DNA Minor Groove Binding Ligands Research Articles

Dimeric bisbenzimidazoles DB(n) in combination with ionizing radiation decrease number and clonogenic activity of MCF-7 breast cancer stem cells

Purpose of the study was search for agents to eliminate the pool of cancer stem cells (CSCs) and increase their radiosensitivity among DNA minor groove binding ligands, synthetic dimeric bisbenzimidazoles of the DB(n) series, where n is the number of methylene groups in the linker connecting two bisbenzimidazole residues (n = 1, 3, 5, 7, 9, 11). The investigation was concerned with MCF-7 breast cancer cells in vitro. Six compounds were synthesized, and their binding to cells and subcellular distribution were studied using flow cytometry and laser scanning microscopy. DB(5) and DB(7) demonstrated the highest binding and accumulation in cell nuclei and were selected for studying single and combined effects with γ-radiation at a dose of 4 Gy on number, clonogenic activity of CSCs and expression of vimentin, one of the major markers of epithelial to mesenchymal transition (EMT). Radiation exposure of MCF-7 cells increased the relative and absolute numbers of CD44+СD24−/low CSCs by a factor of 1.7 (р = 0.04) and 1.6 (р = 0.008), respectively, compared with the control. The combined exposure to DB(5) or DB(7) and γ-radiation led to a considerable reduction of CSC pool compared to control and single irradiation. Particularly, the CSC absolute number decreased by a factor of 16.6 and 14.1, respectively, after combined exposure compared to the radiation alone (р = 0.006 in both cases). The combined effects on the clonogenic activity were synergistic in case of CSCs, and additive or subadditive in case of non-CSCs. The combined exposure to DB(5) or DB(7) and γ-radiation resulted in inhibiting the radiation-induced EMT. Thus, DB(5, 7) were capable of removing the stimulating effect of γ-radiation on the CSC population. The suppression of radiation-induced EMT under the influence of these compounds caused a considerable reduction of CSC pool compared to both radiation and control.

Dynamic self-assembly of DNA minor groove-binding ligand DB921 into nanotubes triggered by an alkali halide.

We describe a novel self-assembling supramolecular nanotube system formed by a heterocyclic cationic molecule which was originally designed for its potential as an antiparasitic and DNA sequence recognition agent. Our structural characterisation work indicates that the nanotubes form via a hierarchical assembly mechanism that can be triggered and tuned by well-defined concentrations of simple alkali halide salts in water. The nanotubes assembled in NaCl have inner and outer diameters of ca. 22 nm and 26 nm respectively, with lengths that reach into several microns. Our results suggest the tubes consist of DB921 molecules stacked along the direction of the nanotube long axis. The tubes are stabilised by face-to-face π-π stacking and ionic interactions between the charged amidinium groups of the ligand and the negative halide ions. The assembly process of the nanotubes was followed using small-angle X-ray and neutron scattering, transmission electron microscopy and ultraviolet/visible spectroscopy. Our data demonstrate that assembly occurs through the formation of intermediate ribbon-like structures that in turn form helices that tighten and compact to form the final stable filament. This assembly process was tested using different alkali-metal salts, showing a strong preference for chloride or bromide anions and with little dependency on the type of cation. Our data further demonstrates the existence of a critical anion concentration above which the rate of self-assembly is greatly enhanced.

Accelerated flexible protein-ligand docking using Hamiltonian replica exchange with a repulsive biasing potential.

A molecular dynamics replica exchange based method has been developed that allows rapid identification of putative ligand binding sites on the surface of biomolecules. The approach employs a set of ambiguity restraints in replica simulations between receptor and ligand that allow close contacts in the reference replica but promotes transient dissociation in higher replicas. This avoids long-lived trapping of the ligand or partner proteins at nonspecific, sticky, sites on the receptor molecule and results in accelerated exploration of the possible binding regions. In contrast to common docking methods that require knowledge of the binding site, exclude solvent and often keep parts of receptor and ligand rigid the approach allows for full flexibility of binding partners. Application to peptide-protein, protein-protein and a drug-receptor system indicate rapid sampling of near-native binding regions even in case of starting far away from the native binding site outperforming continuous MD simulations. An application on a DNA minor groove binding ligand in complex with DNA demonstrates that it can also be used in explicit solvent simulations.

