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An increasingly comprehension of the folding intermediate states of DNA G-quadruplexes (G4s) is currently an important scientific challenge, especially for the human telomeric (h-tel) G4s-forming sequences, characterized by a highly polymorphic nature. Despite the G-triplex conformation was proposed as one of the possible folding intermediates for the antiparallel and hybrid h-tel G4s, for the parallel h-tel topology with an all-anti guanine orientation, a vertical strand-slippage involving the G-triplets was proposed in previous works through microseconds-long standard molecular dynamics simulations (MDs). Here, in order to get further insights into the vertical strand-slippage and the folding intermediate states of the parallel h-tel G4s, we have carried out a Well-Tempered Metadynamics simulation (WT-MetaD), which allowed us to retrieve an ensemble of six G4s having two/G-tetrad conformations derived by the G-triplets vertical slippage. The insights highlighted in this work are aimed at rationalizing the mechanistic characterisation of the parallel h-tel G4 folding process.

Background: Recent findings demonstrated that, in mammalian cells, telomere DNA (Tel) is transcribed into telomeric repeat-containing RNA (TERRA), which is involved in fundamental biological processes, thus representing a promising anticancer target. For this reason, the discovery of dual (as well as selective) Tel/TERRA G-quadruplex (G4) binders could represent an innovative strategy to enhance telomerase inhibition., Methods: Initially, docking simulations of known Tel and TERRA active ligands were performed on the 3D coordinates of bimolecular G4 Tel DNA (Tel 2 ) and TERRA (TERRA 2 ). Structure-based pharmacophore models were generated on the best complexes and employed for the virtual screening of ~257,000 natural compounds. The 20 best candidates were submitted to biophysical assays, which included circular dichroism and mass spectrometry at different K + concentrations., Results: Three hits were here identified and characterized by biophysical assays. Compound 7 acts as dual Tel 2 /TERRA 2 G4-ligand at physiological KCl concentration, while hits 15 and 17 show preferential thermal stabilization for Tel 2 DNA. The different molecular recognition against the two targets was also discussed., Conclusions: Our successful results pave the way to further lead optimization to achieve both increased selectivity and stabilizing effect against TERRA and Tel DNA G4s., General Significance: The current study combines for the first time molecular modelling and biophysical assays applied to bimolecular DNA and RNA G4s, leading to the identification of innovative ligand chemical scaffolds with a promising anticancer profile. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio., (Copyright © 2016 Elsevier B.V. All rights reserved.)

G-quadruplexes (G4s) are higher-order DNA structures typically present at promoter regions of genes and telomeres. Here, the G4 formation decreases the replicative DNA at each cell cycle, finally leading to apoptosis. The ability to control this mitotic clock, particularly in cancer cells, is fascinating and passes through a rational understanding of the ligand/G4 interaction. We demonstrate that an accurate description of the ligand/G4 binding mechanism is possible using an innovative free-energy method called funnel-metadynamics (FM), which we have recently developed to investigate ligand/protein interaction. Using FM simulations, we have elucidated the binding mechanism of the anticancer alkaloid berberine to the human telomeric G4 ( d [AG 3 (T 2 AG 3 ) 3 ]), computing also the binding free-energy landscape. Two ligand binding modes have been identified as the lowest energy states. Furthermore, we have found prebinding sites, which are preparatory to reach the final binding mode. In our simulations, the ions and the water molecules have been explicitly represented and the energetic contribution of the solvent during ligand binding evaluated. Our theoretical results provide an accurate estimate of the absolute ligand/DNA binding free energy ([Formula: see text] = -10.3 ± 0.5 kcal/mol) that we validated through steady-state fluorescence binding assays. The good agreement between the theoretical and experimental value demonstrates that FM is a most powerful method to investigate ligand/DNA interaction and can be a useful tool for the rational design also of G4 ligands.

