Determination of intracellular protein-ligand binding affinity by competition binding in-cell NMR.

Enrico Luchinat,Claudiu T Supuran,Alessio Nocentini,Matteo Pennestri,Lucia Banci,Letizia Barbieri,Matteo Cremonini

doi:10.1107/s2059798321009037

Abstract

Structure-based drug development suffers from high attrition rates due to the poor activity of lead compounds in cellular and animal models caused by low cell penetrance, off-target binding or changes in the conformation of the target protein in the cellular environment. The latter two effects cause a change in the apparent binding affinity of the compound, which is indirectly assessed by cellular activity assays. To date, direct measurement of the intracellular binding affinity remains a challenging task. In this work, in-cell NMR spectroscopy was applied to measure intracellular dissociation constants in the nanomolar range by means of protein-observed competition binding experiments. Competition binding curves relative to a reference compound could be retrieved either from a series of independent cell samples or from a single real-time NMR bioreactor run. The method was validated using a set of sulfonamide-based inhibitors of human carbonic anhydrase II with known activity in the subnanomolar to submicromolar range. The intracellular affinities were similar to those obtained in vitro, indicating that these compounds selectively bind to the intracellular target. In principle, the approach can be applied to any soluble intracellular target that gives rise to measurable chemical shift changes upon ligand binding.

Highlights

Structure-based drug-design approaches rely on knowledge of the three-dimensional structure of the target protein to develop effective drugs
We provide two alternative approaches to obtain intracellular competition binding curves: (i) by conventional ‘closed-tube’ in-cell Nuclear magnetic resonance (NMR), in which several independent cell samples, each of which is treated with two competing ligands at different doses, are analyzed separately for a short acquisition time to preserve cell viability, and (ii) by time-resolved in-cell NMR through the use of an NMR bioreactor (Kubo et al, 2013; Breindel et al, 2018; Luchinat, Barbieri, Campbell et al, 2020), in which a single sample of cells is kept viable and metabolically active for a prolonged period of time, during which a test ligand is added at increasing concentrations in a stepwise manner together with a reference compound kept at a constant concentration
Carbonic anhydrases (CAs) II can be overexpressed in human cells at NMRdetectable levels and is free from interactions with slowtumbling cellular components, and it gives rise to well resolved signals in in-cell NMR spectra (Luchinat, Barbieri, Cremonini et al, 2020a)

Summary

Introduction

Structure-based drug-design approaches rely on knowledge of the three-dimensional structure of the target protein to develop effective drugs. In the preclinical studies that follow, the best-performing candidates in vitro are screened for in-cell or in vivo activity by cell-based assays in vitro and/or in animal models At this stage of drug development, the efficacy of the compounds is evaluated from enzymatic assays on cell cultures, or more indirectly from other cellular responses, such as cell death, proliferation, invasiveness or metabolic activity (Hughes et al., 2011; Kepp et al, 2011). In these trials, there is often no direct readout of the protein–ligand interaction, and of the binding affinity, in the cellular environment.

Methods

Results

Conclusion