P88: Multiple mechanisms of DNA minor groove binding ligand epigenetic effects

Purpose DNA minor groove is the main target of small molecules, which noncovalently and to a certain extent site-specifically bind to appropriate nucleotide sequences. Study of these substances can give rise to understanding the mechanistic relationship between sites of interaction and activity of appropriate enzymes with “houskeeping” function including helicases, topoisomerases, methyltransferases, demethylases and DNA/RNA-polymerases. Results We revealed for the first time that AT-specific minor groove binding ligands (MGBLs), in particular bisbenzimidazoles (Hoechst33258 and its derivatives), widely used in molecular and cell biology for DNA-staining, induce loss of heterozygosity at high frequency while point mutations and chromosome deletions at insignificant levels. Moreover, we demonstrated that the agents realized their genotoxic blastomogenic effects via homologous recombination mechanism exclusively. Lately the same mechanism of genotoxicity has been shown for MGBL carbazole derivative Curaxin, which is toxic for a broad range of tumor cell lines in vitro and inhibit tumor growth in different mouse models of cancer in vivo. Moreover, powerful antitumor activity has been demonstrated for Trabectedin, which binds to the DNA’s minor groove and alkylate guanine residues. All this provided a framework for wide-ranging investigation of cell response to MGBLs exposure, molecular mechanisms of their recombinogenic as also their anticancer activity. A special interest is paid to epigenetic mechanisms of MGBs action. Our study aimed to examine the epigenetic effects of recombinogenic (Hoechst33342, Hoechst33258) and non-recombinogenic (DAPI, Diminazene, Pentamidine and Netropsin) MGBLs. After we unmasked MGBLs’ recombinogenic activity, we hypothesized that their molecular mechanism of indirect DNA damage involves poly(ADP-ribose)polymerase-1 (PARP-1) activation. Surprisingly, we found that all AT-specific MGBLs preventing PARP-1 interaction with DNA inhibit its activation, and hence, the DNA-dependent pathway of PARP-1 activation function. These inhibitors effectively block PARP-1 activity in vivo, as it was demonstrated in a Drosophila experimental system and in human breast cancer-derived BT474 cell line. Further epigenetic effects of these indirect genotoxic carcinogens were analyzed using HeLa cell population with epigenetically suppressed GFP-reporter gene as a model. All compounds had strong GFP- reactivation effect. The obtained results confirm scarce data of previous publications on the ability of DNA minor groove ligands to influence gene transcription process. Statistically significant results of changes of DNA methylation level were detected under 5-azaC and Hoechst 33258 treatment, but it was absent after Hoechst 33342 treatment. For the rest compounds significant loss of promoter region methylation was not observed. The common epigenetic marks of transcription include histone H3 trimethylation in lysine 4 (H3K4me3) and histone H3 and H4 acetylation (acH3/acH4) on promoter regions of genes. We showed that TSA, Hoechst 33342 and DAPI treatment of HeLa-TI cells lead to increased level of histone H3 trimethylation of lysines 4 (H3K4me3), but for all that the level of histone H4 acetylation remains without significant changes. On the contrary, histone H3 acetylation level was stably increased at all samples. The modifications are typical for silent genes and decrease of their amount suggests the transcriptional reactivation. Loss of H4K20me3 mark in comparison with untreated control was demonstrated for MGBLs. Conclusion Taking together, findings of the study will lay the fundamental groundwork for the development of novel anticancer strategies and new chemotherapy effects of small molecules. The mechanism of PARP-1 inhibition by MGBLs and its epigenetic influence on silent genes, via DNA methylation and histone modifications make reasonable further study of these compounds in three prospective directions: (1) as self-acting cytotoxic agents; (2) as a component of combined chemotherapy targeting DNA repair by PARP-1, thereby facilitating DNA damage caused by other anticancer drugs; (3) as an agents reactivating epigenetically repressed genes which silencing occurs during the earliest stages of neoplasia and accumulates with progression toward malignancy.

