Synthesis and Antimicrobial Evaluation of Amixicile-Based Inhibitors of the Pyruvate-Ferredoxin Oxidoreductases of Anaerobic Bacteria and Epsilonproteobacteria.

Amixicile is a promising derivative of nitazoxanide (an antiparasitic therapeutic) developed to treat systemic infections caused by anaerobic bacteria, anaerobic parasites, and members of the Epsilonproteobacteria (Campylobacter and Helicobacter). Amixicile selectively inhibits pyruvate-ferredoxin oxidoreductase (PFOR) and related enzymes by inhibiting the function of the vitamin B1 cofactor (thiamine pyrophosphate) by a novel mechanism. Here, we interrogate the amixicile scaffold, guided by docking simulations, direct PFOR inhibition assays, and MIC tests against Clostridium difficile, Campylobacter jejuni, and Helicobacter pylori Docking simulations revealed that the nitro group present in nitazoxanide interacts with the protonated N4'-aminopyrimidine of thiamine pyrophosphate (TPP). The ortho-propylamine on the benzene ring formed an electrostatic interaction with an aspartic acid moiety (B456) of PFOR that correlated with improved PFOR-inhibitory activity and potency by MIC tests. Aryl substitution with electron-withdrawing groups and substitutions of the propylamine with other alkyl amines or nitrogen-containing heterocycles both improved PFOR inhibition and, in many cases, biological activity against C. difficile Docking simulation results correlate well with mechanistic enzymology and nuclear magnetic resonance (NMR) studies that show members of this class of antimicrobials to be specific inhibitors of vitamin B1 function by proton abstraction, which is both novel and likely to limit mutation-based drug resistance.

Preclinical studies of amixicile, a systemic therapeutic developed for treatment of Clostridium difficile infections that also shows efficacy against Helicobacter pylori.

Amixicile shows efficacy in the treatment of Clostridium difficile infections (CDI) in a mouse model, with no recurrence of CDI. Since amixicile selectively inhibits the action of a B vitamin (thiamine pyrophosphate) cofactor of pyruvate:ferredoxin oxidoreductase (PFOR), it may both escape mutation-based drug resistance and spare beneficial probiotic gut bacteria that do not express this enzyme. Amixicile is a water-soluble derivative of nitazoxanide (NTZ), an antiparasitic therapeutic that also shows efficacy against CDI in humans. In comparative studies, amixicile showed no toxicity to hepatocytes at 200 µM (NTZ was toxic above 10 µM); was not metabolized by human, dog, or rat liver microsomes; showed equivalence or superiority to NTZ in cytochrome P450 assays; and did not activate efflux pumps (breast cancer resistance protein, P glycoprotein). A maximum dose (300 mg/kg) of amixicile given by the oral or intraperitoneal route was well tolerated by mice and rats. Plasma exposure (rats) based on the area under the plasma concentration-time curve was 79.3 h · µg/ml (30 mg/kg dose) to 328 h · µg/ml (100 mg/kg dose), the maximum concentration of the drug in serum was 20 µg/ml, the time to the maximum concentration of the drug in serum was 0.5 to 1 h, and the half-life was 5.6 h. Amixicile did not concentrate in mouse feces or adversely affect gut populations of Bacteroides species, Firmicutes, segmented filamentous bacteria, or Lactobacillus species. Systemic bioavailability was demonstrated through eradication of Helicobacter pylori in a mouse infection model. In summary, the efficacy of amixicile in treating CDI and other infections, together with low toxicity, an absence of mutation-based drug resistance, and excellent drug metabolism and pharmacokinetic metrics, suggests a potential for broad application in the treatment of infections caused by PFOR-expressing microbial pathogens in addition to CDI.

Response to metronidazole and oxidative stress is mediated through homeostatic regulator HsrA (HP1043) in Helicobacter pylori.

