Mjsabulski

Protein expression and localization are often studied in vivo by tagging molecules with green fluorescent protein (GFP), yet subtle changes in protein levels are not easily detected. To develop a sensitive in vivo method to amplify fluorescence signals and allow cell-specific quantification of protein abundance changes, we sought to apply an enzyme-activated cellular fluorescence system in vivo by delivering ester-masked fluorophores to Caenorhabditis elegans neurons expressing porcine liver esterase (PLE). To aid uptake into sensory neuron membranes, we synthesized two novel fluorogenic hydrolase substrates with long hydrocarbon tails. Recombinant PLE activated these fluorophores in vitro. In vivo activation occurred in sensory neurons, along with potent activation in intestinal lysosomes quantifiable by imaging and microplate and partially attributable to gut esterase 1 (GES-1) activity. These data demonstrate the promise of biorthogonal hydrolases and their fluorogenic substrates as in vivo neuronal imaging tools and for characterizing endogenous C. elegans hydrolase substrate specificities.

Working worms: A panel of ester-masked fluorophores was used to label neurons and identify substrate specificities of C. elegans intestinal esterases, demonstrating the utility of the worm as a model for investigating in vivo esterase function, laying groundwork for studies characterizing in vivo specificity of other esterases and supporting development of esterase–substrate pairs as sensitized imaging tools.

In vivo covalent chemical capture by using photoactivatable unnatural amino acids (UAAs) is a powerful tool for the identification of transient protein–protein interactions (PPIs) in their native environment. However, the isolation and characterization of the crosslinked complexes can be challenging. Here, we report the first in vivo incorporation of the bifunctional UAA BPKyne for the capture and direct labeling of crosslinked protein complexes through post-crosslinking functionalization of a bioorthogonal alkyne handle. Using the prototypical yeast transcriptional activator Gal4, we demonstrate that BPKyne is incorporated at the same level as the commonly used photoactivatable UAA pBpa and effectively captures the Gal4–Gal80 transcriptional complex. Post-crosslinking, the Gal4–Gal80 adduct was directly labeled by treatment of the alkyne handle with a biotin-azide probe; this enabled facile isolation and visualization of the crosslinked adduct from whole-cell lysate. This bifunctional amino acid extends the utility of the benzophenone crosslinker and expands our toolbox of chemical probes for mapping PPIs in their native cellular environment.

Using the bifunctional unnatural amino acid, BPKyne, we have developed a strategy to capture and directly label transient protein–protein interactions (PPIs) in their native environment. Click chemical functionalization post-crosslinking with a biotin–azide probe enabled the isolation of transcriptional protein complexes from yeast cells. This amino acid will expand the toolbox for the discovery of new PPIs in live cells.

Multivalent peptide nanofibers have attracted intense attention as promising platforms, but the fabrication of those nanofibers is mainly dependent on the spontaneous assembly of β-sheet peptides. Herein we report an alternative approach to the creation of nanofibers: the polyoxometalate-driven self-assembly of short peptides. The resultant nanofibers with concentrated positive charges are excellent multivalent ligands for binding with bacterial cells and thus lead to a salient improvement in bioactivity.

Fatal attraction: Polyoxometalates were able to drive the self-assembly of short peptides into well-defined nanofibers through multivalent electrostatic attraction. The resulting fibrillar nanostructures with lysine residues concentrated on the surface showed enhanced antimicrobial activity and biological stability (see picture).

Genetic fusion of cargo proteins to a positively supercharged variant of green fluorescent protein enables their quantitative encapsulation by engineered lumazine synthase capsids possessing a negatively charged lumenal surface. This simple tagging system provides a robust and versatile means of creating hierarchically ordered protein assemblies for use as nanoreactors. The generality of the encapsulation strategy and its effect on enzyme function were investigated with eight structurally and mechanistically distinct catalysts.

Caught in a trap: Genetic fusion of cargo proteins to a positively supercharged variant of green fluorescent protein enables their quantitative encapsulation by engineered lumazine synthase capsids possessing a negatively charged lumenal surface.

Epidermal growth factor receptor (EGFR) is a key target in chemotherapy. Some drugs acting on the receptor are currently in use; however, drug resistance, which causes tumour relapse, calls for the discovery of alternative inhibitors. Using docking and receptor hotspot mimicry, we have designed novel peptides directed at EGF, the main growth factor ligand of EGFR. An array of biophysical techniques was used to characterise the structure and interaction of these ligands with the target protein. Both design methods identified peptides able to bind EGF, and the capacity of these peptides to inhibit the interaction between EGF and EGFR was demonstrated in two in vitro systems. Based on targeting the smaller companion of a protein–protein interaction, the new approach described herein can be envisaged as a parallel drug design strategy, and our compounds represent the first in a new class of binders that could serve as complementary compounds in potential multidrug cancer therapy.

A novel EGF–EGFR targeting approach: A new class of peptide ligands against EGF has been designed by using computer-aided docking and EGFR hotspot mimicry. Their binding to EGF was studied in detail, and their ability to disrupt the EGF–EGFR interaction was demonstrated in vitro.

