Exploring Multi-Subsite Binding Pockets in Proteins: DEEP-STD NMR Fingerprinting and Molecular Dynamics Unveil a Cryptic Subsite at the GM1 Binding Pocket of Cholera Toxin B.

Serena Monaco,Inmaculada Robina,Samuel Walpole,Ridvan Nepravishta,Ana T Carmona,Jesus Angulo,Macarena Martínez-Bailén,Hassan Doukani,Javier Ramos-Soriano,Maria Bergström

doi:10.1002/chem.202001723

Abstract

Ligand‐based NMR techniques to study protein–ligand interactions are potent tools in drug design. Saturation transfer difference (STD) NMR spectroscopy stands out as one of the most versatile techniques, allowing screening of fragments libraries and providing structural information on binding modes. Recently, it has been shown that a multi‐frequency STD NMR approach, differential epitope mapping (DEEP)‐STD NMR, can provide additional information on the orientation of small ligands within the binding pocket. Here, the approach is extended to a so‐called DEEP‐STD NMR fingerprinting technique to explore the binding subsites of cholera toxin subunit B (CTB). To that aim, the synthesis of a set of new ligands is presented, which have been subject to a thorough study of their interactions with CTB by weak affinity chromatography (WAC) and NMR spectroscopy. Remarkably, the combination of DEEP‐STD NMR fingerprinting and Hamiltonian replica exchange molecular dynamics has proved to be an excellent approach to explore the geometry, flexibility, and ligand occupancy of multi‐subsite binding pockets. In the particular case of CTB, it allowed the existence of a hitherto unknown binding subsite adjacent to the GM1 binding pocket to be revealed, paving the way to the design of novel leads for inhibition of this relevant toxin.

Highlights

In the context of drug discovery, fragment-based drug discovery (FBDD) has gained momentum over the last decade as a method for lead generation.[1]
We have recently proposed an expansion of the methodology termed Differential Epitope Mapping Saturation transfer difference (STD) NMR (DEEP-Saturation Transfer Difference NMR (STD NMR)) based on the new concept of multi-frequency STD NMR experiments.[8]
We present the synthesis of a new set of cholera toxin subunit B (CTB) ligands to explore the GM1 binding pocket of CTB and propose the differential epitope mapping (DEEP)-STD NMR fingerprinting protocol, to gain orientation

Summary

Introduction

In the context of drug discovery, fragment-based drug discovery (FBDD) has gained momentum over the last decade as a method for lead generation.[1]. Structural and thermodynamic studies on this complex showed that high specificity is given by the presence of two adjacent binding subsites, accommodating the two “arms” of the GM1 ligand, that is, the galactose and sialic acid non-reducing ends.[13,14,15] Grounded in this knowledge, many carbohydrate-based scaffolds have been designed that aim to target both binding subsites.[16,17,18,19,20] Despite these glycomimetics showing very good affinity levels, the challenge has remained to overcome the problem of hydrolyzability. The combination of STD NMR competition experiments,[22] ILOEs,[23] docking,[24,25,26] advanced molecular dynamics,[27] and STD NMR intensity prediction from 3D molecular models of the complexes using CORCEMA-ST,[28] has allowed us to answer the question of how large and flexible ligands like 3 can be accommodated in the GM1 binding pocket of CTB, demonstrating the existence of a hitherto unknown binding subsite adjacent to the GM1 binding pocket, paving the way to the design of novel non-carbohydrate leads for CTB inhibition

Results and Discussion

Conclusion

Conflict of interest