A Screening Protocol for Exploring Loop Length Requirements for the Formation of a Three Cytosine-Cytosine+ Base-Paired i-Motif.

DNA sequences containing at least four runs of repetitive cytosines can fold into tetra-helical structures called i-Motifs (iMs). The interest in these DNA secondary structures is increasing due to their therapeutical and technological applications. Still, limited knowledge of their folding requirements is currently available. We developed a novel step-by-step pipeline for the systematic screening of putative iM-forming model sequences. Focusing on structures comprising only three cytosine-cytosine+ base pairs, we investigated what the minimal lengths of the loops required for formation of an intra-molecular iM are. Our data indicate that two and three nucleotides are required to connect the strands through the minor and majorgrooves of the iM, respectively. Additionally, they highlight an asymmetric behavior according to the distribution of the cytosines. Specifically, no sequence containing a single cytosine in the first and third run was able to fold into intra-molecular iMs with the same stability of those formed when the first and the third run comprise two cytosines. This knowledge represents a step forward toward the development of prediction tools for the proper identification of biologically functional iMs, as well as for the rational design of these secondary structures as technological devices.

Modulation of the tetrameric I-motif folding of C-rich Tetrahymena telomeric sequences by hexitol nucleic acid (HNA) modifications.

I-motifs are non-canonical DNA structures consisting of two parallel strands held together by hemiprotonated cytosine-cytosine+ base pairs, which intercalate to form a ordered column of stacked base pairs. This unique structure covers potential relevance in various fields, including gene regulation and biotechnological applications. A unique structural feature of I-motifs (iM), is the presence of sugar-sugar interactions through their extremely narrow minor grooves. Consistently, oligonucleotides containing pentose derivatives such as ribose, 2'-deoxyribose, arabinose, and 2'-deoxy-2'-fluoroarabinose highlighted a very different attitude to fold into iM. On the other hand, there is significant attention focused on exploring sugar-modifications that can increase nucleic acids resistance to nuclease degradation, a crucial requirement for therapeutic applications. An interesting example, not addressed in the iM field yet, is represented by hexitol nucleic acid (HNA), a metabolically stable six-membered ring analogue compatible with A-like double helix formation. Herein, we selected two DNA C-rich Tetrahymena telomeric sequences whose tetrameric iMs were already resolved by NMR and we investigated the iM folding of related HNA and RNA oligonucleotides by circular dichroism, differential scanning calorimetry and NMR. The comparison of their behaviours vs the DNA counterparts provided interesting insights into the influence of the sugar on iM folding. In particular, ribose and hexitol prevented iM formation. However, by clustering the hexitol-containing residues at the 3'-end, it was possible to modulate the distribution of the different topological species described for the DNA iMs. These data open new avenues for the exploitation of sugar modifications for I-motif characterization and applications.

Selective Ligands for Non-Canonical DNA Structures: Do They Have a Future in Medicinal Chemistry?

Winning the war against cancer represents a major goal currently [...].

G-quadruplexes formation within the promoter of TEAD4 oncogene and their interaction with Vimentin.

G-quadruplexes (G4s) are nucleic acid secondary structures detected within human chromosomes, that cluster at gene promoters and enhancers. This suggests that G4s may play specific roles in the regulation of gene expression. Within a distinct subgroup of G-rich domains, the formation of two or more adjacent G4 units (G4-repeats) is feasible. Recently it was shown that Vimentin, a protein highly expressed within mesenchymal cells, selectively recognizes these arrangements. Putative G4-repeats have been searched within the human gene proximal promoters by the bioinformatics tool QPARSE and they resulted to be enriched at genes related to epithelial-to-mesenchymal transition (EMT). This suggested that Vimentin binding at these sites might be relevant for the maintenance of the mesenchymal phenotype. Among all the identified sequences, in the present study we selected the one located within the promoter of the TEAD4 oncogene. TEAD4 codifies for a transcriptional enhancer factor, TEAD4, that actively promotes EMT, supporting, cell proliferation and migration. Moreover, in colorectal cancer cells TEAD4 directly enhances the expression of Vimentin. Thus, the possible interaction of Vimentin with TEAD4 promoter could highlight a positive feedback loop between these two factors, associated to important tumor metastasis related events. Here, we exploited spectroscopic and electrophoretic measurements under different conditions to address the folding behavior of the selected sequence. This allowed us to validate the folding of TEAD4 promoter into a G4-repeat able to interact with Vimentin.

