Synthesis and antitubercular activity of heterocycle substituted diphenyl ether derivatives

Despite being an ancient disease, tuberculosis (TB) remains the leading single-agent infectious disease killer in the world. The emerging serious problem of TB control and clinical management prompted us to synthesize a novel series of heterocyclic substituted diphenyl ether derivatives and determine their activity against the H37Rv strain of Mycobacterium. All ten compounds inhibited the growth of the H37Rv strain of Mycobacterium at concentrations of 1 μg/mL. This activity was found to be comparable to the reference drugs rifampicin and isoniazid at the same concentration. While the antimicrobial activity of other diphenyl ether analogues, such as triclosan, is associated with the inhibition of enoyl-ACP reductase (ENR), the synthesised substituted diphenyl ether derivatives did not affect this enzyme activity in spite of their structural similarity with triclosan. Therefore, these compounds appear to have a novel mechanism of action against M. tuberculosis, and their structural features should be studied further for their potential as new antitubercular drugs.     

Crystallographic analysis of triclosan bound to enoyl reductase

Molecular genetic studies with strains of Escherichia coli resistant to triclosan, an ingredient of many anti-bacterial household goods, have suggested that this compound works by acting as an inhibitor of enoyl reductase (ENR) and thereby blocking lipid biosynthesis. We present structural analyses correlated with inhibition data, on the complexes of E. coli and Brassica napus ENR with triclosan and NAD(+) which reveal how triclosan acts as a site-directed, picomolar inhibitor of the enzyme by mimicking its natural substrate. Elements of both the protein and the nucleotide cofactor play important roles in triclosan recognition, providing an explanation for the factors controlling its tight binding to the enzyme and for the emergence of triclosan resistance.     

Triclosan inhibits the growth of Plasmodium falciparum and Toxoplasma gondii by inhibition of apicomplexan Fab I

Fab I, enoyl acyl carrier protein reductase (ENR), is an enzyme used in fatty acid synthesis. It is a single chain polypeptide in plants, bacteria, and mycobacteria, but is part of a complex polypeptide in animals and fungi. Certain other enzymes in fatty acid synthesis in apicomplexan parasites appear to have multiple forms, homologous to either a plastid, plant-like single chain enzyme or more like the animal complex polypeptide chain. We identified a plant-like Fab I in Plasmodium falciparum and modelled the structure on the Brassica napus and Escherichia coli structures, alone and complexed to triclosan (5-chloro-2-[2,4 dichlorophenoxy] phenol]), which confirmed all the requisite features of an ENR and its interactions with triclosan. Like the remarkable effect of triclosan on a wide variety of bacteria, this compound markedly inhibits growth and survival of the apicomplexan parasites P. falciparum and Toxoplasma gondii at low (i.e. IC50 congruent with150-2000 and 62 ng/ml, respectively) concentrations. Discovery and characterisation of an apicomplexan Fab I and discovery of triclosan as lead compound provide means to rationally design novel inhibitory compounds.     

Effect of substrate binding loop mutations on the structure, kinetics, and inhibition of enoyl acyl carrier protein reductase from Plasmodium falciparum

Enoyl acyl carrier protein reductase (ENR), which catalyzes the final and rate limiting step of fatty acid elongation, has been validated as a potential drug target. Triclosan is known to be an effective inhibitor for this enzyme. We mutated the substrate binding site residue Ala372 of the ENR of Plasmodium falciparum (PfENR) to Methionine and Valine which increased the affinity of the enzyme towards triclosan to almost double, close to that of Escherichia coli ENR (EcENR) which has a Methionine at the structurally similar position of Ala372 of PfENR. Kinetic studies of the mutants of PfENR and the crystal structure analysis of the A372M mutant revealed that a more hydrophobic environment enhances the affinity of the enzyme for the inhibitor. A triclosan derivative showed a threefold increase in the affinity towards the mutants compared to the wild type, due to additional interactions with the A372M mutant as revealed by the crystal structure. The enzyme has a conserved salt bridge which stabilizes the substrate binding loop and appears to be important for the active conformation of the enzyme. We generated a second set of mutants to check this hypothesis. These mutants showed loss of function, except in one case, where the crystal structure showed that the substrate binding loop is stabilized by a water bridge network.     

