NIHRDataInsights/HRCSData huggingface dataset:
Title: PI3K Signalling in Idiopathic Pulmonary Fibrosis
Funder: Medical Research Council

Abstract: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic parenchymal lung disease, with a median survival of around 3 years. The molecular mechanisms underlying the pathogenesis of IPF are not fully understood. There is emerging evidence that control of phosphatidylinosital-3-kinase (PI3K) signalling is dysregulated in IPF fibroblasts, promoting myofibroblast differentiation and collagen production. I will examine the hypothesis that dysregulated TGF-beta and extracellular matrix driven PI3K signalling play a key role in the development of IPF by influencing myofibroblast differentiation and collagen deposition. To address this hypothesis, I aim to 1) Define PI3K isoform and pAkt immunolocalisation in normal and IPF human lung using the extensive collection of IPF lung biopsies at the Royal Brompton. 2) Define the downstream pathway by which TGF-beta leads to PI3K isoform and Akt activation in primary IPF and control human lung fibroblasts in vitro. 3) Determine the role of PI3K in mediating TGF-beta and increased matrix stiffness-induced myofibroblast differentiation and collagen production in primary IPF and control human lung fibroblasts in vitro. 4) Examine the role of PI3K isoform-specific inhibitors in the bleomycin model of lung injury and fibrosis. Endpoints will be assessed as histological and immunohistochemical analysis of lung sections, ELISA and western blot analysis of phosphoproteins, flow cytometry of fibroblasts, gene expression at the mRNA and protein levels and HPLC for hydroxyproline assessment as an index of collagen deposition. I will also use a novel micro CT approach, developed at UCL in collaboration with GSK, to quantitate fibrosis in the bleomycin model. These studies will shed important light on the mechanisms by which dysregulated PI3K signalling contributes to the development of IPF and potentially lead to the identification of novel therapeutic targets.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFECTION
Title: Identification of a novel oral clinical candidate superior to praziquantel to treat schistosomiasis
Funder: Medical Research Council

Abstract: Schistosomiasis is a severe helminthic disease affecting an estimated 600 million people. Treatment relies on a single drug, praziquantel (PZQ), but this is not ideal since (i) PZQ kills adult worms but not juveniles so cure requires retreatment (ii) emergence of resistance is a major concern following several reports of treatment failure (iii) PZQ is sub-optimal for paediatric use often causing choking. No good starting points for schistosomiasis drug discovery have been reported in the literature. Our recent development of an HTS using Schistosoma mansoni larvae allows, for the first time, screening of large compound collections for quality lead compounds and to optimize SAR. Our aim is to develop a clinical candidate better than PZQ notably in being active against all intra-mammalian stages. The in vitro screening sequence (larval HTS/juvenile worms/adult worms/cytotoxicity/S.haematobium and S.japonicum adults) will identify compounds active against all stages of the important species and with promising parasite/cytotoxicity profiles. Salvensis, our collaborator, has agreement from a large pharmaceutical company to access a refined subset of its proprietary compound file. In addition we have large collections of commercially available inhibitors available. Funding is sought to bridge the translational gaps in NTD drug discovery between academia and pharma and between large compound collections and the first available HTS for schistosome drug discovery. Salvensis, a not-for-profit organisation based on >50 years experience in pharma drug discovery will also use state of the art computational methodology in selecting and designing compounds for screening. Using our in vitro and in vivo (S. mansoni/mouse) assays, LSHTM and Salvensis will use carefully selected third parties to identify the best series and optimise them, through a series of milestones, to an oral clinical candidate which we will aim to license for clinical development.


