Induction of AML cell differentiation using HOXA9/DNA binding inhibitors as a potential therapeutic option for HOXA9-dependent AML.

The mainstay of acute myeloid leukemia (AML) treatment still relies on traditional chemotherapy, with a survival rate of approximately 30% for patients under 65 years of age and as low as 5% for those beyond. This unfavorable prognosis primarily stems from frequent relapses, resistance to chemotherapy, and limited approved targeted therapies for specific AML subtypes. Around 70% of all AML cases show overexpression of the transcription factor HOXA9, which is associated with a poor prognosis, increased chemoresistance, and higher relapse rates. However, direct targeting of HOXA9 in a clinical setting has not been achieved yet. The dysregulation caused by the leukemic HOXA9 transcription factor primarily results from its binding activity to DNA, leading to differentiation blockade. Our previous investigations have identified two HOXA9/DNA binding competitors, namely DB1055 and DB818. We assessed their antileukemic effects in comparison to HOXA9 knockdown or cytarabine treatment. Using human AML cell models, DB1055 and DB818 induced in vitro cell growth reduction, death, differentiation, and common transcriptomic deregulation but did not impact human CD34+ bone marrow cells. Furthermore, DB1055 and DB818 exhibited potent antileukemic activities in a human THP-1 AML in vivo model, leading to the differentiation of monocytes into macrophages. In vitro assays also demonstrated the efficacy of DB1055 and DB818 against AML blasts from patients, with DB1055 successfully reducing leukemia burden in patient-derived xenografts in NSG immunodeficient mice. Our findings indicate that inhibiting HOXA9/DNA interaction using DNA ligands may offer a novel differentiation therapy for the future treatment of AML patients dependent on HOXA9.

X-ray Structure Characterization of the Selective Recognition of AT Base Pair Sequences.

The rational design of small molecules that target specific DNA sequences is a promising strategy to modulate gene expression. This report focuses on a diamidinobenzimidazole compound, whose selective binding to the minor groove of AT DNA sequences holds broad significance in the molecular recognition of AT-rich human promoter sequences. The objective of this study is to provide a more detailed and systematized understanding, at an atomic level, of the molecular recognition mechanism of different AT-specific sequences by a rationally designed minor groove binder. The specialized method of X-ray crystallography was utilized to investigate how the sequence-dependent recognition properties in general, A-tract, and alternating AT sequences affect the binding of diamidinobenzimidazole in the DNA minor groove. While general and A-tract AT sequences give a narrower minor groove, the alternating AT sequences intrinsically have a wider minor groove which typically constricts upon binding. A strong and direct hydrogen bond between the N-H of the benzimidazole and an H-bond acceptor atom in the minor groove is essential for DNA recognition in all sequences described. In addition, the diamidine compound specifically utilizes an interfacial water molecule for its DNA binding. DNA complexes of AATT and AAAAAA recognition sites show that the diamidine compound can bind in two possible orientations with a preference for water-assisted hydrogen bonding at either cationic end. The complex structures of AAATTT, ATAT, ATATAT, and AAAA are bound in a singular orientation. Analysis of the helical parameters shows a minor groove expansion of about 1 Å across all the nonalternating DNA complexes. The results from this systematic approach will convey a greater understanding of the specific recognition of a diverse array of AT-rich sequences by small molecules and more insight into the design of small molecules with enhanced specificity to AT and mixed DNA sequences.

Arylimidamides Have Potential for Chemoprophylaxis against Blood-Transmitted Chagas Disease.

Chagas disease (CD) affects over 6 million people worldwide and can be transmitted iatrogenically. Crystal violet (CV) was previously used for pathogen reduction but has harmful side-effects. In the present study, three arylimidamides (AIAs) and CV were used to sterilize mice blood samples experimentally contaminated with bloodstream trypomastigotes (BT) of Trypanosoma cruzi, at non hemolytic doses. All AIAs were not toxic to mouse blood cells until the highest tested concentration (96 µM). The previous treatment of BT with the AIAs impaired the infection establishment of cardiac cell cultures. In vivo assays showed that pre-incubation of mouse blood samples with the AIAs and CV (96 µM) significantly suppressed the parasitemia peak, but only the AIA DB1831 gave ≥90% animal survival, while vehicle treated samples reached 0%. Our findings support further studies regarding the potential use of AIAs for blood bank purposes.

