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BACKGROUNDThe toxic accumulation of phenylalanine (Phe) in the brain underlies the neurological presentation of phenylketonuria (PKU). Solute carrier family 6 member 19 (SLC6A19) is the major transporter responsible for the (re)absorption of Phe in the kidney and intestine. Here, we describe the characterization of the first small molecule SLC6A19 inhibitor to enter clinical development for the treatment of PKU.METHODSC57Bl/6J WT and Pahenu2 mice were dosed with an inhibitor of SLC6A19 to investigate the effects on urinary amino acids and plasma Phe. In a phase 1 study, healthy human volunteers were dosed with JNT-517, an investigational oral inhibitor of SLC6A19. The primary objective of the study was safety. Secondary objectives included pharmacokinetic and pharmacodynamic studies.RESULTSInhibition of SLC6A19 increased the urinary excretion of Phe in a mouse model of PKU, thereby reducing plasma Phe levels. JNT-517, an investigational oral SLC6A19 inhibitor, was found to be safe and well tolerated and increased the urinary excretion of Phe in a phase 1 healthy volunteer study.CONCLUSIONSThese data indicate that pharmacological inhibition of SLC6A19 presents a promising approach to lower toxic elevated levels of amino acids found in PKU and related amino acid metabolism disorders by facilitating their renal elimination.TRIAL REGISTRATIONAustralian New Zealand Clinical Trials Registry (ANZCTR), ACTRN12622001222730.FUNDINGThe studies in this paper were funded by Jnana Therapeutics.

Background: Urotensin II (U-II) is highly expressed in the human lung and has been implicated in regulating respiratory physiology in preclinical studies. Our objective was to test antagonism of the urotensin (UT) receptor by GSK1440115, a novel, competitive, and selective inhibitor of the UT receptor, as a therapeutic strategy for the treatment of asthma., Methods: Safety, tolerability, and pharmacokinetics (PK) of single doses of GSK1440115 (1-750 mg) were assessed in a Phase I, placebo controlled study in 70 healthy subjects. In a Phase Ib study, 12 asthmatic patients were randomized into a two-period, single-blind crossover study and treated with single doses of 750 mg GSK1440115 or placebo and given a methacholine challenge., Results: Administration of GSK1440115 was safe and well-tolerated in healthy subjects and asthmatic patients. In both studies, there was a high degree of variability in the observed PK following oral dosing with GSK1440115 at all doses. There was a marked food effect in healthy subjects at the 50 mg dose. In the presence of food at the 750 mg dose, the time to maximal concentration was between 2 and 6 h and the terminal half-life was short at approximately 2 h. All asthmatic patients maintained greater than the predicted concentration levels necessary to achieve predicted 96% receptor occupancy for ≥3 h (between 4 and 7 h post-dose). There were no apparent trends or relationships between the systemic plasma exposure of GSK1440115 and pharmacodynamic endpoints, PC20 after methacholine challenge and FEV1, in asthmatics., Conclusion: While GSK1440115 was safe and well-tolerated, it did not induce bronchodilation in asthmatics, or protect against methacholine-induced bronchospasm, suggesting that acute UT antagonism is not likely to provide benefit as an acute bronchodilator in this patient population.

Systematic analysis of the entire two-component signal transduction system (TCSTS) gene complement of Staphylococcus aureus revealed the presence of a putative TCSTS (designated SrhSR) which shares considerable homology with the ResDE His-Asp phospho-relay pair of Bacillus subtilis. Disruption of the srhSR gene pair resulted in a dramatic reduction in growth of the srhSR mutant, when cultured under anaerobic conditions, and a 3-log attenuation in growth when analyzed in the murine pyelonephritis model. To further understand the role of SrhSR, differential display two-dimensional gel electrophoresis was used to analyze the cell-free extracts derived from the srhSR mutant and the corresponding wild type. Proteins shown to be differentially regulated were identified by mass spectrometry in combination with protein database searching. An srhSR deletion led to changes in the expression of proteins involved in energy metabolism and other metabolic processes including arginine catabolism, xanthine catabolism, and cell morphology. The impaired growth of the mutant under anaerobic conditions and the dramatic changes in proteins involved in energy metabolism shed light on the mechanisms used by S. aureus to grow anaerobically and indicate that the staphylococcal SrhSR system plays an important role in the regulation of energy transduction in response to changes in oxygen availability. The combination of proteomics, bio-informatics, and microbial genetics employed here represents a powerful set of techniques which can be applied to the study of bacterial gene function.

A genomics-based approach was used to identify the entire gene complement of putative two-component signal transduction systems (TCSTSs) in Streptococcus pneumoniae. A total of 14 open reading frames (ORFs) were identified as putative response regulators, 13 of which were adjacent to genes encoding probable histidine kinases. Both the histidine kinase and response regulator proteins were categorized into subfamilies on the basis of phylogeny. Through a systematic programme of mutagenesis, the importance of each novel TCSTS was determined with respect to viability and pathogenicity. One TCSTS was identified that was essential for the growth of S. pneumoniaeThis locus was highly homologous to the yycFG gene pair encoding the essential response regulator/histidine kinase proteins identified in Bacillus subtilis and Staphylococcus aureus. Separate deletions of eight other loci led in each case to a dramatic attenuation of growth in a mouse respiratory tract infection model, suggesting that these signal transduction systems are important for the in vivo adaptation and pathogenesis of S. pneumoniae. The identification of conserved TCSTSs important for both pathogenicity and viability in a Gram-positive pathogen highlights the potential of two-component signal transduction as a multicomponent target for antibacterial drug discovery.

