Carbohydrate-Templated Syntheses of Trifluoromethyl-Substituted MEP Analogues for the Study of the Methylerythritol Phosphate Pathway.

Trifluoromethyl analogues of methylerythritol phosphate (MEP) and 2-C-methyl-erythritol 2,4-cyclodiphosphate (MEcPP), natural substrates of key enzymes from the MEP pathway, were prepared starting from d-glucose as the chiral template to secure absolute configurations. The obligate trifluoromethyl group was inserted with complete diastereoselectivity using the Ruppert-Prakash nucleophile. Target compounds were assayed against the corresponding enzymes showing that trifluoro-MEP did not disrupt IspD activity, whereas trifluoro-MEcPP induced 40% inhibition of IspG at 1 mM.

The Reductive Dehydroxylation Catalyzed by IspH, a Source of Inspiration for the Development of Novel Anti-Infectives.

The non-mevalonate or also called MEP pathway is an essential route for the biosynthesis of isoprenoid precursors in most bacteria and in microorganisms belonging to the Apicomplexa phylum, such as the parasite responsible for malaria. The absence of this pathway in mammalians makes it an interesting target for the discovery of novel anti-infectives. As last enzyme of this pathway, IspH is an oxygen sensitive [4Fe-4S] metalloenzyme that catalyzes 2H+/2e- reductions and a water elimination by involving non-conventional bioinorganic and bioorganometallic intermediates. After a detailed description of the discovery of the [4Fe-4S] cluster of IspH, this review focuses on the IspH mechanism discussing the results that have been obtained in the last decades using an approach combining chemistry, enzymology, crystallography, spectroscopies, and docking calculations. Considering the interesting druggability of this enzyme, a section about the inhibitors of IspH discovered up to now is reported as well. The presented results constitute a useful and rational help to inaugurate the design and development of new potential chemotherapeutics against pathogenic organisms.

Methylerythritol Phosphate Pathway: Enzymatic Evidence for a Rotation in the LytB/IspH-Catalyzed Reaction.

IspH/LytB, an oxygen-sensitive [4Fe-4S] enzyme, catalyzes the last step of the methylerythritol phosphate (MEP) pathway, a target for the development of new antimicrobial agents. This metalloenzyme converts (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) into the two isoprenoid precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Here, the synthesis of (S)-[4-2 H1 ]HMBPP and (R)-[4-2 H1 ]HMBPP is reported together with a detailed NMR analysis of the products formed after their respective incubation with E. coli IspH/LytB in the presence of the biological reduction system used by E. coli to reduce the [4Fe-4S] center. (S)-[4-2 H1 ]HMBPP was converted into [4-2 H1 ]DMAPP and (E)-[4-2 H1 ]IPP, whereas (R)-[4-2 H1 ]HMBPP yielded [4-2 H1 ]DMAPP and (Z)-[4-2 H1 ]IPP, hence providing the direct enzymatic evidence that the mechanism catalyzed by IspH/LytB involves a rotation of the CH2 OH group of the substrate to display it away from the [4Fe-4S].

Synthesis and Kinetic evaluation of an azido analogue of methylerythritol phosphate: a Novel Inhibitor of E. coli YgbP/IspD.

As multidrug resistant pathogenic microorganisms are a serious health menace, it is crucial to continuously develop novel medicines in order to overcome the emerging resistance. The methylerythritol phosphate pathway (MEP) is an ideal target for antimicrobial development as it is absent in humans but present in most bacteria and in the parasite Plasmodium falciparum. Here, we report the synthesis and the steady-state kinetics of a novel potent inhibitor (MEPN3) of Escherichia coli YgbP/IspD, the third enzyme of the MEP pathway. MEPN3 inhibits E. coli YgbP/IspD in mixed type mode regarding both substrates. Interestingly, MEPN3 shows the highest inhibitory activity when compared to known inhibitors of E. coli YgbP/IspD. The mechanism of this enzyme was also studied by steady-state kinetic analysis and it was found that the substrates add to the enzyme in sequential manner.

Further Insight into Crystal Structures of Escherichia coli IspH/LytB in Complex with Two Potent Inhibitors of the MEP Pathway: A Starting Point for Rational Design of New Antimicrobials.

IspH, also called LytB, a protein involved in the biosynthesis of isoprenoids through the methylerythritol phosphate pathway, is an attractive target for the development of new antimicrobial drugs. Here, we report crystal structures of Escherichia coli IspH in complex with the two most potent inhibitors: (E)-4-mercapto-3-methylbut-2-en-1-yl diphosphate (TMBPP) and (E)-4-amino-3-methylbut-2-en-1-yl diphosphate (AMBPP) at 1.95 and 1.7 Šresolution, respectively. The structure of the E. coli IspH:TMBPP complex exhibited two conformers of the inhibitor. This unexpected feature was exploited to design and evolve new antimicrobial candidates in silico.

Isoprenoid Biosynthesis in Pathogenic Bacteria: Nuclear Resonance Vibrational Spectroscopy Provides Insight into the Unusual [4Fe-4S] Cluster of the E.†coli LytB/IspH Protein.

The LytB/IspH protein catalyzes the last step of the methylerythritol phosphate (MEP) pathway which is used for the biosynthesis of essential terpenoids in most pathogenic bacteria. Therefore, the MEP pathway is a target for the development of new antimicrobial agents as it is essential for microorganisms, yet absent in humans. Substrate-free LytB has a special [4Fe-4S](2+) cluster with a yet unsolved structure. This motivated us to use synchrotron-based nuclear resonance vibrational spectroscopy (NRVS) in combination with quantum chemical-molecular mechanical (QM/MM) calculations to gain more insight into the structure of substrate-free LytB. The apical iron atom of the [4Fe-4S](2+) is clearly linked to three water molecules. We additionally present NRVS data of LytB bound to its natural substrate, (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) and to the inhibitors (E)-4-amino-3-methylbut-2-en-1-yl diphosphate and (E)-4-mercapto-3-methylbut-2-en-1-yl diphosphate.