Metronidazole (MTZ) is often used in combination therapies to treat infections caused by the gastric pathogen Helicobacter pylori. Resistance to MTZ results from loss-of-function mutations in genes encoding RdxA and FrxA nitroreductases. MTZ-resistant strains, when cultured at sub-MICs of MTZ (5 to 20 µg/ml), show dose-dependent defects in bacterial growth; depressed activities of many Krebs cycle enzymes, including pyruvate:ferredoxin oxidoreductase (PFOR); and low transcript levels of porGDAB (primer extension), phenotypes consistent with an involvement of a transcriptional regulator. Using a combination of protein purification steps, electrophoretic mobility shift assays (EMSAs), and mass spectrometry analyses of proteins bound to porG promoter sequences, we identified HP1043, an essential homeostatic global regulator (HsrA [for homeostatic stress regulator]). Competition EMSAs and supershift analyses with HsrA-enriched protein fractions confirmed specific binding to porGDAB and hsrA promoter sequences. Exposure to MTZ resulted in >10-fold decreases in levels of HsrA and in levels of the HsrA-regulated gene products PFOR and TlpB. Exposure to paraquat (PQ), hydrogen peroxide, or organic peroxides showed near equivalence with MTZ, revealing a common oxidative stress response pathway. Finally, direct superoxide dismutase (SOD) assays showed an inverse relationship between HsrA levels and SOD activity, suggesting that HsrA may serve as a repressor of sodB. As a homeostatic sentinel, HsrA appears to be ideally positioned to enable rapid shutdown of genes associated with metabolism and growth while activating (directly or indirectly) oxidative defense genes in response to low levels of toxic metabolites (MTZ or oxygen) before they reach DNA-damaging levels.

Mutations to essential orphan response regulator HP1043 of Helicobacter pylori result in growth-stage regulatory defects.

Helicobacter pylori establishes lifelong infections of the gastric mucosa, a niche considered hostile to most microbes. While responses to gastric acidity and local inflammation are understood, little is known as to how they are integrated into homeostatic control of cell division and growth-stage gene expression. Here we investigate the essential orphan response regulator HP1043, a member of the OmpR/PhoB subfamily of transcriptional regulators that is unique to the Epsilonproteobacteria and that lacks phosphorylation domains. To test the hypothesis that conformational changes in the homodimer might lead to defects in gene expression, we sought mutations that might alter DNA-binding efficiency. Two introduced mutations (C215S, C221S) C terminal to the DNA-binding domain of HP1043 (HP1043CC11) resulted in a 2-fold higher affinity for its own promoter by footprinting. Modeling studies with the crystal structure of HP1043 suggested that C215S might affect the helix-turn-helix domain. Genomic replacement of the hp1043 allele with the hp1043CC11 mutant allele resulted in a 2-fold decrease in protein levels, despite a dramatic increase in mRNA. The mutations did not affect in vitro growth rates or colonization efficiency in a mouse model. Proteomic profiling (CC11 mutant strain versus wild type) identified many expression differences, and quantitative PCR further revealed that 11 out of 12 examined genes had lost growth-stage regulation and that 6 of the genes contained HP1043 binding consensus sequences within the promoter regions (fur, cagA, cag23, flhA, flip, and napA). Our studies show that mutations that affect DNA-binding affinity can be used to identify new members of the HP1043 regulon.

Structure of RdxA--an oxygen-insensitive nitroreductase essential for metronidazole activation in Helicobacter pylori.

UNLABELLED: The RdxA oxygen-insensitive nitroreductase of the human gastric pathogen Helicobacter pylori is responsible for the susceptibility of this organism to the redox active prodrug metronidazole [2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethanol]. Loss-of-function mutations in rdxA are primarily responsible for resistance to this therapeutic. RdxA exhibits potent NADPH oxidase activity under aerobic conditions and metronidazole reductase activity under strictly anaerobic conditions. In the present study, we report the crystal structure of RdxA, which is a homodimer exhibiting domain swapping and containing two molecules of FMN bound at the dimer interface. We have found a gap between the side chain of Tyr47 and the isoalloxazine ring of FMN that appears to be appropriate for substrate binding. The structure does not include residues 97-128, which correspond to a locally unstable part of the NTR from Escherichia coli, and might be involved in cofactor binding. Comparison of H. pylori RdxA with other oxidoreductases of known structure suggests that RdxA may belong to a new subgroup of oxidoreductases in which a cysteine side chain close to the FMN cofactor could be involved in the reductive activity. In this respect, the mutation of C159 to A or S (C159A/S) has resulted in a loss of metronidazole reductase activity but not NADPH oxidase activity. The RdxA structure enables the interpretation of the many loss-of-function mutations described previously, including those affecting C159, a residue whose interaction with FMN is required for the nitroreduction of metronidazole. The present studies provide unique insights into the redox behaviour of the flavin in this key enzyme for metronidazole activation, including a potential use in gene therapy. DATABASE: Structural data have been deposited in the Protein Data Bank under accession number 3QDL.

Amixicile, a novel inhibitor of pyruvate: ferredoxin oxidoreductase, shows efficacy against Clostridium difficile in a mouse infection model.