Histone deacetylase inhibitors potentiate photochemotherapy in cutaneous T-cell lymphoma MyLa cells

Cutaneous T cell lymphomas (CTCL) represent rare extranodal non-Hodgkin’s lymphomas, which are characterised by pleomorphic skin lesions and distinct T-cell markers. CTCL is a relatively benign disease in its early stages, but survival rates decrease significantly with progression. Histone deacetylase inhibitors (HDACi) have recently emerged as a new class of targeted anticancer therapies for CTCL, which have been shown to induce growth inhibition, terminal differentiation and apoptosis in various cancers in vitro and in vivo. In addition to the intrinsic anticancer properties of HDACi, recent studies have demonstrated its ability to synergise with phototherapy. In particular, we examine the therapeutic potential of HDACi in combination with ultraviolet A (UV-A) phototherapy, employing a halogenated DNA minor groove binding ligand called UVASens as a photosensitiser. In vitro studies have demonstrated that UVASens is approximately 1000-fold more potent than current psoralens. The extreme photopotency of UVASens allows the use of lower radiation doses minimising the carcinogenic risks associated with the long-term use of phototherapy. Considering, previous findings using the photosensitiser UVASens and potential synergy of HDACi with phototherapy, it was hypothesised that HDACi will augment photochemotherapy-induced cytotoxicity in CTCL MyLa cells. The findings indicated that combinations of UVASens/UV-A photochemotherapy and HDACi significantly decreased cell viability and increased apoptosis and DNA double-strand breaks in MyLa cells.

Abstract 4549: Use of molecular dynamic simulations to explore changes in conformation and sequence preference for DNA-binding biaryl polyamide structures.

Abstract As a class, minor-groove non-covalent binding small molecules generally show A/T selectivity. The lack of G/C selectivity is thought to relate to the wider minor groove in G/C regions and the presence of the exocyclic 2-amino groups of guanines which project into the minor groove and prevent multiple close van der Waals contacts with the G/C-rich minor groove. AT-selective structures such as distamycin and netropsin orientate in the minor groove in an “NH down” conformation, with their NH groups pointing into the minor groove to enhance the isohelicity of the structures and produce favourable van der Waal interactions with the groove floor below. Using a distamycin scaffold as a starting point we introduced biaryl building blocks in place of pyrroles to change the curvature and van der Waals interaction potential of this novel class of polyamides. We started by designing a library of fragments to span two DNA base pairs based on simple phenyl-substituted heterocycles of sufficient length. With each library member containing one heterocyclic and one carbocyclic component, sufficient diversification was achieved through modifications to, and substitutions within, both components. This allowed the production of libraries of building blocks possessing different curvatures and hydrogen-bonding abilities with the potential to complement functional groups and hydrogen bond donors and acceptors in the walls and floor of the minor groove. A fluorescent intercalator displacement (FID) assay was then used to select molecules with a preference for GC-rather than AT-rich sequences which identified the 4-(1-methyl-1H-pyrrol-3-yl)benzenamine (MPB) motif. Molecular dynamics simulations were undertaken using the AMBER v11 software in implicit solvent over a time-scale of 20ns with the structures in both a “carbonyl-in” and “NH-in” conformation. This demonstrated that the MPB component within the biaryl polyamides undergoes a switch in orientation, with a “carbonyl down” conformation preferred which induces GC specificity. As well as occupying an idealised hydrogen-bonding distance between two side-by-side guanine residues, MPB units contained within the MPB biaryl polyamide structures showed superior hydrogen bonding interactions with GC compared to AT sequences, with each carbonyl of the MPB unit hydrogen bonding to a guanine residue. This was further supported by free-energy of binding calculations undertaken using AMBER-MMPBSA, where the MPB units showed a distinct preference for GC sequences (a difference of -13kcal/mol in favour of “carbonyl -in” over “NH-in” structures). This discovery has been applied to the design and synthesis of more-complex MPB-containing DNA minor-groove binding ligands with a high level of GC-selectivity and significant in vitro and in vivo antitumour activity. Citation Format: Paul J M Jackson, Colin M. James, Khondaker M. Rahman, David E. Thurston. Use of molecular dynamic simulations to explore changes in conformation and sequence preference for DNA-binding biaryl polyamide structures. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4549. doi:10.1158/1538-7445.AM2013-4549