Investigation of the effect of structure modification of furamidine on the DNA minor groove binding and antiprotozoal activity.

New analogs of the antiprotozoal agent Furamidine were prepared utilizing Stille coupling reactions and amidation of the bisnitrile intermediate using lithium bis-trimethylsilylamide. Both the phenyl groups and the furan moiety of furamidine were replaced by heterocycles including thiophene, selenophene, indole or benzimidazole. Based upon the ΔTm and the CD results, the new compounds showed strong binding to the DNA minor groove. The new analogues are also more active both in vitro and in vivo than furamidine. Compounds 7a, 7b, and 7f showed the highest activity in vivo by curing 75% of animals, and this merits further evaluation.

Restoring and Enhancing the Potency of Existing Antibiotics against Drug-Resistant Gram-Negative Bacteria through the Development of Potent Small-Molecule Adjuvants.

The rapid and persistent emergence of drug-resistant bacteria poses a looming public health crisis. The possible task of developing new sets of antibiotics to replenish the existing ones is daunting to say the least. Searching for adjuvants that restore or even enhance the potency of existing antibiotics against drug-resistant strains of bacteria represents a practical and cost-effective approach. Herein, we describe the discovery of potent adjuvants that extend the antimicrobial spectrum of existing antibiotics and restore their effectiveness toward drug-resistant strains including mcr-1-expressing strains. From a library of cationic compounds, MD-100, which has a diamidine core structure, was identified as a potent antibiotic adjuvant against Gram-negative bacteria. Further optimization efforts including the synthesis of ∼20 compounds through medicinal chemistry work led to the discovery of a much more potent compound MD-124. MD-124 was shown to sensitize various Gram-negative bacterial species and strains, including multidrug resistant pathogens, toward existing antibiotics with diverse mechanisms of action. We further demonstrated the efficacy of MD-124 in an ex vivo skin infection model and in an in vivo murine systemic infection model using both wild-type and drug-resistant Escherichia coli strains. MD-124 functions through selective permeabilization of the outer membrane of Gram-negative bacteria. Importantly, bacteria exhibited low-resistance frequency toward MD-124. In-depth computational investigations of MD-124 binding to the bacterial outer membrane using equilibrium and steered molecular dynamics simulations revealed key structural features for favorable interactions. The very potent nature of such adjuvants distinguishes them as very useful leads for future drug development in combating bacterial drug resistance.

Drug design and DNA structural research inspired by the Neidle laboratory: DNA minor groove binding and transcription factor inhibition by thiophene diamidines.

The understanding of sequence-specific DNA minor groove interactions has recently made major steps forward and as a result, the goal of development of compounds that target the minor groove is an active research area. In an effort to develop biologically active minor groove agents, we are preparing and exploring the DNA interactions of diverse diamidine derivatives with a 5'-GAATTC-3' binding site using a powerful array of methods including, biosensor-SPR methods, and X-ray crystallography. The benzimidazole-thiophene module provides an excellent minor groove recognition component. A central thiophene in a benzimidazole-thiophene-phenyl aromatic system provides essentially optimum curvature for matching the shape of the minor groove. Comparison of that structure to one with the benzimidazole replaced with an indole shows that the two structures are very similar, but have some interesting and important differences in electrostatic potential maps, the DNA minor groove binding structure based on x-ray crystallographic analysis, and inhibition of the major groove binding PU.1 transcription factor complex. The binding KD for both compounds is under 10 nM and both form amidine H-bonds to DNA bases. They both have bifurcated H-bonds from the benzimidazole or indole groups to bases at the center of the -AATT- binding site. Analysis of the comparative results provides an excellent understanding of how thiophene compounds recognize the minor groove and can act as transcription factor inhibitors.

