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.

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.

Second Generation G-Quadruplex Stabilizing Trimethine Cyanines.

G-Quadruplex DNA has been recognized as a highly appealing target for the development of new selective chemotherapeutics, which could result in markedly reduced toxicity toward normal cells. In particular, the cyanine dyes that bind selectively to G-quadruplex structures without targeting duplex DNA have attracted attention due to their high amenability to structural modifications that allows fine-tuning of their biomolecular interactions. We have previously reported pentamethine and symmetric trimethine cyanines designed to effectively bind G-quadruplexes through end stacking interactions. Herein, we are reporting a second generation of drug candidates, the asymmetric trimethine cyanines. These have been synthesized and evaluated for their quadruplex binding properties. Incorporating a benz[c,d]indolenine heterocyclic unit increased overall quadruplex binding, and elongating the alkyl length increases the quadruplex-to-duplex binding specificity.

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.

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.

Imino proton NMR guides the reprogramming of A•T specific minor groove binders for mixed base pair recognition.

Sequence-specific binding to DNA is crucial for targeting transcription factor-DNA complexes to modulate gene expression. The heterocyclic diamidine, DB2277, specifically recognizes a single G•C base pair in the minor groove of mixed base pair sequences of the type AAAGTTT. NMR spectroscopy reveals the presence of major and minor species of the bound compound. To understand the principles that determine the binding affinity and orientation in mixed sequences of DNA, over thirty DNA hairpin substrates were examined by NMR and thermal melting. The NMR exchange dynamics between major and minor species shows that the exchange is much faster than compound dissociation determined from biosensor-surface plasmon resonance. Extensive modifications of DNA sequences resulted in a unique DNA sequence with binding site AAGATA that binds DB2277 in a single orientation. A molecular docking result agrees with the model representing rapid flipping of DB2277 between major and minor species. Imino spectral analysis of a (15)N-labeled central G clearly shows the crucial role of the exocyclic amino group of G in sequence-specific recognition. Our results suggest that this approach can be expanded to additional modules for recognition of more sequence-specific DNA complexes. This approach provides substantial information about the sequence-specific, highly efficient, dynamic nature of minor groove binding agents.

YM155 inhibits topoisomerase function.

YM155 (sepantronium bromide) has been evaluated in clinical trials as a survivin suppressant, but despite positive signals from early work, later studies were negative. Clarification of the mechanism of action of YM155 is important for its further development. YM155 affects cells in a cell cycle-specific manner. When cells are in G1, YM155 prevented their progression through the S phase, leaving the cells at G1/S when exposed to YM155. Passage through mitosis from G2 is also defective following YM155 exposure. In this study, YM155 did not behave like a typical DNA intercalator in viscosity, circular dichroism, and absorption spectroscopy studies. In addition, molecular modeling experiments ruled out YM155 DNA interaction to produce DNA intercalation. We show that YM155 inhibited topoisomerase 2α decatenation and topoisomerase 1-mediated cleavage of DNA, suggesting that YM155 inhibits the enzyme function. Consistent with these findings, DNA double-strand break repair was also inhibited by YM155.

Systematic synthetic and biophysical development of mixed sequence DNA binding agents.

It is now well established that, although only about 5% of the human genome codes for protein, most of the DNA has some function, such as synthesis of specific, functional RNAs and/or control of gene expression. These functional sequences open immense possibilities in both biotechnology and therapeutics for the use of cell-permeable, small molecules that can bind mixed-base pair sequences of DNA for regulation of genomic functions. Unfortunately very few types of modules have been designed to recognize mixed DNA sequences and for progress in targeting specific genes, it is essential to have additional classes of compounds. Compounds that can be rationally designed from established modules and which can bind strongly to mixed base pair DNA sequences are especially attractive. Based on extensive experience in design of minor-groove agents for AT recognition, a small library of compounds with two AT specific binding modules, connected through linkers which can recognize the G·C base pairs, were prepared. The compound-DNA interactions were evaluated with a powerful array of biophysical methods and the results show that some pyridyl-linked compounds bind with the target sequence with sub-nanomolar KD, with very slow dissociation kinetics and 200 times selectivity over the related sequence without a G·C base pair. Interestingly, a set of compounds with AT module connected by different linkers shows cooperative dimer recognition of related sequences. This type of design approach can be expanded to additional modules for recognition of a wide variety of sequences.

