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Chemical zymogens of three different types were established herein around protein cysteinome, in each case converting the protein thiol into a disulfide linkage: zero length Z0, polyethylene glycol based ZPEG, and ZLA that features a fast-depolymerizing fuse polymer. The latter was a polydisulfide based on a naturally occurring water-soluble lipoic acid. Three zymogen designs were applied to cysteinyl proteases and a kinase and 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 enzyme active site. This afforded first-in-class chemical zymogens that are activated via protein-protein interactions. For ZLA, we also document a “chain transfer” bioconjugation mechanism and a unique zymogen exchange reaction between two proteins.

Signal transduction through sealed biological membranes is among the most important evolutionary achievements. Herein, we focus on the development of artificial signal transduction mechanisms and engineer a bionic receptor with capacity of transduction of biological signals across biological membranes using tools of chemistry. The bionic receptor described in this work exhibits similarity with the natural counterpart in the most essential characteristics: in having an exofacial ligand for signal capture, in being membrane anchored, and in featuring a releasable secondary messenger molecule, which performs enzyme activation in the endo volume. The main difference with the natural receptors is that signal transduction across the lipid bilayer was performed using the tools of organic chemistry, namely a self-immolative linker. The highest novelty of our work is that the artificial signalling cascade designed herein achieved transmembrane activation of enzymatic activity, as is the hallmark of activity by natural signalling receptors.

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.

Polymeric biomaterials capable of delivering nitric oxide (NO) topically can be used to enhance skin blood flow (SkBF) and accelerate wound healing. Herein, we used reversible addition-fragmentation chain transfer radical (RAFT) polymerization to synthesize the first poly(vinyl alcohol) (PVA) functionalized with terminal NO-releasing S-nitrosothiol (RSNO) groups for topical NO delivery. This strategy was based on the synthesis of a precursor amino-terminated PVA (PVA-NH2), which was next functionalized with iminothiolane yielding 4-imino-4-amino-PVA-butane-1-thiol (PVA-SH), and finally S-nitrosated yielding S-nitroso 4-imino-4-amino-PVA-butane-1-thiol (PVA-SNO). Real-time chemiluminescence NO detection showed that blended films of pure PVA with PVA-SNO with mass ratios 30:70, 50:50 and 70:30 release NO with initial rates ranging from 1 to 12 nmol g−1 min−1, and lead to a 2 to 10-fold dose-response increase in the SkBF, after topical application on the ventral forearm of volunteers. These results show that PVA-SNO is a potential platform for topical NO delivery in biomedical applications.

Macromolecular (pro)drugs hold much promise as broad-spectrum antiviral agents as either microbicides or carriers for intracellular delivery of antiviral drugs. Intriguing opportunity exists in combining the two modes of antiviral activity in the same polymer structure such that the same polymer acts as a microbicide and also serves to deliver the conjugated drug (ribavirin) into the cells. We explore this opportunity in detail and focus on the polymer backbone as a decisive constituent of such formulations. Fourteen polyanions (polycarboxylates, polyphosphates and polyphosphonates, and polysulfonates) were analyzed for blood pro/anti coagulation effects, albumin binding and albumin aggregation, inhibitory activity on polymerases, cytotoxicity, and anti-inflammatory activity in stimulated macrophages. Ribavirin containing monomers were designed to accommodate the synthesis of macromolecular prodrugs with disulfide-exchange triggered drug release. Kinetics of drug release was fast in all cases however enhanced hydrophobicity of the polymer significantly slowed release of ribavirin. Results of this study present a comprehensive view on polyanions as backbone for macromolecular prodrugs of ribavirin as broad-spectrum antiviral agents., Graphical abstract Unlabelled Image

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., Artificial receptors are robust and simple (as small as 1 kDa), are installed into cells in under 2 h with minimal cell handling, and afford nanomolar potency of targeted drug delivery in primary T cells.

Viral pathogens continue to constitute a heavy burden on healthcare and socioeconomic systems. Efforts to create antiviral drugs repeatedly lag behind the advent of pathogens and growing understanding is that broad‐spectrum antiviral agents will make strongest impact in future antiviral efforts. This work performs selection of synthetic polymers as novel broadly active agents and demonstrates activity of these polymers against Zika, Ebola, Lassa, Lyssa, Rabies, Marburg, Ebola, influenza, herpes simplex, and human immunodeficiency viruses. Results presented herein offer structure–activity relationships for these pathogens in terms of their susceptibility to inhibition by polymers, and for polymers in terms of their anionic charge and hydrophobicity that make up broad‐spectrum antiviral agents. The identified leads cannot be predicted based on prior data on polymer‐based antivirals and represent promising candidates for further development as preventive microbicides., Broad spectrum antiviral agents are designed through systematic variation of the polymer composition, specifically the nature of the anionic charge of the polymer and hydrophobicity of the backbone. Structure–function relationship reveals unexpected lead candidates with inhibitory antiviral activity against all tested enveloped viruses.

Synthetic polymers make strong contributions as tools for delivery of biological drugs and chemotherapeutics. The most praised characteristic of polymers in these applications is complete lack of pharmacological function such as to minimize the side effects within the human body. In contrast, synthetic polymers with curative pharmacological activity are truly rare. Moreover, such activity is typically nonspecific rather than structure-defined. In this work, we present the discovery of poly(ethylacrylic acid) (PEAA) as a polymer with a suit of structure-defined, unexpected, pharmacological, and pharmacokinetic properties not observed in close structural analogues. Specifically, PEAA reveals capacity to bind to albumin with ensuing natural hepatic deposition in vivo and exhibits concurrent inhibitory activity against the hepatitis C virus and inflammation in hepatic cells. Our findings provide a view on synthetic polymers as curative, functional agents and present PEAA as a unique biomedical tool with applications related to health of the human liver.

Ice active bacteria can catalyze water freezing at high subzero temperatures using ice nucleating proteins (INPs) located at their outer cell walls. INPs are the most effective ice nucleators known and are of significant interest for agriculture, climate research, and freeze/antifreeze technologies. The aggregation of INPs into large ice nucleation sites is a key step for effective ice nucleation. It has been proposed that ice active bacteria can drive the aggregation of INPs and thereby trigger ice nucleation. However, the mechanism of INP aggregate assembly and the molecular processes behind the activation are still unclear. Both biochemical pathways and activation through electrostatics have been proposed based on experiments with lysed ice active bacteria. For a more direct view on the assembly of INPs, we follow the structure and water interactions of a synthetic model INP of the well-studied ice bacterium Pseudomonas syringae at the air-water interface as a function of the subphase pH. By combining sum frequency generation spectroscopy with two-dimensional infrared spectra, we conclude that self-assembly and electrostatic interactions drive the formation of ordered INP structures capable of aligning interfacial water.