Amyloid-β (Aβ1-42) prefibrillar aggregates are considered the most neurotoxic amyloid species, yet their transient and heterogeneous nature makes selective detection challenging. Many fluorescent probes also fail to discriminate Aβ from homologous peptides such as IAPP, leading to poor specificity. We report a peptide-guided late-stage diversification strategy to generate BODIPY-based probes highly selective for prefibrillar Aβ1-42. A rationally engineered cyclic peptide derived from the C-terminal region of Aβ1-42 provides conformational rigidity and precise molecular recognition. Conjugation to BODIPY fluorophores afforded peptide-dye hybrids systematically evaluated for selectivity and photophysical response. A controlled aggregation protocol enabling reproducible generation of prefibrillar Aβ species was established to validate probe performance. A Sonogashira-derived conjugate (probe 8) showed strong fluorescence turn-on and selective affinity for prefibrillar Aβ1-42, with no response to IAPP aggregates. In neuronal cells, probe 8 outperformed conventional antibodies, supporting its potential for mechanistic studies and early Alzheimer's disease diagnostics.

ABSTRACT Conventional activity‐based probes in activity‐based protein profiling (ABPP) require enrichment or reporter tags for detection, which limits sensitivity and multiplexing. Here, we present an enrichment‐free chemoproteomic approach that enables direct mass spectrometric detection by Matrix‐Assisted Laser Desorption/Ionization (MALDI) of active proteases. An active‐site–directed affinity probe transfers, through a proximity‐induced reaction, a MALDI‐detectable α‐cyano‐4‐hydroxycinnamic acid (CHCA) tag exclusively to catalytically active forms of matrix metalloproteases (MMPs). The CHCA label enhances ionization efficiency and markedly improves signal‐to‐noise ratios, allowing confident identification of CHCA‐labelled peptides under discriminating analytical conditions. Each active metalloprotease is thereby, associated with a distinct set of CHCA signature peptides, defining its activity fingerprint. This workflow achieves multiplexed and quantitative activity profiling of MMPs, directly in complex proteomes. This design expands ABPP into the mass spectrometry domain and establishes a robust platform for activity‐based enzyme detection.

The accumulation of intracellular aggregates of Tau protein is one main hallmark of Alzheimer's disease (AD) and is the consequence of Tau conformational changes, increased phosphorylation, and self-association to form fibrillar aggregates. This pathological process prevents the physiological interaction of Tau with microtubules to the detriment of the structural integrity of neurons. In healthy cells, aberrant protein misfolding and aggregation are counteracted by chaperone proteins whose protective capacity decreases with age. The role of the chaperone Hsp90 and the mechanism by which it can prevent Tau aggregation are controversial. In this work, the strategy of mimicking Hsp90 through the design of the β-hairpin like peptidomimetic β-Hsp90, inspired by two Hsp90/Tau interaction sequences, is presented. β-Hsp90 inhibits Tau aggregation both in vitro and in cells, restoring Tau's physiological interaction with microtubules. β-Hsp90, which interacts with the P1 region of Tau, is more effective than individual peptide sequences from the chaperone HSP90 and another β-hairpin mimic based on Tau sequences. Moreover, β-Hsp90 reduces AD-associated Aβ1-42 aggregation, offering the development of a dual inhibitor. This work paves the way for the design of new drugs targeting devastating untreated amyloid diseases, by mimicking physiological chaperones with small synthetic peptide drugs.

Despite the availability of antibiotics, tuberculosis (TB), caused by Mycobacterium tuberculosis, was once again declared the world's leading cause of death from a single infectious agent in 2023. Furthermore, the rising prevalence of drug-resistant strains of M. tuberculosis, coupled with the limitations of existing therapeutics, underscores the urgent need for new antimicrobial agents that act through different mechanisms, thereby providing novel therapeutic options. From this perspective, antimicrobial peptides (AMPs) derived from the bovine rumen microbiome have shown promise against many resistant pathogens and may therefore offer a promising alternative against TB. Here, we evaluated the efficacy of AMPs from bovine rumen microbiome, namely the Lynronne 1, 2 & 3 and P15s as well as their all-D amino acid enantiomers, against non-tuberculous (M. abscessus, M. marinum and M. smegmatis) and tuberculous (M. bovis BCG, M. tuberculosis) mycobacteria. In particular, their antimycobacterial activity was assessed against extracellularly and intracellularly replicating M. tuberculosis H37Rv pathogenic strain. Their innocuity was further studied by determining their respective cytotoxicity against human cell lines and hemolytic activity on human erythrocytes. Finally, their mechanism of action was investigated by a membrane permeabilization assay and a lipid insertion assay via surface pressure measurement. Although all-D enantiomers showed increased cytotoxicity to human cell lines, they still offer a good therapeutic window with improved activity compared to their L-form counterparts, especially Lynronne 2Dall and P15sDall which emerged as the best growth inhibitors of all mycobacteria. Remarkably, the all-D enantiomers also demonstrated activity against intramacrophagic replicating M. tuberculosis H37Rv, with very limited toxicity towards human cells and no hemolytic activity at their respective minimum inhibitory concentration. Membrane permeabilization and monolayer lipid insertion assays suggested that these peptides mostly act by insertion into the mycobacterial membrane resulting in a rapid membranolytic effect. These findings highlight the potential of the all-D enantiomers of Lynronne peptides, as attractive candidates for the development of new anti-TB drugs. Their effective antibacterial properties combined with low toxicity underscore Lynronne 2Dall and P15sDall as building blocks for the development of promising alternatives to conventional antibiotics in the treatment of mycobacterial infections, particularly against M. tuberculosis.

