The Development of a Fluorescence‐Based Competitive Assay Enabled the Discovery of Dimeric Cyclic Peptide Modulators of Ubiquitin Chains

Abstract

Development of modulators targeting specific interactions of ubiquitin‐based conjugates with their partners is a formidable task since it requires a suitable screening assay and homogeneous ubiquitin conjugates. We developed a novel high‐throughput strategy for screening ligands for Lys48‐linked tetraubiquitin chain in a relatively simple, fast, and affordable manner. This approach combined with a state‐of‐the‐art toolbox of chemical protein synthesis and a specially optimized Cys deprotection protocol enabled us to design highly potent, Lys48‐linked tetraubiquitin chain selective “next generation” dimeric peptide modulators. The dimeric peptide exhibited cancer cell permeability and induced cell death with higher efficiency compared to its monocyclic analogue. These features make our dimeric peptide a promising candidate for further studies using in vivo models. Our assay can be adopted for other various ubiquitin chains in their free or anchored forms as well as conjugates for Ub‐like modifiers.

Highly Efficient Cyclization Approach of Propargylated Peptides via Gold(I)-Mediated Sequential C–N, C–O, and C–C Bond Formation

“…Recently, our group discovered cyclic peptides which are capable of binding specifically Lys48-linked Ub chains and therefore modulating in vitro and in vivo their activities. − Ub chains were used as targets for the RaPID system (Random Nonstandard Peptide Integrated Discovery) to select for high-binding cyclic peptides. Despite our successful design and screening, the cyclization chemistry is largely limited to the thioether linkage. , Preparing new analogues of the cyclic peptide using different cyclization methods could further influence their cell permeability, activity, and pharmacological properties.…”

“…With both peptides in hand ( 5 and 6 ), we examined the binding efficiency using our florescence-based competitive assay . We observed a 32% increase in the binding affinity for Lys48-linked tetra-Ub chains and nearly 42% for Lys48-linked di-Ub chains compared to the cyclic peptide having the thioether linkages [ClAc ( 7 ) and m -ClBz ( 8 )].…”

Combining Chemical Protein Synthesis and Random Nonstandard Peptides Integrated Discovery for Modulating Biological Processes

“…In addition, the binding affinity of the dimeric form of Ub4ix cyclic peptide to the Lys48-linked tetra-Ub and their cellular activity are interesting. 44 The dimer of Ub4ix with a cleavable disulfide bond, (Ub4ix_S7C) 2 , and with a stable thioetherbased linker, (Ub4ix_S7C) 2 CH 2 (Figure 7F), was found to bind tightly with the Lys48-linked tetra-Ub in vitro. Notably, these dimeric peptides are cell-permeable, particularly (Ub4ix_S7C) 2 CH 2 , which induces cell death in Hela cells and exhibits greater early-and late-stage apoptosis in comparison to pG1 Ub4ix.…”

“…In addition, the binding affinity of the dimeric form of Ub4ix cyclic peptide to the Lys48-linked tetra-Ub and their cellular activity are interesting . The dimer of Ub4ix with a cleavable disulfide bond, (Ub4ix_S7C) 2 , and with a stable thioether-based linker, (Ub4ix_S7C) 2 CH 2 (Figure F), was found to bind tightly with the Lys48-linked tetra-Ub in vitro .…”

“…In this regard, we have been targeting various Ub chain linkages for the modulation of related biological processes by utilizing the combination of chemical protein synthesis and RaPID. − , By utilizing the toolbox of chemical protein synthesis, Ub chains of different lengths and linkages with biotin labels can be prepared with high homogeneity and workable quantities. In the following section, we highlight our recent studies in which we have employed the target Ub chain in the RaPID system to select the most effective cyclic peptide ligand(s) that bind with high affinity and specificity and show their biological activities.…”