Dynamic Covalent Michael Acceptors to Penetrate Cells: Thiol‐Mediated Uptake with Tetrel‐Centered Exchange Cascades, Assisted by Halogen‐Bonding Switches

Abstract

Chalcogen‐centered cascade exchange chemistry is increasingly understood to account for thiol‐mediated uptake, that is, the ability of reversibly thiol‐reactive agents to penetrate cells. Here, reversible Michael acceptors are shown to enable and inhibit thiol‐mediated uptake, including the cytosolic delivery of proteins. Dynamic cyano‐cinnamate dimers rival the best chalcogen‐centered inhibitors. Patterns generated in inhibition heatmaps reveal contributions from halogen‐bonding switches that occur independent from the thyroid transporter MCT8. The uniqueness of these patterns supports that the entry of tetrel‐centered exchangers into cells differs from chalcogen‐centered systems. These results expand the chemical space of thiol‐mediated uptake and support the existence of a universal exchange network to bring matter into cells, abiding to be decoded for drug delivery and drug discovery in the broadest sense.

Inclusive Pattern Generation Protocols to Decode Thiol-Mediated Uptake

“…From the massive inhibitor screens realized in the past, ,, only privileged motifs with distinct exchange characteristics were preserved (Figure ). This included all CAXs used in transporters (Figure ), with a negative charge in place of the fluorescent moiety.…”

“…42,56 Reversible Michael acceptors (MACs) 57,58 show good uptake, but suffer from the highest quenching due to the iodine, which is however essential for activity because it contributes a halogenbonding 59,60 switch for pseudocascade exchange. 43 The cellpenetrating activity of OPS [22][23][24]61 was not directly comparable because OPS are oligomers and labeled with a different fluorophore, i.e., Cy5 (Figure 2). 22 CAXs as Inhibitors.…”

Toward a Traceless Tag for the Thiol‐Mediated Uptake of Proteins

“…Inhibitors of TMU have been applied to prove the participation of cell‐surface thiols in the delivery of substrates functionalized with thiol‐reactive transporters. Whilst initial studies relied on a limited and less convincing pool of thiol‐reactive reagents as inhibitors (iodoacetamide, DTNB), recent years have seen the development and application of chemically diverse libraries of thiol‐reactive inhibitors as more sophisticated tools to explore protein networks participating in TMU pathways [20,31–36,63] …”

“…To quantify the separate impacts of esterification and attachment of CAX on protein delivery, the cellular uptake of GFP conjugates 9 and 11 was compared in HeLa-MZ cells by automated high-content high-throughput (AHCHT) microscopy at multiple concentrations following a 4 h incubation. [31,36,63] In initial experiments, cells were imaged both live and after fixation with 3 % PFA, without notable differences in intensity or localization of fluorescent signals. However, as fixation reduced the background signal and removed some fluorescent aggregates which interfered with automated image analysis, subsequent analyses were conducted following PFA fixation (Figure S4).…”

“…Whilst initial studies relied on a limited and less convincing pool of thiolreactive reagents as inhibitors (iodoacetamide, DTNB), recent years have seen the development and application of chemically diverse libraries of thiol-reactive inhibitors as more sophisticated tools to explore protein networks participating in TMU pathways. [20,[31][32][33][34][35][36]63] With this in mind, we screened a small but chemically diverse selection of TMU inhibitors, i. e., 17-22, against the TMU-tagged GFP 11. The reduced GFP uptake was quantified by AHCHT microscopy in HeLa Kyoto cells to assure identical conditions with previous inhibitor screens, while the better quality images accessible with the otherwise very similar HeLa-MZ cells were not needed for AHCHT inhibitor screening (Figure 3g,h).…”