Research activity codes: 5.1 PHARMACEUTICALS
Health categories: INFECTION
Title: MICA: Development and GMP manufacture of a PfRH5 protein vaccine to induce strain-transcending immunity against blood-stage Plasmodium falciparum.
Funder: Medical Research Council

Abstract: The Need: Current estimates suggest Plasmodium falciparum malaria affects 200-300 million people annually, resulting in the death of about 1 million individuals. The most advanced malaria subunit vaccine, RTS,S/AS01, is currently in Phase III clinical trials, with early indications suggesting only 35-50% efficacy against severe disease in young children. Calls have been made for a second generation vaccine to achieve 80% efficacy over four years. New approaches are thus urgently needed. Rationale: No vaccine has yet proven effective against the blood-stage of P. falciparum, which causes the severe manifestations of malaria. Critical road blocks have included the need for exceptionally high antibody titres against known targets to confer protection coupled with problematic levels of antigen polymorphism. However, recent data from the Jenner Institute have identified the full-length P. falciparum Reticulocyte Binding Protein Homologue 5 (PfRH5) antigen as the highest priority vaccine target identified in the field for over a decade. We have shown in preclinical animal studies that antibodies induced by PfRH5 vaccination can block erythrocyte invasion to high efficiency and, critically, cross-inhibit all P. falciparum lines tested to date. Importantly we have also now demonstrated, for the first time, high-level efficacy induced by PfRH5 vaccination against heterologous strain challenge in an in vivo Aotus monkey-P. falciparum challenge model. Solution & Development Plan: We have now identified an S2 drosophila insect cell expression system as a viable GMP-compatible platform for production of this recombinant malaria antigen. This proposal aims to optimise and process develop this system for full-length PfRH5 protein production, prior to progression to GMP manufacture. The product produced by this work will enable subsequent Phase I/IIa clinical trials to assess the impact of high-titre antibodies against PfRH5 on blood-stage malaria infection in humans.


Research activity codes: 5.1 PHARMACEUTICALS
Health categories: INFECTION
Title: Structure-function studies on Clostridium difficile large toxins
Funder: Medical Research Council

Abstract: Clostridium difficile is a major problem as the aetiological agent for antibiotic-associated diarrhoea. The mechanism by which the bacterium colonises the gut during infection is poorly understood, but undoubtedly involves a set of key toxin molecules. Our aim is to further define roles for the key toxins using a detailed structure-function study. While the sequence of amino acids which make up these proteins can provide a great deal of information about the likely roles of the protein, primarily in relation to (and as a result of) other already characterised proteins, it is the tertiary or 3D structure of the protein that reveals the true interactions between proteins and their ligand/receptor interactions. Understanding these complex interactions require data at the atomic level obtained by X-ray crystallography with the aid of other complementary biophysical techniques. To facilitate these studies, preliminary work has created a toolbox of reagents including highly purified C. difficile toxins from a variety of strains, recombinant toxin fragments and panels of monoclonal antibodies to the various toxin domains. Currently, there are considerable gaps in our understanding of these toxins both at a detailed mechanistic level and in respect of their roles in the pathogenesis of C. difficile. Key goals of the proposal are- - Obtaining a detailed structure of the C. difficile to, in particular the central domains and deciphering their role in receptor binding and intracellular translocation. - Obtaining further insights into the mechanism of antibody-mediated toxin neutralisation and the structure and location of key epitopes. Key questions include: why some monoclonal antibodies neutralise toxin activity while others do not and the likely impact of sequence variation on antibody binding. Progress in these areas would improve our understanding of the pathogens of CDI and facilitate the design of improved vaccines, immunotherapeutics and toxin inhibitors.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: A systems approach to elucidation of protein lipidation during virus infection
Funder: Medical Research Council

