Xiyun Ye

Human papillomavirus (HPV) infections account for nearly all cervical cancer cases, which is 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 interface between HPV E6 and E6AP is a promising modality to mitigate HPV-mediated degradation of p53. In this study, we designed a covalent peptide inhibitor, termed reactide, that mimics the E6AP LXXLL binding motif by selectively targeting cysteine 58 in HPV16 E6 with quantitative conversion. This reactide provides a starting point in the development of covalent peptidomimetic inhibitors for intervention against HPV-driven cancers.

α‐Klotho, an aging‐related protein found in the kidney, parathyroid gland, and choroid plexus, acts as an essential co‐receptor with the fibroblast growth factor 23 receptor complex to regulate serum phosphate and vitamin D levels. Decreased levels of α‐Klotho are a hallmark of age‐associated diseases. Detecting or labeling α‐Klotho in biological milieu has long been a challenge, however, hampering the understanding of its role. Here, we developed branched peptides by single‐shot parallel automated fast‐flow synthesis that recognize α‐Klotho with improved affinity relative to their monomeric versions. These peptides were further shown to selectively label Klotho for live imaging in kidney cells. Our results demonstrate that automated flow technology enables rapid synthesis of complex peptide architectures, showing promise for future detection of α‐Klotho in physiological settings.

α‐Klotho, an aging‐related protein found in the kidney, parathyroid gland, and choroid plexus, acts as an essential co‐receptor with the fibroblast growth factor 23 receptor complex to regulate serum phosphate and vitamin D levels. Decreased levels of α‐Klotho are a hallmark of age‐associated diseases. Detecting or labeling α‐Klotho in biological milieu has long been a challenge, however, hampering the understanding of its role. Here, we developed branched peptides by single‐shot parallel automated fast‐flow synthesis that recognize α‐Klotho with improved affinity relative to their monomeric versions. These peptides were further shown to selectively label Klotho for live imaging in kidney cells. Our results demonstrate that automated flow technology enables rapid synthesis of complex peptide architectures, showing promise for future detection of α‐Klotho in physiological settings.

Establishing structure–activity relationships is crucial to understand and optimize the activity of peptide-based inhibitors of protein–protein interactions. Single alanine substitutions provide limited information on the residues that tolerate simultaneous modifications with retention of biological activity. To guide optimization of peptide binders, we use combinatorial peptide libraries of over 4,000 variants—in which each position is varied with either the wild-type residue or alanine—with a label-free affinity selection platform to study protein–ligand interactions. Applying this platform to a peptide binder to the oncogenic protein MDM2, several multi-alanine-substituted analogs with picomolar binding affinity were discovered. We reveal a non-additive substitution pattern in the selected sequences. The alanine substitution tolerances for peptide ligands of the 12ca5 antibody and 14-3-3 regulatory protein are also characterized, demonstrating the general applicability of this new platform. We envision that binary combinatorial alanine scanning will be a powerful tool for investigating structure–activity relationships.