Rapid LC-MS Based High-Throughput Screening Method, Affording No False Positives or False Negatives, Identifies a New Inhibitor for Carbonic Anhydrase

Abstract

Developing effective high-throughput screening (HTS) methods is of paramount importance in the early stage of drug discovery. While rugged and robust assays may be easily developed for certain enzymes, HTS assays designed to identify ligands that block protein binding are much more challenging to develop; attenuating the number of false positives and false negatives under high-throughput screening conditions is particularly difficult. We describe an MS-based HTS workflow that addresses these challenges. The assay mitigates false positives by selectively identifying positive hits exclusively when a ligand at the binding site of interest is displaced; it mitigates false negatives by detecting a reporter compound that ionizes well, not by detecting the ligand binder, which may not ionize. The method was validated by detecting known binders of three proteins, pepsin, maltose binding protein (MBP), and carbonic anhydrase (CA) in the presence of hundreds of non-binders. We also identified a novel CA binder, pifithrin-µ, which could not have been identified by any other MS-based assay because of its poor ionization efficiency. This new method addresses many of the challenges that are currently encountered during high-throughput screening.

Solving the Competitive Binding Equilibria between Many Ligands: Application to High-Throughput Screening and Affinity Optimization

“…A challenge for this approach, however, is that it may lead to false positives depending on the desired affinity for the hits; many compounds with dissociation constants in the range of low pM to high mM were equally identified, with prioritization requiring additional studies. More recently, the group of Desaire proposed a related method that indicates which screening pools may contain a strong binder among the many ligands in each (300–400 compounds) . This method uses a relatively low-affinity control ligand that is displaced by strong binders in the pool, and it is detected in the supernatant after equilibration.…”

“…More recently, the group of Desaire proposed a related method that indicates which screening pools may contain a strong binder among the many ligands in each (300−400 compounds). 50 This method uses a relatively low-affinity control ligand that is displaced by strong binders in the pool, and it is detected in the supernatant after equilibration. The main limitation, aside from requiring a suitable known ligand, is that it does not inform which ligand (or ligands) in a given pool is the strongest binder, for which additional experiments are needed (see below).…”

The Application of High‐Throughput Approaches in Identifying Novel Therapeutic Targets and Agents to Treat Diabetes

“…[86] Other structure-based assays also have HTS formats, especially MS-based approaches that are widely used in discovering enzyme inhibitors. [87][88][89][90] One limitation of structure-based assays, however, is that no information about the effects of hits on targets, like inhibition or potentiation, can be collected. [49] Thus, structure-based assays are often incorporated as a subsequent step of functional HTS to interrogate the structure of identified hits.…”

Schistosomiasis Drug Discovery in the Era of Automation and Artificial Intelligence

“…In addition, IMER can be coupled to different separation systems, such as high performance liquid chromatography (HPLC), which solve the possible problem of product and assayed compounds fluorescing at the same wavelength, since these analytes can be separated and analyzed individually (128). Active anti-cancer compounds (129), enzyme inhibitors (130)(131)(132)(133) and G protein-coupled receptors (GPCR) (134) binders have recently been identified using this approach.…”