Polyphenols are compounds ubiquitously expressed in plants and used for their multiple healthy effects in humans as anti-inflammatory, antimicrobial, antiviral, anticancer and immunomodulatory agents. Due to their ability to modulate the activity of multiple targets involved in carcinogenesis, polyphenols can be employed to inhibit the growth of cancer cells. Several studies reported their high affinity to different G-quadruplex DNA structures, including the oncogene promoters c-myc and bcl-2. In this work we applied a structure-based virtual screening approach in order to screen a database of polyphenolic derivatives and human metabolites against both c-myc and bcl-2 DNA G-quadruplex structures. A Delphinidine derivative was identified as the best "dual" candidate and, after molecular dynamics simulations, resulted able to well stabilize both receptors., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The G-quadruplex DNA structures are mainly present at the terminal portion of telomeres and can be stabilized by ligands able to recognize them in a specific manner. The recognition process is usually related to the inhibition of the enzyme telomerase indirectly involved and over-expressed in a high percentage of human tumors. There are several ligands, characterized by different chemical structures, already reported in the literature for their ability to bind and stabilize the G-quadruplex structures. Using the structural and biological information available on these structures; we performed a high throughput in silico screening of commercially natural compounds databases by means of a structure-based approach followed by docking experiments against the human telomeric sequence d[AG₃(T₂AG₃)₃]. We identified 12 best hits characterized by different chemical scaffolds and conformational and physicochemical properties. All of them were associated to an improved theoretical binding affinity with respect to that of known selective G-binders. Among these hits there is a chalcone derivative; structurally very similar to the polyphenol butein; known to remarkably inhibit the telomerase activity.

Human telomeres are comprised of d(TTAGGG) repeats involved in the formation of G-quadruplex DNA structures. Ligands stabilizing these G-quadruplex DNA structures are potential inhibitors of the cancer cell-associated enzyme telomerase. In human cells, telomerase adds multiple copies of the 5'-GGTTAG-3' motif to the end of the G-strand of the telomere and in the majority of tumor cells it results over-expressed. Several structural studies have revealed a diversity of topologies for telomeric quadruplexes, which are sensitive to the nature of the cations present, to the flanking sequences, and probably also to concentration, as confirmed by the different conformations deposited in the Protein Data Bank (PDB). The existence of different polymorphisms in the DNA quadruplex and the absence of a uniquely precise binding site prompted us to carefully compare the two different docking approaches: MOLINE and Auto- Dock. As target we have selected six different experimental models of the human telomeric sequence d[AG3(T2AG3)3] based on three Gtetrads and as ligands the telomestatin isomers, whose the S enantomer is experimentally known to recognize the G-quadruplex better than the R one. In this communication we discuss the different binding modes of the well known strong telomestatin G-quadruplex binder form the thermodynamic and the geometrical points of view. With respect to this last issue we propose an easy approach to classify binding modes of G-quadruplex ligands based on a single angle descriptor as tool for the quick analysis of the binding modes.

Molecular modeling studies carried out with experimental DNA models with the sequence d[AG(3)(T(2)AG(3))(3)] suggest that the introduction of a net positive charge onto the side chain of a series of fluorenone carboxamides can improve G-quadruplex binding. The terminal morpholino moiety was replaced with a novel N-methylmorpholinium cation starting from two 4-carboxamide compounds. A different substitution on the fluorenone ring was also investigated and submitted to the same quaternarization process. All compounds were analyzed for their DNA binding properties by competition dialysis methods. In vitro antiproliferative tests were carried out against two different tumor cell lines. Docking experiments were conducted by including all four known human repeat unit G-quadruplex DNA sequences (27 experimentally determined conformations) against the most active fluorenone derivatives. The results of theoretical, biophysical, and in vitro experiments indicate two novel derivatives as lead compounds for the development of a new generation of G-quadruplex ligands with greater potency and selectivity.

