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Periodic Report Summary 1 - COOPERA-TB (Hit to lead optimisation of novel anti-TB scaffolds through an academic-industrial partnership)

Publishable Summary

A summary description of the project objectives:

Often associated with poverty, tuberculosis (TB) not only remains rampant in many parts of Africa and Asia but is now also returning to many developed nations. Associated with this resurgence in the number of cases of TB is a growing resistance against currently used antibiotics, which is now one of the most pressing problems affecting the global TB epidemic. Treating TB infections currently requires a drug cocktail given for at least six months, and extending to two years for infections with multi-drug resistant strains of Mycobacterium tuberculosis, the agent causing TB. A largely successful campaign by the World Health Organisation (WHO) to stem the global rise of TB has not diminished the urgent need for new and better drugs and cellular targets to alleviate the devastating impact of this disease.

To meet these challenges, CooperaTB has established a PhD programme for four Early-Stage Researchers (ESRs) in tuberculosis (TB) drug discovery. This focused training research programme seeks to train two biochemists/biologists and two chemists in TB drug discovery. The four ESRs will spend the first 18 months at the University of Birmingham (UoB), U.K., followed by a further 18 months at the Tres Cantos campus of GlaxoSmithKline Diseases of the Developing World (GSK DDW) in Madrid, Spain. In their individual, yet interconnected, research projects, the four ESRs will work on the hit-to-lead optimisation of inhibitors of two novel drug targets, M. tuberculosis DprE1/2 and AspRS, building on preliminary work done at Birmingham in collaboration with GSK DDW. Upon successful completion of the programme, the ESRs will be awarded PhD degree titles by the University of Birmingham and be equipped with the skills and expertise to play a part in seeking solutions to tackling the growing problem of TB and antibiotic resistance more generally.

A description of the work performed since the beginning of the project:

All four ESRs have completed 16 months of their projects at the UoB and 2 months at GSK DDW. In December 2015 (M23), all four ESRs transferred to the Industrial Project Partner site at GSK DDW in Tres Cantos, Madrid, Spain, where they will complete 18 months of their projects before returning to UoB to focus on their PhD dissertations. All four ESRs are employees of UoB and have been enrolled as PhD students, two (ESRs 1 and 3) within the School of Biosciences and two (ESRs 2 and 4) within the School of Chemistry. Although all four ESRs will spend the final half of their fellowships at GSK DDW in Spain, they will remain employees of UoB for the duration. With four ESRs (two chemists and two biologists) and two protein targets, ESRs 1 and 2 have focused on the protein pair, DprE1, a flavin-containing oxidoreductase, and DprE2, an NADH-dependent reductase, which together are responsible for isomerising decaprenylphosphoryl-β-D-ribose to decaprenylphosphoryl-β-D-arabinose. ESR1 has focused on the biology aspects of these enzyme-catalysed processes whilst ESR2 has undertaken a Structure Activity Relationship (SAR) study of a DprE2 inhibitor that was discovered from a high-throughput phenotypic screening campaign undertaken by project partner, GSK DDW. With a similar division of labour, ESRs 3 and 4 have focused their efforts on a new target, AspRS, with ESR3 concentrating on the underlying biology and ESR4 on hit-to-lead optimisation of a hit molecule that was also discovered from a high-throughput phenotypic screening campaign carried out at GSK DDW.

A description of the main results achieved so far:

ESR1 Szilvia Tóth. PhD project title: Development of new tools for screening and characterisation of lead compounds for TB therapy directed at decaprenylphosphoryl-β-D-arabinose synthesis in Mycobacterium tuberculosis.

Szilvia has used single amino acid substitution experiments to show that no individual residue is exclusively responsible for the ligand binding of known inhibitors in a disordered region of the DprE1 enzyme. She has also developed and is currently optimising a biochemical assay for measuring DprE1 enzyme activity. Work on DprE2 has focused on its expression as a fusion protein and optimisation of the protein production process, which has allowed the generation of stable, soluble protein avoiding inclusion bodies or protein aggregation.

ESR2 Giacomo Chiodarelli. PhD project title: Development of new synthetic inhibitors against decaprenylphosphoryl-β-D-arabinose synthesis.

Several series of structural analogues of the initial DprE2 inhibitor have been synthesised in order to probe structure-activity relationships around the hit. Changes have focused on replacing the furan ring, the nitro group and the ethoxy group present in our initial hit to assess their relative importance on enzyme inhibition. These analogues have been tested against BCG whole cells by Szilvia Toth (ESR1) and the results have provided some early indications about how the hit molecule is interacting with the target enzyme. In the absence of a crystal structure of the DprE2 enzyme, more compounds are being synthesised and will be submitted for similar testing in order to elucidate a more detailed pharmacophore model.

ESR3 Ramón Soto Garcia. PhD project title: Development of new tools for screening and characterisation of lead compounds for TB therapy directed to AspRS in Mycobacterium tuberculosis.

Biochemical assays against the M. tuberculosis and human AspRS enzymes and whole-cell activity assays in M. bovis BCG have been used to determine the Minimum Inhibitory Concentration (MIC) of a series of analogues derived from an initial hit, which was identified from a high-throughput phenotypic screening campaign carried out at GSK DDW. In vitro profiling of a series of these compounds containing a thiazolidinone core [synthesised by Bogdan Duma (ESR4)] are implicating an embedded Michael acceptor for binding and therefore activity in both a biochemical assay and a ligand binding experiment. Alongside this work, an AspRS-target-based screening methodology has been developed with the aim of identifying new AspRS inhibitors. Early results have already uncovered three new and structurally distinct compounds, which display potent anti-tubercular activity and binding to AspRS.

ESR4 Bogdan Duma. PhD project title: Development of new synthetic inhibitors against AspRS.

A high-throughput phenotypic screening campaign carried out by the industrial Project Partner led to the disclosure of a range of hits of which one was shown to act against AspRS. The initial hit displays poor solubility and a rhodanine core that is a known PAIN (Pan-Assay INterference) compound scaffold. A number of structural analogues of the initial hit molecule have been synthesised with the aim of enhancing aqueous solubility and increasing potency and with some success; however, clear structure-activity relationships have not yet been determined. Currently the Michael acceptor capabilities of the hit are being assessed and new structures, which move away from the potentially problematic PAIN scaffold whilst still retaining AspRS inhibitory activity, are being explored.

Expected final results and potential impact and use: The guiding principle of this joint academic-industrial PhD programme is to provide the four ESRs with a comprehensive perspective on the drug-discovery process. By the time they graduate, they will not only have acquired in-depth knowledge of drug discovery and development but will also have made significant research contributions to a globally relevant disease area. Finally, through their exposure to both academic and industrial research environments, the ESRs can be expected to become effective advocates for future knowledge transfer and collaboration between academia and industry within Europe. The ESRs have contributed to dissemination activities in terms of poster / oral presentations to specialised and more general audiences at meetings and workshops. A project website [] has been set up as a central contact point, which is helping to publicise this ITN project to the wider scientific community as well as the general public. ESR4 has also set up Twitter [@Coopera_TB] and LinkedIn accounts and Pod-Casts are available on the project web-site, which we are using to publicise our own work and just as importantly, emerging news stories in the broader field of TB and antimicrobial resistance.

Reported by

United Kingdom


Life Sciences
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