Clostridium difficile infection (CDI) is a serious diarrheal disease that often develops following prior antibiotic usage. One of the major problems with current therapies (oral vancomycin and metronidazole) is the high rate of recurrence. Nitazoxanide (NTZ), an inhibitor of pyruvate:ferredoxin oxidoreductase (PFOR) in anaerobic bacteria, parasites, Helicobacter pylori, and Campylobacter jejuni, also shows clinical efficacy against CDI. From a library of ∼250 analogues of NTZ, we identified leads with increased potency for PFOR. MIC screens indicated in vitro activity in the 0.05- to 2-µg/ml range against C. difficile. To improve solubility, we replaced the 2-acetoxy group with propylamine, producing amixicile, a soluble (10 mg/ml), nontoxic (cell-based assay) lead that produced no adverse effects in mice by oral or intraperitoneal (i.p.) routes at 200 mg/kg of body weight/day. In initial efficacy testing in mice treated (20 mg/kg/day, 5 days each) 1 day after receiving a lethal inoculum of C. difficile, amixicile showed slightly less protection than did vancomycin by day 5. However, in an optimized CDI model, amixicile showed equivalence to vancomycin and fidaxomicin at day 5 and there was significantly greater survival produced by amixicile than by the other drugs on day 12. All three drugs were comparable by measures of weight loss/gain and severity of disease. Recurrence of CDI was common for mice treated with vancomycin or fidaxomicin but not for mice receiving amixicile or NTZ. These results suggest that gut repopulation with beneficial (non-PFOR) bacteria, considered essential for protection against CDI, rebounds much sooner with amixicile therapy than with vancomycin or fidaxomicin. If the mouse model is indeed predictive of human CDI disease, then amixicile, a novel PFOR inhibitor, appears to be a very promising new candidate for treatment of CDI.

Synthesis and antimicrobial evaluation of nitazoxanide-based analogues: identification of selective and broad spectrum activity.

A library composed of nitazoxanide-based analogues was synthesized and assayed for increased antibacterial efficacy against the pyruvate-ferredoxin oxidoreductase (PFOR) using microorganisms Helicobacter pylori, Campylobacter jejuni and Clostridium difficile. Derivatives were found to recapitulate and improve activity against these organisms and select analogues were tested for their ability to disrupt the PFOR enzyme directly. The library was also screened for activity against staphylococci and resulted in the identification of analogues capable of inhibiting both staphylococci and all PFOR organisms at low micromolar minimum inhibitory concentrations with low toxicity to human foreskin cells.

Biological activity of modified and exchanged 2-amino-5-nitrothiazole amide analogues of nitazoxanide.

Head group analogues of the antibacterial and antiparasitic drug nitazoxanide (NTZ) are presented. A library of 39 analogues was synthesized and assayed for their ability to suppress growth of Helicobacter pylori, Campylobacter jejuni, Clostridium difficile and inhibit NTZ target pyruvate:ferredoxin oxidoreductase (PFOR). Two head groups assayed recapitulated NTZ activity and possessed improved activity over their 2-amino-5-nitrothiazole counterparts, demonstrating that head group modification is a viable route for the synthesis of NTZ-related antibacterial analogues.

Characterization of the NAD(P)H oxidase and metronidazole reductase activities of the RdxA nitroreductase of Helicobacter pylori.

Metronidazole (MTZ) is widely used in combination therapies against the human gastric pathogen Helicobacter pylori. Resistance to this drug is common among clinical isolates and results from loss-of-function mutations in rdxA, which encodes an oxygen-insensitive nitroreductase. The RdxA-associated MTZ-reductase activity of H. pylori is lost upon cell disruption. Here we provide a mechanistic explanation for this phenomenon. Under aerobic conditions, His6-tagged RdxA protein (purified from Escherichia coli), catalyzed NAD(P)H-dependent reductions of nitroaromatic and quinone substrates including nitrofurazone, nitrofurantoin, furazolidone, CB1954 and 1,4-benzoquinone, but not MTZ. Unlike other nitroreductases, His6-RdxA exhibited potent NAD(P)H-oxidase activity (k(cat) = 2.8 s(-1)) which suggested two possible explanations for the role of oxygen in MTZ reduction: (a) NAD(P)H-oxidase activity promotes cellular hypoxia (nonspecific reduction of MTZ), and (b) molecular oxygen out-competes MTZ for reducing equivalents. The first hypothesis was eliminated upon finding that rdxA expression, although increasing MTZ toxicity in both E. coli and H. pylori constructs, did not increase paraquat toxicity, even though both are of similar redox potential. The second hypothesis was confirmed by demonstrating NAD(P)H-dependent MTZ-reductase activity (apparent K(m) = 122 +/- 58 microM, k(cat) = 0.24 s(-1)) under strictly anaerobic conditions. The MTZ-reductase activity of RdxA was 60 times greater than for NfsB (E. coli NTR), but 10 times lower than the NADPH-oxidase activity. Whether molecular oxygen directly competes with MTZ or alters the redox state of the FMN cofactors is discussed.

