Coupled Enzyme Activity and Thermal Shift Screening of the Maybridge Rule of 3 Fragment Library Against Trypanosoma brucei Choline Kinase; A Genetically Validated Drug Target

In this study we interrogate 630 compounds of the Maybridge Rule of 3 Fragment Library for compounds that interact with, and inhibit TbCK. The Maybridge Rule of 3 Fragment Library is a small collection of quantifiable diverse, pharmacophoric rich, chemical entities that comply with the following criteria; MW ≤ 300, cLogP ≤ 3, H-Bond Acceptors ≤ 3, H-Bond Donors ≤ 3, Rotatable bonds (Flexibility Index) ≤ 3, Polar Surface Area ≤ 60 Å2 and aqueous solubility ≥ 1 mM using LogS and high purity (≥ 95%). Comparisons between two different screening methods, a coupled enzyme activity assay and differential scanning fluorimetry, has allowed identification of compounds that interact and inhibit the T. brucei choline kinase, several of which possess selective trypanocidal activity. Screening of a comparatively small fragment library by two different screening methods has allowed identification of several compounds that interact with and inhibit TbCK, a genetically validated drug target against African sleeping sickness. Some of the inhibitory fragments were also selectively trypanocidal, considering these are relatively simple molecules with no optimization, finding low μΜ inhibitors is very encouraging. Moreover some of the morphological phenotypes of these trypanocidal compounds include cell-cycle arrests similar to those observed for the TbCK conditional knockout grown under permissive conditions

Comparison of characteristics and function of translation termination signals between and within prokaryotic and eukaryotic organisms

Six diverse prokaryotic and five eukaryotic genomes were compared to deduce whether the protein synthesis termination signal has common determinants within and across both kingdoms. Four of the six prokaryotic and all of the eukaryotic genomes investigated demonstrated a similar pattern of nucleotide bias both 5′ and 3′ of the stop codon. A preferred core signal of 4 nt was evident, encompassing the stop codon and the following nucleotide. Codons decoded by hyper-modified tRNAs were over-represented in the region 5′ to the stop codon in genes from both kingdoms. The origin of the 3′ bias was more variable particularly among the prokaryotic organisms. In both kingdoms, genes with the highest expression index exhibited a strong bias but genes with the lowest expression showed none. Absence of bias in parasitic prokaryotes may reflect an absence of pressure to evolve more efficient translation. Experiments were undertaken to determine if a correlation existed between bias in signal abundance and termination efficiency. In Escherichia coli signal abundance correlated with termination efficiency for UAA and UGA stop codons, but not in mammalian cells. Termination signals that were highly inefficient could be made more efficient by increasing the concentration of the cognate decoding release factor

Towards chemical validation of Leishmania infantum ribose 5-phosphate isomerase as a drug target

Funding from the European Community's Seventh Framework Programme under grant agreements No. 602773 (Project KINDRED) was received for all partners in this work. This work also received funds from FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior through the Research Unit No. 4293 and project POCI-01-0145-FEDER-031013 (PTDC/SAUPAR/31013/2017 to NS); Individual funding from FCT through SFRH/BD/133485/2017 (to MS) and CEECIND/02362/2017 (to JT).Neglected tropical diseases caused by kinetoplastid parasites (Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp.) place a significant health and economic burden on developing nations worldwide. Current therapies are largely out-dated, inadequate and facing mounting drug resistance from the causative parasites. Thus, there is an urgent need for drug discovery and development. Target-led drug discovery approaches have focused on the identification of parasite enzymes catalysing essential biochemical processes, which significantly differ from equivalent proteins found in humans, thereby providing potentially exploitable therapeutic windows. One such target is ribose 5-phosphate isomerase B (RpiB), an enzyme involved in the non-oxidative branch of the pentose phosphate pathway, which catalyses the inter-conversion of D-ribose 5-phosphate and D-ribulose 5-phosphate. Although protozoan RpiB has been the focus of numerous targeted studies, compounds capable of selectively inhibiting this parasite enzyme have not been identified. Here, we present the results of a fragment library screening against Leishmania infantum RpiB, performed using thermal shift analysis. Hit fragments were shown to be effective inhibitors of LiRpiB in activity assays, and several were capable of selectively inhibiting parasite growth in vitro. These results support the identification of LiRpiB as a validated therapeutic target. The X-ray crystal structure of apo LiRpiB was also solved, permitting docking studies to assess how hit fragments might interact with LiRpiB to inhibit its activity. Overall, this work will guide structure-based development of LiRpiB inhibitors as anti-leishmanial agents.PostprintPeer reviewe

