Insights into the product release mechanism of dengue virus NS3 helicase.

The non-structural protein 3 helicase (NS3h) is a multifunctional protein that is critical in RNA replication and other stages in the flavivirus life cycle. NS3h uses energy from ATP hydrolysis to translocate along single stranded nucleic acid and to unwind double stranded RNA. Here we present a detailed mechanistic analysis of the product release stage in the catalytic cycle of the dengue virus (DENV) NS3h. This study is based on a combined experimental and computational approach of product-inhibition studies and free energy calculations. Our results support a model in which the catalytic cycle of ATP hydrolysis proceeds through an ordered sequential mechanism that includes a ternary complex intermediate (NS3h-Pi-ADP), which evolves releasing the first product, phosphate (Pi), and subsequently ADP. Our results indicate that in the product release stage of the DENV NS3h a novel open-loop conformation plays an important role that may be conserved in NS3 proteins of other flaviviruses as well.

Solvent effects on the NMR shieldings of stacked DNA base pairs.

Stacking effects are among the most important effects in DNA. We have recently studied their influence in fragments of DNA through the analysis of NMR magnetic shieldings, firstly in vacuo. As a continuation of this line of research we show here the influence of solvent effects on the shieldings through the application of both explicit and implicit models. We found that the explicit solvent model is more appropriate for consideration due to the results matching better in general with experiments, as well as providing clear knowledge of the electronic origin of the value of the shieldings. Our study is grounded on a recently developed theoretical model of our own, by which we are able to learn about the magnetic effects of given fragments of DNA molecules on selected base pairs. We use the shieldings of the atoms of a central base pair (guanine-cytosine) of a selected fragment of DNA molecules as descriptors of physical effects, like π-stacking and solvent effects. They can be taken separately and altogether. The effect of π-stacking is introduced through the addition of some pairs above and below of the central base pair, and now, the solvent effect is considered including a network of water molecules that consist of two solvation layers, which were fixed in the calculations performed in all fragments. We show that the solvent effects enhance the stacking effects on the magnetic shieldings of atoms that belong to the external N-H bonds. The net effect is of deshielding on both atoms. There is also a deshielding effect on the carbon atoms that belong to CO bonds, for which the oxygen atom has an explicit hydrogen bond (HB) with a solvent water molecule. Solvent effects are found to be no higher than a few percent of the total value of the shieldings (between 1% and 5%) for most atoms, although there are few for which such an effect can be higher. There is one nitrogen atom, the acceptor of the HB between guanine and cytosine, that is more highly shielded (around 15 ppm or 10%) when the explicit solvent is considered. In a similar manner, the most external nitrogen atom of cytosine and the hydrogen atom that is bonded to it are highly deshielded (around 10 ppm for nitrogen and around 3 ppm for hydrogen).

How Search Engine Data Enhance the Understanding of Determinants of Suicide in India and Inform Prevention: Observational Study.

BACKGROUND: India is home to 20% of the world's suicide deaths. Although statistics regarding suicide in India are distressingly high, data and cultural issues likely contribute to a widespread underreporting of the problem. Social stigma and only recent decriminalization of suicide are among the factors hampering official agencies' collection and reporting of suicide rates. OBJECTIVE: As the product of a data collaborative, this paper leverages private-sector search engine data toward gaining a fuller, more accurate picture of the suicide issue among young people in India. By combining official statistics on suicide with data generated through search queries, this paper seeks to: add an additional layer of information to more accurately represent the magnitude of the problem, determine whether search query data can serve as an effective proxy for factors contributing to suicide that are not represented in traditional datasets, and consider how data collaboratives built on search query data could inform future suicide prevention efforts in India and beyond. METHODS: We combined official statistics on demographic information with data generated through search queries from Bing to gain insight into suicide rates per state in India as reported by the National Crimes Record Bureau of India. We extracted English language queries on "suicide," "depression," "hanging," "pesticide," and "poison". We also collected data on demographic information at the state level in India, including urbanization, growth rate, sex ratio, internet penetration, and population. We modeled the suicide rate per state as a function of the queries on each of the 5 topics considered as linear independent variables. A second model was built by integrating the demographic information as additional linear independent variables. RESULTS: Results of the first model fit (R2) when modeling the suicide rates from the fraction of queries in each of the 5 topics, as well as the fraction of all suicide methods, show a correlation of about 0.5. This increases significantly with the removal of 3 outliers and improves slightly when 5 outliers are removed. Results for the second model fit using both query and demographic data show that for all categories, if no outliers are removed, demographic data can model suicide rates better than query data. However, when 3 outliers are removed, query data about pesticides or poisons improves the model over using demographic data. CONCLUSIONS: In this work, we used search data and demographics to model suicide rates. In this way, search data serve as a proxy for unmeasured (hidden) factors corresponding to suicide rates. Moreover, our procedure for outlier rejection serves to single out states where the suicide rates have substantially different correlations with demographic factors and query rates.

