Development of Nitroso-Based Probes for Labeling and Regulation of RAS Proteins in Cancer Cells via Sequential Ene-Ligation and Oxime Condensation

Abstract

Prenyl functionalities have been widely discovered in natural products, nucleic acids, and proteins with significant biological roles in both healthy and diseased cells. In this work, we develop a series of new nitroso-based probes for the labeling, enrichment, and regulation of prenylated RAS protein, which is highly associated with ∼20% of human cancers and used to be regarded as an “undruggable” target via a sequential ene-ligation and oxime condensation (SELOC) process. We found that these nitroso species can rapidly react with prenyl-containing molecules through ene-ligation and install a molecular tag for functional applications under physiological conditions. We first investigated this ligation process on two peptide models and demonstrated its labeling efficiency on various proteins such as myoglobin, lysozyme, RNase A, BSA, and HSP40. We further coupled this reactive platform with proteolysis-targeting chimera technology targeting to increase its efficiency and accuracy, as well as to expand its application range. Using the prenylated RAS protein as the model, we demonstrated that RAS could be efficiently decorated with our nitroso probes, which further condensate with oxime and rapidly react with a pomalidomide-containing hydroxylamine probe for protein degradation. As a result, the RAS protein in both HeLa and A549 cell lines has been determined to be efficiently degraded both in vitro and in vivo. This is the first case targeting post-translational modification other than ligand–protein interaction to degrade and regulate RAS proteins. We envision that our SELOC strategy will have great potential in studying the fundamental structures and functions of prenylated biomolecules and developing new drugs based on these unique cellular pathways.

Sequencing, Physiological Regulation, and Representative Disease Research Progress of RNA m6A Modification

“…The impact of these modifications on viral characteristics remains elusive, as well as the types, molecular mechanisms, and targets of these modifications. 136 At present, the role played by these new chemical modifications (NAD, prenyl, and oQ modifications) remains unknown, [137][138][139][140][141][142][143][144][145] although it has been speculated that they may help the virus avoid host attacks (for example m 6 A, m 1 A, and Ψ). Therefore, elucidating the types and characteristics of these chemical modifications is expected to provide new clues for the fight against COVID-19 and many other RNA-relevant viruses.…”

Sequential Azidation/Azolation of Prenylated Derivatives and a Click Reaction Enable Selective Labeling and Degradation of RAS Protein

“…To increase the prenyl’s tool visibility, we aim to develop more practical and versatile methods based on the findings reported previously. ,, We noticed that manipulations of the prenyl group in organic solvents in various conditions have been disclosed, although its usage in living cells remains elusive. Thus, we report here our trials to unlock the selective functionalization of the prenyl group on RAS, and subsequently, by employing the well-designed probes via the click reaction, we realize the labeling, chemical pulldown, enrichment, and degradation of prenylated RAS in an efficient and selective approach.…”

“…Our group has been interested in revealing prenyl’s biological roles in living systems; a series of chemical tools targeting prenyl functionalities on proteins and nucleic acids have been explored and utilized. ,,− To develop more reliable and readily available tools, we report here the chemically selective and efficient approach targeting prenyl functionality. Interestingly, proteolysis-targeting chimera (PROTAC) techniques have also been comprehensively explored for precise degradation of targeted proteins in the past decade, although specific ligand identifications are needed. − The cereblon (CRBN) and von Hippel-Lindau (VHL) proteins are substrate recognition subunits of two ubiquitously expressed and biologically important Cullin RING E3 ubiquitin ligase complexes.…”