Single-dose pharmacokinetic and toxicity analysis of pyrrole–imidazole polyamides in mice

PurposePyrrole–imidazole (Py-Im) polyamides are programmable, sequence-specific DNA minor groove–binding ligands. Previous work in cell culture has shown that various polyamides can be used to modulate the transcriptional programs of oncogenic transcription factors. In this study, two hairpin polyamides with demonstrated activity against androgen receptor signaling in cell culture were administered to mice to characterize their pharmacokinetic properties.MethodsPy-Im polyamides were administered intravenously by tail vein injection. Plasma, urine, and fecal samples were collected over a 24-h period. Liver, kidney, and lung samples were collected postmortem. Concentrations of the administered polyamide in the plasma, excretion, and tissue samples were measured using LC/MS/MS. The biodistribution data were analyzed by both non-compartmental and compartmental pharmacokinetic models. Animal toxicity experiments were also performed by monitoring weight loss after a single subcutaneous (SC) injection of either polyamide.ResultsThe biodistribution profiles of both compounds exhibited rapid localization to the liver, kidneys, and lungs upon injection. Plasma distribution of the two compounds showed distinct differences in the rate of clearance, the volume of distribution, and the AUCs. These two compounds also have markedly different toxicities after SC injection in mice.ConclusionsThe variations in pharmacokinetics and toxicity in vivo stem from a minor chemical modification that is also correlated with differing potency in cell culture. The results obtained in this study could provide a structural basis for further improvement of polyamide activity both in cell culture and in animal models.Electronic supplementary materialThe online version of this article (doi:10.1007/s00280-012-1954-3) contains supplementary material, which is available to authorized users.

Abstract 2731: Detection of blastomogenic activity of DNA minor groove binding ligands by SMART in Drosophila

Abstract The increasing request of chemical safety assessment demands alternative methods to animal experimentation. Internationally accepted short-term assays are capable of detecting five different categories of change: gene mutation; clastogenicity; aneuploidy and non-disjunction; DNA damage as measured by DNA repair; in vitro cell transformation. Recently it was demonstrated that loss of heterozigocity (LOH) through homologous recombination, causing manifestation of a recessive mutant allele, represent frequent DNA rearrangements in tumors of patients with inherited retinoblastoma and Li-Fraumeni syndrome. LOH may be detected easily using the test for somatic mutation and recombination in Drosophila elaborated more than 30 years ago in our laboratory and currently known as SMART. The assay is based on the manifestation of recessive mutant allele after inactivation of wild type one due to somatic point mutation, chromosomal deletion or loss of heterozygocity. Recently we improve SMART to detect blastomogenic activity of chemicals using wtsP4/+ heterozygous larvae. Wts gene is involved in cell cycle regulation and its homologues are tumor suppressor genes in mice and human. Hereby we demonstrate benefits of SMART in the analysis of genotoxic effects of minor groove binding ligands (MGBLs), Hoechst 33258 and Hoechst 33342, and two new bisbenzimidazols: 1-[6-(4-methylpiperazin-1-yl)-1H,3’H-2,5’-bibenzimidazol-2’-yl]methanacetamide N-[6-(4-methylpiperazin-1-yl)-1H,3’H-2,5’-bibenzimidazol-2’-yl]methanacetamide. Hoechst 33258 and Hoechst 33342 are widely used in molecular biology. Evaluation of their genotoxicity is complex due to the mode of its action. They neither alkylate DNA nor form interstrand-crosslinks or DNA adducts and bind DNA via electrostatic, hydrogen and van der Waals bonds and inhibit some enzymes, causing genetic damage in an indirect way. On account of that they are negative in the main Salmonella/ microsome reverse mutation assay using TA 98 and TA 100 strains. Marginal mutagenicity was detected only in a special TA102 strain. In the conventional 6-thioguanine resistance assay on Chinese hamster V79 cells Hoechst 33342 harbors mutagenic activity at highly toxic concentrations only. In our study all bisbenzimidazols produced significant increase of wtsP4 clone frequency at non-toxic concentrations. These dyes exhibited dose-dependent blastomogenic effect, and Hoechst 33342 showed twice higher clone frequency than others that is associated with different cell-membrane penetration. We demonstrate TOPO I inhibition for all MGBLs analyzed. Surprisingly, new derivatives were mutagenic in Ames assay. Thus, our investigation of MGBL mutagenic properties demonstrates the necessity to include the tests for both point mutagenesis and recombinogenic activity determination. Drosophila SMART assay using wtsP4/+ heterozygous larvae seems to be promising in this way. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2731. doi:10.1158/1538-7445.AM2011-2731