Bench to bedside: The ambitious goal of transducing medicinal chemistry from the lab to the clinic.

This paper deals with a critical examination on the possibility of quantitatively predicting the in vivo activity of new chemical entities (NCEs) by making use of in silico and in vitro data including three-dimensional structure of drug-target complex, thermodynamic and crowding parameters, ADME (absorption, distribution, metabolism, excretion) properties, and off-target (toxic) interactions. This formidable challenge is still a dream, given the presently occurring exceedingly high (>95%) attrition rates of NCEs. As a solution we envisage exploiting advanced AI (artificial intelligence) algorithms. In fact, very recent AI implemented programs proved remarkably effective and accurate in predicting the 3D architecture of (any) protein, starting from the amino-acid sequence only. The same accuracy could not be obtained using classical conformational studies. Apart from these breakthrough results, AI algorithms could be profitably used to extract valuable information from the huge amount of data so far accumulated from previous studies. In case of positive results, the drug discovery procedure would be sensibly accelerated, and the relative costs remarkably reduced.

Polymorphic and Higher-Order G-Quadruplexes as Possible Transcription Regulators: Novel Perspectives for Future Anticancer Therapeutic Applications.

In the past two decades, significant efforts have been put into designing small molecules to target selected genomic sites where DNA conformational rearrangements control gene expression. G-rich sequences at oncogene promoters are considered good points of intervention since, under specific environmental conditions, they can fold into non-canonical tetrahelical structures known as G-quadruplexes. However, emerging evidence points to a frequent lack of correlation between small molecule targeting of G-quadruplexes at gene promoters and the expression of the associated protein, which hampers pharmaceutical applications. The wide genomic localization of G-quadruplexes along with their highly polymorphic behavior may account for this scenario, suggesting the need for more focused drug design strategies. Here, we will summarize the G4 structural features that can be considered to fulfill this goal. In particular, by comparing a telomeric sequence with the well-characterized G-rich domain of the KIT promoter, we will address how multiple secondary structures might cooperate to control genome architecture at a higher level. If this holds true, the link between drug-DNA complex formation and the associated cellular effects will need to be revisited.

Vimentin binds to G-quadruplex repeats found at telomeres and gene promoters.

G-quadruplex (G4) structures that can form at guanine-rich genomic sites, including telomeres and gene promoters, are actively involved in genome maintenance, replication, and transcription, through finely tuned interactions with protein networks. In the present study, we identified the intermediate filament protein Vimentin as a binder with nanomolar affinity for those G-rich sequences that give rise to at least two adjacent G4 units, named G4 repeats. This interaction is supported by the N-terminal domains of soluble Vimentin tetramers. The selectivity of Vimentin for G4 repeats versus individual G4s provides an unprecedented result. Based on GO enrichment analysis performed on genes having putative G4 repeats within their core promoters, we suggest that Vimentin recruitment at these sites may contribute to the regulation of gene expression during cell development and migration, possibly by reshaping the local higher-order genome topology, as already reported for lamin B.

Amino-Acid-Anthraquinone Click Chemistry Conjugates Selectively Target Human Telomeric G-Quadruplexes.

Guanine-rich sequences are known to fold into G-quadruplex (G4) arrangements, which are present in oncogenes and in the telomeric regions of chromosomes. In particular, G4s represent an obstacle to functioning of telomerase, an enzyme overexpressed in cancer cells causing their immortalization. Therefore, G4 stabilization using small molecules represents an appealing strategy for the medicinal chemist. Ligands based on an anthraquinone scaffold, to which peptidic side chains were attached by an amide bond, were previously reported. We envisioned improving this ligand concept leveraging the click chemistry approach, which, besides representing a flexible, high yielding synthetic strategy, allows an elongation of the side chains and an increase of π-π stacking and H-bond interactions with the nucleobases through the triazole ring. Compounds were tested for their ability to interact with G4 DNA with a multiple analytical approach, demonstrating an elevated aptitude to stabilize the G4 and high selectivity over double stranded DNA.

Double-stranded flanking ends affect the folding kinetics and conformational equilibrium of G-quadruplexes forming sequences within the promoter of KIT oncogene.