Development of a triclosan scaffold which allows for adaptations on both the A- and B-ring for transport peptides

The enoyl acyl-carrier protein reductase (ENR) enzyme is harbored within the apicoplast of apicomplexan parasites providing a significant challenge for drug delivery, which may be overcome through the addition of transductive peptides, which facilitates crossing the apicoplast membranes. The binding site of triclosan, a potent ENR inhibitor, is occluded from the solvent making the attachment of these linkers challenging. Herein, we have produced 3 new triclosan analogs with bulky A- and B-ring motifs, which protrude into the solvent allowing for the future attachment of molecular transporters for delivery.     

Structural basis and mechanism of enoyl reductase inhibition by triclosan.

The enoyl-acyl carrier protein reductase (ENR) is involved in bacterial fatty acid biosynthesis and is the target of the antibacterial diazaborine compounds and the front-line antituberculosis drug isoniazid. Recent studies suggest that ENR is also the target for the broad-spectrum biocide triclosan. The 1.75 A crystal structure of EnvM, the ENR from Escherichia coli, in complex with triclosan and NADH reveals that triclosan binds specifically to EnvM. These data provide a molecular mechanism for the antibacterial activity of triclosan and substantiate the hypothesis that its activity results from inhibition of a specific cellular target rather than non-specific disruption of the bacterial cell membrane. This has important implications for the emergence of drug-resistant bacteria, since triclosan is an additive in many personal care products such as toothpastes, mouthwashes and soaps. Based on this structure, rational design of triclosan derivatives is possible which might be effective against recently identified triclosan-resistant bacterial strains.     

Novel diphenyl ethers: design, docking studies, synthesis and inhibition of enoyl ACP reductase of Plasmodium falciparum and Escherichia coli

We designed some novel diphenyl ethers and determined their binding energies for Enoyl-Acyl Carrier Protein Reductase (ENR) of Plasmodium falciparum using Autodock. Out of these, we synthesized the promising compounds and tested them for their inhibitory activity against ENRs of P. falciparum as well as Escherichia coli. Some of these compounds show nanomolar inhibition of PfENR and low micromolar inhibition of EcENR. They also exhibit low micromolar potency against in vitro cultures of P. falciparum and E. coli. The study of structure-activity relationship of these compounds paves the way for further improvements in the design of novel diphenyl ethers with improved activity against purified enzyme and the pathogens.     

Targeting tuberculosis and malaria through inhibition of Enoyl reductase: compound activity and structural data

Tuberculosis and malaria together result in an estimated 5 million deaths annually. The spread of multidrug resistance in the most pathogenic causative agents, Mycobacterium tuberculosis and Plasmodium falciparum, underscores the need to identify active compounds with novel inhibitory properties. Although genetically unrelated, both organisms use a type II fatty-acid synthase system. Enoyl acyl carrier protein reductase (ENR), a key type II enzyme, has been repeatedly validated as an effective antimicrobial target. Using high throughput inhibitor screens with a combinatorial library, we have identified two novel classes of compounds with activity against the M. tuberculosis and P. falciparum enzyme (referred to as InhA and PfENR, respectively). The crystal structure of InhA complexed with NAD+ and one of the inhibitors was determined to elucidate the mode of binding. Structural analysis of InhA with the broad spectrum antimicrobial triclosan revealed a unique stoichiometry where the enzyme contained either a single triclosan molecule, in a configuration typical of other bacterial ENR:triclosan structures, or harbored two triclosan molecules bound to the active site. Significantly, these compounds do not require activation and are effective against wild-type and drug-resistant strains of M. tuberculosis and P. falciparum. Moreover, they provide broader chemical diversity and elucidate key elements of inhibitor binding to InhA for subsequent chemical optimization.     

Inhibitor binding studies on enoyl reductase reveal conformational changes related to substrate recognition.

