Identification of druggable host dependency factors shared by multiple SARS-CoV-2 variants of concern.

The high mutation rate of SARS-CoV-2 leads to the emergence of multiple variants, some of which are resistant to vaccines and drugs targeting viral elements. Targeting host dependency factors, e.g. cellular proteins required for viral replication, would help prevent resistance. However, it remains unclear whether different SARS-CoV-2 variants induce conserved cellular responses and exploit the same core host factors. To this end, we compared three variants of concern and found that the host transcriptional response was conserved, differing only in kinetics and magnitude. Through CRISPR screening, we identified host genes required for infection by each variant. Most of the genes were shared by multiple variants. We validated our hits with small molecules and repurposed Food and Drug Administration-approved drugs. All the drugs were highly active against all the variants tested, including new variants that emerged during the study (Delta and Omicron). Mechanistically, we identified reactive oxygen species production as a key step in early virus replication. Antioxidants such as N-acetyl cysteine (NAC) were effective against all the variants in both human lung cells and a humanised mouse model. Our study supports the use of available antioxidant drugs, such as NAC, as a general and effective anti-COVID-19 approach.

Naphthalene Diimide-Tetraazacycloalkane Conjugates Are G-Quadruplex-Based HIV-1 Inhibitors with a Dual Mode of Action.

Human immunodeficiency virus 1 (HIV-1) therapeutic regimens consist of three or more drugs targeting different steps of the viral life cycle to limit the emergence of viral resistance. In line with the multitargeting strategy, here we conjugated a naphthalene diimide (NDI) moiety with a tetraazacycloalkane to obtain novel naphthalene diimide (NDI)-tetraazacycloalkane conjugates. The NDI inhibits the HIV-1 promoter activity by binding to LTR G-quadruplexes, and the tetraazacycloalkane mimics AMD3100, which blocks HIV entry into cells by interfering with the CXCR4 coreceptor. We synthesized, purified, and tested the metal-free NDI-tetraazacycloalkane conjugate and the two derived metal-organic complexes (MOCs) that incorporate Cu2+ and Zn2+. The NDI-MOCs showed enhanced binding to LTR G4s as assessed by FRET and CD assays in vitro. They also showed enhanced activity in cells where they dose-dependently reduced LTR promoter activity and inhibited viral entry only of the HIV-1 strain that exploited the CXCR4 coreceptor. The time of addition assay confirmed the dual targeting at the different HIV-1 steps. Our results indicate that the NDI-MOC conjugates can simultaneously inhibit viral entry, by targeting the CXCR4 coreceptor, and LTR promoter activity, by stabilizing the LTR G-quadruplexes. The approach of combining multiple targets in a single compound may streamline treatment regimens and improve the overall patient outcomes.

Efficient SARS-CoV-2 infection antagonization by rhACE2 ectodomain multimerized onto the Avidin-Nucleic-Acid-NanoASsembly.

Nanodecoy systems based on analogues of viral cellular receptors assembled onto fluid lipid-based membranes of nano/extravescicles are potential new tools to complement classic therapeutic or preventive antiviral approaches. The need for lipid-based membranes for transmembrane receptor anchorage may pose technical challenges along industrial translation, calling for alternative geometries for receptor multimerization. Here we developed a semisynthetic self-assembling SARS-CoV-2 nanodecoy by multimerizing the biotin labelled virus cell receptor -ACE2- ectodomain onto a poly-avidin nanoparticle (NP) based on the Avidin-Nucleic-Acid-NanoASsembly-ANANAS. The ability of the assembly to prevent SARS-CoV-2 infection in human lung cells and the affinity of the ACE2:viral receptor-binding domain (RBD) interaction were measured at different ACE2:NP ratios. At ACE2:NP = 30, 90 % SARS-CoV-2 infection inhibition at ACE2 nanomolar concentration was registered on both Wuhan and Omicron variants, with ten-fold higher potency than the monomeric protein. Lower and higher ACE2 densities were less efficient suggesting that functional recognition between multi-ligand NPs and multi-receptor virus surfaces requires optimal geometrical relationships. In vivo studies in mice showed that the biodistribution and safety profiles of the nanodecoy are potentially suitable for preventing viral infection upon nasal instillation. Viral receptor multimerization using ANANAS is a convenient process which, in principle, could be rapidly adapted to counteract also other viral infections.

Presence, Location and Conservation of Putative G-Quadruplex Forming Sequences in Arboviruses Infecting Humans.

