Preparation, analysis and toxicity characterisation of the redox metabolites of the azo food dye tartrazine.

Tartrazine (E102, FD&C Yellow 5) is a vibrant yellow azo dye added to many processed foods. The safety of this ubiquitous chemical has not been fully elucidated, and it has been linked to allergic reactions and ADHD in some individuals. In our study, bacterial species isolated from human stool decolourised tartrazine and, upon exposure to air, a purple compound formed. Tartrazine is known to undergo reduction in the gut to sulfanilic acid and 4-amino-3-carboxy-5-hydroxy-1-(4-sulfophenyl)pyrazole (SCAP). These metabolites and their derivatives are relevant to the toxicology of tartrazine. The toxicity of sulfanilic acid has been studied before, but the oxidative instability of SCAP has previously prevented full characterisation. We have verified the chemical identity of SCAP and confirmed that the purple-coloured oxidation derivative is 4-(3-carboxy-5-hydroxy-1-(4-sulfophenyl)-1H-pyrazol-4-yl)imino-5-oxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid (purpurazoic acid, PPA), as proposed by Westöö in 1965. A yellow derivative of SCAP is proposed to be the hydrolysed oxidation product, 4,5-dioxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid. SCAP and PPA are moderately toxic to human cells (IC50 89 and 78 µM against HEK-293, respectively), but had no apparent effect on Escherichia coli and Bacillus subtilis bacteria. These results prompt further analyses of the toxicology of tartrazine and its derivatives.

Fusarium verticillioides NAT1 (FDB2) N-malonyltransferase is structurally, functionally and phylogenetically distinct from its N-acetyltransferase (NAT) homologues.

Fusarium endophytes damage cereal crops and contaminate produce with mycotoxins. Those fungi overcome the main chemical defence of host via detoxification by a malonyl-CoA-dependent enzyme homologous to xenobiotic metabolizing arylamine N-acetyltransferase (NAT). In Fusarium verticillioides (teleomorph Gibberella moniliformis, GIBMO), this N-malonyltransferase activity is attributed to (GIBMO)NAT1, and the fungus has two additional isoenzymes, (GIBMO)NAT3 (N-acetyltransferase) and (GIBMO)NAT2 (unknown function). We present the crystallographic structure of (GIBMO)NAT1, also modelling other fungal NAT homologues. Monomeric (GIBMO)NAT1 is distinctive, with access to the catalytic core through two "tunnel-like" entries separated by a "bridge-like" helix. In the quaternary arrangement, (GIBMO)NAT1 monomers interact in pairs along an extensive interface whereby one entry of each monomer is covered by the N-terminus of the other monomer. Although monomeric (GIBMO)NAT1 apparently accommodates acetyl-CoA better than malonyl-CoA, dimerization changes the active site to allow malonyl-CoA to reach the catalytic triad (Cys110, His158 and Asp173) via the single uncovered entry, and anchor its terminal carboxyl-group via hydrogen bonds to Arg109, Asn157 and Thr261. Lacking a terminal carboxyl-group, acetyl-CoA cannot form such stabilizing interactions, while longer acyl-CoAs enter the active site but cannot reach catalytic Cys. Other NAT isoenzymes lack such structural features, with (GIBMO)NAT3 resembling bacterial NATs and (GIBMO)NAT2 adopting a structure intermediate between (GIBMO)NAT1 and (GIBMO)NAT3. Biochemical assays confirmed differential donor substrate preference of (GIBMO)NAT isoenzymes, with phylogenetic analysis demonstrating evolutionary separation. Given the role of (GIBMO)NAT1 in enhancing Fusarium pathogenicity, unravelling the structure and function of this enzyme may benefit research into more targeted strategies for pathogen control.

Synthesis and Initial Evaluation of a Novel Fluorophore for Selective FMDV 3C Protease Detection.

