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Most pathogenic bacteria, apicomplexan parasites and plants rely on the methylerythritol phosphate (MEP) pathway to obtain precursors of isoprenoids. 1-Deoxy-d-xylulose 5-phosphate synthase (DXPS), a thiamine diphosphate (ThDP)-dependent enzyme, catalyses the first and rate-limiting step of the MEP pathway. Due to its absence in humans, DXPS is considered as an attractive target for the development of anti-infectious agents and herbicides. Ketoclomazone is one of the earliest reported inhibitors of DXPS and antibacterial and herbicidal activities have been documented. This study investigated the activity of ketoclomazone on DXPS from various species, as well as the broader ThDP-dependent enzyme family. To gain further insights into the inhibition, we have prepared analogues of ketoclomazone and evaluated their activity in biochemical and computational studies. Our findings support the potential of ketoclomazone as a selective antibacterial agent.
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Pyruvate dehydrogenase complex (PDHc) is suppressed in some cancer types but overexpressed in others. To understand its contrasting oncogenic roles, there is a need for selective PDHc inhibitors. Its E1-subunit (PDH E1) is a thiamine pyrophosphate (TPP)-dependent enzyme and catalyses the first and rate-limiting step of the complex. In a recent study, we reported a series of ester-based thiamine analogues as selective TPP-competitive PDH E1 inhibitors with low nanomolar affinity. However, when the ester linker was replaced with an amide for stability reasons, the binding affinity was significantly reduced. In this study, we show that an amino-oxetane bioisostere of the amide improves the affinity and maintains stability towards esterase-catalysed hydrolysis.
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Complejo Piruvato Deshidrogenasa , Tiamina Pirofosfato , Tiamina , Amidas , Ésteres , Oxidorreductasas , Complejo Piruvato Deshidrogenasa/antagonistas & inhibidores , Complejo Piruvato Deshidrogenasa/metabolismo , Piruvatos , Tiamina/farmacología , Tiamina Pirofosfato/metabolismo , Tiamina Pirofosfato/farmacologíaRESUMEN
A common approach to studying thiamine pyrophosphate (TPP)-dependent enzymes is by chemical inhibition with thiamine/TPP analogues which feature a neutral aromatic ring in place of the positive thiazolium ring of TPP. These are potent inhibitors but their preparation generally involves multiple synthetic steps to construct the central ring. We report efficient syntheses of novel, open-chain thiamine analogues which potently inhibit TPP-dependent enzymes and are predicted to share the same binding mode as TPP. We also report some open-chain analogues that inhibit pyruvate dehydrogenase E1-subunit (PDH E1) and are predicted to occupy additional pockets in the enzyme other than the TPP-binding pockets. This opens up new possibilities for increasing the affinity and selectivity of the analogues for PDH, which is an established anti-cancer target.
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Tiamina Pirofosfato , Tiamina , Tiamina Pirofosfato/farmacología , Tiamina Pirofosfato/metabolismo , Tiamina/farmacología , Tiamina/metabolismo , DifosfatosRESUMEN
Thiamine diphosphate (ThDP), the bioactive form of vitamin B1, is an essential coenzyme needed for processes of cellular metabolism in all organisms. ThDP-dependent enzymes all require ThDP as a coenzyme for catalytic activity, although individual enzymes vary significantly in substrate preferences and biochemical reactions. A popular way to study the role of these enzymes through chemical inhibition is to use thiamine/ThDP analogues, which typically feature a neutral aromatic ring in place of the positively charged thiazolium ring of ThDP. While ThDP analogues have aided work in understanding the structural and mechanistic aspects of the enzyme family, at least two key questions regarding the ligand design strategy remain unresolved: 1) which is the best aromatic ring? and 2) how can we achieve selectivity towards a given ThDP-dependent enzyme? In this work, we synthesise derivatives of these analogues covering all central aromatic rings used in the past decade and make a head-to-head comparison of all the compounds as inhibitors of several ThDP-dependent enzymes. Thus, we establish the relationship between the nature of the central ring and the inhibitory profile of these ThDP-competitive enzyme inhibitors. We also demonstrate that introducing a C2-substituent onto the central ring to explore the unique substrate-binding pocket can further improve both potency and selectivity.
