Selective Recognition of a Single HIV-1 G-Quadruplex by Ultrafast Small-Molecule Screening.

G-quadruplexes (G4s) are implicated in pathological processes such as cancer and infective diseases. Their targeting with G4-ligands has shown therapeutic capacity. Most of the current G4-ligands are planar molecules, do not discriminate among G4s, and have poor druglike properties. The available methods to identify compounds selective for one single G4 are often time-consuming. Here, we describe the development, validation, and application of an affinity-selection mass spectrometry method that employs unlabeled G4 oligonucleotides as targets and allows testing of up to 320 unmodified small molecules in a single tube. As a proof of concept, this method was applied to screen a library of 40 000 druglike molecules against two G4s, transcriptional regulators of the HIV-1 LTR promoter. We identified nonplanar pyrazolopyrimidines that selectively recognize and stabilize the major HIV-1 LTR G4 possibly by fitting and binding through H-bonding in its unique binding pocket. The compounds inhibit LTR promoter activity and HIV-1 replication. We propose this method to prompt the fast development of new G4-based therapeutics.

Structure of an engineered intein reveals thiazoline ring and provides mechanistic insight.

We have engineered an intein which spontaneously and reversibly forms a thiazoline ring at the native N-terminal Lys-Cys splice junction. We identified conditions to stablize the thiazoline ring and provided the first crystallographic evidence, at 1.54 Å resolution, for its existence at an intein active site. The finding bolsters evidence for a tetrahedral oxythiazolidine splicing intermediate. In addition, the pivotal mutation maps to a highly conserved B-block threonine, which is now seen to play a causative role not only in ground-state destabilization of the scissile N-terminal peptide bond, but also in steering the tetrahedral intermediate toward thioester formation, giving new insight into the splicing mechanism. We demonstrated the stability of the thiazoline ring at neutral pH as well as sensitivity to hydrolytic ring opening under acidic conditions. A pH cycling strategy to control N-terminal cleavage is proposed, which may be of interest for biotechnological applications requiring a splicing activity switch, such as for protein recovery in bioprocessing.

A Catalytic and Selective Scissoring Molecular Tool for Quadruplex Nucleic Acids.

A copper complex embedded in the structure of a water-soluble naphthalene diimide has been designed to bind and cleave G-quadruplex DNA. We describe the properties of this ligand, including its catalytic activity in the generation of ROS. FRET melting, CD, NMR, gel sequencing, and mass spectrometry experiments highlight a unique and unexpected selectivity in cleaving G-quadruplex sequences. This selectivity relies both on the binding affinity and structural features of the targeted G-quadruplexes.

Highly Improved Electrospray Ionization-Mass Spectrometry Detection of G-Quadruplex-Folded Oligonucleotides and Their Complexes with Small Molecules.

G-quadruplexes are nucleic acids structures stabilized by physiological concentration of potassium ions. Because low stability G-quadruplexes are hardly detectable by mass spectrometry, we optimized solvent conditions: isopropanol in a triethylamine/hexafluoroisopropanol mixture highly increased G-quadruplex sensitivity with no modification of the physiological G-quadruplex conformation. G-quadruplexes/G-quadruplex-ligand complexes were also correctly detected at concentration as low as 40 nM. Detection of the physiological conformation of G4s and their complexes opens up the possibility to perform high-throughput screening of G-quadruplex ligands for the development of drug molecules effective against critical human diseases.

Virtual Cross-Linking of the Active Nemorubicin Metabolite PNU-159682 to Double-Stranded DNA.

The DNA alkylating mechanism of PNU-159682 (PNU), a highly potent metabolite of the anthracycline nemorubicin, was investigated by gel-electrophoretic, HPLC-UV, and micro-HPLC/mass spectrometry (MS) measurements. PNU quickly reacted with double-stranded oligonucleotides, but not with single-stranded sequences, to form covalent adducts which were detectable by denaturing polyacrylamide gel electrophoresis (DPAGE). Ion-pair reverse-phase HPLC-UV analysis on CG rich duplex sequences having a 5'-CCCGGG-3' central core showed the formation of two types of adducts with PNU, which were stable and could be characterized by micro-HPLC/MS. The first type contained one alkylated species (and possibly one reversibly bound species), and the second contained two alkylated species per duplex DNA. The covalent adducts were found to produce effective bridging of DNA complementary strands through the formation of virtual cross-links reminiscent of those produced by classical anthracyclines in the presence of formaldehyde. Furthermore, the absence of reactivity of PNU with CG-rich sequence containing a TA core (CGTACG), and the minor reactivity between PNU and CGC sequences (TACGCG·CGCGTA) pointed out the importance of guanine sequence context in modulating DNA alkylation.