Guanine quadruplexes (G4s) are non-canonical nucleic acid structures formed by guanine (G)-rich tracts that assemble into a core of stacked planar tetrads. G4s are found in the human genome and in the genomes of human pathogens, where they are involved in the regulation of gene expression and genome replication. G4s have been proposed as novel pharmacological targets in humans and their exploitation for antiviral therapy is an emerging research topic. Here, we report on the presence, conservation and localization of putative G4-forming sequences (PQSs) in human arboviruses. The prediction of PQSs was performed on more than twelve thousand viral genomes, belonging to forty different arboviruses that infect humans, and revealed that the abundance of PQSs in arboviruses is not related to the genomic GC content, but depends on the type of nucleic acid that constitutes the viral genome. Positive-strand ssRNA arboviruses, especially Flaviviruses, are significantly enriched in highly conserved PQSs, located in coding sequences (CDSs) or untranslated regions (UTRs). In contrast, negative-strand ssRNA and dsRNA arboviruses contain few conserved PQSs. Our analyses also revealed the presence of bulged PQSs, accounting for 17-26% of the total predicted PQSs. The data presented highlight the presence of highly conserved PQS in human arboviruses and present non-canonical nucleic acid-structures as promising therapeutic targets in arbovirus infections.

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.

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.

Angiotensin II Promotes SARS-CoV-2 Infection via Upregulation of ACE2 in Human Bronchial Cells.

Blockers of the renin-angiotensin system (RAS) have been reported to increase the angiotensin converting enzyme (ACE)2, the cellular receptor of SARS-CoV-2, and thus the risk and course of COVID-19. Therefore, we investigated if angiotensin (Ang) II and RAS blockers affected ACE2 expression and SARS-CoV-2 infectivity in human epithelial bronchial Calu-3 cells. By infectivity and spike-mediated cell-cell fusion assays, we showed that Ang II acting on the angiotensin type 1 receptor markedly increased ACE2 at mRNA and protein levels, resulting in enhanced SARS-CoV-2 cell entry. These effects were abolished by irbesartan and not affected by the blockade of ACE-1-mediated Ang II formation with ramipril, and of ACE2- mediated Ang II conversion into Ang 1-7 with MLN-4760. Thus, enhanced Ang II production in patients with an activated RAS might expose to a greater spread of COVID-19 infection in lung cells. The protective action of Angiotensin type 1 receptor antagonists (ARBs) documented in these studies provides a mechanistic explanation for the lack of worse outcomes in high-risk COVID-19 patients on RAS blockers.

Multimeric G-quadruplexes: A review on their biological roles and targeting.

In human cells, nucleic acids adopt several non-canonical structures that regulate key cellular processes. Among them, G-quadruplexes (G4s) are stable structures that form in guanine-rich regions in vitro and in cells. G4 folded/unfolded state shapes numerous cellular processes, including genome replication, transcription, and translation. Moreover, G4 folding is involved in genomic instability. G4s have been described to multimerize, forming high-order structures in both DNA and/or RNA strands. Multimeric G4s can be formed by adjacent intramolecular G4s joined by stacking interactions or connected by short loops. Multimeric G4s can also originate from the assembly of guanines embedded on independent DNA or RNA strands. Notably, crucial regions of the human genome, such as the 3'-terminal overhang of the telomeric DNA as well as the open reading frame of genes involved in the preservation of neuron viability in the human central and peripheral nervous system are prone to form multimeric G4s. The biological importance of such structures has been recently described, with multimeric G4s playing potentially protective or deleterious effects in the pathogenic cascade of various diseases. Here, we portray the multifaceted scenario of multimeric G4s, in terms of structural properties, biological roles, and targeting strategies.

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.

Parallel G-quadruplexes recruit the HSV-1 transcription factor ICP4 to promote viral transcription in herpes virus-infected human cells.

G-quadruplexes (G4s) are four-stranded nucleic acid structures abundant at gene promoters. They can adopt several distinctive conformations. G4s have been shown to form in the herpes simplex virus-1 (HSV-1) genome during its viral cycle. Here by cross-linking/pull-down assay we identified ICP4, the major HSV-1 transcription factor, as the protein that most efficiently interacts with viral G4s during infection. ICP4 specific and direct binding and unfolding of parallel G4s, including those present in HSV-1 immediate early gene promoters, induced transcription in vitro and in infected cells. This mechanism was also exploited by ICP4 to promote its own transcription. Proximity ligation assay allowed visualization of G4-protein interaction at the single selected G4 in cells. G4 ligands inhibited ICP4 binding to G4s. Our results indicate the existence of a well-defined G4-viral protein network that regulates the productive HSV-1 cycle. They also point to G4s as elements that recruit transcription factors to activate transcription in cells.

The MDM2 inducible promoter folds into four-tetrad antiparallel G-quadruplexes targetable to fight malignant liposarcoma.