Thermodynamic Factors That Drive Sequence-Specific DNA Binding of Designed, Synthetic Minor Groove Binding Agents.

Ken Breslauer began studies on the thermodynamics of small cationic molecules binding in the DNA minor groove over 30 years ago, and the studies reported here are an extension of those ground-breaking reports. The goals of this report are to develop a detailed understanding of the binding thermodynamics of pyridine-based sequence-specific minor groove binders that have different terminal cationic groups. We apply biosensor-surface plasmon resonance and ITC methods to extend the understanding of minor groove binders in two directions: (i) by using designed, heterocyclic dicationic minor groove binders that can incorporate a G•C base pair (bp), with flanking AT base pairs, into their DNA recognition site, and bind to DNA sequences specifically; and (ii) by using a range of flanking AT sequences to better define molecular recognition of the minor groove. A G•C bp in the DNA recognition site causes a generally more negative binding enthalpy than with most previously used pure AT binding sites. The binding is enthalpy-driven at 25 °C and above. The flanking AT sequences also have a large effect on the binding energetics with the -AAAGTTT- site having the strongest affinity. As a result of these studies, we now have a much better understanding of the effects of the DNA sequence and compound structure on the molecular recognition and thermodynamics of minor groove complexes.

New antiparasitic flexible triaryl diamidines, their prodrugs and aza analogues: Synthesis, in vitro and in vivo biological evaluation, and molecular modelling studies.

Dicationic diamidines have been well established as potent antiparasitic agents with proven activity against tropical diseases like trypanosomiasis and malaria. This work presents the synthesis of new mono and diflexible triaryl amidines (6a-c, 13a,b and 17), their aza analogues (23 and 27) and respective methoxyamidine prodrugs (5, 7, 12a,b, 22 and 26). All diamidines were assessed in vitro against Trypanosoma brucei rhodesiense (T. b. r.) and Plasmodium falciparum (P. f.) where they displayed potent to moderate activities at the nanomolar level with IC50s = 11-378 nM for T. b. r. and 4-323 nM against P. f.. In vivo efficacy testing against T. b. r. STIB900 has shown the monoflexible diamidine 6c as the most potent derivative in this study eliciting 4/4 cures of infected mice for a treatment period of >60 days upon a 4 × 5 mg/kg dose i. p. treatment. Moreover, thermal melting analysis measurement ΔTm for this series of diamidines/poly (dA-dT) complexes fell between 0.5 and 19 °C with 6c showing the highest binding to the DNA minor groove. Finally, a 50 ns molecular dynamics study of an AT-rich DNA dodecamer with compound 6c revealed a strong binding complex supported by vdW and electrostatic interactions.

Engineered modular heterocyclic-diamidines for sequence-specific recognition of mixed AT/GC base pairs at the DNA minor groove.

This report describes a breakthrough in a project to design minor groove binders to recognize any sequence of DNA. A key goal is to invent synthetic chemistry for compound preparation to recognize an adjacent GG sequence that has been difficult to target. After trying several unsuccessful compound designs, an N-alkyl-benzodiimidazole structure was selected to provide two H-bond acceptors for the adjacent GG-NH groups. Flanking thiophenes provide a preorganized structure with strong affinity, DB2831, and the structure is terminated by phenyl-amidines. The binding experimental results for DB2831 with a target AAAGGTTT sequence were successful and include a high ΔT m, biosensor SPR with a K D of 4 nM, a similar K D from fluorescence titrations and supporting competition mass spectrometry. MD analysis of DB2831 bound to an AAAGGTTT site reveals that the two unprotonated N of the benzodiimidazole group form strong H-bonds (based on distance) with the two central G-NH while the central -CH of the benzodiimidazole is close to the -C[double bond, length as m-dash]O of a C base. These three interactions account for the strong preference of DB2831 for a -GG- sequence. Surprisingly, a complex with one dynamic, interfacial water is favored with 75% occupancy.

