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1.
Antimicrob Agents Chemother ; 60(10): 6091-9, 2016 10.
Article in English | MEDLINE | ID: mdl-27480853

ABSTRACT

The increasing global prevalence of drug resistance among many leading human pathogens necessitates both the development of antibiotics with novel mechanisms of action and a better understanding of the physiological activities of preexisting clinically effective drugs. Inhibition of peptidoglycan (PG) biosynthesis and cross-linking has traditionally enjoyed immense success as an antibiotic target in multiple bacterial pathogens, except in Mycobacterium tuberculosis, where it has so far been underexploited. d-Cycloserine, a clinically approved antituberculosis therapeutic, inhibits enzymes within the d-alanine subbranch of the PG-biosynthetic pathway and has been a focus in our laboratory for understanding peptidoglycan biosynthesis inhibition and for drug development in studies of M. tuberculosis During our studies on alternative inhibitors of the d-alanine pathway, we discovered that the canonical alanine racemase (Alr) inhibitor ß-chloro-d-alanine (BCDA) is a very poor inhibitor of recombinant M. tuberculosis Alr, despite having potent antituberculosis activity. Through a combination of enzymology, microbiology, metabolomics, and proteomics, we show here that BCDA does not inhibit the d-alanine pathway in intact cells, consistent with its poor in vitro activity, and that it is instead a mechanism-based inactivator of glutamate racemase (MurI), an upstream enzyme in the same early stage of PG biosynthesis. This is the first report to our knowledge of inhibition of MurI in M. tuberculosis and thus provides a valuable tool for studying this essential and enigmatic enzyme and a starting point for future MurI-targeted antibacterial development.


Subject(s)
Amino Acid Isomerases/chemistry , Antitubercular Agents/pharmacology , Bacterial Proteins/chemistry , Enzyme Inhibitors/pharmacology , Mycobacterium tuberculosis/drug effects , beta-Alanine/analogs & derivatives , Amino Acid Isomerases/antagonists & inhibitors , Amino Acid Isomerases/genetics , Amino Acid Isomerases/metabolism , Amino Acid Sequence , Antitubercular Agents/chemistry , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Catalytic Domain , Cloning, Molecular , Enzyme Inhibitors/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Kinetics , Microbial Sensitivity Tests , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/growth & development , Peptidoglycan/biosynthesis , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Substrate Specificity , beta-Alanine/chemistry , beta-Alanine/pharmacology
2.
PLoS Pathog ; 12(8): e1005783, 2016 08.
Article in English | MEDLINE | ID: mdl-27487182

ABSTRACT

Persistent TH2 cytokine responses following chronic helminth infections can often lead to the development of tissue pathology and fibrotic scarring. Despite a good understanding of the cellular mechanisms involved in fibrogenesis, there are very few therapeutic options available, highlighting a significant medical need and gap in our understanding of the molecular mechanisms of TH2-mediated immunopathology. In this study, we found that the Map3 kinase, TPL-2 (Map3k8; Cot) regulated TH2-mediated intestinal, hepatic and pulmonary immunopathology following Schistosoma mansoni infection or S. mansoni egg injection. Elevated inflammation, TH2 cell responses and exacerbated fibrosis in Map3k8-/-mice was observed in mice with myeloid cell-specific (LysM) deletion of Map3k8, but not CD4 cell-specific deletion of Map3k8, indicating that TPL-2 regulated myeloid cell function to limit TH2-mediated immunopathology. Transcriptional and metabolic assays of Map3k8-/-M2 macrophages identified that TPL-2 was required for lipolysis, M2 macrophage activation and the expression of a variety of genes involved in immuno-regulatory and pro-fibrotic pathways. Taken together this study identified that TPL-2 regulated TH2-mediated inflammation by supporting lipolysis and M2 macrophage activation, preventing TH2 cell expansion and downstream immunopathology and fibrosis.