N-acylhomoserine lactones (AHLs) play a critical role in plant/microbe interactions. The AHL, N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), induces exoenzymes that degrade the plant cell wall by the pathogenic bacterium Erwinia carotovora. Conversely, the antifungal activity of the biocontrol bacterium Pseudomonas aureofaciens 30-84 is due (at least in part) to phenazine antibiotics whose synthesis is regulated by N-hexanoylhomoserine lactone (HHL). Targeting the product of an AHL synthase gene (yenI) from Yersinia enterocolitica to the chloroplasts of transgenic tobacco plants caused the synthesis in plants of the cognate AHL signaling molecules (OHHL and HHL). The AHLs produced by the transgenic plants were sufficient to induce target gene expression in several recombinant bacterial AHL biosensors and to restore biocontrol activity to an HHL-deficient P. aureofaciens strain. In addition, pathogenicity was restored to an E. carotovora strain rendered avirulent as a consequence of a mutation in the OHHL synthase gene, carI. The ability to generate bacterial quorum-sensing signaling molecules in the plant offers novel opportunities for disease control and for manipulating plant/microbe interactions.

In cell-free Yersinia pseudotuberculosis culture supernatants, we have chemically characterized three N-acyl homoserine lactone (AHL) molecules, N-octanoyl homoserine lactone (C8-HSL), N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) and N-hexanoyl homoserine lactone (C6-HSL). We have identified, cloned and sequenced two pairs of LuxR/I homologues termed YpsR/I and YtbR/I. In Escherichia coli at 37 degrees C, YpsI and YtbI both synthesize C6-HSL, although YpsI is responsible for 3-oxo-C6-HSL and YtbI for C8-HSL synthesis respectively. However, in a Y. pseudotuberculosis ypsI-negative background, YtbI appears capable of adjusting the AHL profile from all three AHLs at 37 degrees C and 22 degrees C to the absence of 3-oxo-C6-HSL at 28 degrees C. Insertion deletion mutagenesis of ypsR leads to the loss of C8-HSL at 22 degrees C, which suggests that at this temperature the YpsR protein is involved in the hierarchical regulation of the ytbR/I locus. When compared with the parent strain, the ypsR and ypsI mutants exhibit a number of phenotypes, including clumping (ypsR mutant), overexpression of a major flagellin subunit (ypsR mutant) and increased motility (both ypsR and ypsI mutants). The clumping and motility phenotypes are both temperature dependent. These data are consistent with a hierarchical quorum-sensing cascade in Y. pseudotuberculosis that is involved in the regulation of clumping and motility.

Plasmid reporter vectors have been constructed which respond to activation of LuxR and its homologues LasR and RhlR (VsmR) by N-acyl homoserine lactones (AHLs). The expression of luxCDABE from transcriptional fusions to PluxI, PlasI and PrhlI respectively, occurs in the presence of activating AHLs. A profile of structure/activity relationships is seen where the natural ligand is most potent. The characterisation of individual LuxR homologue/AHL combinations allows a comprehensive evaluation of quorum sensing signals from a test organism.

In bacteria, a network of cellular transduction mechanisms integrate signals from the bacterial environment to control gene expression, and thereby the bacterial phenotype. 'Quorum sensing' describes one such signalling mechanism in response to population density. It relies on the accumulation of small extracellular signalling molecules to modulate the transcription of target operons.

Yersinia enterocolitica produces compounds capable of transcriptionally activating the Photobacterium fischeri bioluminescence (lux) operon. Using high-performance liquid chromatography, high resolution tandem mass spectrometry in conjunction with chemical synthesis, two signal molecules were identified and shown to be N-hexanoyl-L-homoserine lactone (HHL) and N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). A gene (yenI) was isolated from Y. enterocolitica and demonstrated to direct the synthesis of both HHL and OHHL. DNA sequence analysis revealed an open reading frame (ORF) of 642 bp encoding a protein (YenI) of 24.6 kDa with approximately 20% identity to the LuxI family of proteins. Northern blot analysis of yenI expression indicated yenI is transcribed as a single gene and 5' transcript mapping of yenI identified a transcriptional start site 89 bp upstream of the ORF. DNA sequence analysis of the region downstream of yenI located a second ORF, termed yenR, with significant homology to the LuxR family of transcriptional activators. An insertion mutation of yenI abolishes HHL and OHHL production, indicating its central role in N-acylhomoserine lactone synthesis in Y. enterocolitica. Transcriptional analysis using a chromosomal yenI::luxAB fusion has demonstrated that yenI is not subject to autoinduction but is expressed constitutively. Whilst production of the Yop proteins in the wild type and in yenI mutants is indistinguishable, two-dimensional SDS-PAGE analysis of total cell proteins indicated that a number of proteins lack the yenI mutant.

The pheromone N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) regulates expression of bioluminescence in the marine bacterium Vibrio fischeri, the production of carbapenem antibiotic in Erwinia carotovora and exoenzymes in both E. carotovora and Pseudomonas aeruginosa. A characteristic feature of this regulatory mechanism in V. fischeri is that it is cell density-dependent, reflecting the need to accumulate sufficient pheromone to trigger the induction of gene expression. Using a lux plasmid-based bioluminescent sensor for OHHL, pheromone production by E. carotovora, Enterobacter agglomerans, Hafnia alvei, Rahnella aquatilis and Serratia marcescens has been demonstrated and shown also to be cell density-dependent. Production of OHHL implies the presence in these bacteria of a gene equivalent to luxI. Chromosomal banks from all five enteric bacteria have yielded clones capable of eliciting OHHL production when expressed in Escherichia coli. The luxI homologue from both E. carotovora (carI) and E. agglomerans (eagI) were characterized at the DNA sequence level and the deduced protein sequences have only 25% identity with the V. fischeri LuxI. Despite this, carI, eagI and luxI are shown to be biologically equivalent. An insertion mutant of eagI demonstrates that this gene is essential for OHHL production in E. agglomerans.