Well-differentiated liposarcoma (WDLPS) is a malignant neoplasia hard to diagnose and treat. Its main molecular signature is amplification of the MDM2-containing genomic region. The MDM2 oncogene is the master regulator of p53: its overexpression enhances p53 degradation and inhibits apoptosis, leading to the tumoral phenotype. Here, we show that the MDM2 inducible promoter G-rich region folds into stable G-quadruplexes both in vitro and in vivo and it is specifically recognized by cellular helicases. Cell treatment with G-quadruplex-ligands reduces MDM2 expression and p53 degradation, thus stimulating cancer cell cycle arrest and apoptosis. Structural characterization of the MDM2 G-quadruplex revealed an extraordinarily stable, unique four-tetrad antiparallel dynamic conformation, amenable to selective targeting. These data indicate the feasibility of an out-of-the-box G-quadruplex-targeting approach to defeat WDLPS and all tumours where restoration of wild-type p53 is sought. They also point to G-quadruplex-dependent genomic instability as possible cause of MDM2 expansion and WDLPS tumorigenesis.

A dynamic i-motif with a duplex stem-loop in the long terminal repeat promoter of the HIV-1 proviral genome modulates viral transcription.

I-motifs are non-canonical nucleic acids structures characterized by intercalated H-bonds between hemi-protonated cytosines. Evidence on the involvement of i-motif structures in the regulation of cellular processes in human cells has been consistently growing in the recent years. However, i-motifs within non-human genomes have never been investigated. Here, we report the characterization of i-motifs within the long terminal repeat (LTR) promoter of the HIV-1 proviral genome. Biophysical and biochemical analysis revealed formation of a predominant i-motif with an unprecedented loop composition. One-dimensional nuclear magnetic resonance investigation demonstrated formation of three G-C H-bonds in the long loop, which likely improve the structure overall stability. Pull-down experiments combined with mass spectrometry and protein crosslinking analysis showed that the LTR i-motif is recognized by the cellular protein hnRNP K, which induced folding at physiological conditions. In addition, hnRNP K silencing resulted in an increased LTR promoter activity, confirming the ability of the protein to stabilize the i-motif-forming sequence, which in turn regulates the LTR-mediated HIV-1 transcription. These findings provide new insights into the complexity of the HIV-1 virus and lay the basis for innovative antiviral drug design, based on the possibility to selectively recognize and target the HIV-1 LTR i-motif.

Conserved G-Quadruplexes Regulate the Immediate Early Promoters of Human Alphaherpesviruses.

Human Alphaherpesviruses comprise three members, herpes simplex virus (HSV) 1 and 2 and varicella zoster virus (VZV). These viruses are characterized by a lytic cycle in epithelial cells and latency in the nervous system, with lifelong infections that may periodically reactivate and lead to serious complications, especially in immunocompromised patients. The mechanisms that regulate viral transcription have not been fully elucidated, but the master role of the immediate early (IE) genes has been established. G-quadruplexes are non-canonical nucleic-acid structures that control transcription, replication, and recombination in many organisms including viruses and that represent attractive antiviral targets. In this work, we investigate the presence, conservation, folding and activity of G-quadruplexes in the IE promoters of the Alphaherpesviruses. Our analysis shows that all IE promoters in the genome of HSV-1, HSV-2 and VZV contain fully conserved G-quadruplex forming sequences. These comprise sequences with long loops and bulges, and thus deviating from the classic G-quadruplex motifs. Moreover, their location is both on the leading and lagging strand and in some instances they contain exuberant G-tracts. Biophysical and biological analysis proved that all sequences actually fold into G-quadruplex under physiological conditions and can be further stabilized by the G-quadruplex ligand BRACO-19, with subsequent impairment of viral IE gene transcription in cells. These results help shed light on the control of viral transcription and indicate new viral targets to design drugs that impair the early steps of Alphaherpesviruses. In addition, they validate the significance of G-quadruplexes in the general regulation of viral cycles.

Synthesis and photocytotoxic activity of [1,2,3]triazolo[4,5-h][1,6]naphthyridines and [1,3]oxazolo[5,4-h][1,6]naphthyridines.

[1,2,3]Triazolo[4,5-h][1,6]naphthyridines and [1,3]oxazolo[5,4-h][1,6]naphthyridines were synthesized with the aim to investigate their photocytotoxic activity. Upon irradiation, oxazolo-naphtapyridines induced light-dependent cell death at nanomolar/low micromolar concentrations (EC50 0.01-6.59 µM). The most photocytotoxic derivative showed very high selectivity and photocytotoxicity indexes (SI = 72-86, PTI>5000), along with a triplet excited state with exceptionally long lifetime (18.0 µs) and high molar absorptivity (29781 ±â€¯180 M-1cm-1 at λmax 315 nm). The light-induced production of ROS promptly induced an unquenchable apoptotic process selectively in tumor cells, with mitochondrial and lysosomal involvement. Altogether, these results demonstrate that the most active compound acts as a promising singlet oxygen sensitizer for biological applications.

