Improvement of the pharmacokinetics and in vivo antibacterial efficacy of a novel type IIa topoisomerase inhibitor by formulation in liposomes.

Several useful properties of liposome-based formulations of various existing antibacterial drugs have been reported. These properties include lower MICs, improved pharmacokinetics, lower toxicity, selective distribution to infected tissues, and enhanced in vivo efficacy. Here we report in vivo studies of a liposomal formulation of a member of a novel class of antibacterial type II topoisomerase inhibitors, others of which have progressed to early phases of clinical trials. The free (i.e., nonliposomal) compound has broad-spectrum MICs but suboptimal pharmacokinetics in rats and mice, characterized by a high volume of distribution and rapid clearance. The liposomal formulation of the compound had essentially unchanged MICs but greatly reduced volume of distribution and clearance in rats and mice. In an in vivo mouse model of Staphylococcus aureus infection of one thigh, the liposomal compound localized preferentially to the infected thigh, whereas the free compound showed no preference for the infected versus the uninfected thigh. Most importantly, the liposomal compound had enhanced efficacy at clearing the infection compared with the free compound. Delivery of this class of compounds as liposomal formulations may offer clinical advantages compared with free compounds.

A high-throughput, homogeneous, fluorescence resonance energy transfer-based assay for phospho-N-acetylmuramoyl-pentapeptide translocase (MraY).

Peptidoglycan biosynthesis is an essential process in bacteria and is therefore a suitable target for the discovery of new antibacterial drugs. One of the last cytoplasmic steps of peptidoglycan biosynthesis is catalyzed by the integral membrane protein MraY, which attaches soluble UDP-N-acetylmuramoyl-pentapeptide to the membrane-bound acceptor undecaprenyl phosphate. Although several natural product-derived inhibitors of MraY are known, none have the properties necessary to be of clinical use as antibacterial drugs. Here we describe a novel, homogeneous, fluorescence resonance energy transfer-based MraY assay that is suitable for high-throughput screening for novel MraY inhibitors. The assay allows for continuous measurement, or it can be quenched prior to measurement.

A high-throughput fluorescence resonance energy transfer-based assay for DNA ligase.

DNA ligase is the enzyme that catalyzes the formation of the backbone phosphodiester bond between the 5'-PO(4) and 3'-OH of adjacent DNA nucleotides at single-stranded nicks. These nicks occur between Okazaki fragments during replication of the lagging strand of the DNA as well as during DNA repair and recombination. As essential enzymes for DNA replication, the NAD(+)-dependent DNA ligases of pathogenic bacteria are potential targets for the development of antibacterial drugs. For the purposes of drug discovery, a high-throughput assay for DNA ligase activity is invaluable. This article describes a straightforward, fluorescence resonance energy transfer-based DNA ligase assay that is well suited for high-throughput screening for DNA ligase inhibitors as well as for use in enzyme kinetics studies. Its use is demonstrated for measurement of the steady-state kinetic constants of Haemophilus influenzae NAD(+)-dependent DNA ligase and for measurement of the potency of an inhibitor of this enzyme.

Evaluation of pKa estimation methods on 211 druglike compounds.

The pK(a) values of 211 discovery (druglike) compounds were determined experimentally using capillary electrophoresis coupled with ultraviolet spectroscopy and a novel fitting algorithm. These values were compared to those predicted by five different commercially available pK(a) estimation packages: ACDLabs/pK(a), Marvin (ChemAxon), MoKa (Molecular Discovery), Epik (Schrodinger), and Pipeline Pilot (Accelrys). Even though the topological method MoKa was noticeably faster than ACD, the accuracy of those two methods and Marvin was statistically indistinguishable, with a root-mean-squared error of about 1 pK(a) unit compared to experiment. Pipeline Pilot and EpiK both produced pK(a) estimates in significantly worse agreement with the experiment. Interestingly, on a number of compounds, the predictions due to ACD v12 were in poorer agreement with the experiment than ACD v10. Microscopic and "apparent" pK(a) predictions were also compared using ACD v10. Microscopic pK(a)s gave significantly worse agreement with the experiment than the "apparent" values. In all cases, the errors appeared to be randomly distributed across chemical series.

A homogeneous, high-throughput fluorescence resonance energy transfer-based DNA polymerase assay.

A homogeneous, fluorescence resonance energy transfer (FRET)-based DNA polymerase assay that is suitable for high-throughput screening for inhibitors, and can also be used for steady-state kinetic investigations, is described. The activity, kinetic mechanism, and processivity of the isolated alpha subunit of DNA polymerase III, the product of the dnaE gene, from the gram-negative pathogen Haemophilus influenzae were investigated using the FRET assay.