Enoyl acyl carrier protein reductase (ENR) is involved in fatty acid biosynthesis. In Escherichia coli this enzyme is the target for the experimental family of antibacterial agents, the diazaborines, and for triclosan, a broad spectrum antimicrobial agent. Biochemical studies have suggested that the mechanism of diazaborine inhibition is dependent on NAD(+) and not NADH, and resistance of Brassica napus ENR to diazaborines is thought to be due to the replacement of a glycine in the active site of the E. coli enzyme by an alanine at position 138 in the plant homologue. We present here an x-ray analysis of crystals of B. napus ENR A138G grown in the presence of either NAD(+) or NADH and the structures of the corresponding ternary complexes with thienodiazaborine obtained either by soaking the drug into the crystals or by co-crystallization of the mutant with NAD(+) and diazaborine. Analysis of the ENR A138G complex with diazaborine and NAD(+) shows that the site of diazaborine binding is remarkably close to that reported for E. coli ENR. However, the structure of the ternary ENR A138G-NAD(+)-diazaborine complex obtained using co-crystallization reveals a previously unobserved conformational change affecting 11 residues that flank the active site and move closer to the nicotinamide moiety making extensive van der Waals contacts with diazaborine. Considerations of the mode of substrate binding suggest that this conformational change may reflect a structure of ENR that is important in catalysis.     

Structural elucidation of the specificity of the antibacterial agent triclosan for malarial enoyl acyl carrier protein reductase.

The human malaria parasite Plasmodium falciparum synthesizes fatty acids using a type II pathway that is absent in humans. The final step in fatty acid elongation is catalyzed by enoyl acyl carrier protein reductase, a validated antimicrobial drug target. Here, we report the cloning and expression of the P. falciparum enoyl acyl carrier protein reductase gene, which encodes a 50-kDa protein (PfENR) predicted to target to the unique parasite apicoplast. Purified PfENR was crystallized, and its structure resolved as a binary complex with NADH, a ternary complex with triclosan and NAD(+), and as ternary complexes bound to the triclosan analogs 1 and 2 with NADH. Novel structural features were identified in the PfENR binding loop region that most closely resembled bacterial homologs; elsewhere the protein was similar to ENR from the plant Brassica napus (root mean square for Calphas, 0.30 A). Triclosan and its analogs 1 and 2 killed multidrug-resistant strains of intra-erythrocytic P. falciparum parasites at sub to low micromolar concentrations in vitro. These data define the structural basis of triclosan binding to PfENR and will facilitate structure-based optimization of PfENR inhibitors.     

Dynamics of Plasmodium falciparum enoyl‐ACP reductase and implications on drug discovery

Enoyl‐acyl carrier protein reductase (ENR) is a crucial enzyme in the type II fatty acid synthesis pathway of many pathogens such as Plasmodium falciparum, the etiological agent of the most severe form of malaria. Because of its essential function of fatty acid double bond reduction and the absence of a human homologue, PfENR is an interesting drug target. Although extensive knowledge of the protein structure has been gathered over the last decade, comparatively little remains known about the dynamics of this crucial enzyme. Here, we perform extensive molecular dynamics simulations of tetrameric PfENR in different states of cofactor and ligand binding, and with a variety of different ligands bound. A pocket‐volume analysis is also performed, and virtual screening is used to identify potential druggable hotspots. The implications of the results for future drug‐discovery projects are discussed.     

GABPalpha regulates Oct-3/4 expression in mouse embryonic stem cells

There is a dire need for novel therapeutics to treat the virulent malarial parasite, Plasmodium falciparum. Recently, the X-ray crystal structure of enoyl-acyl carrier protein reductase (ENR) in complex with triclosan has been determined and provides an opportunity for the rational design of novel inhibitors targeting the active site of ENR. Here, we report the discovery of several compounds by virtual screening and their experimental validation as high potency PfENR inhibitors.     