Identification of G-quadruplex DNA/RNA binders: Structure-based virtual screening and biophysical characterization.

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 (Tel2) and TERRA (TERRA2). 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 Tel2/TERRA2 G4-ligand at physiological KCl concentration, while hits 15 and 17 show preferential thermal stabilization for Tel2 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.

Nucleolin stabilizes G-quadruplex structures folded by the LTR promoter and silences HIV-1 viral transcription.

Folding of the LTR promoter into dynamic G-quadruplex conformations has been shown to suppress its transcriptional activity in HIV-1. Here we sought to identify the proteins that control the folding of this region of proviral genome by inducing/stabilizing G-quadruplex structures. The implementation of electrophorethic mobility shift assay and pull-down experiments coupled with mass spectrometric analysis revealed that the cellular protein nucleolin is able to specifically recognize G-quadruplex structures present in the LTR promoter. Nucleolin recognized with high affinity and specificity the majority, but not all the possible G-quadruplexes folded by this sequence. In addition, it displayed greater binding preference towards DNA than RNA G-quadruplexes, thus indicating two levels of selectivity based on the sequence and nature of the target. The interaction translated into stabilization of the LTR G-quadruplexes and increased promoter silencing activity; in contrast, disruption of nucleolin binding in cells by both siRNAs and a nucleolin binding aptamer greatly increased LTR promoter activity. These data indicate that nucleolin possesses a specific and regulated activity toward the HIV-1 LTR promoter, which is mediated by G-quadruplexes. These observations provide new essential insights into viral transcription and a possible low mutagenic target for antiretroviral therapy.

Targeting loop adenines in G-quadruplex by a selective oxirane.

Caught in the oxirane: Naphthalene diimides conjugated to a quinone methide and an oxirane have been synthesized and investigated as selective DNA G-quadruplex alkylating agents. The oxirane derivative generates a stable adduct with a G-quadruplex and shows selective alkylation of the loop adenines, as illustrated.

Fused in Liposarcoma Protein, a New Player in the Regulation of HIV-1 Transcription, Binds to Known and Newly Identified LTR G-Quadruplexes.

HIV-1 integrated long terminal repeat (LTR) promoter activity is modulated by folding of its G-rich region into non-canonical nucleic acids structures, such as G-quadruplexes (G4s), and their interaction with cellular proteins. Here, by a combined pull-down/mass spectrometry/Western-blot approach, we identified the fused in liposarcoma (FUS) protein and found it to preferentially bind and stabilize the least stable and bulged LTR G4, especially in the cell environment. The outcome of this interaction is the down-regulation of viral transcription, as assessed in a reporter assay with LTR G4 mutants in FUS-silencing conditions. These data indicate that the complexity and dynamics of HIV-1 LTR G4s are much greater than previously envisaged. The G-rich LTR region, with its diverse G4 landscape and multiple cell protein interactions, stands out as prime sensing center for the fine regulation of viral transcription. This region thus represents a rational antiviral target for inhibiting both the actively transcribing and latent viruses.

Quindoline-derivatives display potent G-quadruplex-mediated antiviral activity against herpes simplex virus 1.

G-quadruplexes (G4s) are non-canonical nucleic acid structures that regulate key biological processes, from transcription to genome replication both in humans and viruses. The herpes simplex virus-1 (HSV-1) genome is prone to form G4s that, along with proteins, regulate its viral cycle. General G4 ligands have been shown to hamper the viral cycle, pointing to viral G4s as original antiviral targets. Because cellular G4s are also normally present in infected cells, the quest for improved anti-HSV-1 G4 ligands is still open. Here, we evaluated a series of new quindoline-derivatives which showed high binding to and stabilization of the viral G4s. They displayed nanomolar-range anti-HSV-1 activity paralleled by negligible cytotoxicity in human cells, thus proving remarkable selectivity. The best-in-class compound inhibited the viral life cycle at the early times post infection up to the step of viral genome replication. In infected human cells, it reduced expression of ICP4, the main viral transcription factor, by stabilizing the G4s embedded in ICP4 promoter. Quindoline-derivatives thus emerge as a new class of G4 ligands with potent dual anti HSV-1 activity.