The Thiophene "Sigma-Hole" as a Concept for Preorganized, Specific Recognition of G⋠C Base Pairs in the DNA Minor Groove.

In spite of its importance in cell function, targeting DNA is under-represented in the design of small molecules. A barrier to progress in this area is the lack of a variety of modules that recognize G⋅C base pairs (bp) in DNA sequences. To overcome this barrier, an entirely new design concept for modules that can bind to mixed G⋅C and A⋅T sequences of DNA is reported herein. Because of their successes in biological applications, minor-groove-binding heterocyclic cations were selected as the platform for design. Binding to A⋅T sequences requires hydrogen-bond donors whereas recognition of the G-NH2 requires an acceptor. The concept that we report herein uses pre-organized N-methylbenzimidazole (N-MeBI) thiophene modules for selective binding with mixed bp DNA sequences. The interaction between the thiophene sigma hole (positive electrostatic potential) and the electron-donor nitrogen of N-MeBI preorganizes the conformation for accepting an hydrogen bond from G-NH2 . The compound-DNA interactions were evaluated with a powerful array of biophysical methods and the results show that N-MeBI-thiophene monomer compounds can strongly and selectively recognize single G⋅C bp sequences. Replacing the thiophene with other moieties significantly reduces binding affinity and specificity, as predicted by the design concept. These results show that the use of molecular features, such as sigma-holes, can lead to new approaches for small molecules in biomolecular interactions.

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.

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.

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.

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.

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.

Structure of an RNA G-quadruplex from the West Nile virus genome.

Potential G-quadruplex sites have been identified in the genomes of DNA and RNA viruses and proposed as regulatory elements. The genus Orthoflavivirus contains arthropod-transmitted, positive-sense, single-stranded RNA viruses that cause significant human disease globally. Computational studies have identified multiple potential G-quadruplex sites that are conserved across members of this genus. Subsequent biophysical studies established that some G-quadruplexes predicted in Zika and tickborne encephalitis virus genomes can form and known quadruplex binders reduced viral yields from cells infected with these viruses. The susceptibility of RNA to degradation and the variability of loop regions have made structure determination challenging. Despite these difficulties, we report a high-resolution structure of the NS5-B quadruplex from the West Nile virus genome. Analysis reveals two stacked tetrads that are further stabilized by a stacked triad and transient noncanonical base pairing. This structure expands the landscape of solved RNA quadruplex structures and demonstrates the diversity and complexity of biological quadruplexes. We anticipate that the availability of this structure will assist in solving further viral RNA quadruplexes and provides a model for a conserved antiviral target in Orthoflavivirus genomes.

Binding to the DNA Minor Groove by Heterocyclic Dications: from AT Specific to GC Recognition Compounds.

Compounds that bind in the DNA minor groove have provided critical information on DNA molecular recognition, have found extensive uses in biotechnology, and are providing clinically useful drugs against diseases as diverse as cancer and sleeping sickness. This review focuses on the development of clinically useful heterocyclic diamidine minor groove binders. These compounds show that the classical model for minor groove binding in AT DNA sequences must be expanded in several ways: compounds with nonstandard shapes can bind strongly to the groove, water can be directly incorporated into the minor groove complex in an interfacial interaction, compounds can be designed to recognize GC and mixed AT/GC base pair sequences, and stacked dimers can form to recognize specific sequences. © 2023 Wiley Periodicals LLC.

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.

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.