G-quadruplexes embedded within promoters play a crucial role in regulating the gene expression. KIT is a widely studied oncogene, whose promoter contains three G-quadruplex forming sequences, c-kit1, c-kit2 and c-kit*. For these sequences available studies cover ensemble and single-molecule analyses, although for kit* the latter were limited to a study on a promoter domain comprising all of them. Recently, c-kit2 has been reported to fold according to a multi-step process involving folding intermediates. Here, by exploiting fluorescence resonance energy transfer, both in ensemble and at the single molecule level, we investigated the folding of expressly designed constructs in which, alike in the physiological context, either c-kit2 or c-kit* are flanked by double stranded DNA segments. To assess whether the presence of flanking ends at the borders of the G-quadruplex affects the folding, we studied under the same protocols oligonucleotides corresponding to the minimal G-quadruplex forming sequences. Data suggest that addition of flanking ends results in biasing both the final equilibrium state and the folding kinetics. A previously unconsidered aspect is thereby unravelled, which ought to be taken into account to achieve a deeper insight of the complex relationships underlying the fine tuning of the gene-regulatory properties of these fascinating DNA structures.

Nanomechanics of G-quadruplexes within the promoter of the KIT oncogene.

G-quadruplexes (G4s) are tetrahelical DNA structures stabilized by four guanines paired via Hoogsteen hydrogen bonds into quartets. While their presence within eukaryotic DNA is known to play a key role in regulatory processes, their functional mechanisms are still under investigation. In the present work, we analysed the nanomechanical properties of three G4s present within the promoter of the KIT proto-oncogene from a single-molecule point of view through the use of magnetic tweezers (MTs). The study of DNA extension fluctuations under negative supercoiling allowed us to identify a characteristic fingerprint of G4 folding. We further analysed the energetic contribution of G4 to the double-strand denaturation process in the presence of negative supercoiling, and we observed a reduction in the energy required for strands separation.

A Gold Nanoparticle Nanonuclease Relying on a Zn(II) Mononuclear Complex.

Similarly to enzymes, functionalized gold nanoparticles efficiently catalyze chemical reactions, hence the term nanozymes. Herein, we present our results showing how surface-passivated gold nanoparticles behave as synthetic nanonucleases, able to cleave pBR322 plasmid DNA with the highest efficiency reported so far for catalysts based on a single metal ion mechanism. Experimental and computational data indicate that we have been successful in creating a catalytic site precisely mimicking that suggested for natural metallonucleases relying on a single metal ion for their activity. It comprises one Zn(II) ion to which a phosphate diester of DNA is coordinated. Importantly, as in nucleic acids-processing enzymes, a positively charged arginine plays a key role by assisting with transition state stabilization and by reducing the pKa of the nucleophilic alcohol of a serine. Our results also show how designing a catalyst for a model substrate (bis-p-nitrophenylphosphate) may provide wrong indications as for its efficiency when it is tested against the real target (plasmid DNA).

Novel, Potent, and Druglike Tetrahydroquinazoline Inhibitor That Is Highly Selective for Human Topoisomerase II α over ß.

We disclose a novel class of 6-amino-tetrahydroquinazoline derivatives that inhibit human topoisomerase II (topoII), a validated target of anticancer drugs. In contrast to topoII-targeted drugs currently in clinical use, these compounds do not act as topoII poisons that enhance enzyme-mediated DNA cleavage, a mechanism that is linked to the development of secondary leukemias. Instead, these tetrahydroquinazolines block the topoII function with no evidence of DNA intercalation. We identified a potent lead compound [compound 14 (ARN-21934) IC50 = 2 µM for inhibition of DNA relaxation, as compared to an IC50 = 120 µM for the anticancer drug etoposide] with excellent metabolic stability and solubility. This new compound also shows ~100-fold selectivity for topoIIα over topoß, a broad antiproliferative activity toward cultured human cancer cells, a favorable in vivo pharmacokinetic profile, and the ability to penetrate the blood-brain barrier. Thus, ARN-21934 is a highly promising lead for the development of novel and potentially safer topoII-targeted anticancer drugs.

A two-quartet G-quadruplex topology of human KIT2 is conformationally selected by a perylene derivative.

