Selective Affimers Recognise the BCL‐2 Family Proteins BCL‐x_L and MCL‐1 through Noncanonical Structural Motifs**

Abstract

The BCL-2 family is a challenging group 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 useful 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-x L , BCL-2, BAK and BAX, then used multiple biophysical characterisation methods to probe the interactions. We established that purified Affimers elicit selective recognition of their target BCL-2 protein. For anti-apoptotic targets BCL-x L and MCL-1, competitive inhibition of their canonical protein-protein interactions is demonstrated. Co-crystal 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 proof-of-concept studies indicate that Affimers could be used as alternative templates to inspire the design of selective BCL-2 family modulators and more generally other protein-protein interaction inhibitors.

“Affimer” synthetic protein scaffolds block oxidized LDL binding to the LOX-1 scavenger receptor and inhibit ERK1/2 activation

“…To date, the majority of applications for Affimers have been as probes to support high-resolution imaging ( 51 , 52 ) or as antibody replacements in diagnostics and biosensor development ( 53 , 54 , 55 ). However, they have also proven to be powerful tools in the study of protein-protein interactions ( 56 , 57 ), with the capability to discriminate between very closely related proteins ( 58 ). Affimers have also been identified that are capable of modulating biological activities, either inhibiting viral replication ( 59 ) or SUMO-mediated protein interactions ( 56 ).…”

Mechanism of activation and the rewired network: New drug design concepts

“…Proteins act through structural changes and drugs aim to block their action. A competitive drug binding mechanism is powerful since it directly blocks ligand docking 125,157–166 . A noncompetitive drug binding can be powerful by altering the active site shape, leading to the same outcome 167–177 .…”

“…A competitive drug binding mechanism is powerful since it directly blocks ligand docking. 125,[157][158][159][160][161][162][163][164][165][166] A noncompetitive drug binding can be powerful by altering the active site shape, leading to the same outcome. [167][168][169][170][171][172][173][174][175][176][177] These drug actions typically target the active conformation.…”

Towards optimizing peptide-based inhibitors of protein–protein interactions: predictive saturation variation scanning (PreSaVS)

“…16 However, generally, sequence space is too great too explore using synthetic chemistry. Biological selection methods are powerful and do allow more space to be searched, [17][18][19] however, these can be experimentally demanding and expensive. For all these reasons, development and improvement of computational approaches to examine and design PPIs are important.…”