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 Lys48linked tetraubiquitin chain in a relatively simple, fast, and affordable manner. This approach combined with a state-ofthe-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.

Gold(I)-Mediated Rapid Cyclization of Propargylated Peptides via Imine Formation

“…With both peptides in hand ( 5 and 6 , Figure A), we investigated the binding efficiency using our fluorescence-based competitive assay . We observed a ∼22% increase in the binding affinity for Lys48-linked di-Ub chains for the imine-cyclized product ( 5 ) and ∼15% for its reduced cyclic product ( 6 ), compared to cyclic peptide having the thioether linkage ( 3 ) (Figure B), and the K d was determined to be 5.30 ± 1.4 nM (Figure C).…”

Selective macrocyclic peptide modulators of Lys63-linked ubiquitin chains disrupt DNA damage repair

“…2a and Supplementary Fig. 3 ) 29 . Therefore, we prepared fluoresceine-tagged cyclic peptide 1 (CP1-FITC) (Supplementary methods) and determined a K D value of 95.8 ± 2.3 nM (Fig.…”

“…With cyclic peptides 2–13 in hand, we then examined their affinity for the Lys63-linked Di-Ub using our fluorescence-based assay 29 . The relative binding of each cyclic peptide was normalized to cyclic peptide 1, as a reference, where the binding affinity of each cyclic peptide is inversely proportional to the FITC fluorescence of CP1-FITC.…”

“…37 ). Subsequently, we examined the rate of apoptosis in HeLa cells treated with cyclic peptide 2, using Annexin V–FITC/PI double staining kit (Methods section) 29 . Our results show that treating HeLa cells with 1 μM of cyclic peptide 2 for 96 h led to an increase of overall Annexin V positive cells (~11.7%) (Fig.…”

“…Sample preparation: Apoptotic cell death was estimated by using the standard MEBCYTO® Apoptosis Kit (MBL) protocol 29 . In brief, HeLa cells, seeded were treated with samples (1 μM of peptide 2 or DMSO) for 96 h at 37 °C with 5% CO 2 .…”