The development and evaluation of a Boc-AL(Boc)Q(Trt)-AMC fluorophore to detect 3C Protease, produced by Foot and Mouth Disease Virus (FMDV) is reported, with a view to a potential use as a rapid screen for FMDV infected livestock The peptide-linked conjugate fluorophore is evaluated in vitro for sensitivity, specificity, stability and rapidity and shows statistically significant increases in fluorescence when exposed to physiologically relevant concentrations of 3C Protease and selectivity when compared with other common proteases likely to be located, typically in the absence of FMDV. The stability of deprotected Boc-AL(Boc)Q(Trt)-AMC is reported as a limitation of this probe.

Corrigendum: Variant Signal Peptides of Vaccine Antigen, FHbp, Impair Processing Affecting Surface Localization and Antibody-Mediated Killing in Most Meningococcal Isolates.

[This corrects the article DOI: 10.3389/fmicb.2019.02847.].

Potential Use of the Maillard Reaction for Pharmaceutical Applications: Gastric and Intestinal Controlled Release Alginate-Albumin Beads.

In this study, bovine serum albumin (BSA) and alginate (ALG) conjugates were synthesized by the Maillard reaction in order to evaluate their potential to develop controlled release drug delivery systems. The progress of the Maillard reaction was evidenced using ultraviolet (UV) absorbance, determination of BSA remaining free amino groups, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). BSA-ALG conjugates possessed enhanced and tunable viscosity, foamability and foam stability. Foam generated from BSA-ALG conjugate solution was used to prepare floating gastroretentive calcium ALG beads. Unlike traditional ALG beads, BSA-ALG foam beads were able to float and sustain the ciprofloxacin (CIP) release in gastric medium. Interestingly, intestinal beads made of ALG, BSA-ALG physical mixture and BSA-ALG conjugate resulted in different release rates and orders of indomethacin (IND) in simulated intestinal fluids; while beads based on a physical mixture of BSA-ALG resulted in a first order sustained release profile, both systems based on ALG and BSA-ALG conjugate displayed zero order sustained release profiles with IND being released at a slower rate from the conjugate beads.

Variant Signal Peptides of Vaccine Antigen, FHbp, Impair Processing Affecting Surface Localization and Antibody-Mediated Killing in Most Meningococcal Isolates.

Meningococcal lipoprotein, Factor H binding protein (FHbp), is the sole antigen of the Trumenba vaccine (Pfizer) and one of four antigens of the Bexsero vaccine (GSK) targeting Neisseria meningitidis serogroup B isolates. Lipidation of FHbp is assumed to occur for all isolates. We show in the majority of a collection of United Kingdom isolates (1742/1895) non-synonymous single nucleotide polymorphisms (SNPs) in the signal peptide (SP) of FHbp. A single SNP, common to all, alters a polar amino acid that abolishes processing: lipidation and SP cleavage. Whilst some of the FHbp precursor is retained in the cytoplasm due to reduced binding to SecA, remarkably some is translocated and further surface-localized by Slam. Thus we show Slam is not lipoprotein-specific. In a panel of isolates tested, the overall reduced surface localization of the precursor FHbp, compared to isolates with an intact SP, corresponded with decreased susceptibility to antibody-mediated killing. Our findings shed new light on the canonical pathway for lipoprotein processing and translocation of important relevance for lipoprotein-based vaccines in development and in particular for Trumenba.

Comparative analysis of xenobiotic metabolising N-acetyltransferases from ten non-human primates as in vitro models of human homologues.