Subject(s)
Cell Differentiation/immunology , Lipolysis/immunology , MAP Kinase Kinase Kinases/immunology , Macrophages/immunology , Proto-Oncogene Proteins/immunology , Schistosoma mansoni/immunology , Schistosomiasis mansoni/immunology , Th2 Cells/immunology , Animals , Cell Differentiation/genetics , Fibrosis , Inflammation/genetics , Inflammation/immunology , Inflammation/pathology , Lipolysis/genetics , MAP Kinase Kinase Kinases/genetics , Macrophages/pathology , Mice , Mice, Knockout , Proto-Oncogene Proteins/genetics , Schistosomiasis mansoni/genetics , Schistosomiasis mansoni/pathology , Th2 Cells/pathology
3.
Int J Pharm ; 379(2): 235-43, 2009 Sep 11.
Article in English | MEDLINE | ID: mdl-19467308

ABSTRACT

Glycerolipidic prodrug is an interesting concept to enhance lymphatic absorption of polar drugs intended to oral delivery such as didanosine (ddI). In order to improve ddI bioavailability, two didanosine glycerolipidic prodrugs, the phosphorylated (ProddIP) and the non-phosphorylated derivatives (ProddINP) were synthesized to follow triglyceride metabolism. The biomimetism approach of these prodrugs has been studied in vitro at two steps. First, liposomal formulation of each prodrug was incubated with a lipolysis model based on pancreatin and analysed using liquid chromatography combined with tandem mass spectrometry (LC-MS/MS). These experiments evidenced that both didanosine prodrugs were recognized by the lipases; as expected, they were cleaved at both positions sn-1 and sn-3 of glycerol. ProddIP was metabolised twice more rapidly than ProddINP suggesting an implication of some phospholipases in ProddIP degradation. Secondly, the detection of dideoxyadenosine triphosphate (ddA-TP) into HIV-1 infected cells after their incubation with ProddINP loaded liposomes evidenced their ability to release ddI that could penetrate into the cells and be metabolised by intracellular kinases. These results confirmed that the synthesized glycerolipidic prodrugs of didanosine could be investigated for a biomimetic approach with final aiming of increasing the drug oral bioavailability by enhancing intestinal absorption.


Subject(s)
Biomimetic Materials/metabolism , Chemistry, Pharmaceutical/methods , Models, Biological , Prodrugs/metabolism , Animals , Biomimetic Materials/chemistry , Humans , Mass Spectrometry/methods , Metabolic Networks and Pathways/physiology , Prodrugs/chemistry
4.
Drug Metab Dispos ; 36(8): 1570-7, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18474674

ABSTRACT

Gemcitabine (2',2'-difluorodeoxyribofuranosylcytosine; dFdC) is an anticancer nucleoside analog active against wide variety of solid tumors. However, this compound is rapidly inactivated by enzymatic deamination and can also induce drug resistance. To overcome the above drawbacks, we recently designed a new squalenoyl nanomedicine of dFdC [4-N-trisnorsqualenoyl-gemcitabine (SQdFdC)] by covalently coupling gemcitabine with the 1,1',2-trisnorsqualenic acid; the resultant nanomedicine displayed impressively greater anticancer activity compared with the parent drug in an experimental murine model. In the present study, we report that SQdFdC nanoassemblies triggered controlled and prolonged release of dFdC and displayed considerably greater t(1/2) (approximately 3.9-fold), mean residence time (approximately 7.5-fold) compared with the dFdC administered as a free drug in mice. It was also observed that the linkage of gemcitabine to the 1,1',2-trisnorsqualenic acid noticeably delayed the metabolism of dFdC into its inactive difluorodeoxyuridine (dFdU) metabolite, compared with dFdC. Additionally, the elimination of SQdFdC nanoassemblies was considerably lower compared with free dFdC, as indicated by lower radioactivity found in urine and kidneys, in accordance with the plasmatic concentrations of dFdU. SQdFdC nanoassemblies also underwent considerably higher distribution to the organs of the reticuloendothelial system, such as spleen and liver (p < 0.05), both after single- or multiple-dose administration schedule. Herein, this paper brings comprehensive pharmacokinetic and biodistribution insights that may explain the previously observed greater efficacy of SQdFdC nanoassemblies against experimental leukemia.


