OpenZika: An IBM World Community Grid Project to Accelerate Zika Virus Drug Discovery

The Zika virus outbreak in the Americas has caused global concern. To help accelerate this fight against Zika, we launched the OpenZika project. OpenZika is an IBM World Community Grid Project that uses distributed computing on millions of computers and Android devices to run docking experiments, in order to dock tens of millions of drug-like compounds against crystal structures and homology models of Zika proteins (and other related flavivirus targets). This will enable the identification of new candidates that can then be tested in vitro, to advance the discovery and development of new antiviral drugs against the Zika virus. The docking data is being made openly accessible so that all members of the global research community can use it to further advance drug discovery studies against Zika and other related flaviviruses.The Zika virus (ZIKV) has emerged as a major public health threat to the Americas as of 2015 [1]. We have previously suggested that it represents an opportunity for scientific collaboration and open scientific exchange [2]. The health of future generations may very well depend on the decisions we make, our willingness to share our findings quickly, and open collaboration to rapidly find a cure for this disease. Since February 1, 2016, when the World Health Organization deemed the cluster of microcephaly cases, Guillain-Barré, and other neurological disorders associated with ZIKV in Latin America and the Caribbean as constituting a Public Health Emergency of International Concern [3] (PHEIC), we have seen a rapid increase in publications (S1 References and main references). We [2] and others [4,5] described steps that could be taken to initiate a drug discovery program on ZIKV. For example, computational approaches, such as virtual screening of chemical libraries or focused screening to repurpose FDA and/or EU-approved drugs, can be used to help accelerate the discovery of an anti-ZIKV drug. An antiviral drug discovery program can be initiated using structure-based design, based on homology models of the key ZIKV proteins. With the lack of structural information regarding the proteins of ZIKV, we built homology models for all the ZIKV proteins, based on close homologs such as dengue virus, using freely available software [6] (S1 Table). These were made available online on March 3, 2016. We also predicted the site of glycosylation of glycoprotein E as Asn154, which was recently experimentally verified [7].Since the end of March 2016, we have now seen two cryo-EM structures and 16 crystal structures of five target classes (S1 Table). These structures, alongside the homology models, represent potential starting points for docking-based virtual screening campaigns to help find molecules that are predicted to have high affinity with ZIKV proteins. These predictions can then be tested against the virus in cell-based assays and/or using individual protein-based assays. There are millions of molecules available that can be assayed, but which ones are likely to work, and how should we prioritize them?In March, we initiated a new open collaborative project called OpenZika (Fig 1), with IBM’s World Community Grid (WCG, worldcommunitygrid.org), which has been used previously for distributed computing projects (S2 Table). On May 18, 2016, the OpenZika project began the virtual screening of ~6 million compounds that are in the ZINC database (Fig 1), as well as the FDA-approved drugs and the NIH clinical collection, using AutoDock Vina and the homology models and crystal structures (S1 Table, S1 Text, S1 References), to discover novel candidate compounds that can potentially be developed into new drugs for treating ZIKV. These will be followed by additional virtual screens with a new ZINC library of ~38 million compounds, and the PubChem database (at most ~90 million compounds), after their structures are prepared for docking.Open in a separate windowFig 1Workflow for the OpenZika project.A. Docking input files of the targets and ligands are prepared, and positive control docking studies are performed. The crystallographic binding mode of a known inhibitor is shown as sticks with dark purple carbon atoms, while the docked binding mode against the NS5 target from HCV has cyan carbons. Our pdbqt files of the libraries of compounds we screen are also openly accessible (http://zinc.docking.org/pdbqt/). B. We have already prepared the docking input files for ~6 million compounds from ZINC (i.e., the libraries that ALP previously used in the GO Fight Against Malaria project on World Community Grid), which are currently being used in the initial set of virtual screens on OpenZika. C. IBM’s World Community Grid is an internet-distributed network of millions of