Fragment screening at AstraZeneca: developing the next generation biophysics fragment set.

Fragment based drug discovery is a critical part of the lead generation toolbox and relies heavily on a readily available, high quality fragment library. Over years of use, the AstraZeneca fragment set had become partially depleted and instances of compound deterioration had been found. It was recognised that a redevelopment was required. This provided an opportunity to evolve our screening sets strategy, whilst ensuring that the quality of the fragment set met the robust requirements of fragment screening campaigns. In this communication we share the strategy employed, in particular highlighting two aspects of our approach that we believe others in the community would benefit from, namely that; (i) fragments were selected with input from Medicinal Chemists at an early stage, and (ii) the library was arranged in a layered format to ensure maximum flexibility on a per target basis.

Flotation-A New Method to Circumvent PCR Inhibitors in the Diagnosis of Lawsonia intracellularis.

The obligate intracellular bacterium Lawsonia intracellularis causes enteritis and poor growth in weaned pigs. Cultivation is difficult and diagnosis ante mortem is mainly based on techniques such as polymerase chain reaction. However, false negative results caused by the presence of PCR-inhibitory factors constitute a problem. This study aimed to develop and evaluate a new technique, flotation, to separate L. intracellularis from inhibitors in faeces prior to PCR. The technique was evaluated by comparison to two previously evaluated and commonly used methods, preparation by boiling lysate combined with nested PCR and preparation by a commercial kit combined with conventional PCR. Continuous density centrifugation of faecal samples containing L. intracellularis suggested the buoyant density of the microbe to be between 1.064 and 1.077 g/mL. Several flotation setups were tested to achieve optimal separation of the microbe from inhibitors and faecal particles. The finally selected setup floated whole L. intracellularis from the application site at the bottom to the upper part of the gradient while inhibitory components mainly remained in the bottom. PCR was performed directly on material recovered from the upper interphase. The method was evaluated on 116 clinical samples. As compared to sample preparation by boiling combined with nested PCR, fewer samples were inhibited but also fewer positives were identified. In comparison to preparation by a commercial kit combined with conventional PCR, presently used for routine diagnosis, similar results were obtained. However, the new method was comparably faster to perform. The new method, based on flotation of Lawsonia intracellularis combined with conventional PCR, was well suited for routine diagnosis.