PubMed | Leibniz Institute for Natural Product Research and Infection Biology, University of Cambridge and Isomerase Therapeutics Ltd.
Type: Journal Article | Journal: Applied and environmental microbiology | Year: 2016
Streptomyces iranensis HM 35 is an alternative rapamycin producer to Streptomyces rapamycinicus Targeted genetic modification of rapamycin-producing actinomycetes is a powerful tool for the directed production of rapamycin derivatives, and it has also revealed some key features of the molecular biology of rapamycin formation in S. rapamycinicus. The approach depends upon efficient conjugational plasmid transfer from Escherichia coli to Streptomyces, and the failure of this step has frustrated its application to Streptomyces iranensis HM 35. Here, by systematically optimizing the process of conjugational plasmid transfer, including screening of various media, and by defining optimal temperatures and concentrations of antibiotics and Ca(2+) ions in the conjugation media, we have achieved exconjugant formation for each of a series of gene deletions in S. iranensis HM 35. Among them were rapK, which generates the starter unit for rapamycin biosynthesis, and hutF, encoding a histidine catabolizing enzyme. The protocol that we have developed may allow efficient generation of targeted gene knockout mutants of Streptomyces species that are genetically difficult to manipulate.The developed protocol of conjugational plasmid transfer from Escherichia coli to Streptomyces iranensis may allow efficient generation of targeted gene knockout mutants of other genetically difficult to manipulate, but valuable, Streptomyces species.
Novoa E.M.,Barcelona Institute for Research in Biomedicine |
Novoa E.M.,Massachusetts Institute of Technology |
Camacho N.,Barcelona Institute for Research in Biomedicine |
Tor A.,Barcelona Institute for Research in Biomedicine |
And 19 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2014
Malaria remains a major global health problem. Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Here we explore the potential of the aminoacyl-tRNA synthetase (ARS) family as a source of antimalarial drug targets. First, a battery of known and novel ARS inhibitors was tested against Plasmodium falciparum cultures, and their activities were compared. Borrelidin, a natural inhibitor of threonyl-tRNA synthetase (ThrRS), stands out for its potent antimalarial effect. However, it also inhibits human ThrRS and is highly toxic to human cells. To circumvent this problem, we tested a library of bioengineered and semisynthetic borrelidin analogs for their antimalarial activity and toxicity. We found that some analogs effectively lose their toxicity against human cells while retaining a potent antiparasitic activity both in vitro and in vivo and cleared malaria from Plasmodium yoelii-infected mice, resulting in 100%mice survival rates. Our work identifies borrelidin analogs as potent, selective, and unexplored scaffolds that efficiently clear malaria both in vitro and in vivo. © 2014, National Academy of Sciences. All rights reserved.
Law B.J.C.,University of Manchester |
Struck A.-W.,University of Manchester |
Bennett M.R.,University of Manchester |
Wilkinson B.,John Innes Center |
And 2 more authors.
Chemical Science | Year: 2015
The methylation of natural products by S-adenosyl methionine (AdoMet, also known as SAM)-dependent methyltransferase enzymes is a common tailoring step in many biosynthetic pathways. The introduction of methyl substituents can affect the biological and physicochemical properties of the secondary metabolites produced. Recently it has become apparent that some AdoMet-dependent methyltransferases exhibit promiscuity and will accept AdoMet analogues enabling the transfer of alternative alkyl groups. In this study we have characterised a methyltransferase, RapM, which is involved in the biosynthesis of the potent immunosuppressive agent rapamycin. We have shown that recombinant RapM regioselectively methylates the C16 hydroxyl group of desmethyl rapamycin precursors in vitro and is promiscuous in accepting alternative co-factors in addition to AdoMet. A coupled enzyme system was developed, including a mutant human enzyme methionine adenosyl transferase (MAT), along with RapM, which was used to prepare alkylated rapamycin derivatives (rapalogs) with alternative ethyl and allyl ether groups, derived from simple S-ethyl or S-allyl methionine analogues. There are two other methyltransferases RapI and RapQ which provide methyl substituents of rapamycin. Consequently, using the enzymatic approach described here, it should be possible to generate a diverse array of alkylated rapalogs, with altered properties, that would be difficult to obtain by traditional synthetic approaches. © The Royal Society of Chemistry 2015.
PubMed | Lund University, Isomerase Therapeutics Ltd, Mitopharm Ltd, Northumbria University and Selcia Ltd
Type: | Journal: Nature communications | Year: 2016
Mitochondrial complex I (CI) deficiency is the most prevalent defect in the respiratory chain in paediatric mitochondrial disease. This heterogeneous group of diseases includes serious or fatal neurological presentations such as Leigh syndrome and there are very limited evidence-based treatment options available. Here we describe that cell membrane-permeable prodrugs of the complex II substrate succinate increase ATP-linked mitochondrial respiration in CI-deficient human blood cells, fibroblasts and heart fibres. Lactate accumulation in platelets due to rotenone-induced CI inhibition is reversed and rotenone-induced increase in lactate:pyruvate ratio in white blood cells is alleviated. Metabolomic analyses demonstrate delivery and metabolism of [(13)C]succinate. In Leigh syndrome patient fibroblasts, with a recessive NDUFS2 mutation, respiration and spare respiratory capacity are increased by prodrug administration. We conclude that prodrug-delivered succinate bypasses CI and supports electron transport, membrane potential and ATP production. This strategy offers a potential future therapy for metabolic decompensation due to mitochondrial CI dysfunction.
Hansson M.J.,NeuroVive Pharmaceutical |
Hansson M.J.,Lund University |
Moss S.J.,Isomerase Therapeutics Ltd. |
Moss S.J.,Biotica Technology Ltd. |
And 18 more authors.
Chemistry and Biology | Year: 2015
Inhibition of host-encoded targets, such as the cyclophilins, provides an opportunity to generate potent high barrier to resistance antivirals for the treatment of a broad range of viral diseases. However, many host-targeted agents are natural products, which can be difficult to optimize using synthetic chemistry alone. We describe the orthogonal combination of bioengineering and semisynthetic chemistry to optimize the drug-like properties of sanglifehrin A, a known cyclophilin inhibitor of mixed nonribosomal peptide/polyketide origin, to generate the drug candidate NVP018 (formerly BC556). NVP018 is a potent inhibitor of hepatitis B virus, hepatitis C virus (HCV), and HIV-1 replication, shows minimal inhibition of major drug transporters, and has a high barrier to generation of both HCV and HIV-1 resistance. © 2015 Elsevier Ltd.
PubMed | Isomerase Therapeutics Ltd, Scripps Research Institute and University of Vermont
Type: | Journal: Scientific reports | Year: 2015
Aminoacyl-tRNA synthetases (AARSs) catalyze an early step in protein synthesis, but also regulate diverse physiological processes in animal cells. These include angiogenesis, and human threonyl-tRNA synthetase (TARS) represents a potent pro-angiogenic AARS. Angiogenesis stimulation can be blocked by the macrolide antibiotic borrelidin (BN), which exhibits a broad spectrum toxicity that has discouraged deeper investigation. Recently, a less toxic variant (BC194) was identified that potently inhibits angiogenesis. Employing biochemical, cell biological, and biophysical approaches, we demonstrate that the toxicity of BN and its derivatives is linked to its competition with the threonine substrate at the molecular level, which stimulates amino acid starvation and apoptosis. By separating toxicity from the inhibition of angiogenesis, a direct role for TARS in vascular development in the zebrafish could be demonstrated. Bioengineered natural products are thus useful tools in unmasking the cryptic functions of conventional enzymes in the regulation of complex processes in higher metazoans.
Agency: GTR | Branch: Innovate UK | Program: | Phase: Feasibility Study | Award Amount: 119.33K | Year: 2015
Generation and analysis of a library of novel Natural Products using a new recombineering technology, with potential for use in pharmaceutical development, as treatments in indications such as infectious diseases, oncology and inflammation, as agrochemicals and fine chemicals.
Agency: GTR | Branch: Innovate UK | Program: | Phase: Feasibility Study | Award Amount: 124.29K | Year: 2015
Development of new tools for rational biological synthesis of novel natural products with potential use in design of improved anti-infectives, anticancer agents, agrochemicals and immune modulating agents.
Agency: GTR | Branch: Innovate UK | Program: | Phase: Collaborative Research & Development | Award Amount: 331.79K | Year: 2016
Confirmation of scaleability of new tools for accessing novel natural products with use in human and animal health and as agrochemicals.
Isomerase Therapeutics Ltd | Date: 2014-07-09
Novel rapamycin analogues and methods for their production with FKBP and/or MIP inhibitory activity with reduced mTOR inhibitory activity with therapeutic potential e.g. as bacterial virulence inhibitors.