Unveiling tetrahydroquinolines as promising BVDV entry inhibitors: Targeting the envelope protein.

Bovine viral diarrhea virus (BVDV) is known to cause financial losses and decreased productivity in the cattle industry worldwide. Currently, there are no available antiviral treatments for effectively controlling BVDV infections in laboratories or farms. The BVDV envelope protein (E2) mediates receptor recognition on the cell surface and is required for fusion of virus and cell membranes after the endocytic uptake of the virus during the entry process. Therefore, E2 is an attractive target for the development of antiviral strategies. To identify BVDV antivirals targeting E2 function, we defined a binding site in silico located in domain IIIc at the interface between monomers in the disulfide linked dimer of E2. Employing a de novo design methodology to identify compounds with the potential to inhibit the E2 function, compound 9 emerged as a promising candidate with remarkable antiviral activity and minimal toxicity. In line with targeting of E2 function, compound 9 was found to block the virus entry into host cells. Furthermore, we demonstrated that compound 9 selectively binds to recombinant E2 in vitro. Molecular dynamics simulations (MD) allowed describing a possible interaction pattern between compound 9 and E2 and indicated that the S enantiomer of compound 9 may be responsible for the antiviral activity. Future research endeavors will focus on synthesizing enantiomerically pure compounds to further support these findings. These results highlight the usefulness of de novo design strategies to identify a novel class of BVDV inhibitors that block E2 function inhibiting virus entry into the host cell.

Thermodynamic and mechanistic analysis of the functional properties of dengue virus NS3 helicase.

The Dengue Virus (DENV) non-structural protein 3 (NS3) is a multi-functional protein critical in the viral life cycle. The DENV NS3 is comprised of a serine protease domain and a helicase domain. The helicase domain itself acts as a molecular motor, either translocating in a unidirectional manner along single-stranded RNA or unwinding double-stranded RNA, processes fueled by the hydrolysis of nucleoside triphosphates. In this brief review, we summarize our contributions and ongoing efforts to uncover the thermodynamic and mechanistic functional properties of the DENV NS3 as an NTPase and helicase.

dockECR: Open consensus docking and ranking protocol for virtual screening of small molecules.

The development of open computational pipelines to accelerate the discovery of treatments for emerging diseases allows finding novel solutions in shorter periods of time. Consensus molecular docking is one of these approaches, and its main purpose is to increase the detection of real actives within virtual screening campaigns. Here we present dockECR, an open consensus docking and ranking protocol that implements the exponential consensus ranking method to prioritize molecular candidates. The protocol uses four open source molecular docking programs: AutoDock Vina, Smina, LeDock and rDock, to rank the molecules. In addition, we introduce a scoring strategy based on the average RMSD obtained from comparing the best poses from each single program to complement the consensus ranking with information about the predicted poses. The protocol was benchmarked using 15 relevant protein targets with known actives and decoys, and applied using the main protease of the SARS-CoV-2 virus. For the application, different crystal structures of the protease, and frames obtained from molecular dynamics simulations were used to dock a library of 79 molecules derived from previously co-crystallized fragments. The ranking obtained with dockECR was used to prioritize eight candidates, which were evaluated in terms of the interactions generated with key residues from the protease. The protocol can be implemented in any virtual screening campaign involving proteins as molecular targets. The dockECR code is publicly available at: https://github.com/rochoa85/dockECR.

Correction: Monitoring of the Venezuelan exodus through Facebook's advertising platform.

[This corrects the article DOI: 10.1371/journal.pone.0229175.].

Migrant mobility flows characterized with digital data.

Monitoring migration flows is crucial to respond to humanitarian crisis and to design efficient policies. This information usually comes from surveys and border controls, but timely accessibility and methodological concerns reduce its usefulness. Here, we propose a method to detect migration flows worldwide using geolocated Twitter data. We focus on the migration crisis in Venezuela and show that the calculated flows are consistent with official statistics at country level. Our method is versatile and far-reaching, as it can be used to study different features of migration as preferred routes, settlement areas, mobility through several countries, spatial integration in cities, etc. It provides finer geographical and temporal resolutions, allowing the exploration of issues not contemplated in official records. It is our hope that these new sources of information can complement official ones, helping authorities and humanitarian organizations to better assess when and where to intervene on the ground.

Monitoring of the Venezuelan exodus through Facebook's advertising platform.