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Tiamina Pirofosfato , Tiamina , Tiamina Pirofosfato/química , Tiamina Pirofosfato/metabolismo , Tiamina/farmacología , Tiamina/química , Especificidad por Sustrato , Coenzimas/química , BiocatálisisRESUMEN
Thiamine is metabolized into the coenzyme thiamine diphosphate (ThDP). Interrupting thiamine utilization leads to disease states. Oxythiamine, a thiamine analogue, is metabolized into oxythiamine diphosphate (OxThDP), which inhibits ThDP-dependent enzymes. Oxythiamine has been used to validate thiamine utilization as an anti-malarial drug target. However, high oxythiamine doses are needed in vivo because of its rapid clearance, and its potency decreases dramatically with thiamine levels. We report herein cell-permeable thiamine analogues possessing a triazole ring and a hydroxamate tail replacing the thiazolium ring and diphosphate groups of ThDP. We characterize their broad-spectrum competitive inhibition of ThDP-dependent enzymes and of Plasmodium falciparum proliferation. We demonstrate how the cellular thiamine-utilization pathway can be probed by using our compounds and oxythiamine in parallel.
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Dickeya solani, a plant-pathogenic bacterium, produces solanimycin, a potent hybrid polyketide/nonribosomal peptide (PKS/NRPS) anti-fungal compound. The biosynthetic gene cluster responsible for synthesis of this compound has been identified. Because of instability, the complete structure of the compound has not yet been elucidated, but LC-MS2 identified that the cluster produces two main compounds, solanimycin A and B, differing by a single hydroxyl group. The fragmentation pattern revealed that the central part of solanimycin A is a hexapeptide, Gly-Dha-Dha-Dha-Dha-Dha (where Dha is dehydroalanine). This is supported by isotopic labeling studies using labeled serine and glycine. The N-terminal group is a polyketide-derived C16 acyl group containing a conjugated hexaene, a hydroxyl, and an amino group. The additional hydroxyl group in solanimycin B is on the α-carbon of the glycine residue. The incorporation of five sequential Dha residues is unprecedented because there is only one NRPS module in the cluster that is predicted to activate and attach serine (which is subsequently dehydrated to Dha), meaning that this NRPS module must act iteratively. While a few other iterative NRPS modules are known, they all involve iteration of two or three modules. We believe that the repetitive use of a single module makes the solanimycin biosynthetic pathway unique among NRPSs so far reported.
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Antifúngicos , Péptido Sintasas , Familia de Multigenes , Péptido Sintasas/metabolismo , Sintasas Poliquetidas/metabolismoRESUMEN
There is an unmet clinical need for imaging agents capable of detecting early evidence of tumor cell death, since the timing, extent, and distribution of cell death in tumors following treatment can give an indication of treatment outcome. We describe here 68Ga-labeled C2Am, which is a phosphatidylserine-binding protein, for imaging tumor cell death in vivo using positron emission tomography (PET). A one-pot synthesis of 68Ga-C2Am (20 min, 25 °C, >95% radiochemical purity) has been developed, using a NODAGA-maleimide chelator. The binding of 68Ga-C2Am to apoptotic and necrotic tumor cells was assessed in vitro using human breast and colorectal cancer cell lines, and in vivo, using dynamic PET measurements in mice implanted subcutaneously with the colorectal tumor cells and treated with a TRAIL-R2 agonist. 68Ga-C2Am showed predominantly renal clearance and low retention in the liver, spleen, small intestine, and bone and generated a tumor-to-muscle (T/m) ratio of 2.3 ± 0.4, at 2 h post probe administration and at 24 h following treatment. 68Ga-C2Am has the potential to be used in the clinic as a PET tracer for assessing early treatment response in tumors.
