Native Mass Spectrometry‐Guided Screening Identifies Hit Fragments for HOP‐HSP90 PPI Inhibition**

Abstract

Contemporary medicinal chemistry considers fragment-based drug discovery (FBDD) and inhibition of protein-protein interactions (PPI) as important means of expanding the volume of druggable chemical space. However, the ability to robustly identify valid fragments and PPI inhibitors is an enormous challenge, requiring the application of sensitive biophysical methodology. Accordingly, in this study, we exploited the speed and sensitivity of nanoelectrospray (nano-ESI) native mass spectrometry to identify a small collection of fragments which bind to the TPR2AB domain of HOP. Follow-up biophysical assessment of a small selection of binding fragments confirmed binding to the single TPR2A domain, and that this binding translated into PPI inhibitory activity between TPR2A and the HSP90 C-terminal domain. An in-silico assessment of binding fragments at the PPI interfacial region, provided valuable structural insight for future fragment elaboration strategies, including the identification of losartan as a weak, albeit dose-dependent inhibitor of the target PPI.

Fragment-to-Lead Medicinal Chemistry Publications in 2022

“…Several 2022 studies report promising new fragment screening techniques to identify hits but do not undertake substantial hit optimization efforts and, therefore, do not meet the criteria for Table inclusion. Interesting examples are the use of photoactivated covalent capture of DNA-encoded fragments (PAC-FragmentDEL) for the discovery of hits against PAK4 and 2-epimerase, nanoliquid capillary chromatography for the identification of acetylcholinesterase inhibitors, a solvatochromic protein binding assay using a peptidic fluoroprobe, and native mass spectrometry to identify fragments that disrupted the HSP90-HOP protein–protein interaction …”

“…Interesting examples are the use of photoactivated covalent capture of DNA-encoded fragments (PAC-FragmentDEL) for the discovery of hits against PAK4 and 2-epimerase, 53 nanoliquid capillary chromatography for the identification of acetylcholinesterase inhibitors, 54 a solvatochromic protein binding assay using a peptidic fluoroprobe, 55 and native mass spectrometry to identify fragments that disrupted the HSP90-HOP protein− protein interaction. 56 X-ray Structures. As with previous years, experimental protein−ligand structural information continues to play an important role in FBDD.…”

Native Mass Spectrometry: Insights and Opportunities for Targeted Protein Degradation

“…Additionally, as new hyphenated MS techniques develop (e.g., MS-cryoEM is on the horizon − ), very powerful combinations of biophysical methods that can be utilized for TPD are envisaged. Because nMS is well developed for fragment screening, ,− and fragment sized starting points are arguably better as E3 recruiter ligands and/or POI ligands, it is conceivable that nMS could also play a strong role in identifying novel E3 recruiter ligands by screening fragment libraries. With TPD and proximity-inducing activities expanding beyond protein targets, it is expected that nMS will easily pivot to investigate these alternate biomolecular systems and play a commanding role in the discovery and development of new degraders.…”

Multiplexed Native Mass Spectrometry Determination of Ligand Selectivity for Fatty Acid-Binding Proteins

“…Native mass spectrometry (nMS) is a rapid, sensitive and high-throughput technique most commonly used to probe noncovalent interactions of biomolecules, particularly the binding stoichiometries and affinities of folded intact proteins and their small-molecule ligands. − While traditional biophysical techniques such as nuclear magnetic resonance (NMR) spectroscopy, protein X-ray crystallography, isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) remain effective in target-based drug discovery, limitations associated with these methods have encouraged increasing applications of nMS as an orthogonal approach for ligand screening. − To elaborate, NMR requires milligram quantities of protein and is challenging for larger proteins; X-ray crystallography can be time-consuming and is often limited by the need for crystallization, ITC is low-throughput and requires moderate quantities of protein, and SPR requires immobilization of one of the binding partners, which may affect the binding site. In contrast, nMS utilizes only picomole quantities of protein and compound, is less restricted by analyte size, and does not require labeling, crystallization or immobilization. , Furthermore, nMS possesses a wide dynamic range, with reported K D values in the low nanomolar and high millimolar range demonstrating good agreement with those generated by SPR and ITC. − While throughput was historically limited owing to the need for manual sample preparation and data acquisition, advances in instrumentation and technology have enabled nMS workflows to be automated, facilitating the use of nMS for fragment screening purposes. ,, …”