| Summary: | The use of peptides as inhibitors for protein-protein interactions (PPI) is an attractive strategy for developing therapeutics. Understanding the structure-activity relationships of peptide-based inhibitors is crucial for optimizing their activity. To guide this optimization, combinatorial peptide libraries with label- free affinity selection platforms have been developed to study protein-ligand interactions. Applying this platform, several multi-alanine-substituted analogs with picomolar binding affinity were discovered for the oncogenic protein MDM2. In addition, tolerance to alanine substitutions for peptide ligands of the 12ca5 antibody and 14-3-3 regulatory protein were characterized.
With the understanding of residue contributions to PPI, we have developed affinity maturation strategies to improve binding affinity from micromolar to low nanomolar. The kidney, parathyroid gland, and choroid plexus express the aging-related transmembrane protein α-Klotho, co-receptor in the fibroblast growth factor 23 receptor complex. Reduced α-Klotho levels indicate chronic kidney disease and other age-related diseases. The long-standing difficulty in detecting or labeling α-Klotho prevented us to understand its biological functions. Here we describe branched multimeric peptides that recognize α-Klotho with high affinity and selectivity in the biological milieu. The branched peptides are prepared in a single-shot synthesis by parallel automated fast-flow synthesis in under one hour. The branched α-Klotho-binding peptides show improvement in affinity relative to the monomeric versions and can be used to label Klotho for live imaging in kidney cells.
In addition, we described the discovery of peptides that recognize α-Klotho with high affinity and selectivity by applying in-solution size exclusion-based affinity selection-mass spectrometry (AS-MS). N-terminal small molecule modifications on these peptides leads to great binding improvement. After two rounds of AS-MS, the affinity-matured peptides have at least 2300-fold increased binding affinity to α-Klotho compared to the initially reported peptide Pep-10. The lead peptide binders were utilized to enrich Klotho from cell lysates and to label Klotho in live kidney cells. Our results further support the utility of in-solution, label-free AS-MS protocols to deliver peptide-based binders to target proteins of interest with high affinity and selectivity, resulting in functional probes for biological studies.
Human papillomavirus (HPV) infections account for nearly all cervical cancer cases, the fourth most common cancer in women worldwide. High-risk variants, including HPV16, drive tumorigenesis in part by promoting the degradation of the tumor suppressor p53. This degradation is mediated by the HPV early protein 6 (E6), which recruits the E3 ubiquitin ligase E6AP and redirects its activity towards ubiquitinating p53. Targeting the protein interaction interfaces between HPV E6 and E6AP is a promising modality to mitigate HPV-mediated degradation of p53. Herein, we designed a covalent peptide inhibitor, termed ‘reactide’, that mimics the E6AP LXXLL peptide by targeting cysteine residue 58 in HPV16 E6 with quantitative conversion and selectivity. This reactide provides a starting point for chemical intervention for HPV-driven cancers.
Covalent molecules have found widespread applications as activity-based probes and as irreversibly inhibitory drugs. Currently, there is no rapid, label-free, and highly diverse affinity selection method to enrich reactive peptides from unnaturally chemical space. Herein, we describe a AS-MS method to identify peptide inhibitors with reversible warhead. The method uses mix disulfides to build reversible peptide- protein conjugates that can ambiguously enrich crosslinked molecules for MS/MS sequencing. Using this approach, we optimized peptide that irreversibly inhibited a viral oncoprotein HPV 16E6 with nanomolar potency and specificity. This approach should enable rapid, enrichment to identify new classes of highly selective covalent inhibitors for diverse molecular targets.
Lastly, a systematic study on the tolerance of non-canonical amino acids of characteristic chirality, steric effects, and backbone structures has been presented. Overall, these studies highlight the potential of peptide-based inhibitors and affinity selection platforms in drug discovery
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