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Suppression of pyruvate dehydrogenase complex (PDHc) is a mechanism for cancer cells to manifest the Warburg effect. However, recent evidence suggests that whether PDHc activity is suppressed or activated depends on the type of cancer. The PDHc E1 subunit (PDH E1) is a thiamine pyrophosphate (TPP)-dependent enzyme, catalysing the first and rate-limiting step of PDHc; thus, there is a need for selective PDH E1 inhibitors. There is, however, inadequate understanding of the structure-activity relationship (SAR) and a lack of inhibitors specific for mammalian PDH E1. Our group have reported TPP analogues as TPP-competitive inhibitors to study the family of TPP-dependent enzymes. Most of these TPP analogues cannot be used to study PDHc in cells because (a) they inhibit all members of the family and (b) they are membrane-impermeable. Here we report derivatives of thiamine/TPP analogues that identify elements distinctive to PDH E1 for selectivity. Based on our SAR findings, we developed a series of furan-based thiamine analogues as potent, selective and membrane-permeable inhibitors of mammalian PDH E1. We envision that our SAR findings and inhibitors will aid work on using chemical inhibition to understand the oncogenic role of PDHc.
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Tiamina Pirofosfato , Tiamina , Animales , Tiamina Pirofosfato/metabolismo , Relación Estructura-Actividad , Piruvato Deshidrogenasa (Lipoamida)/metabolismo , Difosfatos , Piruvatos , Complejo Piruvato Deshidrogenasa/metabolismo , Mamíferos/metabolismoRESUMEN
[This corrects the article DOI: 10.1039/D2MD00085G.].
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Inhibition of thiamine pyrophosphate (TPP)-dependent enzymes with thiamine/TPP analogues that have the central thiazolium ring replaced with other rings is well established, but a limited number of central rings have been reported. We report a novel analogue, pyrrothiamine, with a central pyrrole ring. We further develop pyrrothiamine derivatives as potent and selective inhibitors of pyruvate dehydrogenase, which might have anti-cancer potential.
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Tiamina Pirofosfato , Tiamina , Tiamina/farmacología , Tiamina Pirofosfato/farmacología , Difosfatos , Oxidorreductasas , Piruvatos , Complejo Piruvato DeshidrogenasaRESUMEN
A series of derivatives of a triazole analogue of thiamine has been synthesised. When tested as inhibitors of porcine pyruvate dehydrogenase, the benzoyl ester derivatives proved to be potent thiamine pyrophosphate (TPP) competitive inhibitors, with the affinity of the most potent analogue (K i = 54 nM) almost matching the affinity of TPP itself. When tested as antiplasmodials, most of the derivatives showed modest activity (IC50 value >60 µM), except for a 4'-N-benzyl derivative, which has an IC50 value in the low micromolar range. This activity was not affected by increasing the extracellular concentration of thiamine in the culture medium for any of the compounds (except a modest increase in the IC50 for the unfunctionalized benzoyl ester), nor by overexpressing thiamine pyrophosphokinase in the parasite, making it unlikely to be due to an effect on thiamine transport or metabolism.
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A key step in the biosynthesis of numerous polyketides is the stereospecific formation of a spiroacetal (spiroketal). We report here that spiroacetal formation in the biosynthesis of the macrocyclic polyketides ossamycin and oligomycin involves catalysis by a novel spiroacetal cyclase. OssO from the ossamycin biosynthetic gene cluster (BGC) is homologous to OlmO, the product of an unannotated gene from the oligomycin BGC. The deletion of olmO abolished oligomycin production and led to the isolation of oligomycin-like metabolites lacking the spiroacetal structure. Purified OlmO catalyzed complete conversion of the major metabolite into oligomycin C. Crystal structures of OssO and OlmO reveal an unusual 10-strand ß-barrel. Three conserved polar residues are clustered together in the ß-barrel cavity, and site-specific mutation of any of these residues either abolished or substantially diminished OlmO activity, supporting a role for general acid/general base catalysis in spiroacetal formation.
