Transcription factors (TFs) play a central role in gene regulation, and their malfunction can result in a plethora of severe diseases. TFs are therefore interesting therapeutic targets, but their involvement in protein–protein interaction networks and the frequent lack of well–defined binding pockets render them challenging targets for classical small molecules. As an alternative, peptide–based scaffolds have proven useful, in particular with an α‐helical active conformation. Peptide–based strategies often require extensive structural optimization efforts, which could benefit from a more detailed understanding of the dynamics in inhibitor/protein interactions. In this study, we investigate how truncated stapled α‐helical peptides interact with the transcription factor Nuclear Factor‐Y (NF−Y). We identified a 13‐mer minimal binding core region, for which two crystal structures with an altered C‐terminal peptide conformation when bound to NF−Y were obtained. Subsequent molecular dynamics simulations confirmed that the C‐terminal part of the stapled peptide is indeed relatively flexible while still showing defined interactions with NF−Y. Our findings highlight the importance of flexibility in the bound state of peptides, which can contribute to overall binding affinity.

Over the last two decades, indoleamine 2,3-dioxygenase 1 (IDO1) has attracted wide interest as a key player in immune regulation, fostering the design and development of small molecule inhibitors to restore immune response in tumor immunity. In this framework, biochemical, structural, and pharmacological studies have unveiled peculiar structural plasticity of IDO1, with different conformations and functional states that are coupled to fine regulation of its catalytic activity and non-enzymic functions. The large plasticity of IDO1 may affect its ligand recognition process, generating bias in structure-based drug design campaigns. In this work, we report a screening campaign of a fragment library of compounds, grounding on the use of three distinct conformations of IDO1 that recapitulate its structural plasticity to some extent. Results are instrumental to discuss tips and pitfalls that, due to the large plasticity of the enzyme, may influence the identification of novel and differentiated chemical scaffolds of IDO1 ligands in structure-based screening campaigns.

PD-1/PD-L1 protein complex is attracting a great deal of interest as a drug target for the design of immune therapies able to block its assembly. Although some biologic drugs have entered clinical use, their poor response rate in patients are demanding further efforts to design small molecule inhibitors of PD-1/PD-L1 complex with higher efficacy and optimal physicochemical properties. Dysregulation of pH in the tumor microenvironment is indeed one of the key mechanisms promoting drug resistance and lack of response in cancer therapy. Integrating computational and biophysical approaches, herein we report a screening campaign that has led to identifying VIS310 as a novel ligand of PD-L1, with physicochemical properties enabling a pH-dependent binding potency. Additional optimization efforts by analogue-based screening have been instrumental to disclosing VIS1201, which exhibits improved binding potency against PD-L1 and is able to inhibit PD-1/PD-L1 complex formation in a ligand binding displacement assay. While providing preliminary structure–activity relationships (SARs) of a novel class of PD-L1 ligands, our results lay the foundation for the discovery of immunoregulatory small molecules resilient to tumor microenvironmental conditions for escaping drug-resistance mechanisms.