Abstract: The first objectives are the chemical synthesis of lipid probes and capture reagents which enable purification and detection of lipidated proteins. Probes for each of classes of lipid modification, will be derivatised with azido or alkyne moieties for subsequent covalent ligation in vitro to the capture reagents. The capture reagents will be derivatised with the reciprocal chemical moieties, and also contain a capture agent (biotin) and a fluorescent detection agent (TAMRA). These moieties enable in vitro ligation of a lipid containing protein to the capture reagent by a copper catalysed chemical reaction. Different combinations of probes and capture reagents will be synthesised, and tested for efficiency of protein incorporation in vitro and in vivo for method optimisation. In vivo incorporation will be assessed by cell extraction, in vitro ligation, capture using affinity enrichment on neutravidin beads, and SDS-PAGE. Lipidated proteins will then be detected by in gel fluorescence of the lipidated protein species. Once initial optimisation is achieved, we will use the best performing probe/capture pairs to examine lipidation on a global scale using nano liquid chromatography-tandem mass spectrometry (nLC-MS/MS). After capture, lipidated species will be subject to tryptic digestion and the peptides analysed by nLC-MS/ms We will then utilise the optimal reagents and capture pairs for analysis of protein lipidation in vivo during infection and replication of herpes simplex virus, employing stable isotopic labelling in suitable cell culture models coupled with nLC-MS/MS, to produce the first quantitative global analysis of host and viral protein lipidation during herpesvirus infection. Our final objective will be to validate the study and technical platform by individual analysis of candidate proteins by biochemical and molecular approaches using western blotting and introduction of specific cloned species.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: MICA: Revisiting unexploited natural product antibiotics in the fight against multidrug-resistant bacterial pathogens
Funder: Medical Research Council

Abstract: Resistance to antibacterial drugs among bacterial pathogens severely compromises our ability to treat and cure the infections that they cause, and represents one of the greatest problems currently facing human health. Novel antibacterial drugs are now urgently required, but owing to a confluence of factors, antibacterial drug discovery and development has all but stalled. The present proposal outlines an approach that seeks to deliver novel antibacterial drug candidates into the development pipeline. This approach is characterized by (i) a re-focus on natural product antibiotics, the source of the overwhelming majority of antibacterial drugs in clinical use, and (ii) the decision to pursue only unexploited antibiotic classes which have demonstrated in vivo activity against bacterial pathogens in animal models, a strategy that will circumvent many of the problems encountered in recent antibacterial drug discovery/development programmes. Building on preliminary studies, we propose to ferment and purify five promising natural product antibiotics in sufficient quantities to enable their downstream characterization, conduct a full evaluation of their microbiological activities and modes of action, and examine their pharmacological profiles (including pharmacokinetics, toxicity and in vivo efficacy in mice). Results gained from these studies will provide important fundamental scientific knowledge, and will establish whether one or more of these compounds are suitable for further clinical development and/or chemical improvement. The project will be conducted in collaboration with Cubist Pharmaceuticals, an industry-leader in natural product antibiotics; by harnessing complementary strengths and capabilities in antibiotic research within academia and industry, we aim to establish a synergistic partnership capable of progressing these novel antibacterial drug candidates from bench to bedside.


Research activity codes: 5.1 PHARMACEUTICALS
Health categories: INFECTION
Title: Defining the mechanism of action of the 8-aminoquinolines: A pre-requisite to rationally designed safe antimalarials for the elimination era
Funder: Medical Research Council

Abstract: Despite its development some 60 years ago, the mode of action of primaquine, the only drug proven to be clinically effective against liver and sexual stage malaria parasites, is unknown. This knowledge gap is a major barrier to the development of safer alternative therapies based on rational re-design of molecules that share this mechanism of action. We have new data demonstrating that hydroxylated primaquine metabolites are redox cycled in vitro by P. falciparum ferredoxin-NADP+ reductase (PfFNR) and a novel diflavin reductase (PfCPR). Furthermore spontaneous oxidation of the resulting quinoneimines generates hydrogen peroxide and hydroxyl radicals. Both these enzymes are up-regulated in gametocytes and in liver stages and we hypothesise that the reactive oxygen species generated through these enzymes leads to parasite kill. Significantly, this data is the first demonstration of specific parasite enzymes involved in the mode of action of the 8-aminoquinolines and represents a starting point for detailed mechanism of action studies. The aim of this project is generate definitive evidence of the extent to which PfFNR and PfCPR are involved in the mode of action of the 8-aminoquinolines and to describe the mechanisms underpinning gametocidal and liver-stage activity. PfFNR and PfCPR transgenics (e.g. knock-outs/knockdowns/overexpressing) will be generated to determine the effect of primaquine/tafenoquine metabolites (with support from GSK) against gametocytes and liver stages using the humanised-liver mouse model (Kappe laboratory). Concurrently, we will study the mechanism of enzyme-dependent redox cycling of hydroxylated primaquine and tafenoquine metabolites, towards informing follow-on medicinal chemistry QSAR studies. This information will form the foundation for future rational drug re-design strategies aimed at delivering the next generation of transmission blocking/radical curative antimalarials with utility for the malaria elimination agenda.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;5.1 PHARMACEUTICALS
Health categories: INFECTION
Title: Structure-function analysis of a pH-responsive molecular switch required for fungal pathogenicity
Funder: Medical Research Council