Triclosan susceptibility and co-metabolism--a comparison for three aerobic pollutant-degrading bacteria

The antimicrobial agent triclosan is an emerging and persistent environmental pollutant. This study evaluated the susceptibility and biodegradation potential of triclosan by three bacterial strains (Sphingomonas wittichii RW1, Burkholderia xenovorans LB400 and Sphingomonas sp. PH-07) that are able to degrade aromatic pollutants (dibenzofuran, biphenyl and diphenyl ether, respectively) with structural similarities to triclosan. These strains showed less susceptibility to triclosan when grown in complex and mineral salts media. Biodegradation experiments revealed that only strain PH-07 was able to catabolize triclosan to intermediates that included hydroxylated compounds (monohydroxy-triclosan, and dihydroxy-triclosan) and the ether bond cleavage products (4-chlorophenol and 2,4-dichlorophenol), indicating that the initial dihydroxylation occurred on both aromatic rings of triclosan. Additional growth inhibition tests demonstrated that the main intermediate, 2,4-dichlorophenol, was less toxic to strain PH-07 than was triclosan. Our results indicate that ether bond cleavage might be the primary mechanism of avoiding triclosan toxicity by this strain.     

Mechanism and inhibition of saFabI, the enoyl reductase from Staphylococcus aureus

Approximately one-third of the world's population carries Staphylococcus aureus. The recent emergence of extreme drug resistant strains that are resistant to the "antibiotic of last resort", vancomycin, has caused a further increase in the pressing need to discover new drugs against this organism. The S. aureus enoyl reductase, saFabI, is a validated target for drug discovery. To drive the development of potent and selective saFabI inhibitors, we have studied the mechanism of the enzyme and analyzed the interaction of saFabI with triclosan and two related diphenyl ether inhibitors. Results from kinetic assays reveal that saFabI is NADPH-dependent, and prefers acyl carrier protein substrates carrying fatty acids with long acyl chains. On the basis of product inhibition studies, we propose that the reaction proceeds via an ordered sequential ternary complex, with the ACP substrate binding first, followed by NADPH. The interaction of NADPH with the enzyme has been further explored by site-directed mutagenesis, and residues R40 and K41 have been shown to be involved in determining the specificity of the enzyme for NADPH compared to NADH. Finally, in preliminary inhibition studies, we have shown that triclosan, 5-ethyl-2-phenoxyphenol (EPP), and 5-chloro-2-phenoxyphenol (CPP) are all nanomolar slow-onset inhibitors of saFabI. These compounds inhibit the growth of S. aureus with MIC values of 0.03-0.06 microg/mL. Upon selection for resistance, three novel safabI mutations, A95V, I193S, and F204S, were identified. Strains containing these mutations had MIC values approximately 100-fold larger than that of the wild-type strain, whereas the purified mutant enzymes had K i values 5-3000-fold larger than that of wild-type saFabI. The increase in both MIC and K i values caused by the mutations supports the proposal that saFabI is the intracellular target for the diphenyl ether-based inhibitors.     

A study of the structure-activity relationship for diazaborine inhibition of Escherichia coli enoyl-ACP reductase

Enoyl acyl carrier protein (ACP) reductase catalyses the last reductive step of fatty acid biosynthesis, reducing the enoyl group of a growing fatty acid chain attached to ACP to its acyl product using NAD(P)H as the cofactor. This enzyme is the target for the diazaborine class of antibacterial agents, the biocide triclosan, and one of the targets for the front-line anti-tuberculosis drug isoniazid. The structures of complexes of Escherichia coli enoyl-ACP reductase (ENR) from crystals grown in the presence of NAD+ and a family of diazaborine compounds have been determined. Analysis of the structures has revealed that a mobile loop in the structure of the binary complex with NAD+ becomes ordered on binding diazaborine/NAD+ but displays a different conformation in the two subunits of the asymmetric unit. The work presented here reveals how, for one of the ordered conformations adopted by the mobile loop, the mode of diazaborine binding correlates well with the activity profiles of the diazaborine family. Additionally, diazaborine binding provides insights into the pocket on the enzyme surface occupied by the growing fatty acid chain.     