Helicobacter pylori and mitogen-activated protein kinases mediate activator protein-1 (AP-1) subcomponent protein expression and DNA-binding activity in gastric epithelial cells.

Emerging evidence has suggested a critical role for activator protein-1 (AP)-1 in regulating various cellular functions. The goal of this study was to investigate the effects of Helicobacter pylori and mitogen-activated protein kinases (MAPK) on AP-1 subcomponents expression and AP-1 DNA-binding activity in gastric epithelial cells. We found that H. pylori infection resulted in a time- and dose-dependent increase in the expression of the proteins c-Jun, JunB, JunD, Fra-1, and c-Fos, which make up the major AP-1 DNA-binding proteins in AGS and MKN45 cells, while the expression levels of Fra-2 and FosB remained unchanged. Helicobacter pylori infection and MAPK inhibition altered AP-1 subcomponent protein expression and AP-1 DNA-binding activity, but did not change the overall subcomponent composition. Different clinical isolates of H. pylori showed various abilities to induce AP-1 DNA binding. Mutation of cagA, cagPAI, or vacA, and the nonphosphorylateable CagA mutant (cagA(EPISA)) resulted in less H. pylori-induced AP-1 DNA-binding activity, while mutation of the H. pylori flagella had no effect. extracellular signal-related kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) each selectively regulated AP-1 subcomponent expression and DNA-binding activity. These results provide more insight into how H. pylori and MAPK modulate AP-1 subcomponents in gastric epithelial cells to alter the expression of downstream target genes and affect cellular functions.

Role of nucleoid-associated proteins Hha and H-NS in expression of Salmonella enterica activators HilD, HilC, and RtsA required for cell invasion.

The coordinate expression of Salmonella enterica invasion genes on Salmonella pathogenicity island 1 is under the control of the complex circuits of regulation that involve the AraC/XylS family transcriptional activators HilD, HilC, and RtsA and nucleoid-associated proteins. Single-copy transcription fusions were used to assess the effects of nucleoid-associated proteins Hha and H-NS on hilD, hilC, and rtsA expression. The data show that all three genes, hilD, hilC, and rtsA, were repressed by H-NS and/or Hha. The repression of rtsA was the highest among tested genes. The level of rtsA-lac was equally elevated in hns and hha mutants and was further enhanced in the hns hha double mutant under low-osmolarity conditions. Electrophoretic mobility shift experiments showed that H-NS and Hha directly bind to the rtsA promoter. In addition to the negative control that was exerted by H-NS/Hha under low-osmolarity conditions, the homologous virulence activators HilD, HilC, and RtsA (Hil activators) induced rtsA-lac expression in a high-salt medium. A DNase footprinting assay of the rtsA promoter revealed one common DNA-binding site for all three Hil activators centered at position -54 relative to the transcriptional start site. In the absence of Hha and H-NS, however, osmoregulation of the rtsA promoter was lost, and Hil activators were not required for rtsA transcription. These results taken together suggest that the HilD, HilC, and RtsA proteins induce the transcription of the rtsA promoter by counteracting H-NS/Hha-mediated repression.

Crucial roles of both flanking sequences in silencing of the hilA promoter in Salmonella enterica.

The hilA gene on the Salmonella enterica pathogenicity island-1 encodes the key transcriptional regulator of host cell invasion. Transcription of hilA is regulated by numerous physiological signals, including repression under low osmolarity conditions. To investigate the osmotic control of hilA transcription, promoter truncations that remove sequences flanking the hilA promoter were examined. Expression of the minimal hilA core promoter (-55 to +90, relative to the transcription start site) was 57-times higher than the intact promoter (-242 to +505) in the absence of osmotic stress. Both flanking sequences contributed to the strong silencing effect, which was greatly relieved by the simultaneous loss of the two nucleoid-structuring proteins, H-NS and Hha. Mobility-shift assays revealed the presence of binding sites for the H-NS and Hha proteins, both upstream and downstream of the promoter. Either flanking region depressed expression when it was placed downstream of the lacUV5 promoter, and this inhibition was increased when the other flanking sequence was present upstream of the promoter. These results show that the hilA promoter is highly active without other transcription regulators. Its high activity is strongly depressed in low osmolarity conditions by the nucleoid-structuring proteins H-NS and Hha, possibly by formation of a repressive DNA loop. The hilA activators, HilD and HilC appear to overcome effects of downstream silencing region and disrupt repressive DNA loop. Action of activators requires contact with RNA polymerase from their DNA binding site, centered at position -77, relative to the hilA transcription start site.

