Chemical Zymogens and Transmembrane Activation of Transcription in Synthetic Cells.

In this work, synthetic cells equipped with an artificial signaling pathway that connects an extracellular trigger event to the activation of intracellular transcription are engineered. Learning from nature, this is done via an engineering of responsive enzymes, such that activation of enzymatic activity can be triggered by an external biochemical stimulus. Reversibly deactivated creatine kinase to achieve triggered production of adenosine triphosphate, and a reversibly deactivated nucleic acid polymerase for on-demand synthesis of RNA are engineered. An extracellular, enzyme-activated production of a diffusible zymogen activator is also designed. The key achievement of this work is that the importance of cellularity is illustrated whereby the separation of biochemical partners is essential to resolve their incompatibility, to enable transcription within the confines of a synthetic cell. The herein designed biochemical pathway and the engineered synthetic cells are arguably primitive compared to their natural counterpart. Nevertheless, the results present a significant step toward the design of synthetic cells with responsive behavior, en route from abiotic to life-like cell mimics.

Artificial Receptor in Synthetic Cells Performs Transmembrane Activation of Proteolysis.

The design of artificial, synthetic cells is a fundamentally important and fast-developing field of science. Of the diverse attributes of cellular life, artificial transmembrane signaling across the biomolecular barriers remains a high challenge with only a few documented successes. Herein, the study achieves signaling across lipid bilayers and connects an exofacial enzymatic receptor activation to an intracellular biochemical catalytic response using an artificial receptor. The mechanism of signal transduction for the artificial receptor relies on the triggered decomposition of a self-immolative linker. Receptor activation ensues its head-to-tail decomposition and the release of a secondary messenger molecule into the internal volume of the synthetic cell. Transmembrane signaling is demonstrated in synthetic cells based on liposomes and mammalian cell-sized giant unilamellar vesicles and illustrates receptor performance in cell mimics with a diverse size and composition of the lipid bilayer. In giant unilamellar vesicles, transmembrane signaling connects exofacial receptor activation with intracellular activation of proteolysis. Taken together, the results of this study take a step toward engineering receptor-mediated, responsive behavior in synthetic cells.

Transmembrane signaling by a synthetic receptor in artificial cells.

Signal transduction across biological membranes is among the most important evolutionary achievements. Herein, for the design of artificial cells, we engineer fully synthetic receptors with the capacity of transmembrane signaling, using tools of chemistry. Our receptors exhibit similarity with their natural counterparts in having an exofacial ligand for signal capture, being membrane anchored, and featuring a releasable messenger molecule that performs enzyme activation as a downstream signaling event. The main difference from natural receptors is the mechanism of signal transduction, which is achieved using a self-immolative linker. The receptor scaffold is modular and can readily be re-designed to respond to diverse activation signals including biological or chemical stimuli. We demonstrate an artificial signaling cascade that achieves transmembrane enzyme activation, a hallmark of natural signaling receptors. Results of this work are relevant for engineering responsive artificial cells and interfacing them and/or biological counterparts in co-cultures.

Chemical zymogens for the protein cysteinome.

We present three classes of chemical zymogens established around the protein cysteinome. In each case, the cysteine thiol group was converted into a mixed disulfide: with a small molecule, a non-degradable polymer, or with a fast-depolymerizing fuse polymer (ZLA). The latter was a polydisulfide based on naturally occurring molecule, lipoic acid. Zymogen designs were applied to cysteine proteases and a kinase. In each case, enzymatic activity was successfully masked in full and reactivated by small molecule reducing agents. However, only ZLA could be reactivated by protein activators, demonstrating that the macromolecular fuse escapes the steric bulk created by the protein globule, collects activation signal in solution, and relays it to the active site of the enzyme. This afforded first-in-class chemical zymogens that are activated via protein-protein interactions. We also document zymogen exchange reactions whereby the polydisulfide is transferred between the interacting proteins via the "chain transfer" bioconjugation mechanism.

Synthetic chemical ligands and cognate antibodies for biorthogonal drug targeting and cell engineering.

A vast range of biomedical applications relies on the specificity of interactions between an antigen and its cognate receptor or antibody. This specificity can be highest when said antigen is a non-natural (synthetic) molecule introduced into a biological setting as a bio-orthogonal ligand. This review aims to present the development of this methodology from the early discovery of haptens a century ago to the recent clinical trials. We discuss such methodologies as antibody recruitment, artificial internalizing receptors and chemically induced dimerization, present the use of chimeric receptors and/or bispecific antibodies to achieve drug targeting and transcytosis, and illustrate how these platforms most impressively found use in the engineering of therapeutic cells such as the chimeric antigen receptor cells. This review aims to be of interest to a broad scientific audience and to spur the development of synthetic artificial ligands for biomedical applications.

Synthetic Artificial Apoptosis-Inducing Receptor for On-Demand Deactivation of Engineered Cells.

The design of a fully synthetic, chemical "apoptosis-inducing receptor" (AIR) molecule is reported that is anchored into the lipid bilayer of cells, is activated by the incoming biological input, and responds with the release of a secondary messenger-a highly potent toxin for cell killing. The AIR molecule has four elements, namely, an exofacial trigger group, a bilayer anchor, a toxin as a secondary messenger, and a self-immolative scaffold as a mechanism for signal transduction. Receptor installation into cells is established via a robust protocol with minimal cell handling. The synthetic receptor remains dormant in the engineered cells, but is effectively triggered externally by the addition of an activating biomolecule (enzyme) or in a mixed cell population through interaction with the surrounding cells. In 3D cell culture (spheroids), receptor activation is accessible for at least 5 days, which compares favorably with other state of the art receptor designs.

Chemical Artificial Internalizing Receptors for Primary T Cells.

The newest generation of cell-based technologies relies heavily on methods to communicate to the engineered cells using artificial receptors, specifically to deactivate the cells administered to a patient in the event of adverse effects. Herein, artificial synthetic internalizing receptors are engineered that function in mammalian cells in 2D and in 3D and afford targeted, specific intracellular drug delivery with nanomolar potency in the most challenging cell type, namely primary, donor-derived T cells. Receptor design comprises a lipid bilayer anchor for receptor integration into cell membrane and a small xenobiotic molecule as a recognition ligand. Artificial receptors are successfully targeted by the corresponding antibody-drug conjugate (ADC) and exhibit efficient cargo cell entry with ensuing intracellular effects. Receptor integration into cells is fast and robust and affords targeted cell entry in under 2 h. Through a combination of the receptor design and the use of ADC, combined benefits previously made available by chimeric artificial receptors (performance in T cells) and the chemical counterpart (robustness and simplicity) in a single functional platform is achieved. Artificial synthetic receptors are poised to facilitate the maturation of engineered cells as tools of biotechnology and biomedicine.