G-quadruplex forming sequences in the genome of all known human viruses: A comprehensive guide.

G-quadruplexes are non-canonical nucleic-acid structures that control transcription, replication, and recombination in organisms. G-quadruplexes are present in eukaryotes, prokaryotes, and viruses. In the latter, mounting evidence indicates their key biological activity. Since data on viruses are scattered, we here present a comprehensive analysis of potential quadruplex-forming sequences (PQS) in the genome of all known viruses that can infect humans. We show that occurrence and location of PQSs are features characteristic of each virus class and family. Our statistical analysis proves that their presence within the viral genome is orderly arranged, as indicated by the possibility to correctly assign up to two-thirds of viruses to their exact class based on the PQS classification. For each virus we provide: i) the list of all PQS present in the genome (positive and negative strands), ii) their position in the viral genome, iii) the degree of conservation among strains of each PQS in its genome context, iv) the statistical significance of PQS abundance. This information is accessible from a database to allow the easy navigation of the results: http://www.medcomp.medicina.unipd.it/main_site/doku.php?id=g4virus. The availability of these data will greatly expedite research on G-quadruplex in viruses, with the possibility to accelerate finding therapeutic opportunities to numerous and some fearsome human diseases.

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.

Pyrrolo[3',2':6,7]cyclohepta[1,2-b]pyridines with potent photo-antiproliferative activity.

Pyrrolo[3',2':6,7]cyclohepta[1,2-b]pyridines were synthesized as a new class of tricyclic system in which the pyridine ring is annelated to a cycloheptapyrrole scaffold, with the aim of obtaining new photosensitizing agents with improved antiproliferative activity and lower undesired toxic effects. A versatile synthetic pathway was approached, which allowed the isolation of derivatives of the title ring system with a good substitution pattern on the pyrrole moiety. Photobiological studies revealed that the majority of the new compounds showed a potent cytotoxic effect upon photoactivation with light of the proper wavelength, especially when decorated with a 2-ethoxycabonyl group and a N-benzyl substituted moiety, with EC50 values reaching the submicromolar level. The mechanism of action was evaluated.

Synthesis and antiproliferative mechanism of action of pyrrolo[3',2':6,7] cyclohepta[1,2-d]pyrimidin-2-amines as singlet oxygen photosensitizers.

A new series of pyrrolo[3',2':6,7]cyclohepta[1,2-d]pyrimidin-2-amines, was conveniently prepared using a versatile and high yielding multistep sequence. A good number of derivatives was obtained and the cellular photocytotoxicity was evaluated in vitro against three different human tumor cell lines with EC50 (0.08-4.96 µM) values reaching the nanomolar level. Selected compounds were investigated by laser flash photolysis. The most photocytotoxic derivative, exhibiting a fairly long-lived triplet state (τ ∼ 7 µs) and absorbance in the UV-Vis, was tested in the photo-oxidations of 9,10-anthracenedipropionic acid (ADPA) by singlet oxygen. The photosentizing properties are responsible for the compounds' ability to photoinduce massive cell death with involvement of mitochondria.

Serotype epidemiology and multidrug resistance patterns of Salmonella enterica infecting humans in Italy.

BACKGROUND: Salmonella enterica is the zoonotic agent most frequently responsible for foodborne infections in humans worldwide. In this work the presence of S. enterica was investigated in 734 unique enteropathogenic isolates collected from human patients between 2011 and 2012. RESULTS: All Salmonella spp. isolates were subjected to serotyping and antimicrobial susceptibility testing. Isolates displaying phenotypes and antimicrobial susceptibility profiles different from the reference strains were genotipically characterized. Several plasmid-embedded resistance determinants were identified and characterized. Non-typhoidal serotypes were most frequently diagnosed; monophasic Salmonella typhimurium 1,4 [5],12:i- and S. typhimurium represented the most prevalent serovars. Five isolates displayed phenotypes with extremely reduced susceptibility to antimicrobials: we detected multidrug resistance elements belonging to Ambler class A and class C in two non-typhoidal S. enterica serovars, i.e. Rissen and monophasic S. typhimurium 1,4 [5],12:i-, and in one typhoidal serovar, i.e., Paratyphi B. These resistance determinants have been so far almost exclusively associated with non-Salmonella members of the Enterobacteriaceae family. Alarmingly, two colistin resistant Salmonella enteritidis were also found. CONCLUSIONS: This work draws the attention to the still low, but rising, percentage of multidrug resistant Salmonella isolates infecting humans in Italy. Our results suggest that prompt monitoring of Salmonella serovar dissemination and resistance to antimicrobials is highly required.