Accurate automated logPo/w measurement by gradient-flow liquid-liquid partition chromatography. Part 1. Neutral compounds.

There is a continuing need for a method which can measure logPoctanol/water accurately and precisely. Liquid-liquid partition chromatography is ideally suited for this as the retention time is a direct function of the partition or distribution coefficient. However, the range of measurements using isocratic-flow conditions is limited by the log-linear relation between run time and logPo/w. The use of gradient-flow programming and short columns gives a more versatile procedure, so that appropriate data points can be collected at both the low and high ends of the chromatographic run, corresponding to logPo/w values between 1 and 4. At the same time the precision, accuracy and versatility of the method are kept. This study is a validation of the procedure and measures the logPo/w on 23 representative pharmaceuticals, comprising a mixed set of neutral compounds. The linear plot fitting micropartition logPo/w and those found in literature shows a slope close to unity and a residuum close to zero. This indicates that gradient-flow micropartition chromatography is ruled by partitioning between octanol and the aqueous phase.

MraY Inhibitors as Novel Antibacterial Agents.

MraY presents all necessary biological requirements to be considered as a target of interest for the discovery of novel antibacterials. Furthermore, several inhibitors aimed at this enzyme have been discovered. Amphomycin, which is currently used as a topical antibacterial in the veterinary industry is one of them, but the major source of future developments resides in the nucleoside based inhibitors group. This group has been subdivided into classes: Tunicamycins, Ribosamino-uridines, Uridylpeptides and Capuramycins. Analysis of pharmacological behaviours observed with several compounds within these classes, shows that broad-spectrum antibacterial activity, including relevant resistant strains and in vivo efficacy without toxicity are achievable. Among them, Caprazamycins, Muraymycins, Riburamycins and Capuramycins present the most promising profiles.

Fluorescence detection-based functional assay for high-throughput screening for MraY.

We have developed a novel assay specific to MraY, which catalyzes the first membrane step in the biosynthesis of bacterial cell wall peptidoglycan. This was accomplished by using UDP-MurNAc-N(epsilon)-dansylpentapeptide, a fluorescent derivative of the MraY nucleotide substrate, and a partially purified preparation of MraY solubilized from membranes of an Escherichia coli overproducing strain. Two versions of the assay were developed, one consisting of the high-pressure liquid chromatography separation of the substrate and product (dansylated lipid I) and the other, without separation and adapted to the high-throughput format, taking advantage of the different fluorescence properties of the nucleotide and lipid I in the reaction medium. The latter assay was validated with a set of natural and synthetic MraY inhibitors.