Discovery of Selective Cannabinoid CB2 Receptor Agonists by High-Throughput Screening.

The endocannabinoid system (ECS) plays a diverse role in human physiology ranging from the regulation of mood and appetite to immune modulation and the response to pain. Drug development that targets the cannabinoid receptors (CB1 and CB2) has been explored; however, success in the clinic has been limited by the psychoactive side effects associated with modulation of the neuronally expressed CB1 that are enriched in the CNS. CB2, however, are expressed in peripheral tissues, primarily in immune cells, and thus development of CB2-selective drugs holds the potential to modulate pain among other indications without eliciting anxiety and other undesirable side effects associated with CB1 activation. As part of a collaborative effort among industry and academic laboratories, we performed a high-throughput screen designed to discover selective agonists or positive allosteric modulators (PAMs) of CB2. Although no CB2 PAMs were identified, 167 CB2 agonists were discovered here, and further characterization of four select compounds revealed two with high selectivity for CB2 versus CB1. These results broaden drug discovery efforts aimed at the ECS and may lead to the development of novel therapies for immune modulation and pain management with improved side effect profiles.

HPLC biogram analysis: a powerful tool used for hit confirmation in early drug discovery.

High-performance liquid chromatography (HPLC) biogram methodology is a powerful pharmaceutical screening hit confirmation strategy that couples analytical HPLC data with functional bioassay data. It is used primarily for screening hit chemical validation and triaging in support of early phase discovery programs and enables further investigation of the source of bioactivity in screening hits. The process combines semi-preparative separation technologies, automated compound handling and distribution, high-throughput biological screening, and informatics tools. The final output is an HPLC retention time versus bioactivity graphical overlay report. In this manner, biograms allow the analyst to determine which component in the sample is responsible for the biological activity, enabling decision making toward chemotype selection and prioritization from a pool of potential candidates. Another powerful aspect of the biogram assay lies in its utility in investigating biological activity in atypical samples, such as degraded samples or mixtures, for detection of minor active impurities or in addressing lot-to-lot activity discrepancies for a given test compound. Biograms are employed to track, isolate, and identify the source of biological activity in such samples, often yielding important information for program decisions.

High-throughput screening and rapid inhibitor triage using an infectious chimeric Hepatitis C virus.

The recent development of a Hepatitis C virus (HCV) infectious virus cell culture model system has facilitated the development of whole-virus screening assays which can be used to interrogate the entire virus life cycle. Here, we describe the development of an HCV growth assay capable of identifying inhibitors against all stages of the virus life cycle with assay throughput suitable for rapid screening of large-scale chemical libraries. Novel features include, 1) the use of an efficiently-spreading, full-length, intergenotypic chimeric reporter virus with genotype 1 structural proteins, 2) a homogenous assay format compatible with miniaturization and automated liquid-handling, and 3) flexible assay end-points using either chemiluminescence (high-throughput screening) or Cellomics ArrayScan™ technology (high-content screening). The assay was validated using known HCV antivirals and through a large-scale, high-throughput screening campaign that identified novel and selective entry, replication and late-stage inhibitors. Selection and characterization of resistant viruses provided information regarding inhibitor target and mechanism. Leveraging results from this robust whole-virus assay represents a critical first step towards identifying inhibitors of novel targets to broaden the spectrum of antivirals for the treatment of HCV.