Xenobiotic metabolising N-acetyltransferases (NATs) perform biotransformation of drugs and carcinogens. Human NAT1 is associated with endogenous metabolic pathways of cells and is a candidate drug target for cancer. Human NAT2 is a well-characterised polymorphic xenobiotic metabolising enzyme, modulating susceptibility to drug-induced toxicity. Human NATs are difficult to express to high purification yields, complicating large-scale production for high-throughput screens or use in sophisticated enzymology assays and crystallography. We undertake comparative functional investigation of the NAT homologues of ten non-human primates, to characterise their properties and evaluate their suitability as models of human NATs. Considering the amount of generated recombinant protein, the enzymatic activity and thermal stability, the NAT homologues of non-human primates are demonstrated to be a much more effective resource for in vitro studies compared with human NATs. Certain NAT homologues are proposed as better models, such as the NAT1 of macaques Macaca mulatta and M. sylvanus, the NAT2 of Erythrocebus patas, and both NAT proteins of the gibbon Nomascus gabriellae which show highest homology to human NATs. This comparative investigation will facilitate in vitro screens towards discovery and optimisation of candidate pharmaceutical compounds for human NAT isoenzymes, while enabling better understanding of NAT function and evolution in primates.

Drug metabolism and antibiotic resistance in micro-organisms.

LINKED ARTICLES: This article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro-organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc.

Expression and regulation of type 2A protein phosphatases and alpha4 signalling in cardiac health and hypertrophy.

Cardiac physiology and hypertrophy are regulated by the phosphorylation status of many proteins, which is partly controlled by a poorly defined type 2A protein phosphatase-alpha4 intracellular signalling axis. Quantitative PCR analysis revealed that mRNA levels of the type 2A catalytic subunits were differentially expressed in H9c2 cardiomyocytes (PP2ACß > PP2ACα > PP4C > PP6C), NRVM (PP2ACß > PP2ACα = PP4C = PP6C), and adult rat ventricular myocytes (PP2ACα > PP2ACß > PP6C > PP4C). Western analysis confirmed that all type 2A catalytic subunits were expressed in H9c2 cardiomyocytes; however, PP4C protein was absent in adult myocytes and only detectable following 26S proteasome inhibition. Short-term knockdown of alpha4 protein expression attenuated expression of all type 2A catalytic subunits. Pressure overload-induced left ventricular (LV) hypertrophy was associated with an increase in both PP2AC and alpha4 protein expression. Although PP6C expression was unchanged, expression of PP6C regulatory subunits (1) Sit4-associated protein 1 (SAP1) and (2) ankyrin repeat domain (ANKRD) 28 and 44 proteins was elevated, whereas SAP2 expression was reduced in hypertrophied LV tissue. Co-immunoprecipitation studies demonstrated that the interaction between alpha4 and PP2AC or PP6C subunits was either unchanged or reduced in hypertrophied LV tissue, respectively. Phosphorylation status of phospholemman (Ser63 and Ser68) was significantly increased by knockdown of PP2ACα, PP2ACß, or PP4C protein expression. DNA damage assessed by histone H2A.X phosphorylation (γH2A.X) in hypertrophied tissue remained unchanged. However, exposure of cardiomyocytes to H2O2 increased levels of γH2A.X which was unaffected by knockdown of PP6C expression, but was abolished by the short-term knockdown of alpha4 expression. This study illustrates the significance and altered activity of the type 2A protein phosphatase-alpha4 complex in healthy and hypertrophied myocardium.

Investigation of the mycobacterial enzyme HsaD as a potential novel target for anti-tubercular agents using a fragment-based drug design approach.