Abstract: Invasive fungal infections (IFIs) pose a severe and fatal threat to patients undergoing organ or stem cell transplantation, anti-cancer therapy, or suffering from AIDs. On a global scale IFIs cause equivalent mortality to TB and malaria, but our ability to cure IFIs remains critically restricted by the limited repertoire and suboptimal efficacy of licensed antifungal drugs. Fungi use a GPCR-like protein (called PalH or Rim21), absent in humans, to sense external pH. In pathogenic fungi the loss of PalH, or the transcription factor (PacC) it communicates with, prevents fungal survival in the mammalian host. Recently, a major mould pathogen of humans, Aspergillus fumigatus, we found that loss of pH signalling impacts multiple processes associated with fungal infection including penetration of the lung lining. The simplest model for PalH-mediated pH sensing would be one in which the PalH pH sensor exists in multiple conformational states, each having a different affinity for, and/or ability to activate, the PalF arrrestin partner. Plausible pH-dependent triggers of altered conformational equilibrium might include binding of a ligand, altered protonation state of amino acid side chains, modulation of interactions between PalH amino terminus and extracellular loops (ECLs), PalH oligomerisation, altered lipid-protein interactions, or alteration of physical characteristics of the cell such as cell volume, or cell wall composition. To distinguish between these multiple possibilities, we will probe the relative contributions of particular amino acid side chains, and carboxy- and amino-terminal domains to functionality of the PalH receptor. We will then redirect mutant constructs into recombinant protein expression vectors, yeast membrane two hybrid (YTMH) vectors, and A. fumigatus FRET and bimolecular fluorescence complementation strains to interrogate the effects of mutations on protein structure, arrestin engagement and receptor oligomerisation.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Identifying and Characterising Developmental Regulators of Human Infectious Leishmania
Funder: Medical Research Council

Abstract: My aim is to use Leishmania mexicana as a cultured, genetically manipulable model to isolate and characterise post-transcriptional regulators essential to Leishmania spp. differentiation and virulence. Host immune cells provide the environmental cues, and signal the parasites to differentiate to virulent amastigote stage human infective forms Factors that regulate and promote this differentiation enable the cells to adapt to the new host environment, survive and spread. Essential regulators may serve as potential drug targets as Leishmania spp. proteomes are very divergent from human. I will initiate the investigation by uv-crosslinking Leishmania parasites of different stages and performing polysomal gradients to isolate putative mRNPs that associate with the translational machinery and are unique to human-infective lifecycle stages. Non-basal polysomal RNA-binding proteins will present potential trans-regulators while stage-specific polysomal mRNAs present putative targets and virulence factors to investigate. In addition, I will tag UTRs of known virulence factors to trap associating RNA binding proteins and potential regulators. Candidate regulatory proteins will be epitope-tagged to immunoprecipitate associating proteins (IP) and RNAs (RNA IP); validating isolated protein:transcript interactions and novel mRNPs. The effect of candidate regulators on target transcripts and differentiation will be genetically tested by ectopic overexpression and protein depletion methods. The regulation of associating transcripts will be examined for impact on the protein level using reporter:UTR constructs and antibodies to promising trans-regulators and transcript targets. To determine the location and requirements of protein:transcript associations I will use uv-crosslinking IP (CLiP) techniques. I will build upon significant findings of this work and investigate homologous factors in Leishmania donovani parasites, causative agent of deadly visceral Leishmaniasis.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Extracellular Acidosis and Tissue Destruction in Tuberculosis
Funder: Medical Research Council