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Policétidos , Antibacterianos , Catálisis , Familia de Multigenes , Oligomicinas , Policétidos/química , Metabolismo SecundarioRESUMEN
Methylcyclopropene (Cyoc)-tagged tetra-acetylated monosaccharides, and in particular mannosamine derivatives, are promising tools for medical imaging of cancer using metabolic oligosaccharide engineering and the extremely fast inverse electron-demand Diels-Alder bioorthogonal reaction. However, the in vivo potential of these monosaccharide derivatives has yet to be fully explored due to their low aqueous solubility. To address this issue, we sought to vary the extent of acetylation of Cyoc-tagged monosaccharides and probe its effect on the extent of glycan labeling in various cancer cell lines. We demonstrate that, in the case of AcxManNCyoc, tri- and diacetylated derivatives generated significantly enhanced cell labeling compared to the tetra-acetylated monosaccharide. In contrast, for the more readily soluble azide-tagged sugars, a decrease in acetylation led to decreased glycan labeling. Ac3ManNCyoc gave better labeling than the azido-tagged Ac4ManNAz and has significant potential for in vitro and in vivo imaging of glycosylated cancer biomarkers.
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Neoplasias , Coloración y Etiquetado , Acetilación , Monosacáridos/metabolismo , Neoplasias/diagnóstico por imagen , Polisacáridos/metabolismo , Coloración y Etiquetado/métodosRESUMEN
Pyrophosphates have important functions in living systems and thus pyrophosphate-containing molecules and their more stable bisphosphonate analogues have the potential to be used as drugs for treating many diseases including cancer and viral infections. Both pyrophosphates and bisphosphonates are polyanionic at physiological pH and, whilst this is essential for their biological activity, it also limits their use as therapeutic agents. In particular, the high negative charge density of these compounds prohibits cell entry other than by endocytosis, prevents transcellular oral absorption and causes sequestration to bone. Therefore, prodrug strategies have been developed to temporarily disguise the charges of these compounds. This review examines the various systems that have been used to mask the phosphorus-containing moieties of pyrophosphates and bisphosphonates and also illustrates the utility of such prodrugs.
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A biosynthetic pathway for the red-antibiotic, prodigiosin, was proposed over a decade ago but not all the suggested intermediates could be detected experimentally. Here we show that a thioester that was not originally included in the pathway is an intermediate. In addition, the enzyme PigE was originally described as a transaminase but we present evidence that it also catalyses the reduction of the thioester intermediate to its aldehyde substrate.
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INTRODUCTION: Trialing novel cancer therapies in the clinic would benefit from imaging agents that can detect early evidence of treatment response. The timing, extent and distribution of cell death in tumors following treatment can give an indication of outcome. We describe here an 18F-labeled derivative of a phosphatidylserine-binding protein, the C2A domain of Synaptotagmin-I (C2Am), for imaging tumor cell death in vivo using PET. METHODS: A one-pot, two-step automated synthesis of N-(5-[18F]fluoropentyl)maleimide (60 min synthesis time, > 98% radiochemical purity) has been developed, which was used to label the single cysteine residue in C2Am within 30 min at room temperature. Binding of 18F-C2Am to apoptotic and necrotic tumor cells was assessed in vitro, and also in vivo, by dynamic PET and biodistribution measurements in mice bearing human tumor xenografts treated with a TRAILR2 agonist or with conventional chemotherapy. C2Am detection of tumor cell death was validated by correlation of probe binding with histological markers of cell death in tumor sections obtained immediately after imaging. RESULTS: 18F-C2Am showed a favorable biodistribution profile, with predominantly renal clearance and minimal retention in spleen, liver, small intestine, bone and kidney, at 2 h following probe administration. 18F-C2Am generated tumor-to-muscle (T/m) ratios of 6.1 ± 2.1 and 10.7 ± 2.4 within 2 h of probe administration in colorectal and breast tumor models, respectively, following treatment with the TRAILR2 agonist. The levels of cell death (CC3 positivity) following treatment were 12.9-58.8% and 11.3-79.7% in the breast and colorectal xenografts, respectively. Overall, a 20% increase in CC3 positivity generated a one unit increase in the post/pre-treatment tumor contrast. Significant correlations were found between tracer uptake post-treatment, at 2 h post-probe administration, and histological markers of cell death (CC3: Pearson R = 0.733, P = 0.0005; TUNEL: Pearson R = 0.532, P = 0.023). CONCLUSION: The rapid clearance of 18F-C2Am from the blood pool and low kidney retention allowed the spatial distribution of cell death in a tumor to be imaged during the course of therapy, providing a rapid assessment of tumor treatment response. 18F-C2Am has the potential to be used in the clinic to assess early treatment response in tumors.