computers (Mac, Windows, and Linux) and Android-based tablets or smartphones in over 80 countries. Over 715,000 volunteers donate their dormant computer time (that would otherwise be wasted) towards different projects that are both (a) run by an academic or nonprofit research institute, and (b) are devoted to benefiting humanity. D. OpenZika is harnessing World Community Grid to dock millions of commercially available compounds against multiple ZIKV homology models and crystal structures (and targets from related viruses) using AutoDock Vina (AD Vina). This ultimately produces candidates (virtual hits that produced the best docking scores and displayed the best interactions with the target during visual inspection) against individual proteins, which can then be prioritized for in vitro testing by collaborators. After it is inspected, all computational data against ZIKV targets will be made open to the public on our website (http://openzika.ufg.br/experiments/#tab-id-7), and OpenZika results are also available upon request. The computational and experimental data produced will be published as quickly as possible.Initially, compounds are being screened against the ZIKV homologs of drug targets that have been well-validated in research against dengue and hepatitis C viruses, such as NS5 and Glycoprotein E (S1 Table, S1 Text, S1 References). These may allow us to identify broad-spectrum antivirals against multiple flaviviruses, such as dengue virus, West Nile virus, and yellow fever virus. In addition, docking against the crystal structure of a related protein from a different pathogen can sometimes discover novel hits against the pathogen of interest [8].As well as applying docking-based filters, the compounds virtually screened on OpenZika will also be filtered using machine learning models (S1 Text, S1 References). These should be useful selection criteria for subsequent tests by our collaborators in whole-cell ZIKV assays, to verify their antiviral activity for blocking ZIKV infection or replication. Since all OpenZika docking data will be in the public domain soon after they are completed and verified, we and other labs can then advance the development of some of these new virtual candidates into experimentally validated hits, leads, and drugs through collaborations with wet labs.This exemplifies open science, which should help scientists around the world as they address the long and arduous process of discovering and developing new drugs. Screening millions of compounds against many different protein models in this way would take far more resources and time than any academic researcher could generally obtain or spend. As of August 16, 2016, we have submitted 894 million docking jobs. Over 6,934 CPU years have been donated to us, enabling over 439 million different docking jobs. We recently selected an initial batch of candidates for NS3 helicase (data openly available at http://openzika.ufg.br/experiments/#tab-id-7), for in vitro testing. Without the unique community of volunteers and tremendous resources provided by World Community Grid, this project would have been very difficult to initiate in a reasonable time frame at this scale.The OpenZika project will ultimately generate several billion docking results, which could make it the largest computational drug discovery project ever performed in academia. The potential challenges we foresee will be finding laboratories with sufficient funding to pursue compounds, synthesize analogs, and develop target-based assays to validate our predictions and generate SAR (Structure-Activity Relationship) data to guide the process of developing the new hits into leads and then drugs. Due to the difficult nature of drug discovery and the eventual evolution of drug resistance, funding of ZIKV research once initiated will likely need to be sustained for several years, if not longer (e.g., HIV research has been funded for decades). As with other WCG projects, once scientists identify experimentally validated leads, finding a company to license them and pursue them in clinical trials and beyond will need incentives such as the FDA Tropical Disease Priority voucher, [9] which has a financial value on the open market [10].By working together and opening our research to the scientific community, many other labs will also be able to take promising molecular candidates forward to accelerate progress towards defeating the ZIKV outbreak. We invite any interested researcher to join us (send us your models or volunteer to assay the candidates we identify through this effort against any of the flaviviruses), and we hope new volunteers in the general public will donate their dormant, spare computing cycles to this cause. We will ultimately report the full computational and experimental results of this collaboration.