Evaluation of the importance of C18/C19 stereochemistry of azinomycin A/B epoxyamide partial structures with respect to DNA alkylation sequence selectivity is reported using a unique assay with a DNA oligomer containing imbedded normal (5'-GGC-3'/3'-CCG-5') and inverted (5'-CGG-3'/3'-GCC-5') azinomycin consensus cross-linking sequences. Both species were found to have unique selectivity profiles and alkylate DNA in a manner distinct from azinomycin B. Computational docking experiments support altered binding modes for the enantiomers.

A series of fluorenone-carboxamide compounds was analyzed with regard to DNA binding properties by UV spectroscopy and competition dialysis methods. The morpholino derivative 10 provided interesting results in terms of affinity and specificity toward the DNA G-tetraplex structures. Interactions against this target were evaluated by a comparative molecular modeling study in agreement with the experimental data, proposing a model for the rational design of new agents with potent and selective DNA tetraplex binding properties.

In this work, we present molecular modeling studies carried out using six DNA sequences and six azinomycin analogues, including the naturally occurring compound azinomycin B, selected on the basis of known cell cytotoxicity and structural analogies (epoxide and aziridine alkylating moieties). Among several computational methods the Stochastic Dynamics with Energy Minimization (SDEM) approach yielded results superior to the others with the natural compound (r(2) > 0.9) and was adopted for studying other DNA adducts, obtaining good correlation between the average theoretical cross-linking properties and the antitumor activity scale. As a result, some interesting SAR considerations have been highlighted and a cross-linking conformation different from that of the azinomycin was identified in a less potent, simplified analogue.

A new set of charges specifically developed for biologically relevant N7-alkylated purine adducts have been implemented in the AMBER force field of the MacroModel package and applied to the conformational search of azinomycin B-DNA interactions. To perform a sequence dependent reactivity relationship study, four DNA triplets known to interact differently with the drug, 5'-GCT-3', 5'-GCC-3', 5'-GTC-3', and 5'-GTT-3', have been modeled in B-form and intercalative conformations. Monte Carlo simulations of all possible monoadducts and intercalative complexes have been carried out and analyzed using a filtering criterion that estimates the probability of covalent bond formation and covalent cross-linking. We observed a good correlation between existing experimental data and our computational estimations that validate the approach. The comparison of the conformational properties of the drug-DNA monoadducts and complexes confirms the most probable mechanism of action involving an initial aziridine and subsequent epoxide alkylation. The different hydrogen bond network in the monoadducts and in the intercalative complexes between the drug and the three base-pair receptor is the primary reason for the different cross-linking reactivity. In addition, steric hindrance of the major groove exposed methyl group of central thymine-based triplets plays an important role in the lack of the reactivity of these sequences. Synthetic work on the azinomycins and the information coming from this computational study will be important for the design of more potent or DNA sequence-selective agents based on the azinomycin skeleton.

Background: Dolutegravir, an integrase strand-transfer inhibitor (STI), shows a high genetic barrier to resistance. Dolutegravir is reported to be effective against viruses resistant to raltegravir and elvitegravir. In this study, we report the case of a patient treated with dolutegravir monotherapy. Failure of dolutegravir treatment was observed concomitant with the appearance of N155H-K211R-E212T mutations in the integrase (IN) gene in addition to the polymorphic K156N mutation that was present at baseline in this patient.Methods: The impact of N155H-K156N-K211R-E212T mutations was studied in cell-free, culture-based assays and by molecular modelling.Results: Cell-free and culture-based assays confirm that selected mutations in the patient, in the context of the polymorphic mutation K156N present at the baseline, lead to high resistance to dolutegravir requiring that the analysis be done at timepoints longer than usual to properly reveal the results. Interestingly, the association of only N155H and K156N is sufficient for significant resistance to dolutegravir. Modelling studies showed that dolutegravir is less stable in IN/DNA complexes with respect to the WT sequence.Conclusions: Our results indicate that the stability of STI IN/DNA complexes is an important parameter that must be taken into account when evaluating dolutegravir resistance. This study confirms that a pathway including N155H can be selected in patients treated with dolutegravir with the help of the polymorphic K156N that acts as a secondary mutation that enhances the resistance to dolutegravir. [ABSTRACT FROM AUTHOR]