Inhibition of IspH, a [4Fe-4S]2+ enzyme involved in the biosynthesis of isoprenoids via the methylerythritol phosphate pathway.

The MEP pathway, which is absent in animals but present in most pathogenic bacteria, in the parasite responsible for malaria and in plant plastids, is a target for the development of antimicrobial drugs. IspH, an oxygen-sensitive [4Fe-4S] enzyme, catalyzes the last step of this pathway and converts (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) into the two isoprenoid precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A crucial step in the mechanism of this enzyme is the binding of the C4 hydroxyl of HMBPP to the unique fourth iron site in the [4Fe-4S](2+) moiety. Here, we report the synthesis and the kinetic investigations of two new extremely potent inhibitors of E. coli IspH where the OH group of HMBPP is replaced by an amino and a thiol group. (E)-4-Mercapto-3-methylbut-2-en-1-yl diphosphate is a reversible tight-binding inhibitor of IspH with K(i) = 20 ± 2 nM. A detailed kinetic analysis revealed that (E)-4-amino-3-methylbut-2-en-1-yl diphosphate is a reversible slow-binding inhibitor of IspH with K(i) = 54 ± 19 nM. The slow binding behavior of this inhibitor is best described by a one-step mechanism with the slow step consisting of the formation of the enzyme-inhibitor (EI) complex.

NADH oxidase activity of Bacillus subtilis nitroreductase NfrA1: insight into its biological role.

NfrA1 nitroreductase from the Gram-positive bacterium Bacillus subtilis is a member of the NAD(P)H/FMN oxidoreductase family. Here, we investigated the reactivity, the structure and kinetics of NfrA1, which could provide insight into the unclear biological role of this enzyme. We could show that NfrA1 possesses an NADH oxidase activity that leads to high concentrations of oxygen peroxide and an NAD(+) degrading activity leading to free nicotinamide. Finally, we showed that NfrA1 is able to rapidly scavenge H(2)O(2) produced during the oxidative process or added exogenously.

Carbohydrate-containing components of biofilms produced in vitro by some staphylococcal strains related to orthopaedic prosthesis infections.

The capacity of coagulase-negative staphylococci to colonize implanted medical devices is generally attributed to their ability to produce biofilms. Biofilm of the model strain of Staphylococcus epidermidis RP62A was shown to contain two carbohydrate-containing moieties, a linear poly-beta-(1-->6)-N-acetyl-D-glucosamine (PNAG) and teichoic acid. In the present study, we investigated several biofilm-producing staphylococci isolated from infected orthopaedic implants and characterized the composition of the laboratory-grown biofilms using chemical analysis and 1H nuclear magnetic resonance spectroscopy. Extracellular teichoic acid was produced by all strains studied. Some of the clinical strains were shown to produce biofilms with compositions similar to that of the model strain, containing a varying amount of PNAG. The chemical structure of PNAG of the clinical strains was similar to that previously described for the model strains S. epidermidis RP62A and Staphylococcus aureus MN8m, differing only in the amount of charged groups. Biofilms of the strains producing a substantial amount of PNAG were detached by dispersin B, a PNAG-degrading enzyme, while being unsusceptible to proteinase K treatment. On the other hand, some strains produced biofilms without any detectable amount of PNAG. The biofilms of these strains were dispersed by proteinase K, but not by dispersin B.

Biofilms of clinical strains of Staphylococcus that do not contain polysaccharide intercellular adhesin.

Staphylococcus aureus and coagulase-negative staphylococci, primarily Staphylococcus epidermidis, are recognized as a major cause of nosocomial infections associated with the use of implanted medical devices. The capacity of S. epidermidis to form biofilms, allowing it to evade host immune defence mechanisms and antibiotic therapy, is considered to be crucial in colonizing the surfaces of medical implants and dissemination of infection. It has previously been demonstrated that the biofilm of a model strain S. epidermidis RP62A comprises two carbohydrate-containing moieties, a polysaccharide having a structure of a linear poly-N-acetyl-(1-->6)-beta-D-glucosamine and teichoic acid. In the present paper we show that, unlike this model strain, certain clinical isolates of coagulase-negative staphylococci produce biofilms that do not contain detectable amounts of poly-N-acetyl-(1-->6)-beta-D-glucosamine. In contrast to that of S. epidermidis RP62A, these biofilms are not detached with metaperiodate, while proteinase K causes their partial dispersal.

Photochemical reactivity of trifluoromethyl aromatic amines: the example of 3,5-diamino-trifluoromethyl-benzene (3,5-DABTF).

This work presents the application of an on-line photoreactor to the detection of 3,5-diamino-trifluoromethyl-benzene (3,5-DABTF) in aqueous solutions. When irradiated at 310 nm, this compound is defluorinated to 3,5-diaminobenzoic acid by a nucleophilic substitution of the fluoride by water. Concomitantly, defluorination intermediates polymerize through amide bonds to give dark-colored compounds. We take advantage of the photocatalyzed defluorination and the subsequent decrease in pH to develop an original and specific photoreactor. Continuous recording of pH and temperature in the outlet of the reactor by a dual electrode gives us an opportunity to optimize the system. In the photoreactor, 3,5-DABTF is immediately and totally transformed as attested by the rapid drop of the flowing solution pH from 6.2 to 3.2 and the chromatographic analysis of the outgoing solutions. The detection remains effective from 1 to 1000 parts per million.