DNA Conformation Induces Adaptable Binding by Tandem Zinc Finger Proteins.

Tandem zinc finger (ZF) proteins are the largest and most rapidly diverging family of DNA-binding transcription regulators in mammals. ZFP568 represses a transcript of placental-specific insulin like growth factor 2 (Igf2-P0) in mice. ZFP568 binds a 24-base pair sequence-specific element upstream of Igf2-P0 via the eleven-ZF array. Both DNA and protein conformations deviate from the conventional one finger-three bases recognition, with individual ZFs contacting 2, 3, or 4 bases and recognizing thymine on the opposite strand. These interactions arise from a shortened minor groove caused by an AT-rich stretch, suggesting adaptability of ZF arrays to sequence variations. Despite conservation in mammals, mutations at Igf2 and ZFP568 reduce their binding affinity in chimpanzee and humans. Our studies provide important insights into the evolutionary and structural dynamics of ZF-DNA interactions that play a key role in mammalian development and evolution.

Context-Dependent Gene Regulation by Homeodomain Transcription Factor Complexes Revealed by Shape-Readout Deficient Proteins.

Eukaryotic transcription factors (TFs) form complexes with various partner proteins to recognize their genomic target sites. Yet, how the DNA sequence determines which TF complex forms at any given site is poorly understood. Here, we demonstrate that high-throughput in vitro DNA binding assays coupled with unbiased computational analysis provide unprecedented insight into how different DNA sequences select distinct compositions and configurations of homeodomain TF complexes. Using inferred knowledge about minor groove width readout, we design targeted protein mutations that destabilize homeodomain binding both in vitro and in vivo in a complex-specific manner. By performing parallel systematic evolution of ligands by exponential enrichment sequencing (SELEX-seq), chromatin immunoprecipitation sequencing (ChIP-seq), RNA sequencing (RNA-seq), and Hi-C assays, we not only classify the majority of in vivo binding events in terms of complex composition but also infer complex-specific functions by perturbing the gene regulatory network controlled by a single complex.

RNA polymerase II trapped on a molecular treadmill: Structural basis of persistent transcriptional arrest by a minor groove DNA binder.

Elongating RNA polymerase II (Pol II) can be paused or arrested by a variety of obstacles. These obstacles include DNA lesions, DNA-binding proteins, and small molecules. Hairpin pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA in a sequence-specific manner and induce strong transcriptional arrest. Remarkably, this Py-Im-induced Pol II transcriptional arrest is persistent and cannot be rescued by transcription factor TFIIS. In contrast, TFIIS can effectively rescue the transcriptional arrest induced by a nucleosome barrier. The structural basis of Py-Im-induced transcriptional arrest and why TFIIS cannot rescue this arrest remain elusive. Here we determined the X-ray crystal structures of four distinct Pol II elongation complexes (Pol II ECs) in complex with hairpin Py-Im polyamides as well as of the hairpin Py-Im polyamides-dsDNA complex. We observed that the Py-Im oligomer directly interacts with RNA Pol II residues, introduces compression of the downstream DNA duplex, prevents Pol II forward translocation, and induces Pol II backtracking. These results, together with biochemical studies, provide structural insight into the molecular mechanism by which Py-Im blocks transcription. Our structural study reveals why TFIIS fails to promote Pol II bypass of Py-Im-induced transcriptional arrest.

Spectroscopic, biochemical and computational studies of bioactive DNA minor groove binders targeting 5'-WGWWCW-3' motif.

Spectroscopic, biochemical, and computational modelling studies have been used to assess the binding capability of a set of minor groove binding (MGB) ligands against the self-complementary DNA sequences 5'-d(CGCACTAGTGCG)-3' and 5'-d(CGCAGTACTGCG)-3'. The ligands were carefully designed to target the DNA response element, 5'-WGWWCW-3', the binding site for several nuclear receptors. Basic 1D 1H NMR spectra of the DNA samples prepared with three MGB ligands show subtle variations suggestive of how each ligand associates with the double helical structure of both DNA sequences. The variations among the investigated ligands were reflected in the line shape and intensity of 1D 1H and 31P-{1H} NMR spectra. Rapid visual inspection of these 1D NMR spectra proves to be beneficial in providing valuable insights on MGB binding molecules. The NMR results were consistent with the findings from both UV DNA denaturation and molecular modelling studies. Both the NMR spectroscopic and computational analyses indicate that the investigated ligands bind to the minor grooves as antiparallel side-by-side dimers in a head-to-tail fashion. Moreover, comparisons with results from biochemical studies offered valuable insights into the mechanism of action, and antitumor activity of MGBs in relation to their structures, essential pre-requisites for future optimization of MGBs as therapeutic agents.