Characterisation of coupling products formed by biotransformation of biphenyl and diphenyl ether by the white rot fungus Pycnoporus cinnabarinus

Cells of the white rot fungus Pycnoporus cinnabarinus grown in glucose were able to hydroxylate biphenyl and diphenyl ether, although growth was inhibited by these substrates at concentrations above 250 microM. 2- and 4-Hydroxybiphenyl were detected as products of biphenyl metabolism and 2- and 4-hydroxydiphenyl ether as products of diphenyl ether metabolism in the culture media. After addition of 2-hydroxydiphenyl ether and 2-hydroxybiphenyl to cell-free supernatants containing laccase as the only ligninolytic enzyme, different coloured precipitates were formed. HPLC analysis revealed the formation of additional hydrophobic metabolites with one major product per transformation. Mass spectrometric analysis of the methyl derivatives of the polymer mixture indicated dimers and trimers with different binding types. The main products were identified as dimers with carbon-carbon bonds in para-position to the hydroxyl group of the monomers by mass spectroscopy and nuclear magnetic resonance spectroscopy.     

Enoyl-ACP reductase (FabI) of Haemophilus influenzae: steady-state kinetic mechanism and inhibition by triclosan and hexachlorophene

Steady-state kinetics, equilibrium binding, and primary substrate kinetic isotope effect studies revealed that the reduction of crotonyl-CoA by NADH, catalyzed by Haemophilus influenzae enoyl-ACP reductase (FabI), follows a rapid equilibrium random kinetic mechanism with negative interaction among the substrates. Two biphenyl inhibitors, triclosan and hexachlorophene, were studied in the context of the kinetic mechanism. IC(50) values for triclosan in the presence and absence of NAD(+) were 0.1 +/- 0.02 and 2.4 +/- 0.02 microM, respectively, confirming previous observations that the E-NAD(+) complex binds triclosan more tightly than the free enzyme. Preincubation of the enzyme with triclosan and NADH suggested that the E-NADH complex is the active triclosan binding species as well. These results were reinforced by measurement of binding kinetic transients. Intrinsic protein fluorescence changes induced by binding of 20 microM triclosan to E, E-NADH, E-NAD(+), and E-crotonyl-CoA occur at rates of 0.0124 +/- 0.001, 0.0663 +/- 0.002, 0.412 +/- 0.01, and 0.0069 +/- 0.0001 s(-1), respectively. The rate of binding decreased with increasing crotonyl-CoA concentrations in the E-crotonyl-CoA complex, and the extrapolated rate at zero concentration of crotonyl-CoA corresponded to the rate observed for the binding to the free enzyme. This suggests that triclosan and the acyl substrate share a common binding site. Hexachlorophene inhibition, on the other hand, was NAD(+)- and time-independent; and the calculated IC(50) value was 2.5 +/- 0.4 microM. Steady-state inhibition patterns did not allow the mode of inhibition to be unambiguously determined, but binding kinetics suggested that free enzyme, E-NAD(+), and E-crotonyl-CoA have similar affinity for hexachlorophene, since the k(obs)s were in the same range of 20-24 s(-1). When the E-NADH complex was mixed with hexachlorophene ligand, concentration-independent fluorescence quenching at 480 nm was observed, suggesting at least partial competition between NADH and hexachlorophene for the same binding site. Mutual exclusivity studies, together with the above-discussed results, indicate that triclosan and hexachlorophene bind at different sites of H. influenzae FabI.     

Chemical interference of pathogen-associated molecular pattern-triggered immune responses in Arabidopsis reveals a potential role for fatty-acid synthase type II complex-derived lipid signals