Contribution of the RpoA C-terminal domain to stimulation of the Salmonella enterica hilA promoter by HilC and HilD.

Expression of invasion genes in Salmonella pathogenicity island 1 (SPI-1) is mainly driven by the transcriptional activator HilA. Transcription of hilA is subject to complex control and is stimulated by the SPI-1-encoded HilC and HilD proteins. The C-terminal domain of RpoA contributes to hilA activation by HilC/D under certain inducing conditions.

DNA-binding activities of the HilC and HilD virulence regulatory proteins of Salmonella enterica serovar Typhimurium.

The HilC and HilD proteins of Salmonella enterica serovar Typhimurium are members of the AraC/XylS family of transcription regulators. They are encoded on Salmonella pathogenicity island 1 (SPI1) and control expression of the hilA gene, which encodes the major transcriptional activator for many genes encoded on SPI1 and elsewhere that contribute to invasion of host cells. Gel electrophoretic shift and DNase footprinting assays revealed that purified HilC and HilD proteins can bind to multiple regions in the hilA and hilC promoters and to a single region in the hilD promoter. Although both HilC and -D proteins can bind to the same DNA regions, they showed different dependencies on the sequence and lengths of their DNA targets. To identify the binding-sequence specificity of HilC and HilD, a series of single base substitutions changing each position in a DNA fragment corresponding to positions -92 to -52 of the hilC promoter was tested for binding to HilC and HilD in a gel shift DNA-binding assay. This mutational analysis in combination with sequence alignments allowed deduction of consensus sequences for binding of both proteins. The consensus sequences overlap but differ so that HilC can bind to both types of sites but HilD only to one. The hilA and hilC promoters contain multiple binding sites of each type, whereas the hilD promoter contains a site that binds HilC but not HilD without additional binding elements. The HilC and HilD proteins had no major effect on transcription from the hilA or hilD promoters using purified proteins in vitro but changed the choice of promoter at hilC. These results are consistent with a model derived from analysis of lacZ fusions stating that HilC and HilD enhance hilA expression by counteracting a repressing activity.

Mutational scanning and affinity cleavage analysis of UhpA-binding sites in the Escherichia coli uhpT promoter.

UhpA, a member of the NarL family of response regulators, activates transcription of the Escherichia coli uhpT gene for the sugar phosphate transporter UhpT in response to extracellular glucose-6-phosphate. UhpA binds with different affinities to adjacent regions in the uhpT promoter, termed the strong-binding (S) region from -80 to -50 and the weak-binding (W) region from -50 to -32. Transcription activation by UhpA is stimulated by the catabolite gene activator protein (CAP)-cyclic AMP complex and depends on the C-terminal domains of the RNA polymerase RpoA and RpoD subunits. Because single-base substitutions in the UhpA-binding region had little effect on promoter activity, nucleotide substitutions in successive 4-bp blocks throughout this region were examined for their effects on promoter activation and UhpA binding. Changes in three of four blocks within the W region substantially impaired the ability of UhpA to bind to this region, to drive expression of a uhpT-lacZ reporter, and to support UhpA-dependent in vitro transcription. These W region variant promoters were strongly stimulated by CAP. Changes in several parts of the S region impaired UhpA binding to both the S and W regions and decreased promoter activity in vivo and in vitro. Thus, binding of UhpA to the W region is crucial for UhpA-dependent activation and depends on occupancy of the S region. None of these substitutions eliminated promoter function. The orientation of UhpA-binding sites was assessed by the affinity cleavage method. The iron chelate FeBABE [iron (S)-1-(p-bromoacetamidobenzyl) EDTA] was covalently attached to engineered cysteine residues near the DNA-binding region in UhpA. Hydroxyl radicals generated by the iron chelate attached at position 187 resulted in DNA strand cleavages in two clusters of sites located in the middle of the S and W regions. These results are consistent with the binding of two dimers of UhpA. Each dimer binds to an inverted repeat of monomer-binding sites with the consensus sequence CCTGRR, where R is A or G, and each is separated by 6 bp. It is likely that members of the NarL family bind to dyad targets, in contrast to the binding of OmpR family response regulators to direct-repeat targets.