Successful bench-to-bedside translation of nanomedicine relies heavily on the development of nanocarriers with superior therapeutic efficacy and high biocompatibility. However, the optimal strategy for improving one aspect often conflicts with the other. Herein, we report a tactic of designing tumor-pH-labile linkage-bridged copolymers of clinically validated poly(d,l-lactide) and poly(ethylene glycol) (PEG-Dlink_m-PDLLA) for safe and effective drug delivery. Upon arriving at the tumor site, PEG-Dlink_m-PDLLA nanoparticles will lose the PEG layer and increase zeta potential by responding to tumor acidity, which significantly enhances cellular uptake and improves the in vivo tumor inhibition rate to 78.1 % in comparison to 47.8 % of the non-responsive control. Furthermore, PEG-Dlink_m-PDLLA nanoparticles show comparable biocompatibility with the clinically used PEG-b-PDLLA micelle. The improved therapeutic efficacy and safety demonstrate great promise for our strategy in future translational studies.

PEG-detachable delivery micelles: A chemotherapeutic vector with superior therapeutic efficacy and high biocompatibility is obtained by designing bridged PEGylated polylactide-containing tumor-acidity-responsive linkages. The decreased PEGylation and increased zeta potential in the tumor matrix enhanced cellular uptake of the vector, enabling safe and effective antitumor drug delivery.

Antibodies are currently the fastest-growing class of therapeutics. Although naked antibodies have proven valuable as pharmaceutical agents, they have some limitations, such as low tissue penetration and a long circulatory half-life. They have been conjugated to toxic payloads, PEGs, or radioisotopes to increase and optimize their therapeutic efficacy. Although nonspecific conjugation is suitable for most in vitro applications, it has become evident that site specifically modified antibodies may have advantages for in vivo applications. Herein we describe a novel approach in which the antibody fragment is tagged with two handles: one for the introduction of a fluorophore or ¹⁸F isotope, and the second for further modification of the fragment with a PEG moiety or a second antibody fragment to tune its circulatory half-life or its avidity. Such constructs, which recognize Class II MHC products and CD11b, showed high avidity and specificity. They were used to image cancers and could detect small tumors.

Improve your image! Dual labeling of antibody fragments with a fluorophore or ¹⁸F isotope for multimodal imaging and with a PEG moiety or a second antibody fragment to improve circulatory half-life or avidity led to constructs that recognized Class II MHC products (see picture) and CD11b with high specificity. PET imaging with the constructs enabled the detection of tumors as small as a few millimeters in size.

In this work, we use a double-layered stack of TiO₂ nanotubes (TiNTs) to construct a visible-light-triggered drug delivery system. The key for visible light drug release is a hydrophobic cap on the nanotubes containing Au nanoparticles (AuNPs). The AuNPs allow for a photocatalytic scission of the hydrophobic chain under visible light. To demonstrate this principle, we loaded ampicillin (AMP) into the lower part of the TiO₂ nanotube stack, triggered visible-light-induced release, and carried out antibacterial studies. The release from the platform becomes most controllable if the drug is silane-grafted in the hydrophilic bottom layer for drug storage. Thus, visible light photocatalysis can also determine the release kinetics of the active drug from the nanotube wall.

A visible-light-triggered drug delivery system is constructed based on a double-layered stack of TiO₂ nanotubes. The key for visible light drug release is a hydrophobic cap on the nanotubes containing Au nanoparticles, where SPR with the TiO₂ conduction band provides the active species for chain scission. The system was tested in antibacterial experiments against E. coli.

Pseudomonas aeruginosa uses N-acylated l-homoserine lactone signals and a triumvirate of LuxR-type receptor proteins—LasR, RhlR, and QscR—for quorum sensing (QS). Each of these receptors can contribute to QS activation or repression and, thereby, the control of myriad virulence phenotypes in this pathogen. LasR has traditionally been considered to be at the top of the QS receptor hierarchy in P. aeruginosa; however, recent reports suggest that RhlR plays a more prominent role in infection than originally predicted, in some circumstances superseding that of LasR. Herein, we report the characterization of a set of synthetic, small-molecule agonists and antagonists of RhlR. Using E. coli reporter strains, we demonstrated that many of these compounds can selectively activate or inhibit RhlR instead of LasR and QscR. Moreover, several molecules maintain their activities in P. aeruginosa at concentrations analogous to native RhlR signal levels. These compounds represent useful chemical probes to study the role of RhlR in the complex QS circuitry of P. aeruginosa, its direct (and indirect) effects on virulence, and its overall merit as a target for anti-infective therapy.

Virulence control: P. aeruginosa uses multiple LuxR-type receptors and lactone signals in its quorum sensing (QS) system. The receptor hierarchy is complex, but the RhlR receptor appears to play a more prominent role in regulating QS and, therefore, virulence phenotypes, than originally predicted. We have characterized non-native, lactone signal analogues that strongly and selectively agonize or antagonize RhlR.

A supramolecular antibiotic switch is described that can reversibly “turn-on” and “turn-off” its antibacterial activity on demand, providing a proof-of-concept for a way to regulate antibacterial activity of biotics. The switch relies on supramolecular assembly and disassembly of cationic poly(phenylene vinylene) derivative (PPV) with cucurbit[7]uril (CB[7]) to regulate their different interactions with bacteria. This simple but efficient strategy does not require any chemical modification on the active sites of the antibacterial agent, and could also regulate the antibacterial activity of classical antibiotics or photosensitizers in photodynamic therapy. This supramolecular antibiotic switch may be a successful strategy to fight bacterial infections and decrease the emergence of bacterial resistance to antibiotics from a long-term point of view.

A supramolecular antibiotic switch to reversibly “turn-on” and “turn-off” antibacterial activity on demand was developed as a proof-of-concept to regulate antibacterial activity. The switch relies on the supramolecular assembly and disassembly of a poly(phenylene vinylene) derivative (PPV) with cucurbit[7]uril (CB[7]). This strategy does not require any chemical modification on the active sites of the antibacterial agent.