Photosensitization by iodinated DNA minor groove binding ligands: Evaluation of DNA double-strand break induction and repair

Iodinated DNA minor groove binding bibenzimidazoles represent a unique class of UVA photosensitizer and their extreme photopotency has been previously characterized. Earlier studies have included a comparison of three isomers, referred to as ortho-, meta- and para-iodoHoechst, which differ only in the location of the iodine substituent in the phenyl ring of the bibenzimidazole. DNA breakage and clonogenic survival studies in human erythroleukemic K562 cells have highlighted the higher photo-efficiency of the ortho-isomer (subsequently designated UV A Sens) compared to the meta- and para-isomers. In this study, the aim was to compare the induction and repair of DNA double-strand breaks induced by the three isomers in K562 cells. Further, we examined the effects of the prototypical broad-spectrum histone deacetylase inhibitor, Trichostatin A, on ortho-iodoHoechst/UVA-induced double-strand breaks in K562 cells. Using γH2AX as a molecular marker of the DNA lesions, our findings indicate a disparity in the induction and particularly, in the repair kinetics of double-strand breaks for the three isomers. The accumulation of γH2AX foci induced by the meta- and para-isomers returned to background levels within 24 and 48 h, respectively; the number of γH2AX foci induced by ortho-iodoHoechst remained elevated even after incubation for 96 h post-irradiation. These findings provide further evidence that the extreme photopotency of ortho-iodoHoechst is due to not only to the high quantum yield of dehalogenation, but also to the severity of the DNA lesions which are not readily repaired. Finally, our findings which indicate that Trichostatin A has a remarkable potentiating effect on ortho-iodoHoechst/UVA-induced DNA lesions are encouraging, particularly in the context of cutaneous T-cell lymphoma, for which a histone deacetylase inhibitor is already approved for therapy. This finding prompts further evaluation of the potential of combination therapies.

Hoechst 33342 induces radiosensitization in malignant glioma cells via increase in mitochondrial reactive oxygen species

Mitochondrial DNA plays an important role in cellular sensitivity to cancer therapeutic agents. Hoechst 33342, a DNA minor groove binding ligand, has shown radiosensitizing effects in different cancer cell lines. In the present study, the possible binding of Hoechst 33342 with mitochondrial DNA, isolated from human cerebral glioma (BMG-1) cells, was investigated and consequences of this binding on excessive reactive oxygen species (ROS) generation in irradiated BMG-1 cells were studied. Alteration in the fluorescence spectroscopic characteristics of Hoechst 33342 suggested binding of Hoechst 33342 with isolated mitochondria and mitochondrial DNA. Persistent increase in level of ROS in the presence of Hoechst 33342 has been observed, which was further enhanced in irradiated cells. Investigations using inhibitors of ETC complex I suggested that mitochondrial bound Hoechst 33342 contributed to increased ROS, which was associated with alteration in ΔΨm and antioxidant machinery. These factors appeared to contribute in potentiating radiation-induced cell death in BMG-1 cells. The finding from these studies will be useful in designing better anti-cancer strategies.

