Development of R7BP inhibitors through cross-linking coupled mass spectrometry and integrated modeling

Abstract

Protein-protein interaction (PPI) networks are known to be valuable targets for therapeutic intervention; yet the development of PPI modulators as next-generation drugs to target specific vertices, edges, and hubs has been impeded by the lack of structural information of many of the proteins and complexes involved. Building on recent advancements in cross-linking mass spectrometry (XL-MS), we describe an effective approach to obtain relevant structural data on R7BP, a master regulator of itch sensation, and its interfaces with other proteins in its network. This approach integrates XL-MS with a variety of modeling techniques to successfully develop antibody inhibitors of the R7BP and RGS7/Gβ5 duplex interaction. Binding and inhibitory efficiency are studied by surface plasmon resonance spectroscopy and through an R7BP-derived dominant negative construct. This approach may have broader applications as a tool to facilitate the development of PPI modulators in the absence of crystal structures or when structural information is limited.

Application of the Nicoya OpenSPR to Studies of Biomolecular Binding: A Review of the Literature from 2016 to 2022

“…SPR is a powerful tool for real-time, label-free measurement of binding affinity and kinetics, and the vast majority of papers citing the use of the OpenSPR fit neatly underneath this umbrella [ 3 , 5 , 7 , 9 , 15 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 ,…”

“…SPR is an extremely useful and versatile technique. In addition to the oft-cited use of the OpenSPR instrument to measure binding kinetics of ligand–analyte pairs, the SPR can be used to measure a variety of other phenomenon, including ligand aggregation and aggregation-based inhibition [ 213 ], assembly and disassembly of holotoxin complexes [ 214 , 215 ], drug release [ 216 ], collaborative binding [ 211 , 212 ], competition studies [ 217 , 218 ], and inhibition assays [ 15 , 219 ]. SPR has also been used to characterize the immobilization of an affinity reagent, rather than testing binding to the target itself.…”

“…SPR is a powerful tool for real-time, label-free measurement of binding affinity and kinetics, and the vast majority of papers citing the use of the OpenSPR fit neatly underneath this umbrella [3,5,7,9,15,73,.…”

“…The analytes it can measure span a broad range including small molecules [ 3 , 4 ], nucleic acids [ 5 , 6 ], proteins [ 7 , 8 ], antibodies [ 9 ], and cells [ 10 ]. It is a versatile technique and has also been used for epitope mapping [ 11 , 12 ], quantitative detection of biomarkers [ 13 , 14 ], and inhibition studies [ 9 , 15 ].…”

Identifying and characterising Thrap3, Bclaf1 and Erh interactions using cross-linking mass spectrometry

“…Cross-linking mass spectrometry (XL-MS) is a powerful technique that enables the identification of proximal amino acid residues within a single protein as well as residues in close proximity in interacting proteins. Intramolecular crosslinks provide distance constraint parameters that can support homology-based tertiary structure modelling or even guide de novo modelling ( Adikaram et al , 2019 ; Kahraman et al , 2013 ; Liu et al , 2020 ; Orbán-Németh et al , 2018 ; O'Reilly & Rappsilber, 2018 ). Moreover, intermolecular cross-linking information has been used to determine spatial orientation and elucidate the topology of protein complex subunits ( Gaik et al , 2015 ; Herzog et al , 2012 ; Leitner et al , 2016 ; Politis et al , 2014 ).…”