BrainBike peptidomimetic enables efficient transport of proteins across brain endothelium.

Protein therapeutics cannot reach the brain in sufficient amounts because of their low permeability across the blood-brain barrier. Here we report a new family of bicyclic peptide shuttles, BrainBikes, capable of increasing transport of proteins, including antibody derivatives, in a human cell-based model of the blood-brain barrier.

Site-specific antibody masking enables conditional activation with different stimuli.

Antibody therapeutics show great potential to treat a variety of diseases. Often, the dose that can be safely administered is limited by side effects that arise from the interaction with the target outside the diseased tissue. Conditionally-active antibodies provide an additional layer of selectivity to improve safety. Distinct external stimuli or internal cues enable different control strategies and applications. However, current antibody masking strategies have low transferability across stimuli. Here we propose a versatile approach to conditionally mask antibody derivatives and its application to a single chain variable fragment (scFv) against a receptor expressed on cancer stem cells in several tumours. Our strategy relies on the site-specific conjugation of a polymer to an engineered cysteine residue through a chemically-synthesised linker that can be cleaved in response to the target stimulus. We show that the masking efficiency depends on the conjugation site and the size of the mask. An optimised mask decreases antigen binding by up to 20-fold and affinity can be fully recovered upon activation by exposure to light at 365 nm or by incubation with matrix metalloproteinases overexpressed in solid tumours. This approach opens up the possibility to rapidly engineer antibodies activatable with any internal or external stimulus.

Cytosolic localization and in vitro assembly of human de novo thymidylate synthesis complex.

De novo thymidylate synthesis is a crucial pathway for normal and cancer cells. Deoxythymidine monophosphate (dTMP) is synthesized by the combined action of three enzymes: serine hydroxymethyltransferase (SHMT1), dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS), with the latter two being targets of widely used chemotherapeutics such as antifolates and 5-fluorouracil. These proteins translocate to the nucleus after SUMOylation and are suggested to assemble in this compartment into the thymidylate synthesis complex. We report the intracellular dynamics of the complex in cancer cells by an in situ proximity ligation assay, showing that it is also detected in the cytoplasm. This result indicates that the role of the thymidylate synthesis complex assembly may go beyond dTMP synthesis. We have successfully assembled the dTMP synthesis complex in vitro, employing tetrameric SHMT1 and a bifunctional chimeric enzyme comprising human thymidylate synthase and dihydrofolate reductase. We show that the SHMT1 tetrameric state is required for efficient complex assembly, indicating that this aggregation state is evolutionarily selected in eukaryotes to optimize protein-protein interactions. Lastly, our results regarding the activity of the complete thymidylate cycle in vitro may provide a useful tool with respect to developing drugs targeting the entire complex instead of the individual components.

Protease-Resistant Peptides for Targeting and Intracellular Delivery of Therapeutics.

Peptides show high promise in the targeting and intracellular delivery of next-generation bio- and nano-therapeutics. However, the proteolytic susceptibility of peptides is one of the major limitations of their activity in biological environments. Numerous strategies have been devised to chemically enhance the resistance of peptides to proteolysis, ranging from N- and C-termini protection to cyclization, and including backbone modification, incorporation of amino acids with non-canonical side chains and conjugation. Since conjugation of nanocarriers or other cargoes to peptides for targeting and cell penetration may already provide some degree of shielding, the question arises about the relevance of using protease-resistant sequences for these applications. Aiming to answer this question, here we provide a critical review on protease-resistant targeting peptides and cell-penetrating peptides (CPPs). Two main approaches have been used on these classes of peptides: enantio/retro-enantio isomerization and cyclization. On one hand, enantio/retro-enantio isomerization has been shown to provide a clear enhancement in peptide efficiency with respect to parent L-amino acid peptides, especially when applied to peptides for drug delivery to the brain. On the other hand, cyclization also clearly increases peptide transport capacity, although contribution from enhanced protease resistance or affinity is often not dissected. Overall, we conclude that although conjugation often offers some degree of protection to proteolysis in targeting peptides and CPPs, modification of peptide sequences to further enhance protease resistance can greatly increase homing and transport efficiency.

The Masking Game: Design of Activatable Antibodies and Mimetics for Selective Therapeutics and Cell Control.

The high selectivity and affinity of antibody binding have made antibodies all-pervasive tools in therapy, diagnosis, and basic science. A plethora of chemogenetic approaches has been devised to make antibodies responsive to stimuli ranging from light to enzymatic activity, temperature, pH, ions, and effector molecules. Within a single decade, the field of activatable antibodies has yielded marketed therapeutics capable of engaging antigens that could not be targeted with traditional antibodies, as well as new tools to control intracellular protein location and investigate biological processes. Many opportunities remain untapped, waiting for more efficient and generally applicable masking strategies to be developed at the interface between chemistry and biotechnology.

The catalytic activity of serine hydroxymethyltransferase is essential for de novo nuclear dTMP synthesis in lung cancer cells.

Cancer cells reprogramme one-carbon metabolism (OCM) to sustain growth and proliferation. Depending on cell demands, serine hydroxymethyltransferase (SHMT) dynamically changes the fluxes of OCM by reversibly converting serine and tetrahydrofolate (THF) into 5,10-methylene-THF and glycine. SHMT is a tetrameric enzyme that mainly exists in three isoforms; two localize in the cytosol (SHMT1/SHMT2α) and one (SHMT2) in the mitochondria. Both the cytosolic isoforms can also translocate to the nucleus to sustain de novo thymidylate synthesis and support cell proliferation. Finally, the expression levels of the different isoforms are regulated to a certain extent by a yet unknown crosstalk mechanism. We have designed and fully characterized a set of three SHMT1 mutants, which uncouple the oligomeric state of the enzyme from its catalytic activity. We have then investigated the effects of the mutations on SHMT1 nuclear localization, cell viability and crosstalk in lung cancer cells (A549; H1299). Our data reveal that in these cell lines de novo thymidylate synthesis requires SHMT1 to be active, regardless of its oligomeric state. We have also confirmed that the crosstalk between the cytosolic and mitochondrial SHMT actually takes place and regulates the expression of the two isoforms. Apparently, the crosstalk mechanism is independent from the oligomeric state and the catalytic activity of SHMT1. DATABASE: Structural data are available in the PDB under the accession number 6FL5.