Groove Binding Ligands for the Interaction with Parallel-Strandedps-Duplex DNA and Triplex DNA

DNA adopts different conformations not only based on novel base pairs, but also with different chain polarities. Besides several duplex structures (A, B, Z, parallel stranded (ps)-DNA, etc.), DNA also forms higher-order structures like triplex, tetraplex, and i-motif. Each of these structures has its own biological significance. The ps-duplexes have been found to be resistant to certain nucleases and endonucleases. Molecules that promote triple-helix formation have significant potential. These investigations have many therapeutic advantages which may be useful in the regulation of the expression of genes responsible for certain diseases by locking either their transcription (antigene) or translation (antisense). Each DNA minor groove binding ligand (MGBL) interacts with DNA through helical minor groove recognition in a sequence-specific manner, and this interferes with several DNA-associated processes. Incidentally, these ligands interact with some non-B-DNA and with higher-order DNA structures including ps-DNA and triplexes. While the design and recognition of minor grooves of duplex DNA by specific MGBLs have been a topic of many reports, limited information is available on the binding behavior of MGBLs with nonduplex DNA. In this review, we summarize various attempts of the interaction of MGBLs with ps-DNA and DNA triplexes.

The biological in vitro effect and selectivity of aromatic dicationic compounds on Trypanosoma cruzi

Trypanosoma cruzi is a parasite that causes Chagas disease, which affects millions of individuals in endemic areas of Latin America. One hundred years after the discovery of Chagas disease, it is still considered a neglected illness because the available drugs are unsatisfactory. Aromatic compounds represent an important class of DNA minor groove-binding ligands that exhibit potent antimicrobial activity. This study focused on the in vitro activity of 10 aromatic dicationic compounds against bloodstream trypomastigotes and intracellular forms of T. cruzi. Our data demonstrated that these compounds display trypanocidal effects against both forms of the parasite and that seven out of the 10 compounds presented higher anti-parasitic activity against intracellular parasites compared with the bloodstream forms. Additional assays to determine the potential toxicity to mammalian cells showed that the majority of the dicationic compounds did not considerably decrease cellular viability. Fluorescent microscopy analysis demonstrated that although all compounds were localised to a greater extent within the kinetoplast than the nucleus, no correlation could be found between compound activity and kDNA accumulation. The present results stimulate further investigations of this class of compounds for the rational design of new chemotherapeutic agents for Chagas disease.

Netropsin, a Minor Groove Binding Ligand: A Potential Radioprotective Agent

Minimizing radiation-induced damages in DNA is an important aspect in the development of chemical radioprotectors. The aim of this study was to evaluate the possible radioprotective ability of the DNA minor groove binding ligand netropsin in an aqueous solution of plasmid DNA (pBR322) and to compare its efficacy with that of Hoechst 33258, a known radioprotector. The radiochemical parameters D(0), G(SSB) and DMF were calculated in pBR322 DNA. Based on a comparison of the DMFs of netropsin and Hoechst 33258, netropsin appeared to be the better radioprotector. The ligand binding site accessibility of the restriction enzyme EcoRI at the ligand-pBR322 complex was assessed using a restriction-digestion assay in irradiated solutions. A distinct ligand-bound site protection in netropsin-DNA was observed in irradiated solutions. However, no site protection was observed in the presence of Hoechst 33258. The possible role of ligand-induced structural stabilization in irradiated aqueous solutions was also investigated using netropsin-calf thymus DNA melting temperature measurements. The greater radioprotective ability of netropsin in solutions of DNA was suggested to be due to its higher binding affinity and its ability to provide higher structural stabilization. EcoRI digestion revealed that hydroxyl radical (OH*) generated by ionizing radiation is not able to radiolyse the netropsin-DNA complex. These results will help in developing better radioprotectors.

