A cell-based screening method using an intracellular antibody for discovering small molecules targeting the translocation protein LMO2.

Nicolas Bery,Sabine Milhas,Florence Raynaud,Angela Hayes,Terence H Rabbitts,Hanna Tulmin,Carole J.R Bataille,Sneha Anand,Ami Miller,Angela Russell

doi:10.1126/sciadv.abg1950

Abstract

Intracellular antibodies are tools that can be used directly for target validation by interfering with properties like protein-protein interactions. An alternative use of intracellular antibodies in drug discovery is developing small-molecule surrogates using antibody-derived (Abd) technology. We previously used this strategy with an in vitro competitive surface plasmon resonance method that relied on high-affinity antibody fragments to obtain RAS-binding compounds. We now describe a novel implementation of the Abd method with a cell-based intracellular antibody-guided screening method that we have applied to the chromosomal translocation protein LMO2. We have identified a chemical series of anti-LMO2 Abd compounds that bind at the same LMO2 location as the inhibitory anti-LMO2 intracellular antibody combining site. Intracellular antibodies could therefore be used in cell-based screens to identify chemical surrogates of their binding sites and potentially be applied to any challenging proteins, such as transcription factors that have been considered undruggable.

Highlights

Intracellular antibodies are a class of reagent that bind targets in the cellular environment [1, 2]
LIM domain only protein 2 (LMO2) is the nanomolar range, rather than picomolar as the anti-RAS, and LMO2 can be expressed in Escherichia coli only when in complex with the LID domain of LIM domain binding 1 (LDB1) [24] or with the intracellular domain antibodies (iDAbs) [23], the implementation of the competitive surface plasmon resonance (cSPR) Abd method to
Intracellular antibody fragments interact with proteins at any antigenic site or where natural partner proteins are involved in protein- protein interaction (PPI)

Summary

Introduction

Intracellular antibodies are a class of reagent that bind targets in the cellular environment [1, 2]. The advantages of intracellular antibody–based reagents are that the natural properties of antibodies such as their high affinity and specificity can be exploited Their relatively quick selection processes with methods such as intracellular antibody capture [13] allow their use to investigate their effects on a target disease in relevant preclinical models (target validation) [9, 10, 15]. We used a competitive surface plasmon resonance (cSPR) method to select compounds from a fragment library overlapping the antibody-binding site on HRASG12V [19]. This selection method yielded RAS-binding fragment hits that were developed by structure-guided design to nanomolar interacting compounds that inhibited RAS-effector interactions [19]. New versatile methods that enable the rapid discovery of compounds targeting challenging proteins, such as the product of chromosomal translocations or transcription factors, would be valuable as these have been considered to be extremely difficult drug targets

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