Hot-Spotting with Thermal Scanning: A Ligand- and Structure-Independent Assessment of Target Ligandability

Abstract

Evaluating the ligandability of a protein target is a key component when defining hit-finding strategies or when prioritize among drug targets. Computational as well as biophysical approaches based on nuclear magnetic resonance (NMR) fragment screening are powerful approaches but suffer from specific constraints that limit their usage. Here, we demonstrate the applicability of high-throughput thermal scanning (HTTS) as a simple and generic biophysical fragment screening method to reproduce assessments from NMR-based screening. By applying this method to a large set of proteins we can furthermore show that the assessment is predictive of the success of high-throughput screening (HTS). The few divergences for targets of low ligandability originate from the sensitivity differences of the orthogonal biophysical methods. We thus applied a new strategy making use of modulations in the solvent structure to improve assay sensitivity. This novel approach enables improved ligandability assessments in accordance with NMR-based assessments and more importantly positions the methodology as a valuable option for biophysical fragment screening.

Multimodal small-molecule screening for human prion protein binders

“…We could also extend our fragment-based drug design strategy by using chemoproteomics 76 to directly assess PrP ligandability on the cell surface. Multiple approaches may be necessary, because targets with low NMR and thermal shift hit rates are reported to, on average, also have lower hit-to-lead development success rates 77 .…”

Multimodal small-molecule screening for human prion protein binders

“…We could also extend our fragment-based drug design strategy by using chemoproteomics 71 to directly assess PrP ligandability on the cell surface. Multiple approaches may be necessary, because targets with low NMR and thermal shift hit rates are reported to, on average, also have lower hit-to-lead development success rates 72 .…”

Creating and screening natural product libraries

“…119,120 This concept has been recently described by the work of Edfeldt and colleagues at AstraZeneca who have retrospectively examined more than 30 biochemical high throughput screening campaigns and attempted to develop tools to better predict a priori which campaigns were likely to yield productive drug candidates. 120,121 Originally this was done using NMR based fragment screening of small libraries (<2000 substances) of simple chemical scaffolds (<200 Da) and scoring which targets bound the most number of substances at a screening concentration of 1 mM. 119,120 Under these constraints, targets were assigned low, medium and high ligandability based on the percent of compounds which bound the target.…”

“…Subsequently, Edfelt and colleagues extended this observation to show a similar outcome when carrying out fragment based ligandability experiments using a thermal shi assay (discussed in detail below) rather than an NMR based approach. 121 Adoption of this kind of biochemical target assessment in the eld of natural products can be seen in the recent extension of ligandability methodology to "native mass spectroscopy" experiments from the research group of R. J. Quinn. 122 An additional body of literature assessing which classes of both biochemical targets and chemical scaffolds have been most and least successful for HTS development has also recently emerged.…”

“…122 An additional body of literature assessing which classes of both biochemical targets and chemical scaffolds have been most and least successful for HTS development has also recently emerged. 121,[123][124][125] These "target-class" assessments highlight potential limitations of a target or scaffold; but may not predict the behavior of either a novel target or a wellannotated target in a novel assay system. The emergent nature of both ligandability and target-class assessments suggests that due diligence prior to undertaking assay development for a biochemical screen is critical.…”