Structural insights and cytotoxicity evaluation of benz[e]indole pyrazolyl-substituted amides.

Five new compounds of benz[e]indole pyrazolyl-substituted amides (2a-e) were synthesised in low to good yields via the direct amide-coupling reaction between a pyrazolyl derivative containing a carboxylic acid and several amine substrates. The molecular structures were determined by various spectroscopic methods, such as NMR (1H, 13C and 19F), FT-IR and high-resolution mass spectrometry (HRMS). X-ray crystallographic analysis on the 4-fluorobenzyl derivative (2d) reveals the amide-O atom to reside to the opposite side of the molecule to the pyrazolyl-N and pyrrolyl-N atoms; in the molecular packing, helical chains feature amide-N‒H⋯N(pyrrolyl) hydrogen bonds. Density-functional theory (DFT) at the geometry-optimisation B3LYP/6-31G(d) level on the full series shows general agreement with the experimental structures. While the LUMO in each case is spread over the benz[e]indole pyrazolyl moiety, the HOMO spreads over the halogenated benzo-substituted amide moieties or is localised near the benz[e]indole pyrazolyl moieties. The MTT assay showed that 2e, exhibited the highest toxicity against a human colorectal carcinoma (HCT 116 cell line) without appreciable toxicity towards the normal human colon fibroblast (CCD-18Co cell line). Based on molecular docking calculations, the probable cytotoxic mechanism of 2e is through the DNA minor groove binding.

[DNA Sequence-Specific Ligands. 19. Synthesis, Spectral Properties, Virological and Biochemical Studies of DB3(n) Fluorescent Dimeric Trisbenzimidazoles].

In this work, we synthesized and characterized the properties of a series of new fluorescent DB3(n) narrow-groove ligands. DB3(n) compounds based on dimeric trisbenzimidazoles have the ability to bind to the AT regions of DNA. The synthesis of DB3(n), whose trisbenzimidazole fragments are linked by oligomethylene linkers of different lengths (n = 1, 5, 9), is based on the condensation of the MB3 monomeric trisbenzimidazole with α,ω-alkyldicarboxylic acids. DB3 (n) proved to be effective inhibitors of the catalytic activity of HIV-1 integrase at submicromolar concentrations (0.20-0.30 µM). DB3(n) was found to inhibit the catalytic activity of DNA topoisomerase I at low micromolar concentrations.

Insights into protein-DNA interactions from hydrogen bond energy-based comparative protein-ligand analyses.

Hydrogen bonds play important roles in protein folding and protein-ligand interactions, particularly in specific protein-DNA recognition. However, the distributions of hydrogen bonds, especially hydrogen bond energy (HBE) in different types of protein-ligand complexes, is unknown. Here we performed a comparative analysis of hydrogen bonds among three non-redundant datasets of protein-protein, protein-peptide, and protein-DNA complexes. Besides comparing the number of hydrogen bonds in terms of types and locations, we investigated the distributions of HBE. Our results indicate that while there is no significant difference of hydrogen bonds within protein chains among the three types of complexes, interfacial hydrogen bonds are significantly more prevalent in protein-DNA complexes. More importantly, the interfacial hydrogen bonds in protein-DNA complexes displayed a unique energy distribution of strong and weak hydrogen bonds whereas majority of the interfacial hydrogen bonds in protein-protein and protein-peptide complexes are of predominantly high strength with low energy. Moreover, there is a significant difference in the energy distributions of minor groove hydrogen bonds between protein-DNA complexes with different binding specificity. Highly specific protein-DNA complexes contain more strong hydrogen bonds in the minor groove than multi-specific complexes, suggesting important role of minor groove in specific protein-DNA recognition. These results can help better understand protein-DNA interactions and have important implications in improving quality assessments of protein-DNA complex models.