Dissecting Dynamic and Hydration Contributions to Sequence-Dependent DNA Minor Groove Recognition.

Sequence selectivity is a critical attribute of DNA-binding ligands and underlines the need for detailed molecular descriptions of binding in representative sequence contexts. We investigated the binding and volumetric properties of DB1976, a model bis(benzimidazole)-selenophene diamidine compound with emerging therapeutic potential in acute myeloid leukemia, debilitating fibroses, and obesity-related liver dysfunction. To sample the scope of cognate DB1976 target sites, we evaluated three dodecameric duplexes spanning >103-fold in binding affinity. The attendant changes in partial molar volumes varied substantially, but not in step with binding affinity, suggesting distinct modes of interactions in these complexes. Specifically, whereas optimal binding was associated with loss of hydration water, low-affinity binding released more hydration water. Explicit-atom molecular dynamics simulations showed that minor groove binding perturbed the conformational dynamics and hydration at the termini and interior of the DNA in a sequence-dependent manner. The impact of these distinct local dynamics on hydration was experimentally validated by domain-specific interrogation of hydration with salt, which probed the charged axial surfaces of oligomeric DNA preferentially over the uncharged termini. Minor groove recognition by DB1976, therefore, generates dynamically distinct domains that can make favorable contributions to hydration release in both high- and low-affinity binding. Because ligand binding at internal sites of DNA oligomers modulates dynamics at the termini, the results suggest both short- and long-range dynamic effects along the DNA target that can influence their effectiveness as low-MW competitors of protein binding.

Positively selected modifications in the pore of TbAQP2 allow pentamidine to enter Trypanosoma brucei.

Mutations in the Trypanosoma brucei aquaporin AQP2 are associated with resistance to pentamidine and melarsoprol. We show that TbAQP2 but not TbAQP3 was positively selected for increased pore size from a common ancestor aquaporin. We demonstrate that TbAQP2's unique architecture permits pentamidine permeation through its central pore and show how specific mutations in highly conserved motifs affect drug permeation. Introduction of key TbAQP2 amino acids into TbAQP3 renders the latter permeable to pentamidine. Molecular dynamics demonstrates that permeation by dicationic pentamidine is energetically favourable in TbAQP2, driven by the membrane potential, although aquaporins are normally strictly impermeable for ionic species. We also identify the structural determinants that make pentamidine a permeant although most other diamidine drugs are excluded. Our results have wide-ranging implications for optimising antitrypanosomal drugs and averting cross-resistance. Moreover, these new insights in aquaporin permeation may allow the pharmacological exploitation of other members of this ubiquitous gene family.

African sleeping sickness is a potentially deadly illness caused by the parasite Trypanosoma brucei. The disease is treatable, but many of the current treatments are old and are becoming increasingly ineffective. For instance, resistance is growing against pentamidine, a drug used in the early stages in the disease, as well as against melarsoprol, which is deployed when the infection has progressed to the brain. Usually, cases resistant to pentamidine are also resistant to melarsoprol, but it is still unclear why, as the drugs are chemically unrelated. Studies have shown that changes in a water channel called aquaglyceroporin 2 (TbAQP2) contribute to drug resistance in African sleeping sickness; this suggests that it plays a role in allowing drugs to kill the parasite. This molecular 'drain pipe' extends through the surface of T. brucei, and should allow only water and a molecule called glycerol in and out of the cell. In particular, the channel should be too narrow to allow pentamidine or melarsoprol to pass through. One possibility is that, in T. brucei, the TbAQP2 channel is abnormally wide compared to other members of its family. Alternatively, pentamidine and melarsoprol may only bind to TbAQP2, and then 'hitch a ride' when the protein is taken into the parasite as part of the natural cycle of surface protein replacement. Alghamdi et al. aimed to tease out these hypotheses. Computer models of the structure of the protein were paired with engineered changes in the key areas of the channel to show that, in T. brucei, TbAQP2 provides a much broader gateway into the cell than observed for similar proteins. In addition, genetic analysis showed that this version of TbAQP2 has been actively selected for during the evolution process of T. brucei. This suggests that the parasite somehow benefits from this wider aquaglyceroporin variant. This is a new resistance mechanism, and it is possible that aquaglyceroporins are also larger than expected in other infectious microbes. The work by Alghamdi et al. therefore provides insight into how other germs may become resistant to drugs.