BACKGROUND AND PURPOSE: With the emergence of extensively drug-resistant tuberculosis, there is a need for new anti-tubercular drugs that work through novel mechanisms of action. The meta cleavage product hydrolase, HsaD, has been demonstrated to be critical for the survival of Mycobacterium tuberculosis in macrophages and is encoded in an operon involved in cholesterol catabolism, which is identical in M. tuberculosis and M. bovis BCG. EXPERIMENTAL APPROACH: We generated a mutant strain of M. bovis BCG with a deletion of hsaD and tested its growth on cholesterol. Using a fragment based approach, over 1000 compounds were screened by a combination of differential scanning fluorimetry, NMR spectroscopy and enzymatic assay with pure recombinant HsaD to identify potential inhibitors. We used enzymological and structural studies to investigate derivatives of the inhibitors identified and to test their effects on growth of M. bovis BCG and M. tuberculosis. KEY RESULTS: The hsaD deleted strain was unable to grow on cholesterol as sole carbon source but did grow on glucose. Of seven chemically distinct 'hits' from the library, two chemical classes of fragments were found to bind in the vicinity of the active site of HsaD by X-ray crystallography. The compounds also inhibited growth of M. tuberculosis on cholesterol. The most potent inhibitor of HsaD was also found to be the best inhibitor of mycobacterial growth on cholesterol-supplemented minimal medium. CONCLUSIONS AND IMPLICATIONS: We propose that HsaD is a novel therapeutic target, which should be fully exploited in order to design and discover new anti-tubercular drugs. LINKED ARTICLES: This article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro-organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc.

Azoreductases in drug metabolism.

Azoreductases are flavoenzymes that have been characterized in a range of prokaryotes and eukaryotes. Bacterial azoreductases are associated with the activation of two classes of drug, azo drugs for the treatment of inflammatory bowel disease and nitrofuran antibiotics. The mechanism of reduction of azo compounds is presented; it requires tautomerisation of the azo compound to a quinoneimine and provides a unifying mechanism for the reduction of azo and quinone substrates by azoreductases. The importance of further work in the characterization of azoreductases from enteric bacteria is highlighted to aid in the development of novel drugs for the treatment of colon related disorders. Human azoreductases are known to play a crucial role in the metabolism of a number of quinone-containing cancer chemotherapeutic drugs. The mechanism of hydride transfer to quinones, which is shared not only between eukaryotic and prokaryotic azoreductases but also the wider family of NAD(P)H quinone oxidoreductases, is outlined. The importance of common single nucleotide polymorphisms (SNPs) in human azoreductases is described not only in cancer prognosis but also with regard to their effects on the efficacy of quinone drug-based cancer chemotherapeutic regimens. This highlights the need to screen patients for azoreductase SNPs ahead of treatment with these regimens. LINKED ARTICLES: This article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro-organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc.

Identification of novel members of the bacterial azoreductase family in Pseudomonas aeruginosa.

Azoreductases are a family of diverse enzymes found in many pathogenic bacteria as well as distant homologues being present in eukarya. In addition to having azoreductase activity, these enzymes are also suggested to have NAD(P)H quinone oxidoreductase (NQO) activity which leads to a proposed role in plant pathogenesis. Azoreductases have also been suggested to play a role in the mammalian pathogenesis of Pseudomonas aeruginosa. In view of the importance of P. aeruginosa as a pathogen, we therefore characterized recombinant enzymes following expression of a group of putative azoreductase genes from P. aeruginosa expressed in Escherichia coli. The enzymes include members of the arsenic-resistance protein H (ArsH), tryptophan repressor-binding protein A (WrbA), modulator of drug activity B (MdaB) and YieF families. The ArsH, MdaB and YieF family members all show azoreductase and NQO activities. In contrast, WrbA is the first enzyme to show NQO activity but does not reduce any of the 11 azo compounds tested under a wide range of conditions. These studies will allow further investigation of the possible role of these enzymes in the pathogenesis of P. aeruginosa.

Identification of NAD(P)H quinone oxidoreductase activity in azoreductases from P. aeruginosa: azoreductases and NAD(P)H quinone oxidoreductases belong to the same FMN-dependent superfamily of enzymes.