Methyl N-(tert-butoxycarbonyl)pyridine-2-carbimidothioate: A new reagent for the synthesis of N-phenylpyridinecarboxamidine ("arylimidamide") DNA-minor groove binders from poorly nucleophilic amines.

We report the synthesis and use of methyl N-(tert-butoxycarbonyl)pyridine-2-carbimidothioate as new reagent for the preparation of N-phenylpyridinecarboxamidines ("arylimidamides"), a class of DNA minor groove binding molecules with antiprotozoal activity. This versatile reagent allowed the access to electron-deficient halogen-containing bis(arylimidamides) that could not be obtained with the classical methods reported in the literature. With this two-step protocol, the N-Boc-protected arylimidamide intermediate, which is soluble in organic solvents, can be purified by centrifugal preparative thin layer chromatography on silica and/or by reverse-phase (C-18) chromatography. The target N-phenylpyridinecarboxamidines are obtained as salts by smooth hydrolysis of the Boc-protecting group with TFA. This methodology allows the synthesis of a pharmaceutically important class of antiparasitic compounds otherwise inaccessible.

More than forty years of nucleic acid structural science.

As scientists who have worked with Stephen Neidle over many years and stages of his career, we present our perspective of his contributions to nucleic acid structural science. We trace some of the highlights of his research on nucleic acid drug interactions and the unique insights about the importance of hydration.

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.

Synthesis, bio-physical and anti-leishmanial studies of some novel indolo[3,2-a]phenanthridine derivatives.

Eight indolo[3,2-a]phenanthridine derivatives have been synthesized in a regioselective manner involving intramolecular Heck-type arylation as a key step. The compounds display interesting photophysical proprties and hence evaluated for their ability to interact with ct-DNA. Preliminary biophysical studies via UV and Fluorescence spectrophotometric titration with ct-DNA, and dye displacement studies with well known intercalator ethidium bromide and the groove binder Hoechst 33,258 reveal that the binding mode is probably minor groove binding. The prepared indolophenanthridine derivatives have also been evaluated as anti-leishmanial agents for the first time. MTT-assays for cell cytotoxicity against Leishmania promastigotes and Leishmania amastigotes were studied with the compounds 10b-f, 12-14 for the determination of their IC50 values. Cytotoxicity was determined using a murine RAW 264.7 cell line and human embryonic kidney cell line HEK 293. In L. donovani amastigote assay, compounds 10e, 10f and 12 showed good activity with relatively low cytotoxicity against RAW 264.7, resulting in acceptable selectivity indices. Selectivity index determination indicated compounds to be potent anti-leishmanial agents while 10b, 10c and 14 showed moderate selectivity index. Moreover, cell-cycle analysis of four different compounds 10b, 12, 13 and 14, representative of each group, was performed by FACS as an attempt to understand the mechanism of actions of these different sub-classes of the compounds on Leishmania.

Selective Anti-Leishmanial Strathclyde Minor Groove Binders Using an N-Oxide Tail-Group Modification.

The neglected tropical disease leishmaniasis, caused by Leishmania spp., is becoming more problematic due to the emergence of drug-resistant strains. Therefore, new drugs to treat leishmaniasis, with novel mechanisms of action, are urgently required. Strathclyde minor groove binders (S-MGBs) are an emerging class of anti-infective agent that have been shown to have potent activity against various bacteria, viruses, fungi and parasites. Herein, it is shown that S-MGBs have potent activity against L. donovani, and that an N-oxide derivation of the tertiary amine tail of typical S-MGBs leads to selective anti-leishmanial activity. Additionally, using S-MGB-219, the N-oxide derivation is shown to retain strong binding to DNA as a 2:1 dimer. These findings support the further study of anti-leishmanial S-MGBs as novel therapeutics.

