Integrating Enzymatic Self-Assembly and Mitochondria Targeting for Selectively Killing Cancer Cells without Acquired Drug Resistance

Abstract

Targeting organelles by modulating the redox potential of mitochondria is a promising approach to kill cancer cells that minimizes acquired drug resistance. However, it lacks selectivity because mitochondria perform essential functions for (almost) all cells. We show that enzyme-instructed self-assembly (EISA), a bioinspired molecular process, selectively generates the assemblies of redox modulators (e.g., triphenyl phosphinium (TPP)) in the pericellular space of cancer cells for uptake, which allows selectively targeting the mitochondria of cancer cells. The attachment of TPP to a pair of enantiomeric, phosphorylated tetrapeptides produces the precursors (L-1P or D-1P) that form oligomers. Upon dephosphorylation catalyzed by ectophosphatases (e.g., alkaline phosphatase (ALP)) overexpressed on cancer cells (e.g., Saos2), the oligomers self-assemble to form nanoscale assemblies only on the surface of the cancer cells. The cancer cells thus uptake these assemblies of TPP via endocytosis, mainly via a caveolae/raft-dependent pathway. Inside the cells, the assemblies of TPP-peptide conjugates escape from the lysosome, induce dysfunction of mitochondria to release cytochrome c, and result in cell death, while the controls (i.e., omitting TPP motif, inhibiting ALP, or removing phosphate trigger) hardly kill the Saos2 cells. Most importantly, the repeated stimulation of the cancers by the precursors, unexpectedly, sensitizes the cancer cells to the precursors. As the first example of the integration of subcellular targeting with cell targeting, this study validates the spatial control of the assemblies of nonspecific cytotoxic agents by EISA as a promising molecular process for selectively killing cancer cells without inducing acquired drug resistance.

Supramolecular catalysis and dynamic assemblies for medicine

“…EISA is a powerful concept to generate functions. For example, EISA selectively generates supramolecular assemblies of small molecules 13–15 in situ for potential cancer therapy 16, 17 and molecular imaging 18 . Moreover, the combination of enzyme-instructed assembly and disassembly affords a reaction cycle to switch between the monomers and assemblies, 19 as well as an approach to target downregulation (or loss of functions) in cancer cells.…”

“…3B). 17 Designed for both cell and subcellular targeting, precursor 6 consists of an environment-sensitive fluorophore 4-nitro-2,1,3-benzoxadiazole (NBD) for visualizing the assemblies, a self-assembling module (e.g., Phe-Phe-Tyr-Lys (FFYK)), an enzyme substrate (e.g., phosphotyrosine), and a mitochondria targeting motif (e.g., triphenyl phosphinium (TPP)) (Fig. 3A).…”

Enzyme-Instructed Assembly and Disassembly Processes for Targeting Downregulation in Cancer Cells

“…10 We are particularly interested in the use of assemblies of molecules for cancer therapy because a serendipitous discovery 11 of the inverse comorbidity between cancer and neurodegenerative diseases implicates molecular nanofibrils formed by self-assembly in inhibiting cancer cells, either in an animal model 12 or in a human trial. 13 This notion, indeed, is supported by the development of enzyme-instructed self-assembly (EISA), 14 which selectively generates nanoscale assemblies of small molecules (e.g., small peptide derivatives 5,15,16 or carbohydrate derivatives 4 ) in situ on cancer cells to inhibit the cancer cells.…”

Self-assembling prodrugs

“…This method was further improved by conjugating a mitochondria targeting agent to direct self-assembled fibres to the mitochondria, eliminating the potential for acquired drug resistance and further sensitizing cells with increased exposure. 172 The mitochondria targeting agent, triphenyl phosphonium (TPP), was conjugated onto the phosphorylated FFYK tetrapeptide, with an environment-sensitive fluorophore (NBD) for self-assembly tracking taking the place of Nap. Upon contact with the overexpressed alkaline phosphatase (ALP) by cancer cells, dephosphorylation resulted in extracellular self-assembly.…”