Inspiratory Vocal Fry: Anatomical and Physiological Aspects, Application in Speech Therapy, Vocal Pedagogy and Singing. A Pilot study.

OBJECTIVE: Inspiratory Vocal Fry (IVF) is the voice production during inspiration of a sound with vocal fry perceptual characteristics. The existing scientific literature shows a lack of studies on it. The aim of the study is to highlight anatomical and physiological characteristics of IVF, to assess its effects on spoken and singing voice, to confirm the potential usefulness in speech therapy and vocal pedagogy. METHODS: Thirty-two healthy subjects (17 male and 15 female) underwent videolaryngostroboscopy to assess the degree of false vocal folds adduction, pharyngeal wall contraction, and degree of vocal folds stretching in different types of phonation: expiratory and inspiratory phonation, Expiratory Vocal Fry (EVF) and IVF. All these parameters were evaluated by a group of three speech therapists and one phoniatrician not belonging to the research group. In addition, for each subject an electroglottography was performed for all the types of phonation previously mentioned, highlighting Contact Quotient (CQ) and Closing/Closed Quotient (CCQ). Three subjects underwent electromyography for a preliminary study of the muscle activation in IVF. RESULTS: False vocal folds adduction (P value = 0.000005) and pharyngeal wall contraction (P value = 0.001155) were significantly reduced in IVF compared to EVF; on the contrary, vocal folds stretching was significantly higher in IVF (P value = 0.000031). Electroglottographic CQ was significantly higher in IVF compared to EVF (P value = 0.019592) and the other types of phonation. Similar results were obtained considering CCQ, as IVF values for this parameter was significantly higher compared to EVF (P value = 0.013062) and expiratory phonation (P value = 0.001324). As regards electromyography, medial thyroarytenoid (TA) motor units were more recruited in IVF, while lateral TA motor units were more recruited in EVF. According to our results, IVF is characterized by higher elastic tension due to a reduced hypertonic contraction of TA muscle and a higher contraction of cricothyroid muscle. Electroglottographic results showed a wider vibratory cycle with an improved massaging effect on vocal folds mucosa. electromyography preliminary analysis confirmed our findings. CONCLUSION: IVF could be an excellent and useful exercise to reduce muscular hypertonic tension and to facilitate mucosal elasticity. It could be potentially applied in speech therapy approach to dysfunctional and organic dysphonias, post-surgical treatment, in pedagogy and practice of artistic voice.

HIV-1 Nucleocapsid Protein Unfolds Stable RNA G-Quadruplexes in the Viral Genome and Is Inhibited by G-Quadruplex Ligands.

The G-quadruplexes that form in the HIV-1 RNA genome hinder progression of reverse transcriptase in vitro, but not in infected cells. We investigated the possibility that the HIV-1 nucleocapsid protein NCp7, which remains associated with the viral RNA during reverse transcription, modulated HIV-1 RNA G-quadruplex stability. By electrophoresis, circular dichroism, mass spectrometry, and reverse transcriptase stop assays, we demonstrated that NCp7 binds and unfolds the HIV-1 RNA G-quadruplexes and promotes DNA/RNA duplex formation, allowing reverse transcription to proceed. The G-quadruplex ligand BRACO-19 was able to partially counteract this effect. These results indicate NCp7 as the first known viral protein able to unfold RNA G-quadruplexes, and they explain how the extra-stable HIV-1 RNA G-quadruplexes are processed; they also point out that the reverse transcription process is hindered by G-quadruplex ligands at both reverse transcriptase and NCp7 level. This information can lead to the development of more effective anti-HIV-1 drugs with a new mechanism of action.

Construction and structure studies of DNA-bipyridine complexes as versatile scaffolds for site-specific incorporation of metal ions into DNA.

