Marcos Pires

Ras genes are frequently activated in human cancers, but the mutant Ras proteins remain largely “undruggable” through the conventional small-molecule approach owing to the absence of any obvious binding pockets on their surfaces. By screening a combinatorial peptide library, followed by structure–activity relationship (SAR) analysis, we discovered a family of cyclic peptides possessing both Ras-binding and cell-penetrating properties. These cell-permeable cyclic peptides inhibit Ras signaling by binding to Ras-GTP and blocking its interaction with downstream proteins and they induce apoptosis of cancer cells. Our results demonstrate the feasibility of developing cyclic peptides for the inhibition of intracellular protein–protein interactions and of direct Ras inhibitors as a novel class of anticancer agents.

Round, round, get around: Cell-permeable cyclic peptides were developed that act as direct Ras inhibitors by blocking Ras–effector protein interactions. These peptides, named cyclorasins, cause growth inhibition and apoptosis in cancer cells.

Benzene is the simplest aromatic hydrocarbon with a six-membered ring. It is one of the most basic structural units for the construction of π conjugated systems, which are widely used as fluorescent dyes and other luminescent materials for imaging applications and displays because of their enhanced spectroscopic signal. Presented herein is 2,5-bis(methylsulfonyl)-1,4-diaminobenzene as a novel architecture for green fluorophores, established based on an effective push–pull system supported by intramolecular hydrogen bonding. This compound demonstrates high fluorescence emission and photostability and is solid-state emissive, water-soluble, and solvent- and pH-independent with quantum yields of Φ=0.67 and Stokes shift of 140 nm (in water). This architecture is a significant departure from conventional extended π-conjugated systems based on a flat and rigid molecular design and provides a minimum requirement for green fluorophores comprising a single benzene ring.

Evergreen: An effective push–pull system supported by intramolecular hydrogen bonding between amino and sulfonyl groups generated strong fluorescence properties. The achievement of a single-benzene-core fluorescent system (see picture) allowed steady green fluorescence, independent of the surrounding environment such as solvent polarity and pH, as well as extraordinarily large Stokes shifts of up to 140 nm.

The objective of the study was to explore the potential of ring tension in cyclic disulfides for thiol-mediated cellular uptake. Fluorescent probes that cannot enter cells were equipped with cyclic disulfides of gradually increasing ring tension. As demonstrated by flow cytometry experiments, uptake into HeLa Kyoto cells increased with increasing tension. Differences in carbon-sulfur-sulfur-carbon (CSSC) dihedral angles as small as 8° caused significant changes in uptake efficiency. Uptake with high ring tension was better than with inactivated or activated linear disulfides or with thiols. Conversion of thiols on the cell surface into sulfides and disulfides decreased the uptake. Reduction of exofacial disulfides into thiols increased the uptake of transporters with disulfides and inactivated controls with thiols. These results confirm the occurrence of dynamic covalent disulfide-exchange chemistry on cell surfaces. Mechanistic and colocalization studies indicate that endocytosis does not fully account for this cellular uptake with ring tension.

Mounting tension: Ring tension, which has been exploited for the modification of biological systems in many different ways, with click chemistry being a notable example, was applied to thiol-mediated cellular uptake. Fluorescent probes were conjugated to cyclic disulfides with varying degrees of ring tension and uptake into human cells was found to increase with increasing tension.

Virus nanoparticles (VNPs) have been applied as carrier proteins for effective vaccine development. In this paper, we report the usage of tobacco mosaic virus (TMV) as a carrier for the display of the small molecule estriol (E3), a weakly immunogenic hapten. A highly efficient copper (I)-catalyzed azide–alkyne cycloaddition reaction (CuAAC) was performed for the conjugation of E3 onto TMV capsid at tyrosine (Tyr) 139, by which the antigen density could be controlled. The immune properties of these constructs were evaluated in mice. We found that a strong and long-term antibody response was elicited by conjugating a high density of small molecular haptens on TMV through an oligo(ethylene glycol) (OEG) linker, likely due to the effective activation of B-cells. This study suggests that TMV can serve as a promising platform to induce strong humoral immune responses and that the optimized conjugation strategy was critical to produce high quality antibodies.

What's hapten-ing: Copper-catalyzed cycloaddition was used to conjugate estriol (E3) onto a tobacco mosaic virus (TMV) capsid. A high density of these small molecular haptens on TMV with oligo(ethylene glycol) linkers was shown to elicit strong, long-term IgG antibody responses in vivo, thus providing a promising platform for induction of humoral immune responses.

