45 research outputs found
Polyphenolic C-glucosidic ellagitannins present in oak-aged wine inhibit HIV-1 nucleocapsid protein
HIV-1 nucleocapsid protein (NC) is a nucleic acid chaperone implicated in several steps of the virus replication cycle and an attractive new target for drug development. In reverse transcription, NC destabilizes nucleic acid secondary structures and catalyzes the annealing of HIV-1 TAR RNA to its DNA copy (cTAR) to form the heteroduplex TAR/cTAR. A screening program led to the identification of the plant polyphenols acutissimins A and B as potent inhibitors of NC in different assays. These two flavano-ellagitannins, which are found in wine aged in oak barrels, exhibited different mechanisms of protein inhibition and higher potency relatively to their epimers, epiacutissimins A and B, and to simpler structures notably representing hydrolytic fragments and metabolites therefrom
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
Identification of novel 2-benzoxazolinone derivatives with specific inhibitory activity against the HIV-1 nucleocapsid protein
In this report, we present a new benzoxazole derivative endowed with inhibitory activity against the
HIV-1 nucleocapsid protein (NC). NC is a 55-residue basic protein with nucleic acid chaperone properties,
which has emerged as a novel and potential pharmacological target against HIV-1. In the pursuit of novel
NC-inhibitor chemotypes, we performed virtual screening and in vitro biological evaluation of a large
library of chemical entities. We found that compounds sharing a benzoxazolinone moiety displayed
putative inhibitory properties, which we further investigated by considering a series of chemical analogues.
This approach provided valuable information on the structure-activity relationships of these
compounds and, in the process, demonstrated that their anti-NC activity could be finely tuned by the
addition of specific substituents to the initial benzoxazolinone scaffold. This study represents the starting
point for the possible development of a new class of antiretroviral agents targeting the HIV-1 NC protein
Mechanisms of HIV-1 Nucleocapsid Protein Inhibition by Lysyl-Peptidyl-Anthraquinone Conjugates
The Nucleocapsid protein NCp7 (NC) is a nucleic acid chaperone responsible for essential steps of the HIV-1 life cycle and an attractive candidate for drug development. NC destabilizes nucleic acid structures and promotes the formation of annealed substrates for HIV-1 reverse transcription elongation. Short helical nucleic acid segments bordered by bulges and loops, such as the Trans-Activation Response element (TAR) of HIV-1 and its complementary sequence (cTAR), are nucleation elements for helix destabilization by NC and also preferred recognition sites for threading intercalators. Inspired by these observations, we have recently demonstrated that 2,6-disubstituted peptidylanthraquinone-conjugates inhibit the chaperone activities of recombinant NC in vitro, and that inhibition correlates with the stabilization of TAR and cTAR stem-loop structures. We describe here enhanced NC inhibitory activity by novel conjugates that exhibit longer peptidyl chains ending with a conserved Nterminal lysine. Their efficient inhibition of TAR/cTAR annealing mediated by NC originates from the combination of at least three different mechanisms, namely, their stabilizing effects on nucleic acids dynamics by threading intercalation, their ability to target TAR RNA substrate leading to a direct competition with the protein for the same binding sites on TAR, and, finally, their effective binding to the NC protein. Our results suggest that these molecules may represent the stepping-stone for the future development of NC-inhibitors capable of targeting the protein itself and its recognition site in RNA
Non-Natural Linker Configuration in 2,6-Dipeptidyl-Anthraquinones Enhances the Inhibition of TAR RNA Binding/Annealing Activities by HIV-1 NC and Tat Proteins
The HIV-1 nucleocapsid (NC) protein represents an excellent molecular target for the development of antiretrovirals
by virtue of its well-characterized chaperone activities, which play pivotal roles in essential steps of the viral life cycle.
Our ongoing search for candidates able to impair NC binding/annealing activities led to the identification of peptidylanthraquinones
as a promising class of nucleic acid ligands. Seeking to elucidate the inhibition determinants and increase the
potency of this class of compounds, we have now explored the effects of chirality in the linker connecting the planar nucleus to
the basic side chains. We show here that the non-natural linker configuration imparted unexpected TAR RNA targeting
properties to the 2,6-peptidyl-anthraquinones and significantly enhanced their potency. Even if the new compounds were able to
interact directly with the NC protein, they manifested a consistently higher affinity for the TAR RNA substrate and their TARbinding
properties mirrored their ability to interfere with NC-TAR interactions. Based on these findings, we propose that the
viral Tat protein, sharing the same RNA substrate but acting in distinct phases of the viral life cycle, constitutes an additional
druggable target for this class of peptidyl-anthraquinones. The inhibition of Tat-TAR interaction for the test compounds
correlated again with their TAR-binding properties, while simultaneously failing to demonstrate any direct Tat-binding
capabilities. These considerations highlighted the importance of TAR RNA in the elucidation of their inhibition mechanism,
rather than direct protein inhibition. We have therefore identified anti-TAR compounds with dual in vitro inhibitory activity on
different viral proteins, demonstrating that it is possible to develop multitarget compounds capable of interfering with processes
mediated by the interactions of this essential RNA domain of HIV-1 genome with NC and Tat proteins
Hubble Space Telescope Combined Strong and Weak Lensing Analysis of the CLASH Sample: Mass and Magnification Models and Systematic Uncertainties
We present results from a comprehensive lensing analysis in HST data, of the
complete CLASH cluster sample. We identify new multiple-images previously
undiscovered allowing improved or first constraints on the cluster inner mass
distributions and profiles. We combine these strong-lensing constraints with
