Abstract by Flora Alexopoulou

In the early stage of peptide drug discovery projects, the identification of peptide hits with therapeutic potential is of crucial importance. Targeting protein-protein interactions (PPIs) often appears particularly challenging due to complex binding interfaces between protein partners that could hamper their druggability using peptides. Advancements in chemical peptide synthesis as well as the emergence of display technologies utilizing the biological translation machinery has enabled the generation of vast and structurally diverse peptide libraries. Screening of such libraries against biological relevant targets has accelerated the identification of peptide hits as well as their subsequent development as therapeutic agents. Here, we applied both chemically synthesized and genetically encoded peptide libraries for the discovery of novel peptide modulators that target PPIs with diverse therapeutic relevance.

We first investigated the development of peptide inhibitors targeting the glial cell line-derived neurotrophic factor (GDNF) family receptor α like (GFRAL) through the synthesis of chemical peptide libraries based on the sequence of the endogenous GFRAL ligand, namely the growth differentiation factor 15 (GDF15). Screening of SPOT arrays displaying GDF15 peptide fragments indicated binding of the C-terminal region of GDF15 to the extracellular domain (ECD) of GFRAL. The same GDF15 region was found to inhibit GFRAL signaling through functional screening of a complementary GFD15 library of longer peptides synthesized using parallel solid phase peptide synthesis (SPPS). This approach resulted in the identification of GDF15 peptide fragments with inhibitory potency in the micromolar range, In an attempt to obtain more potent peptide antagonists, screening of larger peptide libraries through phage display was investigated. Peptides binding to GFRAL ECD were identified through screening of three phage libraries and binding properties of phage peptides were validated using SPOT arrays. Subsequent functional evaluation validated GFRAL inhibition also in the micromolar range.

Next, screening of a trillion unique cyclic peptides that were synthesized using the Random non standard Peptides Integrated Discovery (RaPID) technology led to the discovery of a novel high-affinity peptide binder to the PDZ2 domain of the postsynaptic density protein-95 (PSD 95). Last, the potential for further optimization of peptide binding affinity was demonstrated by performing a RaPID deep mutational scan and identifying peptide mutants with enhanced PDZ2 binding.

In summary, we demonstrate the application of advanced peptide technologies in the peptide drug discovery process. Further, we report the discovery of novel peptide modulators of PPIs that could serve as templates for future development of peptide drug candidates targeting the associated to the studied PPIs biological pathways.