Evaluation of molecular modelling methods to predict the sequence-selectivity of DNA minor groove binding ligands

The accurate prediction of ligand-receptor interaction energies by molecular modelling methods remains challenging. Predicting and understanding the sequence-selectivity of DNA minor groove-binding ligands constitutes a particularly interesting and potentially valuable aspect of this. Here, we use experimental data on the binding of Hoechst 33258 to the minor groove of various A/T-rich DNA duplexes to evaluate the reliability of a popular class of molecular modelling methods based on the energetic analysis of molecular dynamics (MD) simulations. We examine how performance depends on the use of explicit versus implicit solvent models, on the use of generalised Born versus Poisson-Boltzmann models to evaluate solvent-associated energetic terms, and the use of normal mode analysis to evaluate entropic factors. Quantitatively evaluating many different combinations of methodologies, we find that the most reliable results are obtained when the MD simulations are performed in explicit solvent, when the data is processed using the MM-PB/SA approach, and when normal mode analysis is used to estimate configurational entropy changes.

In Vitro and In Vivo Studies of the Trypanocidal Activity of a Diarylthiophene Diamidine against Trypanosoma cruzi

Aromatic diamidines are DNA minor groove-binding ligands that display excellent antimicrobial activity against fungi, bacteria, and protozoa. Due to the currently unsatisfactory chemotherapy for Chagas' disease and in view of our previous reports regarding the effect of diamidines and analogues against both in vitro and in vivo Trypanosoma cruzi infection, this study evaluated the effects of a diarylthiophene diamidine (DB1362) against both amastigotes and bloodstream trypomastigotes of T. cruzi, the etiological agent of Chagas' disease. The data show the potent in vitro activity of DB1362 against both parasite forms that are relevant for mammalian infection at doses which do not exhibit cytotoxicity. Ultrastructural analysis and flow cytometry studies show striking alterations in the nuclei and mitochondria of the bloodstream parasites. In vivo studies were performed at two different drug concentrations (25 and 50 mg/kg/day) using a 2-day or a 10-day regimen. The best results were obtained when acutely infected mice were treated with two doses at the lower concentration, resulting in 100% survival, compared to the infected and untreated mice. Although it did not display higher efficacy than benznidazole, DB1362 reduced both cardiac parasitism and inflammation, and in addition, it protected against the cardiac alterations (determined by measurements) common in T. cruzi infection. These results support further investigation of diamidines and related compounds as potential agents against Chagas' disease.

Cellular Effects of Reversed Amidines on Trypanosoma cruzi

Aromatic diamidines represent a class of DNA minor groove-binding ligands that exhibit high levels of antiparasitic activity. Since the chemotherapy for Chagas' disease is still an unsolved problem and previous reports on diamidines and related analogues show that they have high levels of activity against Trypanosoma cruzi infection both in vitro and in vivo, our present aim was to evaluate the cellular effects in vitro of three reversed amidines (DB889, DB702, and DB786) and one diguanidine (DB711) against both amastigotes and bloodstream trypomastigotes of T. cruzi, the etiological agent of Chagas' disease. Our data show that the reversed amidines have higher levels of activity than the diguanidine, with the order of trypanocidal activities being as follows: DB889 > DB702 > DB786 > DB711. Transmission electron microscopy analysis showed that the reversed amidines induced many alterations in the nuclear morphology, swelling of the endoplasmic reticulum and Golgi structures, and consistent damage in the mitochondria and kinetoplasts of the parasites. Interestingly, in trypomastigotes treated with the reversed amidine DB889, multiple axoneme structures (flagellar microtubules) were noted. Flow cytometry analysis confirmed that the treated parasites presented an important loss of the mitochondrial membrane potential, as revealed by a decrease in rhodamine 123 fluorescence. Our results show that the reversed amidines have promising activities against the relevant mammalian forms of T. cruzi and display high trypanocidal effects at very low doses. This is especially the case for DB889, which merits further in vivo evaluation.