Inverse-electron-demand Diels–Alder cycloaddition (DA_inv) between strained alkenes and tetrazines is a highly bio-orthogonal reaction that has been applied in the specific labeling of biomolecules. In this work we present a two-step labeling protocol for the site-specific labeling of proteins based on attachment of a highly stable norbornene derivative to a specific peptide sequence by using a mutant of the enzyme lipoic acid ligase A (LplA^W37V), followed by the covalent attachment of tetrazine-modified fluorophores to the norbornene moiety through the bio-orthogonal DA_inv . We investigated 15 different norbornene derivatives for their selective enzymatic attachment to a 13-residue lipoic acid acceptor peptide (LAP) by using a standardized HPLC protocol. Finally, we used this two-step labeling strategy to label proteins in cell lysates in a site-specific manner and performed cell-surface labeling on living cells.

Labeling surface proteins on living cells: A norbornene handle was fused to a signal peptide by first using a mutant of lipoic acid ligase A. In a second step, the norbornene underwent inverse-electron-demand Diels–Alder cycloaddition with tetramethylrhodamine modified with a tetrazine moiety. The norbornene handle, has high stability under live-cell conditions and is synthetically easily accessible.

pHLIP opens the door to the cell: An improved cytosolic transfer of anti-microRNAs (anti-miRs) against onco-miRs paves the way for future cancer therapies. The employed anti-miR–peptide conjugates are based on peptide nucleic acids (PNAs), which are connected with the membrane translocation peptide pHLIP through a disulfide bond. The PNAs are thus transferred into the cell and released by the cleavage of the SS bond (see scheme).

Bacterial peptidoglycan is a mesh-like network comprised of sugars and oligopeptides. Transpeptidases cross-link peptidoglycan oligopeptides to provide vital cell wall rigidity and structural support. It was recently discovered that the same transpeptidases catalyze the metabolic incorporation of exogenous D-amino acids onto bacterial cell surfaces with vast promiscuity for the side-chain identity. It is now shown that this enzymatic promiscuity is not exclusive to side chains, but that C-terminus variations can also be accommodated across a diverse range of bacteria. Atomic force microscopy analysis revealed that the incorporation of C-terminus amidated D-amino acids onto bacterial surfaces substantially reduced the cell wall stiffness. We exploited the promiscuity of bacterial transpeptidases to develop a novel assay for profiling different bacterial species.

Transpeptidase domains of penicillin binding proteins catalyze the incorporation of exogenous D-amino acids onto bacterial cell surfaces. The substrates can have diverse side chains (see picture, pink), and this promiscuous enzyme can also accommodate C-terminus variations (blue). Most importantly, fluorescence labeling differences within and between bacterial species could be profiled using a panel of compounds.

Inspired by the knowledge that most antibodies recognize a conformational epitope because of the epitope’s specific three-dimensional shape rather than its linear structure, we combined scaffold-based peptide design and surface molecular imprinting to fabricate a novel nanocarrier harboring stable binding sites that captures a membrane protein. In this study, a disulfide-linked α-helix-containing peptide, apamin, was used to mimic the extracellular, structured N-terminal part of the protein p32 and then serve as an imprinting template for generating a sub-40 nm-sized polymeric nanoparticle that potently binds to the target protein, recognizes p32-positive tumor cells, and successfully mediates targeted photodynamic therapy in vivo. This could provide a promising alternative for currently used peptide-modified nanocarriers and may have a broad impact on the development of polymeric nanoparticle-based therapies for a wide range of human diseases.

Magic bullet: A peptide served as an “indirect” targeting ligand to mediate active tumor-targeted drug delivery. A disulfide-linked α-helix containing peptide, apamin, was used to mimic the extracellular, structured N-terminal part of the protein p32. The combination with surface molecular imprinting produced a nanocarrier that recognizes p32-positive tumors in vivo.

Oligonucleotide-based molecular circuits offer the exciting possibility to introduce autonomous signal processing in biomedicine, synthetic biology, and molecular diagnostics. Here we introduce bivalent peptide–DNA conjugates as generic, noncovalent, and easily applicable molecular locks that allow the control of antibody activity using toehold-mediated strand displacement reactions. Employing yeast as a cellular model system, reversible control of antibody targeting is demonstrated with low nM concentrations of peptide–DNA locks and oligonucleotide displacer strands. Introduction of two different toehold strands on the peptide–DNA lock allowed signal integration of two different inputs, yielding logic OR- and AND-gates. The range of molecular inputs could be further extended to protein-based triggers by using protein-binding aptamers.

Logic antibody locks: Bivalent peptide–DNA conjugates are presented as generic, noncovalent, and easily applicable molecular locks that allow the control of antibody activity using toehold-mediated strand displacement. By connecting antibody-based molecular recognition and DNA-based computing, this new approach allows the introduction of autonomous signal-processing in antibody-based targeting.