Tetrel‐Centered Exchange Cascades to Decouple Inhibition and Induction of Thiol‐Mediated Uptake: Introducing Cell‐Penetrating Thiolactones, Focus on Reversible Michael Acceptor Dimers

Abstract

This study adds thiolactones to the collection of cascade exchangers (CAXs) and reports with dynamic covalent Michael acceptor dimers the first CAXs that rather inhibit than enable thiol‐mediated uptake. The superior performance of dimers compared to monomeric Michael acceptors as inhibitors might suggest that top inhibitors move along thiol/ate arrays without losing covalent contact, while cell penetrators exchange along disulfide arrays. This decoupling of inhibition and induction of thiol‐mediated uptake is important because the latter is promising for drug delivery, while the former possibly helps to reveal targets associated with antiviral, antithrombotic and antitumor activity, leading to drug discovery. These results are consistent with the working model of thiol‐mediated uptake as a general complex network in charge of bringing matter into cells, and thus support its validity.

Inhibition of Cell Motility by Cell-Penetrating Dynamic Covalent Cascade Exchangers: Integrins Participate in Thiol-Mediated Uptake

“…(B) Similar IC 50 comparison for MDA-MB-231 cell motility on fibronectin (symbols as in Figure J). Uptake data from refs , , − . (C–F) Fluorescence microscopy images of (C, D) wild-type and (E, F) INT β 1 siRNA knockdown HK cells incubated with (C, E) immunofluorescence integrin probe (red) and (D, F) 17 (green) (yellow, blue: Hoechst 33342, nuclei; scale bars, 30 μm).…”

“…This decrease matched the formal wound healing times to close a 1 mm scratch (17 h against 40 h). Because it was important to avoid misinterpretation of cell death as motility inhibition, CAXs and controls 1 – 12 were reconfirmed ,− as nontoxic under the experimental conditions (Figure S3).…”

“…TMU has been realized with many classes of CAX for the cytosolic delivery of small molecules, , antibodies and other proteins, − genome editing machinery and other oligonucleotides, − polymers, liposomes, and nanoparticles ,, into various cellular targets including deep tissue, , living animals, , plant cells, and bacteria . Proteomics data, heatmap patterns, , and literature on oligonucleotide phosphorothioate , and viral uptake ,− all support that multipartner exchange networks are involved in how TMU brings matter into cells . However, the dynamic covalent exchange cascades of TMU are complex and its fleeting intermediates are elusive, which is likely the reason why TMU is not better known and understood …”

“…To test the effects of TMU inhibitors on cellular integrin functions, compounds 1 – 16 were bought or synthesized following reported procedures (Figures and S2). ,,− Cell motility assays were adapted to automated high-content high-throughput (AHCHT) imaging methods. , Before cell seeding, 96-well plates were coated with one of three proteins that interact with different integrins: Collagen I (C), which activates mainly β 1 -containing integrins; fibronectin (F), which activates mainly β 1 - and β 3 -containing integrins; and vitronectin (V), which activates mainly β 3 - and β 5 -containing integrins. − HeLa Kyoto (HK), MCF-7, and MDA-MB-231 cells were then seeded on the protein-coated wells to test their migration behavior when treated with TMU inhibitors. Among many possibilities, highly aggressive MDA-MB-231 and noninvasive MCF-7 breast cancer cells were selected as established standards to explore motility and HK cells as a link to uptake and to probe the power of AHCHT assays to detect small changes accurately.…”

“…To test the effects of TMU inhibitors on cellular integrin functions, compounds 1 – 16 were bought or synthesized following reported procedures ( Figures 2 and S2 ). 44 , 45 , 51 − 55 Cell motility assays 4 were adapted to automated high-content high-throughput (AHCHT) imaging methods. 45 , 51 Before cell seeding, 96-well plates were coated with one of three proteins that interact with different integrins: Collagen I (C), which activates mainly β 1 -containing integrins; fibronectin (F), which activates mainly β 1 - and β 3 -containing integrins; and vitronectin (V), which activates mainly β 3 - and β 5 -containing integrins.…”