Subject(s)
Antineoplastic Agents/pharmacokinetics , Deoxycytidine/analogs & derivatives , Squalene/metabolism , Animals , Antineoplastic Agents/blood , Antineoplastic Agents/metabolism , Chromatography, Liquid , Deoxycytidine/blood , Deoxycytidine/metabolism , Deoxycytidine/pharmacokinetics , Female , Magnetic Resonance Spectroscopy , Mice , Mice, Inbred DBA , Tandem Mass Spectrometry , Tissue Distribution , Gemcitabine
5.
Article in English | MEDLINE | ID: mdl-17851141

ABSTRACT

Gemcitabine-squalene is a new prodrug that self-organizes in water forming nanoassemblies. It exhibits better anti-cancer properties in vitro and in vivo than gemcitabine. A liquid chromatography/tandem mass spectrometry assay of gemcitabine-squalene and gemcitabine was developed in human plasma in order to quantitate gemcitabine and its squalene conjugate. After protein precipitation with acetonitrile/methanol (90/10, v/v), the compounds were analyzed by reversed-phase high performance liquid chromatography and detected by tandem mass spectrometry using multiple reaction monitoring. The method was linear over the concentration range of 10-10,000 ng/ml of human plasma for both compounds with an accuracy lower than 10.4% and a precision below 14.8%. The method showed a lower limit of quantitation of 10 ng/ml of human plasma for dFdC and dFdC-SQ. A preliminary in vivo study in mice was shown as application of the method as no significant difference between human and mice plasma for the analysis of dFdC and dFdC-SQ was demonstrated.


Subject(s)
Chromatography, High Pressure Liquid/methods , Deoxycytidine/analogs & derivatives , Squalene/blood , Tandem Mass Spectrometry/methods , Deoxycytidine/blood , Deoxycytidine/chemistry , Humans , Molecular Structure , Reproducibility of Results , Squalene/chemistry , Gemcitabine
6.
Pharmacogenetics ; 13(3): 145-57, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12618592

ABSTRACT

A genetic dimorphism encodes for either alanine (Ala) or valine (Val) in the mitochondrial targeting sequence (MTS) of human manganese superoxide dismutase (MnSOD) and has been reported to modulate the risk of some cancers, neurodegenerative diseases and severe alcoholic liver disease. Although functional consequences of this dimorphism on MnSOD activity have not been assessed, computer models predict a partial alpha-helix structure for the Ala-MnSOD/MTS, but a beta-sheet structure for the Val-variant, which could hamper mitochondrial import. To investigate this hypothesis, we studied the in-vitro import of chimaeric proteins composed of either one of the MnSOD/MTS fused to the mouse dihydrofolate reductase (DHFR) protein, and the import of the two human MnSOD precursor variants into rat liver mitochondria. Compared to Ala-proteins, the Val-MnSOD/MTS-DHFR precursor and Val-MnSOD precursor were both partly arrested within the inner mitochondrial membrane. The Ala-MnSOD precursor generated 30-40% more of the active, matricial, processed MnSOD homotetramer than the Val-MnSOD precursor. These results show that the Ala-MnSOD/MTS allows efficient MnSOD import into the mitochondrial matrix, while the Val-variant causes partial arrest of the precursor within the inner membrane and decreased formation of the active MnSOD tetramer in the mitochondrial matrix.


Subject(s)
Alanine/genetics , Mitochondria, Liver/enzymology , Polymorphism, Genetic , Superoxide Dismutase/metabolism , Valine/genetics , Animals , Male , Protein Transport , Rats , Rats, Sprague-Dawley , Recombinant Fusion Proteins/metabolism , Submitochondrial Particles/enzymology , Superoxide Dismutase/genetics
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