Biomedical subjects

Peter J Dandliker

Publications and source records attributed to Peter J Dandliker.

9 recordsLinked to original sources

PubMed2007

Solid phase extraction as a faster alternative to HPLC: application to MS analysis of metabolic stability samples.

Due to recent advances in high throughput organic synthesis, discovery teams now need to profile increased numbers of analogs in vitro for their absorption, distribution, metabolism, and excretion (ADME) properties. Consequently, pharmaceutical companies are developing lower cost and higher throughput methods for ADME testing. As demands for metabolic stability testing have increased in our laboratory, the time required to analyze samples using high-pressure liquid chromatography-mass spectrometry (HPLC-MS) has grown rapidly and ultimately limited our data output. In this study we show that solid phase extraction-mass spectrometry (SPE-MS) is a viable alternative to HPLC-MS for monitoring small molecule stability in liver microsomes. Using the SPE-MS approach, samples can be analyzed in 24 s compared to 2.5 min on the HPLC-MS without compromising data quality, thereby alleviating the analytical bottleneck.

Animals↗

PubMed2006

Thienopyridine urea inhibitors of KDR kinase.

A series of substituted thienopyridine ureas was prepared and evaluated for enzymatic and cellular inhibition of KDR kinase activity. Several of these analogs, such as 2, are potent inhibitors of KDR (<10 nM) in both enzymatic and cellular assays. Further characterization of inhibitor 2 indicated that this analog possessed excellent in vivo potency (ED50 2.1 mg/kg) as measured in an estradiol-induced mouse uterine edema model.

Animals↗

PubMed2005

Synthesis and antibacterial activity of 5-methoxy- and 5-hydroxy-6-fluoro-1,8-naphthyridone-3-carboxylic acid derivatives.

A series of 5-methoxy- and 5-hydroxy-6-fluoro-1,8-naphthyridone-3-carboxylic acid derivatives were prepared and evaluated for cell-free bacterial protein synthesis inhibition and whole cell antibacterial activity. When compared to the analogous 5-hydrogen compounds, the presence of the 5-OH group negatively affects biochemical potency. However, a tolerance of the 5-methoxy group is indicated. Only moderate whole cell antibacterial activity is seen, but this could be due to poor cellular penetration. Because only a few 7-position variants were made for this study, further investigation into this novel series combining a broader range of 7-amino derivatives with these 5-position modifications is warranted.

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

Novel inhibitors of bacterial protein synthesis: structure-activity relationships for 1,8-naphthyridine derivatives incorporating position 3 and 4 variants.

Structure-activity relationships for a recently discovered novel ribosome inhibitor (NRI) class of antibacterials were investigated. Preliminary efforts to optimize protein synthesis inhibitory activity of the series through modification of positions 3 and 4 of the naphthyridone lead template resulted in the identification of several biochemically potent analogues. A lack of corresponding whole cell antibacterial activity is thought to be a consequence of poor cellular penetration as evidenced by the enhancement of activity observed for a lead analogue tested in the presence of a cell permeabilizing agent.

Anti-Bacterial Agents↗

PubMed2004

SOS-NMR: a saturation transfer NMR-based method for determining the structures of protein-ligand complexes.

An NMR-based alternative to traditional X-ray crystallography and NMR methods for structure-based drug design is described that enables the structure determination of ligands complexed to virtually any biomolecular target regardless of size, composition, or oligomeric state. The method utilizes saturation transfer difference (STD) NMR spectroscopy performed on a ligand complexed to a series of target samples that have been deuterated everywhere except for specific amino acid types. In this way, the amino acid composition of the ligand-binding site can be defined, and, given the three-dimensional structure of the protein target, the three-dimensional structure of the protein-ligand complex can be determined. Unlike earlier NMR methods for solving the structures of protein-ligand complexes, no protein resonance assignments are necessary. Thus, the approach has broad potential applications--especially in cases where X-ray crystallography and traditional NMR methods have failed to produce structural data. The method is called SOS-NMR for structural information using Overhauser effects and selective labeling and is validated on two protein-ligand complexes: FKBP complexed to 2-(3'-pyridyl)-benzimidazole and MurA complexed to uridine diphosphate N-acetylglucosamine.

Alkyl and Aryl Transferases↗

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↗