Peptide-Bismuth Bicycles

Abstract

Bicyclic peptides arise as next-generation therapeutics. We introduce peptide-bismuth bicycles as new class of constrained peptides. Similar, to alkylating agents, currently in use, bismuth(III) can link three thiols in peptides. Unlike conventional methods, peptide-bismuth bicycles form instantaneously at physiological pH, enabling in-situ access to highly constrained peptides, directly in biological assays. Initial screening campaigns targeted proteases of Zika and West Nile virus. Subsequent screening campaigns introduced 8 additional NS2B-NS3 proteases, including three unreported ones. These efforts revealed a lead compound which, in comparison to its linear precursor, is up to 130 times more active and 19 times more proteolytically resistant against its drug target (West Nile virus). The lead compound is stable at physiological pH over several weeks and tolerates glutathione, the predominant intracellular thiol, at 100-fold access over two days at 37 degrees Celsius. Subsequent studies with rhodamine-labelled analogues revealed a potent series of bicyclic peptides that penetrate human cells at concentrations as low as 0.01 uM without significant cytotoxicity at 10 uM. In comparison to their linear precursors, peptide-bismuth bicycles enter mammalian cells 10 times more effectively and outcompete known cell- penetrating peptides such as Tat(49-57). To enable widespread application of this new compound class, we enhanced our purification protocol by introducing a final ion-exchange step as alternative to laborious HPLC purification, enabling simultaneous bench-top purification of multiple bicyclic peptides. Leveraging the unique architecture of peptide-bismuth bicycles, we introduced inductively coupled plasma- mass spectrometry (ICP-MS) as orthogonal quantification method. Unlike conventional methods, such as fluorescence-activated cell sorting (FACS), ICP-MS enables quantification of unlabelled bismuth-bicycles, hence allowing for direct comparison of labelled and unlabelled peptides, to examine a cargo's effect on cellular uptake. Finally, we explored multimodal imaging using X-ray fluorescence microscopy (XFM) and optical fluorescence microscopy to gain a deeper understanding of uptake, subcellular localisation, and potential changes in the endogenous element distribution upon exposure to cell-penetrating peptide-bismuth bicycles. The innovations described herein expand our understanding of a chemical space around an emerging class of pharmaceuticals and may pave the way for the development of novel therapeutics. Show more