The pyridostatin ( PDS) represents the lead compound of a family of G-quadruplex (G4) stabilizing synthetic small molecules based on a N,N′-bis(quinolinyl)pyridine-2,6-dicarboxamide scaffold. Its mechanism of action involves the induction of telomere dysfunction by competing for binding with telomere-associated proteins, such as human POT1. Recently, through a template-directed 'in situ ' click chemistry approach, a PDS derivative, the carboxypyridostatin ( cPDS), was discovered. It has the peculiarity to exhibit high molecular specificity for RNA over DNA G4, while PDS is a good generic RNA and DNA G4-interacting small molecule. Structural data on the binding modes of these compounds are not available, and the selectivity mode of cPDS toward TERRA G4 is unknown too. Therefore, this work is aimed at rationalizing the selectivity of cPDS versus TERRA G4 by means of molecular dynamics and docking simulations, coupled to better understand the binding mode of these compounds to telomeric G4 structures. The comprehensive analysis of cPDS binding mode and its conformational behavior demonstrates the importance of the ligand conformation properties coupled with a remarkable solvation contribution. This work is expected to provide valuable clues for further rational design of novel and selective TERRA G4 binders. [ABSTRACT FROM AUTHOR]

Several ligands can bind to the non-canonical G-quadruplex DNA structures thereby stabilizing them. These molecules can act as effective anticancer agents by stabilizing the telomeric regions of DNA or by regulating oncogene expression. In order to better interact with the quartets of G-quadruplex structures, G-binders are generally characterized by a large aromatic core involved in p-p stacking. Some natural flexible cyclic molecules from Traditional Chinese Medicine have shown high binding affinity with G-quadruplex, such as berbamine and many other alkaloids. Using the structural information available on G-quadruplex structures, we performed a high throughput in silico screening of commercially available alkaloid derivative databases by means of a structure-based approach based on docking and molecular dynamics simulations against the human telomeric sequence d[AG3(T2AG3)3] and the c-myc promoter structure. We identified 69 best hits reporting an improved theoretical binding affinity with respect to the active set. Among them, a berberine derivative, already known to remarkably inhibit telomerase activity, was related to a better theoretical affinity versus c-myc. [ABSTRACT FROM AUTHOR]

Nowadays, it has been demonstrated that DNA G-quadruplexarrangementsare involved in cellular aging and cancer, thus boosting the discoveryof selective binders for these DNA secondary structures. By takingadvantage of available structural and biological information on thesestructures, we performed a high throughput in silico screening ofcommercially available molecules databases by merging ligand- andstructure-based approaches by means of docking experiments. Compoundsselected by the virtual screening procedure were then tested for theirability to interact with the human telomeric G-quadruplex foldingby circular dichroism, fluorescence spectroscopy, and photodynamictechniques. Interestingly, our screening succeeded in retrieving anew promising scaffold for G-quadruplex binders characterized by apsoralen moiety. [ABSTRACT FROM AUTHOR]

Abstract: Human telomeres are comprised of d(TTAGGG) repeats involved in the formation of G-quadruplex DNA structures. Ligands that stabilize these G-quadruplex DNA structures are potential inhibitors of the cancer cell-associated enzyme telomerase. In human cells, telomerase adds multiple copies of the 5′-GGTTAG-3′ motif to the end of the G-strand of the telomere and in the majority of tumor cells it results over-expressed. Several structural studies have revealed a diversity of topologies for telomeric quadruplexes, as confirmed by the different conformations deposited in the Protein Data Bank. In recent years an increasing number of chemically diverse telomerase inhibitors have been identified, including both natural and synthetic compounds. Thus telomerase has been regarded as one of the most attractive targets in cancer treatment. In this manuscript, with the aim to rationalize the different experimental activities of known telomerase inhibitors, a computational study was carried out to investigate their conformational properties and the relationships between the target affinity and the ligands solvent-accessible surface area. Among the analyzed different scaffolds of G-quadruplex binders, such a descriptor provided helpful preliminary information to discriminate end-stacking ligand binding affinities, revealing itself as a useful predictive tool in drug design and lead optimization processes. [Copyright &y& Elsevier]

