A novel method using nuclear magnetic resonance for plasma protein binding assessment in drug discovery programs.

A new methodology based on Nuclear Magnetic Resonance (NMR) was developed to determine plasma protein binding (PPB) of drug candidates in drug discovery programs. A strong correlation was found between the attenuation of NMR signals of diverse drugs in the presence of different plasma concentrations and their fraction bound (fb) reported in the literature. Based on these results, a protocol for a rapid calculation of fb of small molecules was established. The advantage of using plasma instead of purified recombinant proteins and the possibility of pool analysis to increase throughput were also evaluated. This novel methodology proved to be very versatile, cost-effective, fast and suitable for automation. As a plus, it contemporarily provides a quality check and solubility of the compound.

High-throughput microsomal stability assay for screening new chemical entities in drug discovery.

In this work, the authors present a novel, robotic, automated protocol for assessing a metabolic stability protocol assembled on a Hamilton platform and a new strategy for pooling samples (cassette analysis). To increase the high throughput of the liquid chromatography (LC) step, fast chromatography and automated liquid chromatography tandem mass spectrometry (LC/MS/MS) analytical methods were also developed, and a rapid data analysis system was generated that converts peak areas obtained by LC/MS/MS in intrinsic clearance values. All of the steps of the microsomal stability assay were carefully studied and optimized. Standard errors and confidence intervals of the measured clearances were also automatically generated in the process to allow an immediate evaluation of the significance of observed values. Methods based on pooling analysis of 2 and 4 different analytes were compared with a standard method without pooling. A simple statistical treatment was used to show their equivalence. The different protocols developed were analyzed in terms of the best compromise between accuracy and high-throughput capabilities.

Discovery of a Selective Series of Inhibitors of Plasmodium falciparum HDACs.

The identification of a new series of P. falciparum growth inhibitors is described. Starting from a series of known human class I HDAC inhibitors a SAR exploration based on growth inhibitory activity in parasite and human cells-based assays led to the identification of compounds with submicromolar inhibition of P. falciparum growth (EC50 < 500 nM) and good selectivity over the activity of human HDAC in cells (up to >50-fold). Inhibition of parasital HDACs as the mechanism of action of this new class of selective growth inhibitors is supported by hyperacetylation studies.