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In the biosynthesis of the tripyrrolic pigment prodigiosin, PigB is a predicted flavin-dependent oxidase responsible for the formation of 2-methyl-3-amylpyrrole (MAP) from a dihydropyrrole. To prove which dihydropyrrole is the true intermediate, both possibilities, 5-methyl-4-pentyl-3,4-dihydro-2H-pyrrole (5 a, resulting from transamination of the aldehyde of 3-acetyloctanal) and 2-methyl-3-pentyl-3,4-dihydro-2H-pyrrole (6, resulting from transamination of the ketone), were synthesised. Only 5 a restored pigment production in a strain of Serratia sp. ATCC 39006 blocked earlier in MAP biosynthesis. PigB is membrane-associated and inactive when its transmembrane domain was deleted, but HapB, its homologue in Hahella chejuensis, lacks the transmembrane domain and is active in solution. Two colourimetric assays for PigB and HapB were developed, and the HapB-catalysed reaction was kinetically characterised. Ten analogues of 5 a were synthesised, varying in the C2 and C3 side chains, and tested as substrates of HapB inâ vitro and for restoration of pigment production in Serratia ΔpigD inâ vivo. All lengths of side chain tested at C3 were accepted, but only short side chains at C2 were accepted. The knowledge that 5 a is an intermediate in prodigiosin biosynthesis and the ease of synthesis of analogues of 5 a makes a range of prodigiosin analogues readily available by mutasynthesis.
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Antibacterianos/biosíntesis , Proteínas Bacterianas/química , Gammaproteobacteria/enzimología , Monoaminooxidasa/química , Prodigiosina/biosíntesis , Serratia/enzimología , Especificidad por SustratoRESUMEN
Bottromycin A2 is a structurally unique ribosomally synthesized and post-translationally modified peptide (RiPP) that possesses potent antibacterial activity towards multidrug-resistant bacteria. The structural novelty of bottromycin stems from its unprecedented macrocyclic amidine and rare ß-methylated amino acid residues. The N-terminus of a precursor peptide (BtmD) is converted into bottromycin A2 by tailoring enzymes encoded in the btm gene cluster. However, little was known about key transformations in this pathway, including the unprecedented macrocyclization. To understand the pathway in detail, an untargeted metabolomic approach that harnesses mass spectral networking was used to assess the metabolomes of a series of pathway mutants. This analysis has yielded key information on the function of a variety of previously uncharacterized biosynthetic enzymes, including a YcaO domain protein and a partner protein that together catalyze the macrocyclization.
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Metabolómica , Conformación Molecular , Péptidos Cíclicos/biosíntesis , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , EstereoisomerismoRESUMEN
Glycosylation is a ubiquitous post-translational modification, present in over 50 % of the proteins in the human genome,1 with important roles in cell-cell communication and migration. Interest in glycome profiling has increased with the realization that glycans can be used as biomarkers of many diseases,2 including cancer.3 We report here the first tomographic imaging of glycosylated tissues in live mice by using metabolic labeling and a gadolinium-based bioorthogonal MRI probe. Significant N-azidoacetylgalactosamine dependent T1â contrast was observed inâ vivo two hours after probe administration. Tumor, kidney, and liver showed significant contrast, and several other tissues, including the pancreas, spleen, heart, and intestines, showed a very high contrast (>10-fold). This approach has the potential to enable the rapid and non-invasive magnetic resonance imaging of glycosylated tissues inâ vivo in preclinical models of disease.