We describe an experimental setup using submerged cultures of Arabidopsis seedlings in 96-well microtiter plates that permits chemical intervention of rapid elicitor-mediated immune responses. Screening of a chemical library comprising 120 small molecules with known biological activities revealed four compounds reducing cellulysin- or flg22-activated gene expression of the early pathogen-associated molecular patterns (PAMP)-responsive ATL2 gene. One chemical, oxytriazine, was found to induce ATL2 gene expression in the absence of PAMP. By monitoring additional flg22-triggered immediate early plant responses, we present evidence that two compounds, triclosan and fluazinam, interfere with the accumulation of reactive oxygen species and internalization of the activated plasma membrane resident FLS2 immune receptor. Using triclosan structure types and enzyme activity inhibition assays, Arabidopsis MOD1 enoyl-acyl carrier protein reductase, a subunit of the fatty-acid synthase type II (FAS II) complex, was identified as a likely cellular target of triclosan. Inhibition of all tested elicitor-triggered early immune responses by triclosan indicates a potential role for signaling lipids in flg22-triggered immunity. Chemical profiling of eca mutants, each showing deregulated ATL2 gene expression, with the identified compounds revealed mutantspecific response patterns and allowed us to deduce tentative action sites of ECA genes relative to the compound targets.     

Inhibition of mammalian 5-lipoxygenase by aromatic disulfides

As a primary step in leukotriene biosynthesis, arachidonic acid is converted into 5-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid by 5-lipoxygenase. This enzyme is studied in the supernatant fraction from sonified RBL-1 cells, a preparation that converts [1-14C]arachidonic acid to 5-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid and several 5,12-dihydroxyeicosatetraenoic acids including LTB4. In order to examine the reversibility of inhibitors, the supernatant fraction can be depleted of low molecular weight constituents by vacuum filtration. The 5-lipoxygenase is irreversibly inhibited by 500 microM N-ethyl-maleimide or 300 microM methyl methanethiolsulfonate, reagents that react covalently with protein sulfhydryl groups. In contrast, diphenyl disulfide reversibly inhibits this enzyme at 1-5 microM, irrespective of the GSH concentration in the supernatant. KCN also inhibits 5-lipoxygenase at 4 mM, suggesting the presence of a metal-containing prosthetic group. These observations imply that diphenyl disulfide and similar molecules with electron-releasing substituents on the aromatic rings could inhibit by binding to an electrophilic metallic center, the binding being stabilized by hydrophobic interactions between the enzyme and the aromatic groups on the flexible disulfide. Even though diphenyl disulfide does not inhibit soybean 15-lipoxygenase or endoperoxide synthase in cell-free systems, this compound does suppress prostaglandin as well as leukotriene synthesis in intact murine peritoneal macrophages and CXBG cells. Since lipoxygenases are susceptible to peroxide activation and peroxidase deactivation, changes in the redox state of the cell may alter arachidonic acid metabolism as effectively as actual enzyme inhibition.     

Molecular mechanism of cardiotoxin action on axonal membranes.

Cardiotoxin isolated from Naja mossambica mossambica selectively deactivates the sodium-potassium activated adenosine triphosphatase of axonal membranes. Tetrodotoxin binding and acetylcholinesterase activities are unaffected by cardiotoxin treatment. The details of association of cardiotoxin with the axonal membrane were studied by following the deactivation of the sodium-potassium activated adenosine triphosphatase and by direct binding measurements with a tritiated derivative of the native cardiotoxin. The maximal binding capacity of the membrane is 42-50 nmol of cardiotoxin/mg of membrane protein. The high amount of binding suggests association of the toxin with the lipid phase of the membrane. It has been shown that cardiotoxin first associates rapidly and reversibly to membrane lipids, then, in a second step, it induces a rearrangement of the membrane structure which produces and irreversible deactivation of the sodium-potassium activated adenosine triphosphatase. Solubilization of the membrane-bound ATPase with Lubrol WX gives an active enzyme species that is resistant to cardiotoxin-induced deactivation. Cardiotoxin binding to the membrane is prevented by high concentrations of Ca 2+ and dibucaine. Although cardiotoxins and neurotoxins of cobra venom have large sequence homologies, their mode of action on membranes is very different. The cardiotoxin seems to bind to the lipid phase of the axonal membrane and inhibits the sodium-potassium activated adenosine triphosphatase, whereas the neurotoxin associates with a protein receptor in the post-synaptic membrane and blocks acetylcholine transmission.