Venezuela is going through the worst economical, political and social crisis in its modern history. Basic products like food or medicine are scarce and hyperinflation is combined with economic depression. This situation is creating an unprecedented refugee and migrant crisis in the region. Governments and international agencies have not been able to consistently leverage reliable information using traditional methods. Therefore, to organize and deploy any kind of humanitarian response, it is crucial to evaluate new methodologies to measure the number and location of Venezuelan refugees and migrants across Latin America. In this paper, we propose to use Facebook's advertising platform as an additional data source for monitoring the ongoing crisis. We estimate and validate national and sub-national numbers of refugees and migrants and break-down their socio-economic profiles to further understand the complexity of the phenomenon. Although limitations exist, we believe that the presented methodology can be of value for real-time assessment of refugee and migrant crises world-wide.

Design and Optimization of Quinazoline Derivatives: New Non-nucleoside Inhibitors of Bovine Viral Diarrhea Virus.

Bovine viral diarrhea virus (BVDV) belongs to the Pestivirus genus (Flaviviridae). In spite of the availability of vaccines, the virus is still causing substantial financial losses to the livestock industry. In this context, the use of antiviral agents could be an alternative strategy to control and reduce viral infections. The viral RNA-dependent RNA polymerase (RdRp) is essential for the replication of the viral genome and constitutes an attractive target for the identification of antiviral compounds. In a previous work, we have identified potential molecules that dock into an allosteric binding pocket of BVDV RdRp via a structure-based virtual screening approach. One of them, N-(2-morpholinoethyl)-2-phenylquinazolin-4-amine [1, 50% effective concentration (EC50) = 9.7 ± 0.5 µM], was selected to perform different chemical modifications. Among 24 derivatives synthesized, eight of them showed considerable antiviral activity. Molecular modeling of the most active compounds showed that they bind to a pocket located in the fingers and thumb domains in BVDV RdRp, which is different from that identified for other non-nucleoside inhibitors (NNIs) such as thiosemicarbazone (TSC). We selected compound 2-[4-(2-phenylquinazolin-4-yl)piperazin-1-yl]ethanol (1.9; EC50 = 1.7 ± 0.4 µM) for further analysis. Compound 1.9 was found to inhibit the in vitro replication of TSC-resistant BVDV variants, which carry the N264D mutation in the RdRp. In addition, 1.9 presented adequate solubility in different media and a high-stability profile in murine and bovine plasma.

De novo design approaches targeting an envelope protein pocket to identify small molecules against dengue virus.

Dengue fever is a mosquito-borne viral disease that has become a major public health concern worldwide. This disease presents with a wide range of clinical manifestations, from a mild cold-like illness to the more serious hemorrhagic dengue fever and dengue shock syndrome. Currently, neither an approved drug nor an effective vaccine for the treatment are available to fight the disease. The envelope protein (E) is a major component of the virion surface. This protein plays a key role during the viral entry process, constituting an attractive target for the development of antiviral drugs. The crystal structure of the E protein reveals the existence of a hydrophobic pocket occupied by the detergent n-octyl-ß-d-glucoside (ß-OG). This pocket lies at the hinge region between domains I and II and is important for the low pH-triggered conformational rearrangement required for the fusion of the virion with the host's cell. Aiming at the design of novel molecules which bind to E and act as virus entry inhibitors, we undertook a de novo design approach by "growing" molecules inside the hydrophobic site (ß-OG). From more than 240000 small-molecules generated, the 2,4 pyrimidine scaffold was selected as the best candidate, from which one synthesized compound displayed micromolar activity. Molecular dynamics-based optimization was performed on this hit, and thirty derivatives were designed in silico, synthesized and evaluated on their capacity to inhibit dengue virus entry into the host cell. Four compounds were found to be potent antiviral compounds in the low-micromolar range. The assessment of drug-like physicochemical and in vitro pharmacokinetic properties revealed that compounds 3e and 3h presented acceptable solubility values and were stable in mouse plasma, simulated gastric fluid, simulated intestinal fluid, and phosphate buffered saline solution.

Quantum Chemical Approaches in Structure-Based Virtual Screening and Lead Optimization.

Today computational chemistry is a consolidated tool in drug lead discovery endeavors. Due to methodological developments and to the enormous advance in computer hardware, methods based on quantum mechanics (QM) have gained great attention in the last 10 years, and calculations on biomacromolecules are becoming increasingly explored, aiming to provide better accuracy in the description of protein-ligand interactions and the prediction of binding affinities. In principle, the QM formulation includes all contributions to the energy, accounting for terms usually missing in molecular mechanics force-fields, such as electronic polarization effects, metal coordination, and covalent binding; moreover, QM methods are systematically improvable, and provide a greater degree of transferability. In this mini-review we present recent applications of explicit QM-based methods in small-molecule docking and scoring, and in the calculation of binding free-energy in protein-ligand systems. Although the routine use of QM-based approaches in an industrial drug lead discovery setting remains a formidable challenging task, it is likely they will increasingly become active players within the drug discovery pipeline.