Extending the σ-Hole Motif for Sequence-Specific Recognition of the DNA Minor Groove.

The majority of current drugs against diseases, such as cancer, can bind to one or more sites in a protein and inhibit its activity. There are, however, well-known limits on the number of druggable proteins, and complementary current drugs with compounds that could selectively target DNA or RNA would greatly enhance the availability of cellular probes and therapeutic progress. We are focusing on the design of sequence-specific DNA minor groove binders that, for example, target the promoter sites of transcription factors involved in a disease. We have started with AT-specific minor groove binders that are known to enter human cells and have entered clinical trials. To broaden the sequence-specific recognition of these compounds, several modules that have H-bond acceptors that strongly and specifically recognize G·C base pairs were identified. A lead module is a thiophene-N-alkyl-benzimidazole σ-hole-based system with terminal phenyl-amidines that have excellent affinity and selectivity for a G·C base pair in the minor groove. Efforts are now focused on optimizing this module. In this work, we are evaluating modifications to the compound aromatic system with the goal of improving GC selectivity and affinity. The lead compounds retain the thiophene-N-alkyl-BI module but have halogen substituents adjacent to an amidine group on the terminal phenyl-amidine. The optimum compounds must have strong affinity and specificity with a residence time of at least 100 s.

Small Sequence-Sensitive Compounds for Specific Recognition of the G⋠C Base Pair in DNA Minor Groove.

A series of small diamidines with thiophene and modified N-alkylbenzimidazole σ-hole module represent specific binding to single G⋅C base pair (bp) DNA sequence. The variation of N-alkyl or aromatic rings were sensitive to microstructures of the DNA minor groove. Thirteen new compounds were synthesized to test their binding affinity and selectivity. The dicyanobenzimidazoles needed to synthesize the target diamidines were made via condensation/cyclization reactions of different aldehydes with different 3-amino-4-(alkyl- or phenyl-amino) benzonitriles. The final diamidines were synthesized using lithium bis-trimethylsilylamide (LiN[Si(CH3 )3 ]2 ) or Pinner methods. The newly synthesized compounds showed strong binding and selectivity to AAAGTTT compared to similar sequences AAATTT and AAAGCTTT investigated by several biophysical methods including biosensor-SPR, fluorescence spectroscopy, DNA thermal melting, ESI-MS spectrometry, circular dichroism, and molecular dynamics. The binding affinity results determined by fluorescence spectroscopy are in accordance with those obtained by biosensor-SPR. These small size single G⋅C bp highly specific binders extend the compound database for future biological applications.

Biosensor-surface plasmon resonance: A strategy to help establish a new generation RNA-specific small molecules.

Biosensor surface plasmon resonance (SPR) is a highly sensitive technique and is most commonly used to decipher the interactions of biological systems including proteins and nucleic acids. Throughout the years, there have been significant efforts to develop SPR assays for studying protein-protein interactions, protein-DNA interactions, as well as small molecules to target DNAs that are of therapeutic interest. With the explosion of discovery of new RNA structures and functions, it is time to review the applications of SPR to RNA interaction studies, which have actually extended over a long time period. The primary advantage of SPR is its ability to measure affinities and kinetics in real time, along with being a label-free technique and utilizing relatively small quantities of materials. Recently, developments that use SPR to analyze the interactions of different RNA sequences with proteins and small molecules demonstrate the versatility of SPR as a powerful method in the analysis of the structure-function relationships, not only for biological macromolecules but also for potential drug candidates. This chapter will guide the reader through some background material followed by an extensive assay development to dissect the interactions of small molecules and RNA sequences using SPR as the critical method. The protocol includes (i) fundamental concepts of SPR, (ii) experimental design and execution, (iii) the immobilization of RNA using the streptavidin-biotin capturing method, and (iv) affinities and kinetics analyses of the interactions using specific example samples. The chapter also contains useful notes to address situations that might arise during the process. This assay demonstrates SPR as a valuable quantitative method used in the search for potential therapeutic agents that selectively target RNA.

