Friday, April 4, 2025
4/4/2025
Covalent PD1 Antagonists: Discovery of Protein-Protein Interaction (PPI) Inhibitors in Cancer Immunotherapy - 7. Project summary. Protein–protein interactions (PPIs) play a pivotal role in regulating a plethora of biological processes and their misregulations have been associated with a variety of diseases. Modern proteomic tools enabled over 600,000 PPIs to be identified as potential targets, which can be validated at an incredible pace by programmable gene-editing point-mutations in mammalian cells. Given these technological advances, the modulation of intra- and extracellular PPIs has emerged as one of the most exciting strategies towards next- generation therapeutics. Yet, the development of PPIs inhibitors is lagging far behind the other successful drug modalities (antibodies and small molecules) due to the challenges associated with the large, shallow, often dynamic, and water-exposed binding interfaces. Therefore, we propose to study novel PPI inhibitors modalities via -hairpin peptides featuring large binding-surface areas inspired by the long non-canonical loops forming antibody paratopes. Our goals are to preserve the high-affinity and binding specificity of antibodies by mimicking the binding-competent conformation of CDR-H3 loops sequence to disrupt PPIs. This research proposes to exploit -hairpin scaffolds synthesized in our lab to recreate antibody loops of conceptually any sequence, length, and conformational plasticity (aim 1). As a proof-of-concept (PoC), a structure-guided approach from X-ray crystal structures of protein–antibody drug complexes will be followed to rationally design PPI inhibitors of the programmed cell death 1 (PD1) immunoreceptor and its ligand 1 (PDL1). Indeed, the PD1/PDL1 interaction inhibits T-cell effector function in a process known as tumor immune evasion, and its blockade has proven to be an effective strategy to restore T-cell proliferation in different cancer-types. To validate our PoC, covalent inhibitors will be designed by crafting electrophilic warheads at specific loop positions of the most potent -hairpin binders (aim 2). Overall, our strategy combines the synthesis of -hairpins bearing strained loops (N > 12- residues) that closely mimic the large binding contacts of antibodies CDR-H3 loops to create high-affinity inhibitors with the positioning of electrophilic warheads for covalent binding at the PPI interface. A series of novel electrophilic warheads will be evaluated to target a distinctive lysine residue (K131) located at the center of PD1 binding epitope. A structure–activity optimization will also be achieved by alanine-scanning to validate the most promising cell-penetrating hairpin peptides in living cells (PBMCs). This strategy of CDR-H3 mimicry into covalent -hairpins could potentially be a game-changer by revisiting peptide drugs that are not able to bind tightly and selectively to extra- and/or intracellular proteins and often abandoned during preclinical studies due to a lack of in vivo activity. As a result, a large number of protein targets associated with various diseases could become druggable and offer new possibilities in the space of PPI inhibitors for drug discovery.
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