Chemical Approaches to the Development of Artificial Transcription Factors Based on Pyrrole-Imidazole Polyamides.

To maintain the functions of living organisms, cells have developed complex gene regulatory networks. Transcription factors have a central role in spatiotemporal control of gene expression and this has motivated us to develop artificial transcription factors that mimic their function. We found that three functions could be mimicked by applying our chemical approaches: i) efficient delivery into organelles that contain target DNA, ii) specific DNA binding to the target genomic region, and iii) regulation of gene expression by interaction with other transcription coregulators. We chose pyrrole-imidazole polyamides (PIPs), sequence-selective DNA binding molecules, as DNA binding domains, and have achieved each of the required functions by introducing other functional moieties. The developed artificial transcription factors have potential as chemical tools that can be used to artificially modulate gene expression to enable cell fate control and to correct abnormal gene regulation for therapeutic purposes.

Structure of HIV-1 reverse transcriptase cleaving RNA in an RNA/DNA hybrid.

HIV-1 reverse transcriptase (RT) contains both DNA polymerase and RNase H activities to convert the viral genomic RNA to dsDNA in infected host cells. Here we report the 2.65-Å resolution structure of HIV-1 RT engaging in cleaving RNA in an RNA/DNA hybrid. A preferred substrate sequence is absolutely required to enable the RNA/DNA hybrid to adopt the distorted conformation needed to interact properly with the RNase H active site in RT. Substituting two nucleotides 4 bp upstream from the cleavage site results in scissile-phosphate displacement by 4 Å. We also have determined the structure of HIV-1 RT complexed with an RNase H-resistant polypurine tract sequence, which adopts a rigid structure and is accommodated outside of the nuclease active site. Based on this newly gained structural information and a virtual drug screen, we have identified an inhibitor specific for the viral RNase H but not for its cellular homologs.

Molecular dynamics simulation of non-covalent interactions between polynuclear platinum(II) complexes and DNA.

Several studies with substitution-inert polynuclear platinum(II) complexes (SI-PPC) have been carried out in recent years due to the form of DNA binding presented by these compounds. This form of bonding is achieved by molecular recognition through the formation of non-covalent structures, commonly called phosphate clamps and forks, which generate small extensions of the major and minor grooves. In this work, we use molecular dynamics simulations (MD) to study the formation of these cyclical structures between six different SI-PPCs and a double DNA dodecamer, here called 24_bp_DNA. The results showed the influence of the complex expressed on the number of phosphate clamps and forks formed. Based on the conformational characterization of the DNA fragment, we show that the studied SI-PPCs interact preferentially in the minor groove, causing groove spanning, except for two of them, Monoplatin and AH44. The phosphates of C-G pairs are the main sites for such non-covalent interactions. The Gibbs interaction energy of solvated species points out to AH78P, AH78H, and TriplatinNC as the most probable ones when coupled with DNA. As far as we know, this work is the very first one related to SI-PPCs which brings MD simulations and a complete analysis of the non-covalent interactions with a double DNA dodecamer.

New minor groove covering DNA binding mode of dinuclear Pt(II) complexes with various pyridine-linked bridging ligands and dual anticancer-antiangiogenic activities.

New anticancer platinum(II) compounds simultaneously targeting tumor cells and tumor-derived neoangiogenesis, with new DNA interacting mode and large therapeutic window are appealing alternative to improve efficacy of clinical platinum chemotherapeutics. Herein, we describe three novel dinuclear [{Pt(en)Cl}2(µ-L)]2+ complexes with different pyridine-like bridging ligands (L), 4,4'-bipyridine (Pt1), 1,2-bis(4-pyridyl)ethane (Pt2) and 1,2-bis(4-pyridyl)ethene (Pt3), which highly, positively charged aqua derivatives, [{Pt(en)(H2O)}2(µ-L)]4+, interact with the phosphate backbone forming DNA-Pt adducts with an unique and previously undescribed binding mode, called a minor groove covering. The results of this study suggested that the new binding mode of the aqua-Pt(II) complexes with DNA could be attributed to the higher anticancer activities of their chloride analogues. All three compounds, particularly complex [{Pt(en)Cl}2(µ-4,4'-bipy)]Cl2·2H2O (4,4'-bipy is 4,4'-bipyridine) (Pt1), overcame cisplatin resistance in vivo in the zebrafish-mouse melanoma xenograft model, showed much higher therapeutic potential than antiangiogenic drug sunitinib malate, while effectively blocking tumor neovascularization and melanoma cell metastasis. Overall therapeutic profile showed new dinuclear Pt(II) complexes could be novel, effective and safe anticancer agents. Finally, the correlation with the structural characteristics of these complexes can serve as a useful tool for developing new and more effective anticancer drugs.