G-quadruplexes are promising targets for innovative anticancer therapy. Hence, many efforts are being made to find selective ligands. Drug design is often based on the available high-resolution structures, obtained for the thermodynamically stable forms. However, the complexity of the G-quadruplex folding landscape has clearly emerged in recent years, with the discovery of intermediate conformations that persist on the second to the minute time scale. In the case of the KIT2 G-quadruplex forming sequence, found within human c-KIT promoter, we recently identified a long-lived folding intermediate, characterized by guanine stacking in alternating orientation (as determined by circular dichroism). Given the rate of transcriptional processes, a physiological role of this arrangement should not be excluded. In the present study, we applied circular dichroism (CD) spectroscopy, native electrospray ionization mass spectrometry (ESI-MS) and electrophoretic mobility shift assays (EMSA) to show that a perylene derivative (K20) selects this topology. Interestingly, ESI-MS spectra revealed the presence of a single specifically coordinated K+ ion in the structure, which is thus presumably composed of only two consecutive G-quartets. The parent ligand PIPER failed to promote the same conformational selection, which is therefore a process strictly dependent on the perylene side chains composition. The greater affinity of K20 for the two-quartet antiparallel topology, compared to PIPER, was finally corroborated by evaluating their binding to the KIT∗ G-quadruplex, which is also found within the human promoter of c-KIT.

Modulation of relaxation activity of human topoisomerases by Pt(II)-based complexes.

The clinical efficiency of Pt(II)-based drugs is founded on articulate mechanisms of action. Indeed it depends on a balanced combination of metal ion reactivity towards proteins and nucleic acids. Here we analysed the effect of two trans-platinum planar amines in comparison to cisplatin and transplatin on the DNA processivity by human topoisomerases I and IIα. Each tested metal complex produces DNA adducts with unique geometrical features and, consistently, they exert different effects on the activity of tested enzymes. Moreover, our results highlighted more subtle consequences on the enzymatic activity by the tested metal complexes which derive from a combination of preferential DNA or protein platination. Moreover, we observed that it is not possible to predict the overall output based only on the cis- vs trans- geometry of the tested metal complexes. This variable behaviour reflects the chemical reactivity profile of each single metal complex and can be usefully addressed to describe their different properties in the complex physiological environment.

Targeting topoisomerase II with trypthantrin derivatives: Discovery of 7-((2-(dimethylamino)ethyl)amino)indolo[2,1-b]quinazoline-6,12-dione as an antiproliferative agent and to treat cancer.

Drugs targeting human topoisomerase II (topoII) are used in clinical practice since decades. Nevertheless, there is an urgent need for new and safer topoII inhibitors due to the emergence of secondary malignancies and the appearance of resistance mechanisms upon treatment with topoII-targeted anticancer drugs. In the present investigation, we report the discovery of a new topoII inhibitor, whose design was based on the structure of the natural product trypthantrin, a natural alkaloid containing a basic indoloquinazoline moiety. This new topoII inhibitor, here numbered compound 5, is found to inhibit topoII with an IC50 of 26.6 ± 4.7 µM. Notably, compound 5 is more potent than the template compound trypthantrin, and even than the widely used topoII-targeted clinical drug etoposide. In addition, compound 5 also exhibits high water solubility, and a promising antiproliferative activity on different tumor cell lines such as acute leukemia, colon, and breast cancer. In light of these results, compound 5 represents a promising lead for developing new topoII inhibitors as anti-cancer therapeutic agents.

Coumarins from Magydaris pastinacea as inhibitors of the tumour-associated carbonic anhydrases IX and XII: isolation, biological studies and in silico evaluation.

In an in vitro screening for human carbonic anhydrase (hCA) inhibiting agents from higher plants, the petroleum ether and ethyl acetate extracts of Magydaris pastinacea seeds selectively inhibited hCA IX and hCA XII isoforms. The phytochemical investigation of the extracts led to the isolation of ten linear furocoumarins (1-10), four simple coumarins (12-15) and a new angular dihydrofurocoumarin (11). The structures of the isolated compounds were elucidated based on 1 D and 2 D NMR, MS, and ECD data analysis. All isolated compounds were inactive towards the ubiquitous cytosolic isoform hCA I and II (Ki > 10,000 nM) while they were significantly active against the tumour-associated isoforms hCA IX and XII. Umbelliprenin was the most potent coumarin inhibiting hCA XII isoform with a Ki of 5.7 nM. The cytotoxicity of the most interesting compounds on HeLa cancer cells was also investigated.

G-Quadruplex Modulation of SP1 Functional Binding Sites at the KIT Proximal Promoter.