The facile construction of metal-DNA complexes using 'Click' reactions is reported here. A series of 2'-propargyl-modified DNA oligonucleotides were initially synthesized as structure scaffolds and were then modified through 'Click' reaction to incorporate a bipyridine ligand equipped with an azido group. These metal chelating ligands can be placed in the DNA context in site-specific fashion to provide versatile templates for binding various metal ions, which are exchangeable using a simple EDTA washing-and-filtration step. The constructed metal-DNA complexes were found to be thermally stable. Their structures were explored by solving a crystal structure of a propargyl-modified DNA duplex and installing the bipyridine ligands by molecular modeling and simulation. These metal-DNA complexes could have wide applications as novel organometallic catalysts, artificial ribonucleases, and potential metal delivery systems.

A core extended naphtalene diimide G-quadruplex ligand potently inhibits herpes simplex virus 1 replication.

G-quadruplexes (G4s) are nucleic acids secondary structures, epigenetic regulators in cells and viruses. In herpes simplex virus 1 (HSV-1)-infected cells, G4s are massively present during viral replication. We here aimed at investigating the possibility to target the HSV-1 G4s by a core extended naphtalene diimide (c-exNDI) G4 ligand. Biophysical and biomolecular analysis proved that c-exNDI stabilized the HSV-1 G4s in a concentration dependent manner. In MS competition assays, c-exNDI preferentially recognized HSV-1 G4s over cellular telomeric G4s, the most represented G4s within cells; other less abundant cellular G4s were also recognized. Treatment of HSV-1 infected cells with c-exNDI at low nanomolar concentrations induced significant virus inhibition with no cytotoxicity. The mechanism of action was ascribed to G4-mediated inhibition of viral DNA replication, with consequent impairment of viral genes transcription. Our data suggest that the observed potent antiviral activity and low cytotoxicity mainly depend on a combination of c-exNDI affinity for HSV-1 G4s and their massive presence during infection. HSV-1 G4s may thus represent new effective antiviral targets: the fact that no current antiherpetic drug exploits them and their presence at the viral genome, responsible for both active and latent HSV infections, makes them particularly attracting.

The cellular protein hnRNP A2/B1 enhances HIV-1 transcription by unfolding LTR promoter G-quadruplexes.

G-quadruplexes are four-stranded conformations of nucleic acids that act as cellular epigenetic regulators. A dynamic G-quadruplex forming region in the HIV-1 LTR promoter represses HIV-1 transcription when in the folded conformation. This activity is enhanced by nucleolin, which induces and stabilizes the HIV-1 LTR G-quadruplexes. In this work by a combined pull-down/mass spectrometry approach, we consistently found hnRNP A2/B1 as an additional LTR-G-quadruplex interacting protein. Surface plasmon resonance confirmed G-quadruplex specificity over linear sequences and fluorescence resonance energy transfer analysis indicated that hnRNP A2/B1 is able to efficiently unfold the LTR G-quadruplexes. Evaluation of the thermal stability of the LTR G-quadruplexes in different-length oligonucleotides showed that the protein is fit to be most active in the LTR full-length environment. When hnRNP A2/B1 was silenced in cells, LTR activity decreased, indicating that the protein acts as a HIV-1 transcription activator. Our data highlight a tightly regulated control of transcription based on G-quadruplex folding/unfolding, which depends on interacting cellular proteins. These findings provide a deeper understanding of the viral transcription mechanism and may pave the way to the development of drugs effective against the integrated HIV-1, present both in actively and latently infected cells.

Change in the Molecular Dimension of a RAGE-Ligand Complex Triggers RAGE Signaling.

The weak oligomerization exhibited by many transmembrane receptors has a profound effect on signal transduction. The phenomenon is difficult to characterize structurally due to the large sizes of and transient interactions between monomers. The receptor for advanced glycation end products (RAGE), a signaling molecule central to the induction and perpetuation of inflammatory responses, is a weak constitutive oligomer. The RAGE domain interaction surfaces that mediate homo-dimerization were identified by combining segmental isotopic labeling of extracellular soluble RAGE (sRAGE) and nuclear magnetic resonance spectroscopy with chemical cross-linking and mass spectrometry. Molecular modeling suggests that two sRAGE monomers orient head to head forming an asymmetric dimer with the C termini directed toward the cell membrane. Ligand-induced association of RAGE homo-dimers on the cell surface increases the molecular dimension of the receptor, recruiting Diaphanous 1 (DIAPH1) and activating signaling pathways.