A New Generation of Minor-Groove-Binding-Heterocyclic Diamidines That Recognize G·C Base Pairs in an AT Sequence Context.

We review the preparation of new compounds with good solution and cell uptake properties that can selectively recognize mixed A·T and G·C bp sequences of DNA. Our underlying aim is to show that these new compounds provide important new biotechnology reagents as well as a new class of therapeutic candidates with better properties and development potential than other currently available agents. In this review, entirely different ways to recognize mixed sequences of DNA by modifying AT selective heterocyclic cations are described. To selectively recognize a G·C base pair an H-bond acceptor must be incorporated with AT recognizing groups as with netropsin. We have used pyridine, azabenzimidazole and thiophene-N-methylbenzimidazole GC recognition units in modules crafted with both rational design and empirical optimization. These modules can selectively and strongly recognize a single G·C base pair in an AT sequence context. In some cases, a relatively simple change in substituents can convert a heterocyclic module from AT to GC recognition selectivity. Synthesis and DNA interaction results for initial example lead modules are described for single G·C base pair recognition compounds. The review concludes with a description of the initial efforts to prepare larger compounds to recognize sequences of DNA with more than one G·C base pairs. The challenges and initial successes are described along with future directions.

PU.1 controls fibroblast polarization and tissue fibrosis.

Fibroblasts are polymorphic cells with pleiotropic roles in organ morphogenesis, tissue homeostasis and immune responses. In fibrotic diseases, fibroblasts synthesize abundant amounts of extracellular matrix, which induces scarring and organ failure. By contrast, a hallmark feature of fibroblasts in arthritis is degradation of the extracellular matrix because of the release of metalloproteinases and degrading enzymes, and subsequent tissue destruction. The mechanisms that drive these functionally opposing pro-fibrotic and pro-inflammatory phenotypes of fibroblasts remain unknown. Here we identify the transcription factor PU.1 as an essential regulator of the pro-fibrotic gene expression program. The interplay between transcriptional and post-transcriptional mechanisms that normally control the expression of PU.1 expression is perturbed in various fibrotic diseases, resulting in the upregulation of PU.1, induction of fibrosis-associated gene sets and a phenotypic switch in extracellular matrix-producing pro-fibrotic fibroblasts. By contrast, pharmacological and genetic inactivation of PU.1 disrupts the fibrotic network and enables reprogramming of fibrotic fibroblasts into resting fibroblasts, leading to regression of fibrosis in several organs.

Heterocyclic Diamidine DNA Ligands as HOXA9 Transcription Factor Inhibitors: Design, Molecular Evaluation, and Cellular Consequences in a HOXA9-Dependant Leukemia Cell Model.

Most transcription factors were for a long time considered as undruggable targets because of the absence of binding pockets for direct targeting. HOXA9, implicated in acute myeloid leukemia, is one of them. To date, only indirect targeting of HOXA9 expression or multitarget HOX/PBX protein/protein interaction inhibitors has been developed. As an attractive alternative by inhibiting the DNA binding, we selected a series of heterocyclic diamidines as efficient competitors for the HOXA9/DNA interaction through binding as minor groove DNA ligands on the HOXA9 cognate sequence. Selected DB818 and DB1055 compounds altered HOXA9-mediated transcription in luciferase assays, cell survival, and cell cycle, but increased cell death and granulocyte/monocyte differentiation, two main HOXA9 functions also highlighted using transcriptomic analysis of DB818-treated murine Hoxa9-transformed hematopoietic cells. Altogether, these data demonstrate for the first time the propensity of sequence-selective DNA ligands to inhibit HOXA9/DNA binding both in vitro and in a murine Hoxa9-dependent leukemic cell model.