The regulation of conformational arrangements of gene promoters is a physiological mechanism that has been associated with the fine control of gene expression. Indeed, it can drive the time and the location for the selective recruitment of proteins of the transcriptional machinery. Here, we address this issue at the KIT proximal promoter where three G-quadruplex forming sites are present (kit1, kit2 and kit*). On this model, we focused on the interplay between G-quadruplex (G4) formation and SP1 recruitment. By site directed mutagenesis, we prepared a library of plasmids containing mutated sequences of the WT KIT promoter that systematically exploited different G4 formation attitudes and SP1 binding properties. Our transfection data showed that the three different G4 sites of the KIT promoter impact on SP1 binding and protein expression at different levels. Notably, kit2 and kit* structural features represent an on-off system for KIT expression through the recruitment of transcription factors. The use of two G4 binders further helps to address kit2-kit* as a reliable target for pharmacological intervention.

DNA interaction of a fluorescent, cytotoxic pyridinimino platinum(II) complex.

New pyridinimino complexes of platinum(II) [PtCl2(N^N-R)] (N^N = 2-pyridylmethanimino, R = -(CH2)2O(CH2)2OH, -(CH)2O(CH2)2OCH2Pyr), Pyr = pyren-1-yl) have been prepared. They are characterized by a dioxygenated alkyl side chain and, in one case, by a fluorescent terminal 1-pyrenyl residue. The complexes were characterized by elemental analysis, IR, 1H-, 13C-and 195Pt NMR spectroscopies. For [PtCl2(N^N-(CH2)2O(CH2)2OH] the molecular structure was determined by single crystal X-ray diffraction. The complexes are soluble and stable in DMSO/H2O (80/20, v/v). The pyrenyl terminated compound was tested as antiproliferative agent against selected human cancer cell lines. Comparable cytotoxic effect was obtained on human ovarian carcinoma A-2780 and A-2780cis cells, thus suggesting a certain ability to circumvent cisplatin resistance. The interaction of this complex with DNA was investigated by linear flow dichroism and by spectrophotometric (absorbance and fluorescence) titrations. Both techniques enlightened the presence of a complex mode of interaction with DNA, involving both groove binding and intercalation.

Targeting multiple G-quadruplex-forming DNA sequences: Design, biophysical and biological evaluations of indolo-naphthyridine scaffold derivatives.

It is well recognized that the non-canonical DNA structures known as G-quadruplexes (G4s) have a potential anticancer significance and several compounds have been discovered and evaluated as promising G4 binders with anticancer activity. Here, starting from a promising hit with an indolo-naphthyridine scaffold, a small series of five indolo-naphthyridine based derivatives have been designed and evaluated as G4-targeting compounds. FRET biophysical studies were performed on multiple DNA G4 structures, leading to the identification of a multi-target G4 stabilizer with a slight preference for the c-KIT1 and a good G4 over duplex selectivity. The good affinity of this compound against c-KIT1 G4 was also confirmed by SPR and MST experiments, while biological assays revealed its cytotoxic activity on tumour cells. Finally, Molecular Dynamics simulations helped to elucidate the stabilization effect of the selected compound against the c-KIT1 G4.

Distinct biological responses of metastatic castration resistant prostate cancer cells upon exposure to G-quadruplex interacting naphthalenediimide derivatives.

Small molecules able to bind non-canonical G-quadruplex DNA structures (G4) have been recently tested as novel potential agents for the treatment of prostate cancer thanks to their repression of aberrant androgen receptor gene. However, metastatic castration-resistant prostate cancer (mCRPC), a letal form of prostate cancer, is still incurable. Here we tested two naphthalenediimide derivatives, previously reported as multitarget agents, on a couple of relevant mCRPC cell models (DU145 and PC-3). We showed that these compounds interfere with the RAS/MEK/ERK and PI3K/AKT pathways. Interestingly, both these two biological processes depend upon Epidermal Growth Factor Receptor (EGFR) activation. By means of biological and analytical tools we showed that our compounds are efficient inducers of the structural transition of the EGFR promoter towards a G-quadruplex conformation, ultimately leading to a reduction of the receptor production. The overall result is an interesting cytotoxic profile for these two derivatives. Thanks to their activity at different steps, these compounds can open the way to novel therapeutic approaches for mCRPC that could contribute to escape resistance to selective treatments.