De novo design of miniproteins binding to PD-L1

De novo design of miniproteins, ideal drug candidates that cover the space between small molecule drugs and antibodies, has emerged in the past 40 years as an excellent tool for the development, with atomic accuracy, of bioactive molecules.However, a significant challenge remains due to the absence of tools capable of efficiently design sequences incorporating non-canonical amino acids.In the present work, we intend to de novo design a miniprotein with a EHEE topology (Eextended; H -Helix) targeting programmed cell death ligand 1 protein (PD-L1).The design approach allows the incorporation of three -amino acids in an pattern, ensuring that the structural integrity and binding capabilities of the design remain unaffected.(1S,2S) Aminocyclopentanecarboxylic acid (trans-ACPC) Entry T m (C) K D (nM) BABB_1 73.7 1.1* 961 11.6 BABB_T1 64.7 0.4* 192 2.2 Objectives Materials and methodsMiniprotein design was performed combining Rosetta blue-print design with fast design protocol.Designs were filtered using the Rosetta scores, AlphaFold2, Rosetta ab initio calculations and molecular dynamics (MD).Designs were synthesized by Solid Phase Peptide Synthesis, purified by High Performance Liquid Chromatography (HPLC), and characterized with mass spectrometry, analytical HPLC and Circular Dichroism (CD).The affinity studies towards PD-L1 are done with BioLayer Interferometry (BLI). ConclusionsHere, we successfully designed a novel PD-L1 miniprotein binder with a EHEE topology.Moreover, the design approach allowed to incorporate three -amino acids in the helical region while preserving the structural integrity and improving the binding capacity for the target by 5-fold.