Biophysics: for HTS hit validation, chemical lead optimization, and beyond

Abstract

There are many challenges to the drug discovery process, including the complexity of the target, its interactions, and how these factors play a role in causing the disease. Traditionally, biophysics has been used for hit validation and chemical lead optimization. With its increased throughput and sensitivity, biophysics is now being applied earlier in this process to empower target characterization and hit finding. Areas covered: In this article, the authors provide an overview of how biophysics can be utilized to assess the quality of the reagents used in screening assays, to validate potential tool compounds, to test the integrity of screening assays, and to create follow-up strategies for compound characterization. They also briefly discuss the utilization of different biophysical methods in hit validation to help avoid the resource consuming pitfalls caused by the lack of hit overlap between biophysical methods. Expert opinion: The use of biophysics early on in the drug discovery process has proven crucial to identifying and characterizing targets of complex nature. It also has enabled the identification and classification of small molecules which interact in an allosteric or covalent manner with the target. By applying biophysics in this manner and at the early stages of this process, the chances of finding chemical leads with novel mechanisms of action are increased. In the future, focused screens with biophysics as a primary readout will become increasingly common.

Creating and screening natural product libraries

“…In addition to enzymatic biochemical screens, new cell-free technologies have recently emerged that allow for the direct interrogation of the interaction between biomolecules and small molecules in the context of a high throughput NPD screen. [153][154][155] For many disease-associated molecular targets that are not enzymes but do have structural features that can be probed for small molecule binding biophysical screening techniques are useful. Since these newly developed HTS technologies do not rely on biochemically catalyzed reactions but rather on a physical interaction between the small molecule and the target protein/nucleic acid, they are considered biophysical high throughput assays.…”

“…156,157 Another biophysical assay that has been adopted for HTS assays is the thermal shi assay (TSA). 153,155 This assay measures the thermal stability of a target biomolecule (proteins initially but more recently nucleic acids) across a temperature gradient by monitoring the unfolding dependent binding of a uorogenic dye. 153,155 As the biomolecule slowly unfolds with increasing temperature, more of the dye is incorporated into the structure so that a characteristic melting curve is generated and a specic melting temperature is calculated (T M ).…”

“…153,155 This assay measures the thermal stability of a target biomolecule (proteins initially but more recently nucleic acids) across a temperature gradient by monitoring the unfolding dependent binding of a uorogenic dye. 153,155 As the biomolecule slowly unfolds with increasing temperature, more of the dye is incorporated into the structure so that a characteristic melting curve is generated and a specic melting temperature is calculated (T M ). Comparing the deviations of the T M in the presence of a test substance has been shown to indicate specic interactions (stabilizing or destabilizing) between the substance and the target.…”

“…Although implementation of this screening modality is both convenient and relatively cheap, it does have a few potential limitations. 153,158 As it depends on uorescence detection, a TSA can suffer from uorescent interference from test compounds. In addition, deections of the T M are not necessarily indicative of binding affinities (greater changes in T M do not equate to higher or lower affinity).…”

TANGO: A high through‐put conformation generation and semiempirical method‐based optimization tool for ligand molecules

“…Computational approaches in building lead library provide helpful insight to the experimentalists, thereby reducing the time and cost required for this procedure. Various tools and services are available in the drug discovery paradigm that deals with the screening and optimization of the small molecules . Most of the information for these small molecules is either available in 1D SMILES or in 2D SDF formats .…”