Stops making sense: translational trade-offs and stop codon reassignment

Background Efficient gene expression involves a trade-off between (i) premature termination of protein synthesis; and (ii) readthrough, where the ribosome fails to dissociate at the terminal stop. Sense codons that are similar in sequence to stop codons are more susceptible to nonsense mutation, and are also likely to be more susceptible to transcriptional or translational errors causing premature termination. We therefore expect this trade-off to be influenced by the number of stop codons in the genetic code. Although genetic codes are highly constrained, stop codon number appears to be their most volatile feature. Results In the human genome, codons readily mutable to stops are underrepresented in coding sequences. We construct a simple mathematical model based on the relative likelihoods of premature termination and readthrough. When readthrough occurs, the resultant protein has a tail of amino acid residues incorrectly added to the C-terminus. Our results depend strongly on the number of stop codons in the genetic code. When the code has more stop codons, premature termination is relatively more likely, particularly for longer genes. When the code has fewer stop codons, the length of the tail added by readthrough will, on average, be longer, and thus more deleterious. Comparative analysis of taxa with a range of stop codon numbers suggests that genomes whose code includes more stop codons have shorter coding sequences. Conclusions We suggest that the differing trade-offs presented by alternative genetic codes may result in differences in genome structure. More speculatively, multiple stop codons may mitigate readthrough, counteracting the disadvantage of a higher rate of nonsense mutation. This could help explain the puzzling overrepresentation of stop codons in the canonical genetic code and most variants

Exploring the impact of selection bias in observational studies of COVID-19: a simulation study

BACKGROUND: Non-random selection of analytic subsamples could introduce selection bias in observational studies. We explored the potential presence and impact of selection in studies of SARS-CoV-2 infection and COVID-19 prognosis. METHODS: We tested the association of a broad range of characteristics with selection into COVID-19 analytic subsamples in the Avon Longitudinal Study of Parents and Children (ALSPAC) and UK Biobank (UKB). We then conducted empirical analyses and simulations to explore the potential presence, direction and magnitude of bias due to this selection (relative to our defined UK-based adult target populations) when estimating the association of body mass index (BMI) with SARS-CoV-2 infection and death-with-COVID-19. RESULTS: In both cohorts, a broad range of characteristics was related to selection, sometimes in opposite directions (e.g. more-educated people were more likely to have data on SARS-CoV-2 infection in ALSPAC, but less likely in UKB). Higher BMI was associated with higher odds of SARS-CoV-2 infection and death-with-COVID-19. We found non-negligible bias in many simulated scenarios. CONCLUSIONS: Analyses using COVID-19 self-reported or national registry data may be biased due to selection. The magnitude and direction of this bias depend on the outcome definition, the true effect of the risk factor and the assumed selection mechanism; these are likely to differ between studies with different target populations. Bias due to sample selection is a key concern in COVID-19 research based on national registry data, especially as countries end free mass testing. The framework we have used can be applied by other researchers assessing the extent to which their results may be biased for their research question of interest

Surface functionalized metal-organic frameworks for binding coronavirus proteins

This work was supported by University of St Andrews Restarting Research Funding Scheme (SARRF), funded through the SFC grant reference SFC/AN/08/020 (XRR064) and European Research Council grant ADOR (Advanced Grant 787073). The authors acknowledge the EPSRC Light Element Analysis Facility Grant (EP/T019298/1) and the EPSRC Strategic Equipment Resource Grant (EP/R023751/1).Since the outbreak of SARS-CoV-2, a multitude of strategies have been explored for the means of protection and shielding against virus particles: filtration equipment (PPE) has been widely used in daily life. In this work, we explore another approach in the form of deactivating coronavirus particles through selective binding onto the surface of metal–organic frameworks (MOFs) to further the fight against the transmission of respiratory viruses. MOFs are attractive materials in this regard, as their rich pore and surface chemistry can easily be modified on demand. The surfaces of three MOFs, UiO-66(Zr), UiO-66-NH2(Zr), and UiO-66-NO2(Zr), have been functionalized with repurposed antiviral agents, namely, folic acid, nystatin, and tenofovir, to enable specific interactions with the external spike protein of the SARS virus. Protein binding studies revealed that this surface modification significantly improved the binding affinity toward glycosylated and non-glycosylated proteins for all three MOFs. Additionally, the pores for the surface-functionalized MOFs can adsorb water, making them suitable for locally dehydrating microbial aerosols. Our findings highlight the immense potential of MOFs in deactivating respiratory coronaviruses to be better equipped to fight future pandemics.Publisher PDFPeer reviewe

