Welcome back to the drug discovery series! In this post, we journey through target validation and assay development and validation for compound screening!
As mentioned earlier in the series, most small molecule drugs function by modulating the activity of their particular target protein(s). Targets may be identified in a number of ways, for example, through studying disease pathophysiology to find disease-relevant pathways, or using genome and transcriptome analysis to identify proteins that are differentially expressed and/or aberrantly translated during disease. The early stages of any drug discovery effort must include extensive target validation, ideally through a broad combination of in vitro and in vivo (animal models), cellular and ‘omics’ approaches (1).
Compound Screening – Assay Design and Optimization
Compound screening is often carried out on a large scale, where millions of library compounds are screened. Screening on this scale is known as high throughput screening (HTS). If less than millions of compounds are deployed, one should consider low-to-medium throughput or collections of compounds. During screening, compounds are assayed for interactions with the target, typically in 96-, 384-, or 1536-well format. The nature of the interaction that is assayed between a compound and the target may be inhibition, activation, modulating, or binding.Continue Reading...
Genetically Encoded Biosensors for Research and Drug Discovery
Mitochondrial integrity and function are pivotal to cellular energy production, and mitochondrial dysfunction has been shown to alter the cell cycle, metabolism, cell viability, gene regulation, and other critical aspects of cellular growth and survival.
Mitochondrial Dysfunction – Disease and Cytotoxicity
Mitochondrial dysfunction is associated with a broad spectrum of diseases. For example, in cancer, glycolysis persists to continuously supply ATP for tumor growth while bypassing the need for healthy mitochondria in a phenomenon known as the Warburg Effect (1). Although the underlying genetic reasons for the links between aerobic glycolysis, tumor growth, and hypoxia are not fully understood, the available evidence supports a link between the ability of cancer cells to bypass normal cellular metabolic pathways and mitochondrial dysfunction. Elsewhere, research into neurodegenerative disorders (e.g., Alzheimer’s, amyotrophic lateral sclerosis (ALS), Huntington’s, and Parkinson’s) has revealed the essentiality of mitochondria for neuronal survival, cellular metabolism, and reactive oxygen species production (ROS). Neurons depend on oxidative phosphorylation as a critical source of energy and are very sensitive to intracellular ROS. Consequently, mitochondrial biogenesis and dysfunction are associated with neurodegeneration and aging.Continue Reading...
Gene reporter assays have contributed hugely to our understanding of how genes are regulated during growth and development, for example, through the study of spatiotemporal gene expression patterns, as well as how gene expression is regulated by transcription factors, gene regulatory elements (so-called cis-acting elements), and exogenous regulators (trans-acting factors). Besides investigating gene regulation, gene reporter assays are also useful in transfection experiments, both to optimize and standardize transfection efficiencies and to screen transfected cells in routine workflows. (more…)Continue Reading...