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay allows the rapid identification of HIV-1 nucleocapsid inhibitors.

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Synthesis, Binding and Antiviral Properties of Potent Core-Extended Naphthalene Diimides Targeting the HIV-1 Long Terminal Repeat Promoter G-Quadruplexes.

We have previously reported that stabilization of the G-quadruplex structures in the HIV-1 long terminal repeat (LTR) promoter suppresses viral transcription. Here we sought to develop new G-quadruplex ligands to be exploited as antiviral compounds by enhancing binding toward the viral G-quadruplex structures. We synthesized naphthalene diimide derivatives with a lateral expansion of the aromatic core. The new compounds were able to bind/stabilize the G-quadruplex to a high extent, and some of them displayed clear-cut selectivity toward the viral G-quadruplexes with respect to the human telomeric G-quadruplexes. This feature translated into low nanomolar anti-HIV-1 activity toward two viral strains and encouraging selectivity indexes. The selectivity depended on specific recognition of LTR loop residues; the mechanism of action was ascribed to inhibition of LTR promoter activity in cells. This is the first example of G-quadruplex ligands that show increased selectivity toward the viral G-quadruplexes and display remarkable antiviral activity.

Structure-specific ribonucleases for MS-based elucidation of higher-order RNA structure.

Supported by high-throughput sequencing technologies, structure-specific nucleases are experiencing a renaissance as biochemical probes for genome-wide mapping of nucleic acid structure. This report explores the benefits and pitfalls of the application of Mung bean (Mb) and V1 nuclease, which attack specifically single- and double-stranded regions of nucleic acids, as possible structural probes to be employed in combination with MS detection. Both enzymes were found capable of operating in ammonium-based solutions that are preferred for high-resolution analysis by direct infusion electrospray ionization (ESI). Sequence analysis by tandem mass spectrometry (MS/MS) was performed to confirm mapping assignments and to resolve possible ambiguities arising from the concomitant formation of isobaric products with identical base composition and different sequences. The observed products grouped together into ladder-type series that facilitated their assignment to unique regions of the substrate, but revealed also a certain level of uncertainty in identifying the boundaries between paired and unpaired regions. Various experimental factors that are known to stabilize nucleic acid structure, such as higher ionic strength, presence of Mg(II), etc., increased the accuracy of cleavage information, but did not completely eliminate deviations from expected results. These observations suggest extreme caution in interpreting the results afforded by these types of reagents. Regardless of the analytical platform of choice, the results highlighted the need to repeat probing experiments under the most diverse possible conditions to recognize potential artifacts and to increase the level of confidence in the observed structural information.

Rational design, synthesis, and DNA binding properties of novel sequence-selective peptidyl congeners of ametantrone.

Natural and synthetic compounds characterized by an anthraquinone nucleus represent an important class of anti-neoplastic agents, the mechanism of action of which is related to intercalation into DNA. Ametantrone (AM) is a synthetic 9,10-anthracenedione bearing two (hydroxyethylamino)ethylamino residues at positions 1 and 4; along with other anthraquinones and anthracyclines, it shares a polycyclic intercalating moiety and charged side chains that stabilize DNA binding. All these drugs elicit adverse side effects, which represent a challenge for antitumor chemotherapy. In the present work the structure of AM was augmented with appropriate groups that target well-defined base pairs in the major groove. These should endow AM with DNA sequence selectivity. We describe the rationale for the synthesis and the evaluation of activity of a new series of compounds in which the planar anthraquinone is conjugated at positions 1 and 4 through the side chains of AM or other bioisosteric linkers to appropriate dipeptides. The designed novel AM derivatives were shown to selectively stabilize two oligonucleotide duplexes that both have a palindromic GC-rich hexanucleotide core, but their stabilizing effects on a random DNA sequence was negligible. In the case of the most effective compound, the 1,4-bis-[Gly-(L-Lys)] derivative of AM, the experimental results confirm the predictions of earlier theoretical computations. In contrast, AM had equal stabilizing effects on all three sequences and showed no preferential binding. This novel peptide derivative can be classified as a strong binder regarding the sequences that it selectively targets, possibly opening the exploitation of less cytotoxic conjugates of AM to the targeted treatment of oncological and viral diseases.