Discovery of Novel Bovine Viral Diarrhea Inhibitors Using Structure-Based Virtual Screening on the Envelope Protein E2.

Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus within the family Flaviviridae. BVDV causes both acute and persistent infections in cattle, leading to substantial financial losses to the livestock industry each year. The global prevalence of persistent BVDV infection and the lack of a highly effective antiviral therapy have spurred intensive efforts to discover and develop novel anti-BVDV therapies in the pharmaceutical industry. Antiviral targeting of virus envelope proteins is an effective strategy for therapeutic intervention of viral infections. We performed prospective small-molecule high-throughput docking to identify molecules that likely bind to the region delimited by domains I and II of the envelope protein E2 of BVDV. Several structurally different compounds were purchased or synthesized, and assayed for antiviral activity against BVDV. Five of the selected compounds were active displaying IC50 values in the low- to mid-micromolar range. For these compounds, their possible binding determinants were characterized by molecular dynamics simulations. A common pattern of interactions between active molecules and aminoacid residues in the binding site in E2 was observed. These findings could offer a better understanding of the interaction of BVDV E2 with these inhibitors, as well as benefit the discovery of novel and more potent BVDV antivirals.

The ULK1-FBXW5-SEC23B nexus controls autophagy.

In response to nutrient deprivation, the cell mobilizes an extensive amount of membrane to form and grow the autophagosome, allowing the progression of autophagy. By providing membranes and stimulating LC3 lipidation, COPII (Coat Protein Complex II) promotes autophagosome biogenesis. Here, we show that the F-box protein FBXW5 targets SEC23B, a component of COPII, for proteasomal degradation and that this event limits the autophagic flux in the presence of nutrients. In response to starvation, ULK1 phosphorylates SEC23B on Serine 186, preventing the interaction of SEC23B with FBXW5 and, therefore, inhibiting SEC23B degradation. Phosphorylated and stabilized SEC23B associates with SEC24A and SEC24B, but not SEC24C and SEC24D, and they re-localize to the ER-Golgi intermediate compartment, promoting autophagic flux. We propose that, in the presence of nutrients, FBXW5 limits COPII-mediated autophagosome biogenesis. Inhibition of this event by ULK1 ensures efficient execution of the autophagic cascade in response to nutrient starvation.

Progesterone modulates pro-inflammatory cytokine expression profile after spinal cord injury: Implications for neuropathic pain.

Neuropathic pain is a frequent complication of spinal cord injury (SCI), still refractory to conventional treatment. Glial cell activation and cytokine production contribute to the pathology of central neuropathic syndromes. In this study we evaluated the effects of progesterone, a neuroactive steroid, on pain development and the spinal expression of IL-1ß, its receptors (IL-1RI and IL-1RII) and antagonist (IL-1ra), IL-6 and TNFα, and NR1 subunit of NMDAR. Our results show that progesterone, by modulating the expression of pro-inflammatory cytokines and neuronal IL-1RI/NR1 colocalization, emerges as a promising agent to prevent chronic pain after SCI.

Temporal changes in the expression of the translocator protein TSPO and the steroidogenic enzyme 5α-reductase in the dorsal spinal cord of animals with neuropathic pain: Effects of progesterone administration.

Neuropathic pain is a frequent complication of spinal cord injury (SCI), still refractory to conventional treatment. The presence and biological activity of steroidogenic regulatory proteins and enzymes in the spinal cord suggests that neurosteroids locally generated could modulate pain messages. In this study we explored temporal changes in the spinal expression of the 18kDa translocator protein TSPO, the steroidogenic acute regulatory protein (StAr) and the steroidogenic enzyme 5α-reductase (5α-RI/II) in an experimental model of central chronic pain. Male Sprague-Dawley rats were subjected to a SCI and sacrificed at different time points (1, 14 or 28days). The development of mechanical and cold allodynia was assessed. Injured animals showed an early increase in the mRNA levels of TSPO and 5α-RII, whereas in the chronic phase a significant decrease in the expression of 5α-RI and 5α-RII was observed, coinciding with the presence of allodynic behaviors. Furthermore, since we have shown that progesterone (PG) administration may offer a promising perspective in pain modulation, we also evaluated the expression of steroidogenic proteins and enzymes in injured animals receiving daily injections of the steroid. PG-treated did not develop allodynia and showed a marked increase in the mRNA levels of TSPO, StAR, 5α-RI and 5α-RII 28days after injury. Our results suggest that in the acute phase after SCI, the increased expression of TSPO and 5α-RII may represent a protective endogenous response against tissue injury, which is not maintained in the chronic allodynic phase. PG may favor local steroidogenesis and the production of its reduced metabolites, which could contribute to the antiallodynic effects observed after PG treatment.