In vivo and in vitro (27)Al NMR studies of aluminum(III) chelates of triazacyclononane polycarboxylate ligands.

The metallic radioisotope of a known radiopharmaceutical chelate, (67)Ga(NOTA) (NOTA=1,4,7-triazacyclonane-1,4,7-triacetic acid), used for tumor detection, was substituted by the chemically similar but non radioactive aluminum ion. Our aim was to detect and evaluate the in vivo behavior of the chelate. For this purpose, Al(NOTA) and the related chelate Al(NODASA) (NODASA=1,4,7-triazacyclononane-1-succinic acid-4,7-diacetic acid) were studied using in vitro and in vivo (27)Al NMR spectroscopy in rats. Both chelates showed high stability towards acid catalyzed dissociation and their (27)Al NMR resonances are characteristic of highly symmetrical species, with chemical shifts within the range for octahedral or pseudo-octahedral geometries. The thermodynamic stability constant of the novel chelate Al(NODASA) was estimated using (27)Al NMR. The value obtained suggested that the chelate does not undergo in vivo demetalation by transferrin. The in vivo spectroscopic studies and the analysis of blood and urine samples for Al(III) concentrations indicated that the chelates remain intact under physiological conditions and that they are mainly eliminated from the body through the kidneys.

Discovering antibiotic efficacy biomarkers: toward mechanism-specific high content compound screening.

As current antibiotic therapy is increasingly challenged by emerging drug-resistant bacteria, new technologies are required to identify and develop novel classes of antibiotics. A major bottleneck in today's discovery efforts, however, is a lack of an efficient and standardized method for assaying the efficacy of a drug candidate. We propose a new high content screening approach for identifying efficacious molecules suitable for development of antibiotics. Key to our approach is a new microarray-based efficacy biomarker discovery strategy. We first produced a large dataset of transcriptional responses of Bacillus subtilis to numerous structurally diverse antibacterial drugs. Second we evaluated different protocols to optimize drug concentration and exposure time selection for profiling compounds of unknown mechanism. Finally we identified a surprisingly low number of gene transcripts (approximately 130) that were sufficient for identifying the mechanism of novel substances with reasonable accuracy (approximately 90%). We show that the statistics-based approach reveals a physiologically meaningful set of biomarkers that can be related to major bacterial defense mechanisms against antibiotics. We provide statistical evidence that a parallel measurement of the expression of the biomarkers guarantees optimal performance when using expression systems for screening libraries of novel substances. The general approach is also applicable to drug discovery for medical indications other than infectious diseases.

Identification of antibiotic stress-inducible promoters: a systematic approach to novel pathway-specific reporter assays for antibacterial drug discovery.

As present antibiotics therapy becomes increasingly ineffectual, new technologies are required to identify and develop novel classes of antibacterial agents. An attractive alternative to the classical target-based approach is the use of promoter-inducible reporter assays for high-throughput screening. The wide usage of these assays is, however, limited by the small number of specifically responding promoters that are known at present. This work describes a novel approach for identifying genetic regulators that are suitable for the design of pathway-specific assays. The basis for the proposed strategy is a large set of antibiotics-triggered expression profiles ("Reference Compendium"). Pattern recognition algorithms applied to the expression data pinpoint the relevant transcription-factor-binding sites in whole-genome sequences. Using this technique, we constructed a fatty-acid-pathway-specific reporter assay that is based on a novel stress-inducible promoter. In a proof-of-principle experiment, this assay was shown to enable screening for new small-molecule inhibitors of bacterial growth.

The complete genome sequence of the carcinogenic bacterium Helicobacter hepaticus.

Helicobacter hepaticus causes chronic hepatitis and liver cancer in mice. It is the prototype enterohepatic Helicobacter species and a close relative of Helicobacter pylori, also a recognized carcinogen. Here we report the complete genome sequence of H. hepaticus ATCC51449. H. hepaticus has a circular chromosome of 1,799,146 base pairs, predicted to encode 1,875 proteins. A total of 938, 953, and 821 proteins have orthologs in H. pylori, Campylobacter jejuni, and both pathogens, respectively. H. hepaticus lacks orthologs of most known H. pylori virulence factors, including adhesins, the VacA cytotoxin, and almost all cag pathogenicity island proteins, but has orthologs of the C. jejuni adhesin PEB1 and the cytolethal distending toxin (CDT). The genome contains a 71-kb genomic island (HHGI1) and several genomic islets whose G+C content differs from the rest of the genome. HHGI1 encodes three basic components of a type IV secretion system and other virulence protein homologs, suggesting a role of HHGI1 in pathogenicity. The genomic variability of H. hepaticus was assessed by comparing the genomes of 12 H. hepaticus strains with the sequenced genome by microarray hybridization. Although five strains, including all those known to have caused liver disease, were indistinguishable from ATCC51449, other strains lacked between 85 and 229 genes, including large parts of HHGI1, demonstrating extensive variation of genome content within the species.