Biomedical subjects

Candace Black-Schaefer

Publications and source records attributed to Candace Black-Schaefer.

5 recordsLinked to original sources

PubMed2006

Novel antibacterial class: a series of tetracyclic derivatives.

We describe the synthesis and antibacterial activity of a series of tetracyclic naphthyridones. The members of this series act primarily via inhibition of bacterial translation and belong to the class of novel ribosome inhibitors (NRIs). In this paper we explore the structure-activity relationships (SAR) of these compounds to measure their ability both to inhibit bacterial translation and also to inhibit the growth of bacterial cells in culture. The most active of these compounds inhibit Streptococcus pneumoniae translation at concentrations of <5 microM and have minimum inhibitory concentrations (MICs) of <8 microg/mL against clinically relevant strains of bacteria.

Anti-Bacterial Agents↗

PubMed2005

Mechanism of action of a novel series of naphthyridine-type ribosome inhibitors: enhancement of tRNA footprinting at the decoding site of 16S rRNA.

The novel ribosome inhibitors (NRIs) are a broad-spectrum naphthyridine class that selectively inhibits bacterial protein synthesis (P. J. Dandliker et al., Antimicrob. Agents Chemother. 47:3831-3839, 2003). Footprinting experiments, using a range of NRIs and chemical modification agents on Escherichia coli ribosomes, revealed no evidence for direct protection of rRNA. In the presence of tRNA, however, we found that NRIs enhanced the known ribosomal footprinting pattern of tRNA in a dose-dependent manner. The most prominent increase in protection, at A1492/3 and A1413 in helix-44 of 16S RNA, strictly required the presence of tRNA and poly(U), and the effect was correlated with the potency of the inhibitor. Radioligand binding studies with inhibitor [(3)H]A-424902 showed that the compound binds to tRNA, either in its charged or uncharged form. The dissociation constant for [(3)H]A-424902 binding to Phe-tRNA(Phe) was determined to be 1.8 microM, near its translation inhibition potency of 1.6 muM in a cell-free S. pneumoniae extract assay. The compound did not change the binding of radiolabeled tRNA to the 30S ribosomal subunit. Taken together, these results imply that the NRIs exert their effects on protein synthesis by structurally perturbing the tRNA/30S complex at the decoding site.

Autoradiography↗

PubMed2004

Automatic mass spectrometry method development for drug discovery: application in metabolic stability assays.

High-throughput metabolic screening has been requested routinely to keep pace with high-throughput organic synthesis. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) with a fast gradient has become the method of choice for the task due to its sensitivity and selectivity. We have developed an automated system that consists of a robotic system for in vitro incubation and a commercially available software package for automatic MS/MS method development. A short, generic LC gradient and MS conditions that are applicable to most compounds have been developed to minimize the method development time and data analysis. This system has been used to support a number of in vitro screening assays in early drug discovery phase including microsomal stability and protein binding.

Automation↗

PubMed2004

A strategy for discovery of novel broad-spectrum antibacterials using a high-throughput Streptococcus pneumoniae transcription/translation screen.

The authors report the development of a high-throughput screen for inhibitors of Streptococcus pneumoniae transcription and translation (TT) using a luciferase reporter, and the secondary assays used to determine the biochemical spectrum of activity and bacterial specificity. More than 220,000 compounds were screened in mixtures of 10 compounds per well, with 10,000 picks selected for further study. False-positive hits from inhibition of luciferase activity were an extremely common artifact. After filtering luciferase inhibitors and several known classes of antibiotics, approximately 50 hits remained. These compounds were examined for their ability to inhibit Escherichia coli TT, uncoupled S. pneumoniae translation or transcription, rabbit reticulocyte translation, and in vitro toxicity in human and bacterial cells. One of these compounds had the desired profile of broad-spectrum biochemical activity in bacteria and selectivity versus mammalian biochemical and whole-cell assays.

Anti-Bacterial Agents↗

PubMed2003

Novel antibacterial class.

We report the discovery and characterization of a novel ribosome inhibitor (NRI) class that exhibits selective and broad-spectrum antibacterial activity. Compounds in this class inhibit growth of many gram-positive and gram-negative bacteria, including the common respiratory pathogens Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, and Moraxella catarrhalis, and are nontoxic to human cell lines. The first NRI was discovered in a high-throughput screen designed to identify inhibitors of cell-free translation in extracts from S. pneumoniae. The chemical structure of the NRI class is related to antibacterial quinolones, but, interestingly, the differences in structure are sufficient to completely alter the biochemical and intracellular mechanisms of action. Expression array studies and analysis of NRI-resistant mutants confirm this difference in intracellular mechanism and provide evidence that the NRIs inhibit bacterial protein synthesis by inhibiting ribosomes. Furthermore, compounds in the NRI series appear to inhibit bacterial ribosomes by a new mechanism, because NRI-resistant strains are not cross-resistant to other ribosome inhibitors, such as macrolides, chloramphenicol, tetracycline, aminoglycosides, or oxazolidinones. The NRIs are a promising new antibacterial class with activity against all major drug-resistant respiratory pathogens.

Amino Acyl-tRNA Synthetases↗