containing sequences with sub-nanomolar potency and displays slow dissociation kinetics and high selectivity. A detailed thermodynamic and kinetic study at different experimental salt concentrations and temperatures shows that the binding free energy is salt concentration dependent but essentially temperature independent under our experimental conditions, and binding enthalpy is temperature dependent but salt concentration independent. The results show that in the proper compound structural context novel heterocyclic cations can be designed to strongly recognize complex DNA sequences.