Targeted Subcellular Protein Delivery Using Cleavable Cyclic Cell-Penetrating Peptide-Conjugates.

Proteins conjugated to cyclic cell-penetrating peptides (cCPPs) haAbstractve been shown to be effectively taken up by living cells. Conjugation of proteins to cCPPs in a cleavable manner leads to localization in the cytosol and immediate bioavailability of the protein after uptake. Here we describe how mCherry, a fluorescent protein, can be targeted to a membrane-bound compartment, the nucleus, and a subcellular structure like the actin cytoskeleton after cCPP-mediated uptake into living cells. Targeting peptides are genetically fused to the mCherry protein sequence and the protein is conjugated to the cCPP via a cleavable disulfide bond. Localization of mCherry in the nucleus or the actin skeleton respectively can be observed by live cell confocal fluorescence microscopy.

Cell-Permeable Nanobodies Allow Dual-Color Super-Resolution Microscopy in Untransfected Living Cells.

Super-resolution microscopy in living cells can be restricted by the availability of small molecule probes, which only exist against few targets and genetically encoded tags. Here, we expand the applicability of live-cell STED by engineering cell-permeable and highly fluorescent nanobodies as intracellular targeting agents. To ensure bright fluorescent signals at low concentrations we used the concept of intramolecular photostabilization by ligating a fluorophore along with the photostabilizer trolox to the nanobody using expressed protein ligation (EPL). Furthermore, these semi-synthetic nanobodies are equipped with a cleavable cell-penetrating peptide for efficient cellular entry, which enables super-resolution imaging of GFP and mCherry, as well as two endogenous targets, nuclear lamins and the DNA replication and repair protein PCNA. We monitored cell division and DNA replication via confocal and STED microscopy thus demonstrating the utility of these new intracellular tools for functional analysis.

Cellular uptake of large biomolecules enabled by cell-surface-reactive cell-penetrating peptide additives.

Enabling the cellular delivery and cytosolic bioavailability of functional proteins constitutes a major challenge for the life sciences. Here we demonstrate that thiol-reactive arginine-rich peptide additives can enhance the cellular uptake of protein-CPP conjugates in a non-endocytic mode, even at low micromolar concentration. We show that such thiol- or HaloTag-reactive additives can result in covalently anchored CPPs on the cell surface, which are highly effective at co-delivering protein cargoes. Taking advantage of the thiol reactivity of our most effective CPP additive, we show that Cys-containing proteins can be readily delivered into the cytosol by simple co-addition of a slight excess of this CPP. Furthermore, we demonstrate the application of our 'CPP-additive technique' in the delivery of functional enzymes, nanobodies and full-length immunoglobulin-G antibodies. This new cellular uptake protocol greatly simplifies both the accessibility and efficiency of protein and antibody delivery, with minimal chemical or genetic engineering.

Discovery, X-ray structure and CPP-conjugation enabled uptake of p53/MDM2 macrocyclic peptide inhibitors.

Mouse double minute 2 homolog (MDM2, Hdm2) is an important negative regulator of the tumor suppressor p53. Using a mRNA based display technique to screen a library of >1012 in vitro-translated cyclic peptides, we have identified a macrocyclic ligand that shows picomolar potency on MDM2. X-Ray crystallography reveals a novel binding mode utilizing a unique pharmacophore to occupy the Phe/Trp/Leu pockets on MDM2. Conjugation of a cyclic cell-penetrating peptide (cCPP) to the initially non cell-permeable ligand enables cellular uptake and a pharmacodynamic response in SJSA-1 cells. The demonstrated enhanced intracellular availability of cyclic peptides that are identified by a display technology exemplifies a process for the application of intracellular tools for drug discovery projects.

N-Hydroxysuccinimide-Modified Ethynylphosphonamidates Enable the Synthesis of Configurationally Defined Protein Conjugates.

Herein, the application of N-hydroxysuccinimide-modified phosphonamidate building blocks for the incorporation of cysteine-selective ethynylphosphonamidates into lysine residues of proteins, followed by thiol addition with small molecules and proteins, is reported. It is demonstrated that the building blocks significantly lower undesired homo-crosslinking side products that can occur with commonly applied succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) under physiological pH. The previously demonstrated stability of the phosphonamidate moiety additionally solves the problem of premature maleimide hydrolysis, which can hamper the efficiency of subsequent thiol addition. Furthermore, a method to separate the phosphonamidate enantiomers to be able to synthesize protein conjugates in a defined configuration has been developed. Finally, the building blocks are applied to the construction of functional antibody-drug conjugates, analogously to FDA-approved, SMCC-linked Kadcyla, and to the synthesis of a functional antibody-protein conjugate.

Targeted Subcellular Protein Delivery Using Cleavable Cyclic Cell-Penetrating Peptides.

The delivery of entire functional proteins into living cells is a long-sought goal in science. Cyclic cell-penetrating peptides (cCPPs) have proven themselves to be potent delivery vehicles to carry proteins upon conjugation into the cytosol of living cells with immediate bioavailability via a non-endosomal uptake pathway. With this strategy, we pursue the cytosolic delivery of mCherry, a medium-sized fluorescent protein. Afterward, we achieve subcellular delivery of mCherry to different intracellular loci by genetic fusion of targeting peptides to the protein sequence. We show efficient transport into a membrane-bound compartment, the nucleus, as well as targeting of the actin cytoskeleton, marking one of the first ways to label actin fluorescently in genetically unmodified living cells. Furthermore, we demonstrate that only by conjugation of cCPPs via a disulfide bond, is flawless localization to the target area achieved. This finding underlines the importance of using a cCPP-based delivery vehicle that is cleaved inside cells, for the precise intracellular localization of a protein of interest.

Nanobodies: Chemical Functionalization Strategies and Intracellular Applications.

Nanobodies can be seen as next-generation tools for the recognition and modulation of antigens that are inaccessible to conventional antibodies. Due to their compact structure and high stability, nanobodies see frequent usage in basic research, and their chemical functionalization opens the way towards promising diagnostic and therapeutic applications. In this Review, central aspects of nanobody functionalization are presented, together with selected applications. While early conjugation strategies relied on the random modification of natural amino acids, more recent studies have focused on the site-specific attachment of functional moieties. Such techniques include chemoenzymatic approaches, expressed protein ligation, and amber suppression in combination with bioorthogonal modification strategies. Recent applications range from sophisticated imaging and mass spectrometry to the delivery of nanobodies into living cells for the visualization and manipulation of intracellular antigens.

Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells.

Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.