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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.

SUBMITTER: Vamisetti GB

PROVIDER: S-EPMC8048552 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Inhalation of brevetoxins aerosolized by wind and wave action can lead to asthma-like symptoms in beach goers. Consumption of either shellfish or finfish exposed to K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to activation of voltage-sensitive sodium channels (VSSCs) in cell membranes. Binding of brevetoxin analogs and competitors to site 5 on these channels has historically been measured using a radioligand competition assay that is fraught with difficulty, including slow analysis time, production of radioactive waste, and cumbersome and expensive methods associated with the generation of radioactive labeled ligands. In this study, we describe the development of a novel fluorescent synaptosome binding assay for the brevetoxin receptor. BODIPY(®)-conjugated to PbTx-2 was used as the labeled ligand. The BODIPY(®)-PbTx-2 conjugate was found to displace [(3)H]-PbTx-3 from its binding site on VSSCs on rat brain synaptosomes with an equilibrium inhibition constant of 0.11 nM. We have shown that brevetoxin A and B analogs are all able to compete for binding with the fluorescent ligand. Most importantly, this assay was validated against the current site 5 receptor binding assay standard, the radioligand receptor assay for the brevetoxin receptor using [(3)H]-PbTx-3 as the labeled ligand. The fluorescence based assay yielded equilibrium inhibition constants comparable to the radioligand assay for all brevetoxin analogs. The fluorescence based assay was quicker, far less expensive, and did not generate radioactive waste or need radioactive facilities. As such, this fluorescence-based assay can be used to replace the current radioligand assay for site 5 on voltage-sensitive sodium channels and will be a vital tool for future experiments examining the binding affinity of various ligands for site 5 on sodium channels.

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

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