ABSTRACT
The successful discovery and subsequent development of small molecule inhibitors of drug targets relies on the establishment of robust, cost-effective, quantitative, and physiologically relevant in vitro assays that can support prolonged screening and optimization campaigns. The current study illustrates the process of developing and validating an enzymatic assay for the discovery of small molecule inhibitors using alkaline phosphatase from bovine intestine as model target. The assay development workflow includes an initial phase of optimization of assay materials, reagents, and conditions, continues with a process of miniaturization and automation, and concludes with validation by quantitative measurement of assay performance and signal variability. The assay is further evaluated for dose-response and mechanism-of-action studies required to support structure-activity-relationship studies. Emphasis is placed on the most critical aspects of assay optimization and other relevant considerations, including the technology, assay materials, buffer constituents, reaction conditions, liquid handling equipment, analytical instrumentation, and quantitative assessments. Examples of bottlenecks encountered during assay development and strategies to address them are provided.
ABSTRACT
Multifunctional peptide-polymer hybrid materials have been applied as efficient and biocompatible quantum-dot coating materials. Significant pH responsiveness (e.g., an influence of the pH on the quantum yields of the peptide-polymer/QDs) was found and is attributed to conformational rearrangements of the peptide backbone.
Subject(s)
Coated Materials, Biocompatible/chemistry , Polymers/chemistry , Quantum Dots , Serum Albumin/chemistry , Electrodes , Fluorescence Resonance Energy Transfer , Humans , Hydrogen-Ion Concentration , Models, Molecular , Particle Size , Surface PropertiesABSTRACT
Quantifying metal-binding by force: A quantitative single-molecule force-spectroscopy-based assay is developed to measure the binding affinity of metal ions to proteins. The method uses the unfolding force of a protein as a direct probe to distinguish the apo and metal-ion-bound forms of that protein and quantify the partitioning between the two forms (see figure).