Water soluble quinones are a group of cytotoxic anti-bacterial compounds that are secreted by many species of plants, invertebrates, fungi and bacteria. Studies in a number of species have shown the importance of quinones in response to pathogenic bacteria of the genus Pseudomonas. Two electron reduction is an important mechanism of quinone detoxification as it generates the less toxic quinol. In most organisms this reaction is carried out by a group of flavoenzymes known as NAD(P)H quinone oxidoreductases. Azoreductases have previously been separate from this group, however using azoreductases from Pseudomonas aeruginosa we show that they can rapidly reduce quinones. Azoreductases from the same organism are also shown to have distinct substrate specificity profiles allowing them to reduce a wide range of quinones. The azoreductase family is also shown to be more extensive than originally thought, due to the large sequence divergence amongst its members. As both NAD(P)H quinone oxidoreductases and azoreductases have related reaction mechanisms it is proposed that they form an enzyme superfamily. The ubiquitous and diverse nature of azoreductases alongside their broad substrate specificity, indicates they play a wide role in cellular survival under adverse conditions.

Structure-activity relationships and colorimetric properties of specific probes for the putative cancer biomarker human arylamine N-acetyltransferase 1.

A naphthoquinone inhibitor of human arylamine N-acetyltransferase 1 (hNAT1), a potential cancer biomarker and therapeutic target, has been reported which undergoes a distinctive concomitant color change from red to blue upon binding to the enzyme. Here we describe the use of in silico modeling alongside structure-activity relationship studies to advance the hit compound towards a potential probe to quantify hNAT1 levels in tissues. Derivatives with both a fifty-fold higher potency against hNAT1 and a two-fold greater absorption coefficient compared to the initial hit have been synthesized; these compounds retain specificity for hNAT1 and its murine homologue mNat2 over the isoenzyme hNAT2. A relationship between pKa, inhibitor potency and colorimetric properties has also been uncovered. The high potency of representative examples against hNAT1 in ZR-75-1 cell extracts also paves the way for the development of inhibitors with improved intrinsic sensitivity which could enable detection of hNAT1 in tissue samples and potentially act as tools for elucidating the unknown role hNAT1 plays in ER+ breast cancer; this could in turn lead to a therapeutic use for such inhibitors.

Mechanism-based inhibition of HsaD: a C-C bond hydrolase essential for survival of Mycobacterium tuberculosis in macrophage.

Mycobacterium tuberculosis remains the leading cause of death by a bacterial pathogen worldwide. Increasing prevalence of multidrug-resistant organisms means prioritizing identification of targets for antituberculars. 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (HsaD), part of the cholesterol metabolism operon, is vital for survival within macrophage. The C-C bond hydrolase, HsaD, has a serine protease-like catalytic triad. We tested a range of serine protease and esterase inhibitors for their effects on HsaD activity. As well as providing a potential starting point for drug development, the data provides evidence for the mechanism of C-C bond hydrolysis. This screen also provides a route to initiate development of fragment-based inhibitors.

Purification, quantification, and functional analysis of Complement Factor H.

Complement Factor H (FH) is an abundant, non-enzymic plasma/serum glycoprotein, which has a major role in regulating activation of the complement system. It can be purified from human plasma/serum by affinity chromatography, using a monoclonal anti-FH antibody as ligand. Other affinity chromatography ligands, including cardiolipin and trinitrophenyl-bovine serum albumin (TNP-BSA), can be used to purify human FH and also FH from a wide range of vertebrates, including mammals, birds, bony fish. Human FH protein concentration can be quantified by sandwich ELISA. The activity of FH is generally measured by assays which detect the cleavage, by complement factor I, of the complement protein C3b to form iC3b. Cleavage occurs only in the presence of a cofactor, and FH is one of a small number of cofactors for this reaction.

Crystallographic analysis reveals the structural basis of the high-affinity binding of iophenoxic acid to human serum albumin.