weak-lensing shape measurements within the HST FOV to jointly constrain the
mass distributions. The analysis is performed in two different common
parameterizations (one adopts light-traces-mass for both galaxies and dark
matter while the other adopts an analytical, elliptical NFW form for the dark
matter), to provide a better assessment of the underlying systematics - which
is most important for deep, cluster-lensing surveys, especially when studying
magnified high-redshift objects. We find that the typical (median), relative
systematic differences throughout the central FOV are in the
(dimensionless) mass density, , and in the magnification,
. We show maps of these differences for each cluster, as well as the mass
distributions, critical curves, and 2D integrated mass profiles. For the
Einstein radii () we find that all typically agree within
between the two models, and Einstein masses agree, typically, within
. At larger radii, the total projected, 2D integrated mass profiles
of the two models, within r\sim2\arcmin, differ by . Stacking the
surface-density profiles of the sample from the two methods together, we obtain
an average slope of , in the radial
range [5,350] kpc. Lastly, we also characterize the behavior of the average
magnification, surface density, and shear differences between the two models,
as a function of both the radius from the center, and the best-fit values of
these quantities.Comment: 35 pages (20 main text pages, plus 15 pages for additional figures
and tables); 2 Tables, 17 Figures. V3: accepted version; some minor
corrections and additions made. V4: corrected several entries in Table 2. All
mass models and magnification maps are made publicly available for the
communit
Sex- and age-related differences in the management and outcomes of chronic heart failure: an analysis of patients from the ESC HFA EORP Heart Failure Long-Term Registry
Aims: This study aimed to assess age- and sex-related differences in management and 1-year risk for all-cause mortality and hospitalization in chronic heart failure (HF) patients. Methods and results: Of 16 354 patients included in the European Society of Cardiology Heart Failure Long-Term Registry, 9428 chronic HF patients were analysed [median age: 66 years; 28.5% women; mean left ventricular ejection fraction (LVEF) 37%]. Rates of use of guideline-directed medical therapy (GDMT) were high (angiotensin-converting enzyme inhibitors/angiotensin receptor blockers, beta-blockers and mineralocorticoid receptor antagonists: 85.7%, 88.7% and 58.8%, respectively). Crude GDMT utilization rates were lower in women than in men (all differences: P\ua0 64 0.001), and GDMT use became lower with ageing in both sexes, at baseline and at 1-year follow-up. Sex was not an independent predictor of GDMT prescription; however, age >75 years was a significant predictor of GDMT underutilization. Rates of all-cause mortality were lower in women than in men (7.1% vs. 8.7%; P\ua0=\ua00.015), as were rates of all-cause hospitalization (21.9% vs. 27.3%; P\ua075 years. Conclusions: There was a decline in GDMT use with advanced age in both sexes. Sex was not an independent predictor of GDMT or adverse outcomes. However, age >75 years independently predicted lower GDMT use and higher all-cause mortality in patients with LVEF 6445%
B-CePs as cross-linking probes for the investigation of RNA higher-order structure
Elucidating the structure of RNA and RNA ensembles
is essential to understand biological functions.
In this work, we explored the previously uncharted
reactivity of bis-chloropiperidines (B-CePs) towards
RNA. We characterized at the molecular level the different
adducts induced by the fast reacting compound
B-CeP 1 with RNA. Following an approach
based on solution thermal melting coupled with ESI
mass spectrometry (STHEM-ESI), we proved the ability
of B-CePs to induce inter-molecular cross-links
between guanines in double stranded RNA. These
results open the possibility of using B-CePs as structural
probes for investigating higher-order structures,
such as the kissing loop complex established
by the dimerization initiation site (DIS) of the HIV-1
genome. We confirmed the potential of B-CePs to reveal
the identity of RNA structures involved in longrange
interactions, expecting to benefit the characterization
of samples that are not readily amenable
to traditional high-resolution techniques, and thus
promoting the elucidation of pertinent RNA systems
associated with old and new diseases
In Vitro Evaluation of Bis-3-Chloropiperidines as RNA Modulators Targeting TAR and TAR-Protein Interaction
After a long limbo, RNA has gained its credibility as a druggable target, fully earning its deserved role in the next generation of pharmaceutical R&D. We have recently probed the trans-activation response (TAR) element, an RNA stem–bulge–loop domain of the HIV-1 genome with bis-3-chloropiperidines (B-CePs), and revealed the compounds unique behavior in stabilizing TAR structure, thus impairing in vitro the chaperone activity of the HIV-1 nucleocapsid (NC) protein. Seeking to elucidate the determinants of B-CePs inhibition, we have further characterized here their effects on the target TAR and its NC recognition, while developing quantitative analytical approaches for the study of multicomponent RNA-based interactions
Bis-3-Chloropiperidines Targeting TAR RNA as A Novel Strategy to Impair the HIV-1 Nucleocapsid Protein
Specific RNA sequences regulate functions essential to life. The Trans-Activation Response element (TAR) is an RNA stem\u2013bulge\u2013loop structure involved in several steps of HIV-1 replication. In this work, we show how RNA targeting can inhibit HIV-1 nucleocapsid (NC), a highly conserved protein known to catalyze nucleic acid melting and strand transfers during reverse transcription. Our RNA targeting strategy consists of the employment of bis-3-chloropiperidines (B-CePs) to impair RNA melting through bifunctional alkylation. Specific interactions between B-CePs and TAR RNA were analytically investigated by gel electrophoresis and mass spectrometry, allowing the elucidation of B-CePs\u2019 recognition of TAR, and highlighting an RNA-directed mechanism of protein inhibition. We propose that B-CePs can freeze TAR tridimensional conformation, impairing NC-induced dynamics and finally inhibiting its functions in vitro