Using the Protein Data Bank crystallographic model of paclitaxel with tubulin as reference, a comparative interaction study of the antitumor drug with known macromolecular targets such as β-cyclodextrin and Dickerson''s DNA dodecamer was carried out by molecular modeling techniques. AMBER* united atoms was found to be the most appropriate force field for our study. Conformational search of paclitaxel was performed using a water environment. A large set of conformers was selected for automatic “quasi-flexible” docking calculations performed by the “in-house” software moline. A proper docking protocol was based on a crystallographic model and validated by a remarkable low atomic coordinate deviation. Using this method, a similar pattern via benzamide interaction was established for molecular recognition of paclitaxel cyclodextrin and DNA. The results are supported by our previous observations and other author''s experimental data. [Copyright &y& Elsevier]

Naphthalene diimides (NDIs) have been widely used as scaffold to design DNA-directed agents able to target peculiar DNA secondary arrangements endowed with relevant biochemical roles. Recently, we have reported disubstituted linear- and macrocyclic-NDIs that bind telomeric and non-telomeric G-quadruplex with high degree of affinity and selectivity. Herein, the synthesis, biological evaluation and molecular modelling studies of a series of asymmetrically substituted NDIs are reported. Among these, compound 9 emerges as the most interesting of the series being able to bind telomeric G-quadruplex (ΔTm = 29 °C at 2.5 μM), to inhibit the activity of DNA processing enzymes, such as topoisomerase II and TAQ-polymerase, and to exert antiproliferative effects in the NCI panel of cancer cell lines with GI 50 values in the micro-to nanomolar concentration range ( i.e. SR cell line, GI 50 = 76 nM). Molecular mechanisms of cell death have been investigated and molecular modelling studies have been performed in order to shed light on the antiproliferative and DNA-recognition processes. [ABSTRACT FROM AUTHOR]

Abstract: Human telomeres play a key role in protecting chromosomal ends from fusion events; they are composed of d(TTAGGG) repeats, ranging in size from 3 to 15 kb. They form G-quadruplex DNA structures, stabilized by G-quartets in the presence of cations, and are involved in several biological processes. In particular, a telomere maintenance mechanism is provided by a specialized enzyme called telomerase, a reverse transcriptase able to add multiple copies of the 5′-GGTTAG-3′ motif to the end of the G-strand of the telomere and which is over-expressed in the majority of cancer cells. The central cation has a crucial role in maintaining the stability of the structure. Based on its nature, it can be associated with different topological telomeric quadruplexes, which depend also on the orientation of the DNA strands and the syn/anti conformation of the guanines. Such a polymorphism, confirmed by the different structures deposited in the Protein Data Bank (PDB), prompted us to apply a computational protocol in order to investigate the conformational properties of a set of known G-quadruplex ligands and their molecular recognition against six different experimental models of the human telomeric sequence d[AG3(T2AG3)3]. The average AutoDock correlation between theoretical and experimental data yielded an r 2 value equal to 0.882 among all the studied models. Such a result was always improved with respect to those of the single folds, with the exception of the parallel structure (r 2 equal to 0.886), thus suggesting a key role of this G4 conformation in the stacking interaction network. Among the studied binders, a trisubstituted acridine and a dibenzophenanthroline derivative were well recognized by the parallel and the mixed G-quadruplex structures, allowing the identification of specific key contacts with DNA and the further design of more potent or target specific G-quadruplex ligands. [Copyright &y& Elsevier]