Novel benzothiazole-based ureas as 17β-HSD10 inhibitors, a potential Alzheimer’s disease treatment

Funding: This work was supported by Alzheimer’s Society (specifically The Barcopel Foundation), Scottish Universities Life Science Alliance (SULSA), The Rosetrees Trust, WT-ISSF and RS MacDonald Charitable Trust, Ministry of Education, Youth and Sports of Czech Republic (project ESF no. CZ.02.1.01/0.0/0.0/18_069/0010054), and University of Hradec Kralove (Faculty of Science, no. VT2019-2021, SV2115-2018, and Postdoctoral job positions at UHK).It has long been established, that mitochondrial dysfunction in Alzheimer’s disease (AD) patients can trigger pathological changes in cell metabolism by altering metabolic enzymes such as the mitochondrial 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10) also known as amyloid-binding alcohol dehydrogenase (ABAD). We and others have shown that frentizole and riluzole derivatives can inhibit 17β-HSD10 and that this inhibition is beneficial and holds therapeutic merit for the treatment of AD. Here we evaluate several novel series based on benzothiazolylurea scaffold evaluating key structural and activity relationships required for the inhibition of 17β-HSD10. Results show that the most promising of these compounds have markedly increased potency on our previously published inhibitors, with the most promising exhibiting advantageous features like low cytotoxicity and target engagement in living cells.Publisher PDFPeer reviewe

Being Ready to Treat Ebola Virus Disease Patients

As the outbreak of Ebola virus disease (EVD) in West Africa continues, clinical preparedness is needed in countries at risk for EVD (e.g., United States) and more fully equipped and supported clinical teams in those countries with epidemic spread of EVD in Africa. Clinical staff must approach the patient with a very deliberate focus on providing effective care while assuring personal safety. To do this, both individual health care providers and health systems must improve EVD care. Although formal guidance toward these goals exists from the World Health Organization, Medecin Sans Frontières, the Centers for Disease Control and Prevention, and other groups, some of the most critical lessons come from personal experience. In this narrative, clinicians deployed by the World Health Organization into a wide range of clinical settings in West Africa distill key, practical considerations for working safely and effectively with patients with EVD

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London

Inhibitors of trypanosoma cruzi Sir2 related protein 1 as potential drugs against Chagas disease.

Chagas disease remains one of the most neglected diseases in the world despite being the most important parasitic disease in Latin America. The characteristic chronic manifestation of chagasic cardiomyopathy is the region's leading cause of heart-related illness, causing significant mortality and morbidity. Due to the limited available therapeutic options, new drugs are urgently needed to control the disease. Sirtuins, also called Silent information regulator 2 (Sir2) proteins have long been suggested as interesting targets to treat different diseases, including parasitic infections. Recent studies on Trypanosoma cruzi sirtuins have hinted at the possibility to exploit these enzymes as a possible drug targets. In the present work, the T. cruzi Sir2 related protein 1 (TcSir2rp1) is genetically validated as a drug target and biochemically characterized for its NAD+-dependent deacetylase activity and its inhibition by the classic sirtuin inhibitor nicotinamide, as well as by bisnaphthalimidopropyl (BNIP) derivatives, a class of parasite sirtuin inhibitors. BNIPs ability to inhibit TcSir2rp1, and anti-parasitic activity against T. cruzi amastigotes in vitro were investigated. The compound BNIP Spermidine (BNIPSpd) (9), was found to be the most potent inhibitor of TcSir2rp1. Moreover, this compound showed altered trypanocidal activity against TcSir2rp1 overexpressing epimastigotes and anti-parasitic activity similar to the reference drug benznidazole against the medically important amastigotes, while having the highest selectivity index amongst the compounds tested. Unfortunately, BNIPSpd failed to treat a mouse model of Chagas disease, possibly due to its pharmacokinetic profile. Medicinal chemistry modifications of the compound, as well as alternative formulations may improve activity and pharmacokinetics in the future. Additionally, an initial TcSIR2rp1 model in complex with p53 peptide substrate was obtained from low resolution X-ray data (3.5 Å) to gain insight into the potential specificity of the interaction with the BNIP compounds. In conclusion, the search for TcSir2rp1 specific inhibitors may represent a valuable strategy for drug discovery against T. cruzi