Advantages and Disadvantages of OpenZika

Advantages

• Open Science could accelerate the discovery of new antivirals using docking and virtual screening
• Docking narrows down compounds to test, which saves time and money
• Free to use distributed computing on World Community Grid, and the workflow is simpler than using conventional supercomputers

Disadvantages

• Concern around intellectual property ownership and whether companies will develop drugs coming from effort
• Need for experimental assays will always be a factor
• Testing in vitro and in vivo is not free, nor are the samples of the compounds

     

Optimization of Trichomonas vaginalis Diagnosis during Pregnancy at a University Hospital,Argentina

The aim of this study was to evaluate different methods for Trichomonas vaginalis diagnosis during pregnancy in order to prevent maternal and perinatal complications. A total of 386 vaginal exudates from pregnant women were analyzed. T. vaginalis was investigated by 3 types of microscopic examinations direct wet mount with physiologic saline solution, prolonged May-Grunwald Giemsa (MGG) staining, and wet mount with sodium-acetate-formalin (SAF)/methylene blue method. PCR for 18S rRNA gene as well as culture in liquid medium were performed. The sensitivity and specificity of the microscopic examinations were evaluated considering the culture media positivity or the PCR techniques as gold standard. The frequency of T. vaginalis infection was 6.2% by culture and/or PCR, 5.2% by PCR, 4.7% by culture, 3.1% by SAF/methylene blue method and 2.8% by direct wet smear and prolonged MGG staining. The sensitivities were 83.3%, 75.0%, 50.0%, and 45.8% for PCR, culture, SAF/methylene blue method, and direct wet smear-prolonged MGG staining, respectively. The specificity was 100% for all the assessed methods. Microscopic examinations showed low sensitivity, mainly in asymptomatic pregnant patients. It is necessary to improve the detection of T. vaginalis using combined methods providing higher sensitivity, such as culture and PCR, mainly in asymptomatic pregnant patients, in order to prevent maternal and perinatal complications.     

LuMPIS – A modified luminescence‐based mammalian interactome mapping pull‐down assay for the investigation of protein–protein interactions encoded by GC‐low ORFs

The GC content is highly variable among the genomes of different organisms. It has been shown that recombinant gene expression in mammalian cells is much more efficient when GC‐rich coding sequences of a certain protein are used. In order to study protein–protein interactions in Varicella zoster virus, a GC‐low herpesvirus, we have developed a novel luminescence‐based maltose‐binding protein pull‐down interaction screening system (LuMPIS) that is able to overcome the impaired protein expression levels of GC‐low ORFs in mammalian expression systems.     

SHP-2 regulates myogenesis by coupling to FAK signaling pathway

Transient dephosphorylation of FAK at Tyr-397 is required for cell cycle withdrawal early on during myogenesis. Here, we show that upon serum starvation of C2C12 myoblasts, FAK is transiently dephosphorylated in parallel with SHP-2 activation and association with FAK. SHP-2 knockdown by RNA interference suppressed the transient upregulation of SHP-2 and dephosphorylation of FAK during myogenesis. Furthermore, depletion of SHP-2 retarded the cell cycle withdrawal and the differentiation of serum-starved myoblasts into myotubes. These data provide a mechanistic basis for the reduction in FAK activity in differentiating myoblasts, indicating that myogenesis is critically triggered by FAK/SHP-2 complex.

Structured summary

MINT-7258938: Fak1 (uniprotkb:P34152) physically interacts (MI:0915) with shp2 (uniprotkb:P35235) by anti bait coimmunoprecipitation (MI:0006)     

Perceived Impact of Dental Pain on the Quality of Life of Preschool Children and Their Families

The aim of the present study was to evaluate the perceived impact of dental caries and dental pain on oral health-related quality of life (OHRQoL) among preschool children and their families. A cross-sectional study was conduct with 843 preschool children in Campina Grande, Brazil. Parents/caregivers answered a questionnaire on socio-demographic information, their child’s general/oral health and history of dental pain. The Brazilian version of the Early Childhood Oral Health Impact Scale was administered to determine the perceived impact of caries and dental pain on OHRQoL. The children underwent an oral examination. Logistic regression for complex sample was used to determine associations between the dependent and independent variables (OR: Odds ratio, α = 5%). The independents variables that had a p-value <0.20 in the bivariate analysis were selected for the multivariate model. The prevalence of dental caries and dental pain was 66.3% and 9.4%, respectively. Order of birth of the child, being the middle child (OR: 10.107, 95%CI: 2.008-50.869) and youngest child (OR: 3.276, 95%CI: 1.048-10.284) and dental pain (OR: 84.477, 95%CI: 33.076-215.759) were significant predictors of the perceived impact on OHRQOL for children. Poor perception of oral health was significant predictor of the perceived impact on OHRQOL for family (OR=7.397, 95%CI: 2.190-24.987). Dental caries was not associated with a perceived impact on the ORHQoL of either the children or their families. However, order of child birth and dental pain were indicators of impact of OHRQoL on preschool children and poor perception of oral health was indicators of impact on families.     