DNA recognition by linear indole-biphenyl DNA minor groove ligands.

Linear heterocyclic cations are interesting DNA minor groove ligands due to their lack of isohelical curvature classically associated with groove-binding compounds. We determined the DNA binding properties of four related dications harboring a linear indole-biphenyl core: the diamidine DB1883, a ditetrahydropyrimidine derivative (DB1804), and their monocationic counterparts (DB1944 and DB2627). These compounds exhibit heterogeneity in binding in accordance with their structures. Whereas the monocations exhibit salt-sensitive 1:1 binding to the duplex 5'-CGCGAATTCGCG-3' (A2T2), the dications show a marked preference for a salt-insensitive 2:1 complex. The two binding modes are differentially modulated by salt and specific non-ionic co-solutes. For both dications, 2-methyl-2,4-pentanediol enforces 1:1 binding as observed crystallographically. Fluorescence quenching studies show self-association without DNA in a relative order that is correlated with preference for the 2:1 complex. The data support a structure-binding relationship in which favorable cation-π interactions drive dimer formation via antiparallel stacking of the linear indole-biphenyl cation motif.

Compound Shape Effects in Minor Groove Binding Affinity and Specificity for Mixed Sequence DNA.

AT specific heterocyclic cations that bind in the DNA duplex minor groove have had major successes as cell and nuclear stains and as therapeutic agents which can effectively enter human cells. Expanding the DNA sequence recognition capability of the minor groove compounds could also expand their therapeutic targets and have an impact in many areas, such as modulation of transcription factor biological activity. Success in the design of mixed sequence binding compounds has been achieved with N-methylbenzimidazole ( N-MeBI) thiophenes which are preorganized to fit the shape of the DNA minor groove and H-bond to the -NH of G·C base pairs that project into the minor groove. Initial compounds bind strongly to a single G·C base pair in an AT context with a specificity ratio of 50 ( KD AT-GC/ KD AT) or less and this is somewhat low for biological use. We felt that modifications of compound shape could be used to probe local DNA microstructure in target mixed base pair sequences of DNA and potentially improve the compound binding selectivity. Modifications were made by increasing the size of the benzimidazole N-substituent, for example, by using N-isobutyl instead of N-Me, and by changing the molecular twist by introducing substitutions at specific positions on the aromatic core of the compounds. In both cases, we have been able to achieve a dramatic increase in binding specificity, including no detectible binding to pure AT sequences, without a significant loss in affinity to mixed base pair target sequences.

Indole and Benzimidazole Bichalcophenes: Synthesis, DNA Binding and Antiparasitic Activity.

A novel series of indole and benzimidazole bichalcophene diamidine derivatives were prepared to study their antimicrobial activity against the tropical parasites causing African sleeping sickness and malaria. The dicyanoindoles needed to synthesize the target diamidines were obtained through Stille coupling reactions while the bis-cyanobenzimidazoles intermediates were made via condensation/cyclization reactions of different aldehydes with 4-cyano-1,2-diaminobenzene. Different amidine synthesis methodologies namely, lithium bis-trimethylsilylamide (LiN[Si(CH3)3]2) and Pinner methods were used to prepare the diamidines. Both types (indole and benzimidazole) derivatives of the new diamidines bind strongly with the DNA minor groove and generally show excellent in vitro antitrypanosomal activity. The diamidino-indole derivatives also showed excellent in vitro antimalarial activity while their benzimidazole counterparts were generally less active. Compound 7c was highly active in vivo and cured all mice infected with Trypanosoma brucei rhodesiense, a model that mimics the acute stage of African sleeping sickness, at a low dose of 4 × 5 mg/kg i.p. and hence 7c is more potent in vivo than pentamidine.