Abstract: This project investigates a new hypothesis which is that tissue acidosis arising during innate inflammatory responses to tuberculosis (TB) amplifies matrix metalloproteinase (MMP) gene expression and secretion, worsening tissue destruction. It builds on data obtained in my Academic Clinical Fellowship where I showed that acidosis increases MMP-1,3 & -9 secretion from primary human respiratory epithelial cells & macrophages independent of changes in hypoxia but potentially involving the signalling molecule AMPK. Preliminary data also showed that TB lymph nodes are acidotic. My specific aims are first to investigate effects of extracellular acidosis on MMP gene expression and secretion in TB-infected human monocyte-derived macrophages & respiratory epithelial cells and in a multicellular model of infection. Following kinetic and pH ranging experiments (pH7.4-6.7 based on initial data), I shall use luminex, ELISA, zymography and functional luminescent confocal assays to assess MMP secretion and activity. MMP qPCR will measure gene expression. Effect of acidosis on transcriptional regulators will be investigated using Trans-AM kits, siRNA and ChiP assays. Next, I shall dissect mechanisms by which acidosis regulates MMP gene expression in TB. First, I shall screen for kinase activation using mutilplex assays. I shall focus on the MAPK, PI3 kinase, and AMPK (currently being identified as key in the TB MMP response) paths. The role of the pH sensing receptor TDAG8 which activates Protein Kinase A, in the regulation of MMPs in TB will be investigated. Finally, acidosis and MMP secretion will be examined in patients with TB lymphadenitis measuring pH, MMPs and inhibitory TIMPs in aspirates and dissecting identified signalling and transcriptional control paths by immunohistochemistry. In summary, this project will provide a broad laboratory and clinical research training investigating the effects of acidosis on MMPs in TB, an area in which there is little current data.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Augmentation and regulation of immune responses to Tuberculosis by vitamin D
Funder: Medical Research Council

Abstract: Augmentation of host defences to protect against tuberculosis (TB), by enhancing bacillary clearance coupled to regulatory control of inflammation to minimize immunopathogenesis of active diseases is a global research priority. Vitamin D can enhance intracellular killing of Mycobacterium tuberculosis (Mtb) and promote T cell regulation. Therefore, I aim to test the hypothesis that vitamin D affords the benefits of enhanced Mtb clearance and homeostatic regulation of immune responses in active disease. Immunological control of Mtb is dependent on the interaction between macrophages and T cells, but the influence of vitamin D in this context is not known. I will test the effect of vitamin D on T cell mediated killing of Mtb within macrophages in vitro and identify the changes to host cell response that mediate these effects. I will then test the effect of vitamin D on in vivo immune responses to TB, by genome-wide transcriptional profiling of skin biopsies at the site of tuberculin tests before or after vitamin D supplementation in people with active TB, latent TB or no evidence of infection. This approach will provide comprehensive and unbiased assessment of in vivo immune responses to TB at systems level and with molecular resolution, with which I will test the hypothesis that vitamin D supplementation attenuates adaptive responses in patients with active TB, but not in uninfected patients or those with latent TB, and to define the molecular detail of the effects of vitamin D on cutaneous responses to tuberculin in each group. Finally, I will take advantage of peripheral blood RNA samples collected during the ADJUVIT clinical trial of vitamin D in the treatment of active TB, to test the hypothesis that supplementation of antimicrobial therapy with vitamin D induces qualitatively different gene expression changes in peripheral blood compared to antimicrobial therapy alone, which reflect the immunoregulatory effects of vitamin D.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFECTION