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1.
Angew Chem Int Ed Engl ; : e202407764, 2024 Jun 26.
Article in English | MEDLINE | ID: mdl-38932510

ABSTRACT

Lipopeptides are an important class of biomolecules for drug development. Compared with conventional acylation, a chemoselective lipidation strategy offers a more efficient strategy for late-stage structural derivatisation of a peptide scaffold. It provides access to chemically diverse compounds possessing intriguing and non-native moieties. Utilising an allenamide, we report the first semi-synthesis of antimicrobial lipopeptides leveraging a highly efficient thia-Michael addition of chemically diverse lipophilic thiols. Using chemoenzymatically prepared polymyxin B nonapeptide (PMBN) as a model scaffold, an optimised allenamide-mediated thia-Michael addition effected rapid and near quantitative lipidation, affording vinyl sulfide-linked lipopeptide derivatives. Harnessing the utility of this new methodology, 22 lipophilic thiols of unprecedented chemical diversity were introduced to the PMBN framework. These included alkyl thiols, substituted aromatic thiols, heterocyclic thiols and those bearing additional functional groups (e.g., amines), ultimately yielding analogues with potent Gram-negative antimicrobial activity and substantially attenuated nephrotoxicity. Furthermore, we report facile routes to transform the allenamide into a ß-keto amide on unprotected peptides, offering a powerful "jack-of-all-trades" synthetic intermediate to enable further peptide modification.

2.
J Nat Prod ; 87(4): 764-773, 2024 Apr 26.
Article in English | MEDLINE | ID: mdl-38423998

ABSTRACT

The brevicidines represent a novel class of nonribosomal antimicrobial peptides that possess remarkable potency and selectivity toward highly problematic and resistant Gram-negative pathogenic bacteria. A recently discovered member of the brevicidine family, coined brevicidine B (2), comprises a single amino acid substitution (from d-Tyr2 to d-Phe2) in the amino acid sequence of the linear moiety of brevicidine (1) and was reported to exhibit broader antimicrobial activity against both Gram-negative (MIC = 2-4 µgmL-1) and Gram-positive (MIC = 2-8 µgmL-1) pathogens. Encouraged by this, we herein report the first total synthesis of the proposed structure of brevicidine B (2), building on our previously reported synthetic strategy to access brevicidine (1). In agreement with the original isolation paper, pleasingly, synthetic 2 demonstrated antimicrobial activity toward Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae (MIC = 4-8 µgmL-1). Interestingly, however, synthetic 2 was inactive toward all of the tested Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus strains. Substitution of d-Phe2 with its enantiomer, and other hydrophobic residues, yields analogues that were either inactive or only exhibited activity toward Gram-negative strains. The striking difference in the biological activity of our synthetic 2 compared to the reported natural compound warrants the re-evaluation of the original natural product for purity or possible differences in relative configuration. Finally, the evaluation of synthetic 1 and 2 in a human kidney organoid model of nephrotoxicity revealed substantial toxicity of both compounds, although 1 was less toxic than 2 and polymyxin B. These results indicate that modification to position 2 may afford a strategy to mitigate the nephrotoxicity of brevicidine.


Subject(s)
Anti-Bacterial Agents , Microbial Sensitivity Tests , Structure-Activity Relationship , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Molecular Structure , Pseudomonas aeruginosa/drug effects , Humans , Depsipeptides/pharmacology , Depsipeptides/chemistry , Depsipeptides/chemical synthesis , Klebsiella pneumoniae/drug effects , Escherichia coli/drug effects , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Peptides, Cyclic/pharmacology , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/chemistry
3.
Mol Microbiol ; 120(3): 408-424, 2023 09.
Article in English | MEDLINE | ID: mdl-37475106

ABSTRACT

Antimicrobial tolerance is the ability of a microbial population to survive, but not proliferate, during antimicrobial exposure. Significantly, it has been shown to precede the development of bona fide antimicrobial resistance. We have previously identified the two-component system CroRS as a critical regulator of tolerance to antimicrobials like teixobactin in the bacterial pathogen Enterococcus faecalis. To understand the molecular mechanism of this tolerance, we have carried out RNA-seq analyses in the E. faecalis wild-type and isogenic ∆ croRS mutant to determine the teixobactin-induced CroRS regulon. We identified a 132 gene CroRS regulon and demonstrate that CroRS upregulates biosynthesis of all major components of the enterococcal cell envelope in response to teixobactin. This suggests a coordinating role of this regulatory system in maintaining integrity of the multiple layers of the enterococcal envelope during antimicrobial stress, likely contributing to bacterial survival. Using experimental evolution, we observed that truncation of HppS, a key enzyme in the synthesis of the quinone electron carrier demethylmenaquinone, was sufficient to rescue tolerance in the croRS deletion strain. This highlights a key role for isoprenoid biosynthesis in antimicrobial tolerance in E. faecalis. Here, we propose a model of CroRS acting as a master regulator of cell envelope biogenesis and a gate-keeper between isoprenoid biosynthesis and respiration to ensure tolerance against antimicrobial challenge.


Subject(s)
Anti-Bacterial Agents , Anti-Infective Agents , Anti-Bacterial Agents/pharmacology , Enterococcus faecalis/genetics , Bacterial Proteins/genetics , Homeostasis , Terpenes , Microbial Sensitivity Tests
4.
Article in English | MEDLINE | ID: mdl-34831820

ABSTRACT

BACKGROUND: We describe the requirements and physiological changes when running 10 consecutive marathons in 10 days at the same consistent pace by a female ultra-endurance athlete. METHODS: Sharon Gayter (SG) 54 yrs, 162.5 cm, 49.3 kg maximal oxygen uptake (VO2 max) 53 mL/kg-1/min-1. SG completed 42.195 km on a treadmill every day for 10 days. We measured heart rate (HR), Rating of Perceived Exertion (RPE), oxygen uptake (VO2), weight, body composition, blood parameters, nutrition, and hydration. RESULTS: SG broke the previous record by ~2.5 h, with a cumulative completion time of 43 h 51 min 39 s. Over the 10 days, weight decreased from 51 kg to 48.4 kg, bodyfat mass from 9.1 kg to 7.2 kg (17.9% to 14.8%), and muscle mass from 23.2 kg to 22.8 kg. For all marathons combined, exercise intensity was ~60% VO2 max; VO2 1.6 ± 0.1 L.min-1/32.3 ± 1.1 mL.kg-1.min-1, RER 0.8 ± 0, HR 143 ± 4 b.min-1. Energy expenditure (EE) was 2030 ± 82 kcal/marathon, total EE for 10 days (including BMR) was 33,056 kcal, daily energy intake (EI) 2036 ± 418 kcal (20,356 kcal total), resulting an energy deficit (ED) of 12,700 kcal. DISCUSSION: Performance and pacing were highly consistent across all 10 marathons without any substantial physiological decrements. Although overall EI did not match EE, leading to a significant ED, resulting in a 2.6 kg weight loss and decreases in bodyfat and skeletal muscle mass, this did not affect performance.


Subject(s)
Marathon Running , Running , Energy Metabolism , Exercise Test , Female , Heart Rate , Humans , Oxygen Consumption
5.
Nucleic Acids Res ; 47(14): 7430-7443, 2019 08 22.
Article in English | MEDLINE | ID: mdl-31147703

ABSTRACT

Clonal expansion of mitochondrial DNA (mtDNA) deletions is an important pathological mechanism in adults with mtDNA maintenance disorders, leading to a mosaic mitochondrial respiratory chain deficiency in skeletal muscle. This study had two aims: (i) to determine if different Mendelian mtDNA maintenance disorders showed similar pattern of mtDNA deletions and respiratory chain deficiency and (ii) to investigate the correlation between the mitochondrial genetic defect and corresponding respiratory chain deficiency. We performed a quantitative analysis of respiratory chain deficiency, at a single cell level, in a cohort of patients with mutations in mtDNA maintenance genes. Using the same tissue section, we performed laser microdissection and single cell genetic analysis to investigate the relationship between mtDNA deletion characteristics and the respiratory chain deficiency. The pattern of respiratory chain deficiency is similar with different genetic defects. We demonstrate a clear correlation between the level of mtDNA deletion and extent of respiratory chain deficiency within a single cell. Long-range and single molecule PCR shows the presence of multiple mtDNA deletions in approximately one-third of all muscle fibres. We did not detect evidence of a replicative advantage for smaller mtDNA molecules in the majority of fibres, but further analysis is needed to provide conclusive evidence.


Subject(s)
DNA, Mitochondrial/genetics , Genes, Mitochondrial/genetics , Mitochondria, Muscle/genetics , Mitochondrial Diseases/genetics , Muscle Fibers, Skeletal/metabolism , Adult , Aged , Aged, 80 and over , Cohort Studies , Female , Humans , Male , Middle Aged , Mutation , Sequence Deletion , Single-Cell Analysis
6.
Hum Mol Genet ; 23(17): 4612-20, 2014 Sep 01.
Article in English | MEDLINE | ID: mdl-24740879

ABSTRACT

Large-scale mitochondrial DNA (mtDNA) deletions are an important cause of mitochondrial disease, while somatic mtDNA deletions cause focal respiratory chain deficiency associated with ageing and neurodegenerative disorders. As mtDNA deletions only cause cellular pathology at high levels of mtDNA heteroplasmy, an mtDNA deletion must accumulate to levels which can result in biochemical dysfunction-a process known as clonal expansion. A number of hypotheses have been proposed for clonal expansion of mtDNA deletions, including a replicative advantage for deleted mitochondrial genomes inferred by their smaller size--implying that the largest mtDNA deletions would also display a replicative advantage over smaller mtDNA deletions. We proposed that in muscle fibres from patients with mtDNA maintenance disorders, which lead to the accumulation of multiple mtDNA deletions, we would observe the largest mtDNA deletions spreading the furthest longitudinally through individual muscle fibres by means of a greater rate of clonal expansion. We characterized mtDNA deletions in patients with mtDNA maintenance disorders from a range of 'large' and 'small' cytochrome c oxidase (COX)-deficient regions in skeletal muscle fibres. We measured the size of clonally expanded deletions in 62 small and 60 large individual COX-deficient f regions. No significant difference was observed in individual patients or in the total dataset (small fibre regions mean 6.59 kb--large fibre regions mean 6.51 kb). Thus no difference existed in the rate of clonal expansion throughout muscle fibres between mtDNA deletions of different sizes; smaller mitochondrial genomes therefore do not appear to have an inherent replicative advantage in human muscle.


Subject(s)
DNA, Mitochondrial/genetics , Muscle, Skeletal/pathology , Sequence Deletion/genetics , Aged , Aged, 80 and over , Cytochrome-c Oxidase Deficiency/genetics , Humans , Microdissection , Middle Aged , Muscle Fibers, Skeletal
7.
Brain ; 137(Pt 2): 323-34, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24277717

ABSTRACT

Single, large-scale deletions of mitochondrial DNA are a common cause of mitochondrial disease and cause a broad phenotypic spectrum ranging from mild myopathy to devastating multi-system syndromes such as Kearns-Sayre syndrome. Studies to date have been inconsistent on the value of putative predictors of clinical phenotype and disease progression such as mutation load and the size or location of the deletion. Using a cohort of 87 patients with single, large-scale mitochondrial DNA deletions we demonstrate that a variety of outcome measures such as COX-deficient fibre density, age-at-onset of symptoms and progression of disease burden, as measured by the Newcastle Mitochondrial Disease Adult Scale, are significantly (P < 0.05) correlated with the size of the deletion, the deletion heteroplasmy level in skeletal muscle, and the location of the deletion within the genome. We validate these findings with re-analysis of 256 cases from published data and clarify the previously conflicting information of the value of these predictors, identifying that multiple regression analysis is necessary to understand the effect of these interrelated predictors. Furthermore, we have used mixed modelling techniques to model the progression of disease according to these predictors, allowing a better understanding of the progression over time of this strikingly variable disease. In this way we have developed a new paradigm in clinical mitochondrial disease assessment and management that sidesteps the perennial difficulty of ascribing a discrete clinical phenotype to a broad multi-dimensional and progressive spectrum of disease, establishing a framework to allow better understanding of disease progression.


Subject(s)
DNA, Mitochondrial/genetics , Disease Progression , Gene Deletion , Mitochondrial Diseases/diagnosis , Mitochondrial Diseases/genetics , Aged , Aged, 80 and over , Cohort Studies , DNA, Mitochondrial/antagonists & inhibitors , Female , Humans , Longitudinal Studies , Male , Middle Aged
8.
Hum Mol Genet ; 22(23): 4739-47, 2013 Dec 01.
Article in English | MEDLINE | ID: mdl-23847047

ABSTRACT

Progressive myopathy is a major clinical feature of patients with mitochondrial DNA (mtDNA) disease. There is limited treatment available for these patients although exercise and other approaches to activate muscle stem cells (satellite cells) have been proposed. The majority of mtDNA defects are heteroplasmic (a mixture of mutated and wild-type mtDNA present within the muscle) with high levels of mutated mtDNA and low levels of wild-type mtDNA associated with more severe disease. The culture of satellite cell-derived myoblasts often reveals no evidence of the original mtDNA mutation although it is not known if this is lost by selection or simply not present in these cells. We have explored if the mtDNA mutation is present in the satellite cells in one of the commonest genotypes associated with mitochondrial myopathies (patients with single, large-scale mtDNA deletions). Analysis of satellite cells from eight patients showed that the level of mtDNA mutation in the satellite cells is the same as in the mature muscle but is most often subsequently lost during culture. We show that there are two periods of selection against the mutated form, one early on possibly during satellite cell activation and the other during the rapid replication phase of myoblast culture. Our data suggest that the mutations are also lost during rapid replication in vivo, implying that strategies to activate satellite cells remain a viable treatment for mitochondrial myopathies in specific patient groups.


Subject(s)
DNA, Mitochondrial/genetics , Mitochondria/genetics , Mitochondrial Myopathies/genetics , Satellite Cells, Skeletal Muscle/metabolism , Adult , DNA Copy Number Variations , Female , Gene Deletion , Genetic Variation , Genotype , Humans , Male , Middle Aged , Mitochondria/drug effects , Mitochondria/pathology , Mitochondrial Myopathies/therapy , Muscle Fibers, Skeletal/metabolism , Mutation , NADH Dehydrogenase/genetics , RNA, Ribosomal, 18S/genetics , Real-Time Polymerase Chain Reaction
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