Molecular modelling methods for prediction of sequence-selectivity in DNA recognition

We describe how one can apply molecular modelling methods, based on the molecular mechanics/generalised Born (MM/GB) approach, to the prediction of the relative affinity of DNA minor groove binding ligands for different DNA sequences. We discuss the theoretical background to the technique, some variations in the methodology that can be employed, and illustrate its application through a case study: analysis of the energetics of binding of Hoechst 33258 to the minor groove of various A/T-rich DNA duplexes. We show how the underpinning molecular dynamics (MD) simulations can be set up, how they can be analysed for satisfactory behaviour, and various approaches to extracting thermodynamics of drug binding from them. We find that while certain elaborations to the basic MM/GB method can improve the agreement with experimental data (e.g., calculating the DNA perturbation energy), others have to be analysed with more caution (e.g., calculating configurational entropy changes). Overall, these methodologies can rank the affinity of a ligand for the minor groove of different DNA sequences fairly well, but the calculation of absolute binding affinities is not very reliable.

Entropically-driven binding of mithramycin in the minor groove of C/G-rich DNA sequences

The antitumour antibiotic mithramycin A (MTA) is a DNA minor-groove binding ligand. It binds to C/G-rich tracts as a dimer that forms in the presence of divalent cations such as Mg2+. Differential scanning calorimetry, UV thermal denaturation, isothermal titration calorimetry and competition dialysis were used, together with computations of the hydrophobic free energy of binding, to determine the thermodynamic profile of MTA binding to DNA. The results were compared to those obtained in parallel using the structurally related mithramycin SK (MSK). The binding of MTA to salmon testes DNA determined by UV melting studies (Kobs = 1.2 (±0.3) × 105 M−1) is tighter than that of MSK (2.9 (±1.0) × 104 M−1) at 25°C. Competition dialysis studies showed a tighter MTA binding to both salmon testes DNA (42% C + G) and Micrococcus lysodeikticus DNA (72% C + G). The thermodynamic analysis of binding data at 25°C shows that the binding of MTA and MSK to DNA is entropically driven, dominated by the hydrophobic transfer of the antibiotics from solution to the DNA-binding site. Direct molecular recognition between MTA or MSK and DNA through hydrogen bonding and van der Waals contacts may also contribute significantly to complex formation.

DNA binding ligands targeting drug-resistant Gram-positive bacteria. Part 1: Internal benzimidazole derivatives

Novel DNA minor-groove binding ligands with a promising antibacterial profile are described. Apart from excellent in vitro potency against multiple Gram-positive bacterial strains such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VRE), and penicillin-intermediate Streptococcus pneumoniae (PISP), a small subset of compounds was active against Gram-negative bacteria such as Escherichia coli (E. coli).

Interaction of DAPI with individual strands of trinucleotide repeats. Effects of replication in vitro of the AAT x ATT triplet.

The structural changes produced by the minor-groove binding ligand DAPI (4',6-diamidine-2-phenylindole) on individual strands of trinucleotide repeat sequences were detected by electrophoretic band-shift analysis and related to their effects on DNA replication in vitro. Among the 20 possible single-stranded trinucleotide repeats, only the T-rich strand of the AAT.ATT triplet exhibits an observable fluorescence band and a change in electrophoretic mobility due to the drug binding. This is attributable to the property of DAPI that favours folding of the random coil ATT strand into a fast-migrating hairpin structure by a minor-groove binding mechanism. Electrophoretic characteristics of AAT, ACT, AGT, ATG and ATC are unchanged by DAPI, suggesting the crucial role of T.T with respect to A.A, C.C and G.G mismatch, in favouring the binding properties and the structural features of the ATT-DAPI complexes. Primer extension experiments, using the Klenow fragment of DNA polymerase I, demonstrate that such a selective structural change at ATT targets presents a marked property to stall DNA replication in vitro in comparison with the complementary AAT and a random GC-rich sequence. The results suggest a novel molecular mechanism of action of the DNA minor-groove binding ligand DAPI.