BACKGROUND: Iophenoxic acid is an iodinated radiocontrast agent that was withdrawn from clinical use because of its exceptionally long half-life in the body, which was due in part to its high-affinity binding to human serum albumin (HSA). It was replaced by Iopanoic acid, which has an amino rather than a hydroxyl group at position 3 on the iodinated benzyl ring and, as a result, binds to albumin with lower affinity and is excreted more rapidly from the body. To understand how iophenoxic acid binds so tightly to albumin, we wanted to examine the structural basis of its interaction with HSA. RESULTS: We have determined the co-crystal structure of HSA in complex with iophenoxic acid at 2.75 Å resolution, revealing a total of four binding sites, two of which--in drugs sites 1 and 2 on the protein--are likely to be occupied at clinical doses. High-affinity binding of iophenoxic acid occurs at drug site 1. The structure reveals that polar and apolar groups on the compound are involved in its interactions with drug site 1. In particular, the 3-hydroxyl group makes three hydrogen bonds with the side-chains of Tyr 150 and Arg 257. The mode of binding to drug site 2 is similar except for the absence of a binding partner for the hydroxyl group on the benzyl ring of the compound. CONCLUSIONS: The HSA-iophenoxic acid structure indicates that high-affinity binding to drug site 1 is likely to be due to extensive desolvation of the compound, coupled with the ability of the binding pocket to provide a full set of salt-bridging or hydrogen bonding partners for its polar groups. Consistent with this interpretation, the structure also suggests that the lower-affinity binding of iopanoic acid arises because replacement of the 3-hydroxyl by an amino group eliminates hydrogen bonding to Arg 257. This finding underscores the importance of polar interactions in high-affinity binding to albumin.

Structural basis of binding of fluorescent, site-specific dansylated amino acids to human serum albumin.

Human serum albumin (HSA) has two primary binding sites for drug molecules. These sites selectively bind different dansylated amino acid compounds, which-due to their intrinsic fluorescence-have long been used as specific markers for the drug pockets on HSA. We present here the co-crystal structures of HSA in complex with six dansylated amino acids that are specific for either drug site 1 (dansyl-l-asparagine, dansyl-l-arginine, dansyl-l-glutamate) or drug site 2 (dansyl-l-norvaline, dansyl-l-phenylalanine, dansyl-l-sarcosine). Our results explain the structural basis of the site-specificity of different dansylated amino acids. They also show that fatty acid binding has only a modest effect on binding of dansylated amino acids to drug site 1 and identify the location of secondary binding sites.

Activation of nitrofurazone by azoreductases: multiple activities in one enzyme.

Azoreductases are well known for azo pro-drug activation by gut flora. We show that azoreductases have a wider role in drug metabolism than previously thought as they can also reduce and hence activate nitrofurazone. Nitrofurazone, a nitroaromatic drug, is a broad spectrum antibiotic which has until now been considered as activated in bacteria by nitroreductases. The structure of the azoreductase with nitrofurazone bound was solved at 2.08 Å and shows nitrofurazone in an active conformation. Based on the structural information, the kinetics and stoichiometry of nitrofurazone reduction by azoreductase from P. aeruginosa, we propose a mechanism of activation which accounts for the ability of azoreductases to reduce both azo and nitroaromatic drugs. This mode of activation can explain the cytotoxic side-effects of nitrofurazone through human azoreductase homologues.

A novel mechanism for azoreduction.

Azoreductases are important due to their ability to activate anti-inflammatory azo pro-drugs and to detoxify azo dyes. Three genes encoding azoreductases have been identified in Pseudomonas aeruginosa. We describe here a comparison of the three enzymes. The pure recombinant proteins each have a distinct substrate specificity profile against a range of azo substrates. Using the structure of P. aeruginosa azoreductase (paAzoR) 1 and the homology models of paAzoR2 and paAzoR3, we have identified residues important for substrate specificity. We have defined a novel flavin mononucleotide binding cradle, which is a recurrent motif in many flavodoxin-like proteins. A novel structure of paAzoR1 with the azo pro-drug balsalazide bound within the active site was determined by X-ray crystallography and demonstrates that the substrate is present in a hydrazone tautomer conformation. We propose that the structure with balsalazide bound represents an enzyme intermediate and, together with the flavin mononucleotide binding cradle, we propose a novel catalytic mechanism.