Selective Affimers Recognize BCL-2 Family Proteins Through Non-Canonical Structural Motifs

Abstract

no more than 200 words)The BCL-2 family is a challenging set of proteins to target selectively due to sequence and structural homologies across the family. Selective ligands for the BCL-2 family regulators of apoptosis are desirable as probes to understand cell biology and apoptotic signalling pathways, and as starting points for inhibitor design. We have used phage display to isolate Affimer reagents (non-antibody binding proteins based on a conserved scaffold) to identify ligands for MCL-1, BCL-xL, BCL-2, BAK and BAX, then used multiple biophysical characterisation methods to probe the interactions. We established that purified Affimers elicit selective and potent recognition of their target BCL-2 protein. For anti-apoptotic targets, competitive inhibition of their canonical protein-protein interactions is demonstrated. Cocrystal structures reveal an unprecedented mode of molecular recognition; where a BH3 helix is normally bound, flexible loops from the Affimer dock into the BH3 binding cleft. Moreover, the Affimers induce a change in the target proteins towards a desirable drug bound like conformation. These results indicate Affimers can be used as alternative templates to inspire design of selective BCL-2 family modulators, and provide proof-ofconcept for the elaboration of selective non-antibody binding reagents for use in cell-biology applications. [198 words]

Predicting and Experimentally Validating Hot-Spot Residues at Protein–Protein Interfaces

“…Having experimentally validated the approach with three model systems, we applied the workflow to a novel protein−protein interface with a different topography. For this, we selected a recently described PPI between an Affimer and BCL-x L , 70 which, like MCL-1, is an antiapoptotic member of the BCL-2 family of apoptotic regulators (Figure 7a). 71 In this structure, two nine-residue Predictions for each loop were performed using each of the tools, and the average ΔΔG was determined (Figure 7b).…”

Predicting and Experimentally Validating Hot-Spot Residues at Protein-Protein Interfaces

“…Having experimentally validated the approach with three model systems, we applied the workflow to a novel protein−protein interface with a different topography. For this, we selected a recently described PPI between an Affimer and BCL-x L , 70 which, like MCL-1, is an antiapoptotic member of the BCL-2 family of apoptotic regulators (Figure 7a). 71 In this structure, two nine-residue…”

RAS-inhibiting biologics identify and probe druggable pockets including an SII-α3 allosteric site

“…As some biologics form smaller interfaces 19,23,24 there emerges the tantalising prospect that biologics could be used as tools to identify druggable pockets and novel conformers, and could also have the potential to act as pharmacophore templates for in silico-informed drug discovery. Here, we explore the possibility of using Affimer proteins, an established biologic with a small probe surface formed by two variable regions 24 known to bind at protein interaction 'hotspots' 23,25,26 , to identify and probe RAS for druggable pockets and conformers that might be amenable to small molecule inhibition. Such a direct approach utilising small probe surfaces has not previously been used with biologics and could revolutionise drug discovery, exemplifying a novel pipeline for small molecule design that has the potential to unlock the vast number of currently 'undruggable' proteins.…”