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.

Development and evaluation of a TaqMan MGB RT-PCR assay for detection of H5 and N8 subtype influenza virus.

BACKGROUND: Highly pathogenic influenza A (H5N8) viruses have caused several worldwide outbreaks in birds and are of potential risk to humans. Thus, a specific, rapid and sensitive method for detection is urgently needed. METHODS: In the present study, TaqMan minor groove binder probes and multiplex real-time RT-PCR primers were designed to target the H5 hemagglutinin and N8 neuraminidase genes. A total of 38 strains of avian influenza viruses and other viruses were selected to test the performance of the assay. RESULTS: The results showed that only H5 and N8 avian influenza viruses yielded a positive signal, while all other subtypes avian influenza viruses and other viruses were negative. High specificity, repeatability, and sensitivity were achieved, with a detection limit of 10 copies per reaction. CONCLUSIONS: The developed assay could be a powerful tool for rapid detection of H5N8 influenza viruses in the future.

Novel diindoloazepinone derivatives as DNA minor groove binding agents with selective topoisomerase I inhibition: Design, synthesis, biological evaluation and docking studies.

We present here-in the molecular design and chemical synthesis of a novel series of diindoloazepinone derivatives as DNA minor groove binding agents with selective topoisomerase I inhibition. The in vitro cytotoxicity of the synthesized compounds was evaluated against four human cancer cell lines including DU143, HEPG2, RKO and A549 in addition to non-cancerous immortalized human embryonic kidney cells (HEK-293). Compound 11 showed significant cytotoxicity against all the four human cancer cell lines with IC50 values ranging from 4.2 to 6.59 µM. 11 was also found to display 13-fold selective cytotoxicity towards A549 cancerous cells compared to the non-cancerous cell lines (HEK-293). The decatenation, DNA relaxation and intercalation assays revealed that the investigational compounds 10 and 11 act as highly selective inhibitors of Topo-I with DNA minor groove binding ability which was also supported by the results obtained from circular dichroism (CD), UV-visible spectroscopy and viscosity studies. Apoptosis induced by the lead 11 was observed using morphological observations, AO/EB and DAPI staining procedures. Further, dose-dependent increase in the depolarization of mitochondrial membrane was also observed through JC-1 staining. Annexin V-FITC/PI assay confirmed that 11 induced early apoptosis. Additionally, cell cycle analysis indicated that the cells were arrested at sub-G1 phase. Gratifyingly, in silico studies demonstrated promising interactions of 11 with the DNA and Topo I, thus supporting their potential DNA minor groove binding property with relatively selective Topo I inhibition compared to Topo II.

Hydrophobic Amino Acids as Universal Elements of Protein-Induced DNA Structure Deformation.

Interaction with the DNA minor groove is a significant contributor to specific sequence recognition in selected families of DNA-binding proteins. Based on a statistical analysis of 3D structures of protein-DNA complexes, we propose that distortion of the DNA minor groove resulting from interactions with hydrophobic amino acid residues is a universal element of protein-DNA recognition. We provide evidence to support this by associating each DNA minor groove-binding amino acid residue with the local dimensions of the DNA double helix using a novel algorithm. The widened DNA minor grooves are associated with high GC content. However, some AT-rich sequences contacted by hydrophobic amino acids (e.g., phenylalanine) display extreme values of minor groove width as well. For a number of hydrophobic amino acids, distinct secondary structure preferences could be identified for residues interacting with the widened DNA minor groove. These results hold even after discarding the most populous families of minor groove-binding proteins.