Enzymatic Assemblies of Thiophosphopeptides Instantly Target Golgi Apparatus and Selectively Kill Cancer Cells**

Abstract

Changing an oxygen atom of the phosphoester bond in phosphopeptides by a sulfur atom enables instantly targeting Golgi apparatus (GA) and selectively killing cancer cells by enzymatic self-assembly. Specifically, conjugating cysteamine S-phosphate to the C-terminal of a self-assembling peptide generates a thiophosphopeptide. Being a substrate of alkaline phosphatase (ALP), the thiophosphopeptide undergoes rapid ALP-catalyzed dephosphorylation to form a thiopeptide that self-assembles. The thiophosphopeptide enters cells via caveolin-mediated endocytosis and macropinocytosis and instantly accumulates in GA because of dephosphorylation and formation of disulfide bonds in Golgi by themselves and with Golgi proteins. Moreover, the thiophosphopeptide potently and selectively inhibits cancer cells (HeLa) with the IC 50 (about 3 mM), which is an order of magnitude more potent than that of the parent phosphopeptide.

Enzyme‐Instructed Self‐Assembly of Peptides: From Concept to Representative Applications

“…Although the furin‐triggered localized condensation was previously developed and enables the visualization of GA in a living cell, [68] GA‐targeting EISA for cancer inhibition was still limited. However, it is recently demonstrated that enzymatic assemblies of thiophosphopeptides ( 26 ) instantly accumulate in GA, allowing the selective HeLa inhibition (Figure 6c) [69] . The previous reports already revealed that cysteine‐rich proteins are enriched in GA [70] and a high level of oxidation occurs in GA membrane [71] .…”

“…However, it is recently demonstrated that enzymatic assemblies of thiophosphopeptides (26) in- stantly accumulate in GA, allowing the selective HeLa inhibition (Figure 6c). [69] The previous reports already revealed that cysteine-rich proteins are enriched in GA [70] and a high level of oxidation occurs in GA membrane. [71] Based on these facts, the mechanism for GA targeting was proposed that (i) the 26, uptaken by a HeLa cell, is converted to 27 by the rapid dephosphorylation in GA, (ii) the 27, containing the thiol group, takes part in the self-assembly, (iii) the thiol group of the assemblies forms the disulfide bonds with the cysteine-rich proteins in GA, leading to the accumulation of the cytotoxic assemblies in GA. As the result, the IC 50 value is drastically reduced to about 3 μM.…”

Self-assembling peptides-based nano-cargos for targeted chemotherapy and immunotherapy of tumors: recent developments, challenges, and future perspectives

“…To reactivate the suppressed immune response, immune cell epitopes and/or immune checkpoint inhibitors were conjugated with SAPs (Black et al., 2012 ; Li et al., 2021 ). Cytotoxic peptides conjugate SAPs (Standley et al., 2010 ; Toft et al., 2012 ), and self-assemblies of peptides induced by enzyme or pH in targeted organelle are lethal to cancer cells (Feng et al., 2018 ; Tan et al., 2021 ). To enhance the potential of photodynamic therapy (PDT), SAPs were conjugated with targeting peptide epitopes and photosensitizers (PSs) (Li et al., 2018c ; Tian et al., 2021 ).…”

Synthesis and bioactivity of pyrrole-conjugated phosphopeptides

“…Based on this structure, a considerable number of studies have investigated the physical properties (enzyme-catalyzed hydrogelation or supramolecular structural transformation) [62,70] and biological properties (cell viability, biostability, and cell-selective growth inhibition) [43,62] of the naphthylacetyl N-terminal-capped phosphopeptides. These studies have produced a variety of phosphopeptides that selectively inhibit cancer cells [38,64,65,71,72]. Encouraged by the results from the naphthylacetyl capped phosphopeptides, we decided to use multiple N-methylpyrroles, as the heteroaromatic analog of naphthyl, to cap the N-terminal of phosphopeptides for EISA.…”

“…One of the most explored EISA processes is the use of alkaline phosphatase (ALP) to convert the micelles made of phosphopeptides to the nanofibers of peptides via enzymatic dephosphorylation [20,60]. While ALP-catalyzed EISA has received considerable exploration, the structures of the peptide substrates mainly have centered on naphthylacetyl-capped phosphopeptides (e.g., Nap-ff p y (1)) [36,[61][62][63][64][65][66]. Considering naphthyl is an aromatic group, we decided to explore other aromatic N-terminal capping groups, such as heteroaromatic, N-terminal capping groups of phosphopeptides for EISA because they receive little exploration [67].…”