Hawksbill turtle terra incognita: conservation genetics of?eastern Pacific rookeries

Prior to 2008 and the discovery of several important hawksbill turtle (Eretmochelys imbricata) nesting colonies in the EP (Eastern Pacific), the species was considered virtually absent from the region. Research since that time has yielded new insights into EP hawksbills, salient among them being the use of mangrove estuaries for nesting. These recent revelations have raised interest in the genetic characterization of hawksbills in the EP, studies of which have remained lacking to date. Between 2008 and 2014, we collected tissue samples from 269 nesting hawksbills at nine rookeries across the EP and used mitochondrial DNA sequences (766 bp) to generate the first genetic characterization of rookeries in the region. Our results inform genetic diversity, population differentiation, and phylogeography of the species. Hawksbills in the EP demonstrate low genetic diversity: We identified a total of only seven haplotypes across the region, including five new and two previously identified nesting haplotypes (pooled frequencies of 58.4% and 41.6%, respectively), the former only evident in Central American rookeries. Despite low genetic diversity, we found strong stock structure between the four principal rookeries, suggesting the existence of multiple populations and warranting their recognition as distinct management units. Furthermore, haplotypes EiIP106 and EiIP108 are unique to hawksbills that nest in mangrove estuaries, a behavior found only in hawksbills along Pacific Central America. The detected genetic differentiation supports the existence of a novel mangrove estuary “reproductive ecotype” that may warrant additional conservation attention. From a phylogeographic perspective, our research indicates hawksbills colonized the EP via the Indo‐Pacific, and do not represent relict populations isolated from the Atlantic by the rising of the Panama Isthmus. Low overall genetic diversity in the EP is likely the combined result of few rookeries, extremely small reproductive populations and evolutionarily recent colonization events. Additional research with larger sample sizes and variable markers will help further genetic understanding of hawksbill turtles in the EP.     

Cellular characterisation of Candida tropicalis presenting fluconazole-related trailing growth

We assessed fluconazole susceptibility in 52 Candida tropicalis clinical strains using seven antifungal susceptibility methods, including broth microdilution (BMD) [standard M27 A3 (with neutral and acid pH), ATB Fungus 3, Vitek 2 system and flow cytometric analysis] and agar-based methods (disk diffusion and E-test). Trailing growth, detection of cell-associated secreted aspartic proteases (Saps) and morphological and ultrastructural traits of these clinical strains were also examined. The ranges of fluconazole 24 h-minimum inhibitory concentration (MIC) values were similar among all methods. The essential agreement among the methods used for MIC determinations was excellent and all methods categorised all strains as susceptible, except for one strain that showed a minor error. The presence of the trailing effect was assessed by six methods. Trailing positivity was observed for 86.5-100% of the strains. The exception was the BMD-Ac method where trailing growth was not observed. Morphological and ultrastructural alterations were detected in C. tropicalis trailing cells, including mitochondrial swelling and cell walls with irregular shapes. We tested the production of Saps in 13 C. tropicalis strains expressing trailing growth through flow cytometry. Our results showed that all of the C. tropicalis strains up-regulated surface Sap expression after 24 h or 48 h of exposure to fluconazole, which was not observed in untreated yeast strains. We concluded that C. tropicalis strains expressing trailing growth presented some particular features on both biological and ultrastructural levels.