Overexpression of recombinant Bacillus cereus TSPO (BcTSPO) in E. coli bacteria leads to its recovery with a bound hemin both in bacterial membrane (MB) and inclusion bodies (IB). Unlike mouse TSPO, BcTSPO purified in SDS detergent from IB is well structured and can bind various ligands such as high-affinity PK 11195, protoporphyrin IX (PPIX) and δ-aminolevulinic acid (ALA). For each of the three ligands, 1H–15N HSQC titration NMR experiments suggest that different amino acids of BcTSPO binding cavity are involved in the interaction. PPIX, an intermediate of heme biosynthesis, binds to the cavity of BcTSPO and its fluorescence can be significantly reduced in the presence of light and oxygen. The light irradiation leads to two products that have been isolated and characterized as photoporphyrins. They result from the addition of singlet oxygen to the two vinyl groups hence leading to the formation of hydroxyaldehydes. The involvement of water molecules, recently observed along with the binding heme in Rhodobacter sphaeroides (RsTSPO) is highly probable. Altogether, these results raise the question of the role of TSPO in heme biosynthesis regulation as a possible scavenger of reactive intermediates.

Engrailed2 (En2) is a transcription factor that transfers from cell to cell through unconventional pathways. The poorly understood internalization mechanism of this cationic protein is proposed to require an initial interaction with cell-surface glycosaminoglycans (GAGs). To decipher the role of GAGs in En2 internalization, we have quantified the entry of its homeodomain region in model cells that differ in their content in cell-surface GAGs. The binding specificity to GAGs and the influence of this interaction on the structure and dynamics of En2 was also investigated at the amino acid level. Our results show that a high-affinity GAG-binding sequence (RKPKKKNPNKEDKRPR), upstream of the homeodomain, controls En2 internalization through selective interactions with highly-sulfated heparan sulfate GAGs. Our data underline the functional importance of the intrinsically disordered basic region upstream of En2 internalization domain, and demonstrate the critical role of GAGs as an entry gate, finely tuning homeoprotein capacity to internalize into cells.

Oxytocin (OT) is a neurohypophyseal peptide hormone containing a disulphide-bridged pseudocyclic conformation. The biomedical use of OT peptides is limited amongst others by disadvantageous pharmacokinetic parameters. To increase the stability of OT by replacing the disulphide bridge with the stable and more rigid [1,2,3]triazol-1-yl moiety, we employed the Cu2+-catalysed side chain-to-side chain azide-alkyne 1,3-cycloaddition. Here we report the design, synthesis, conformational analysis, and in vitro pharmacological activity of a homologous series of Cα1-to-Cα6 side chain-to-side chain [1,2,3]triazol-1-yl-containing OT analogues differing in the length of the bridge, location, and orientation of the linking moiety. Exploiting this macrocyclisation approach, it was possible to generate a systematic series of compounds providing interesting insight into the structure-conformation-function relationship of OT. Most analogues were able to adopt similar conformation to endogenous OT in water, namely, a type I β-turn. This approach may in the future generate stabilised pharmacological peptide tools to advance understanding of OT physiology.

Oxytocin (OT) is a neurohypophyseal peptide hormone containing a disulphide-bridged pseudocyclic conformation. The biomedical use of OT peptides is limited amongst others by disadvantageous pharmacokinetic parameters. To increase the stability of OT by replacing the disulphide bridge with the stable and more rigid [1,2,3]triazol-1-yl moiety, we employed the Cu2+-catalysed side chain-to-side chain azide-alkyne 1,3-cycloaddition. Here we report the design, synthesis, conformational analysis, and in vitro pharmacological activity of a homologous series of Cα1-to-Cα6 side chain-to-side chain [1,2,3]triazol-1-yl-containing OT analogues differing in the length of the bridge, location, and orientation of the linking moiety. Exploiting this macrocyclisation approach, it was possible to generate a systematic series of compounds providing interesting insight into the structure-conformation-function relationship of OT. Most analogues were able to adopt similar conformation to endogenous OT in water, namely, a type I β-turn. This approach may in the future generate stabilised pharmacological peptide tools to advance understanding of OT physiology.

Many control objectives can be expressed in terms of a criterion function. Generally, explicit solutions to such optimization problems require full knowledge of the plant and disturbances, and complete freedom in the complexity of the controller. In practice, the plant and the disturbances are seldom known, and it is often desirable to achieve the best possible performance with a controller of prescribed complexity. For example, one may want to tune the parameters of a PID controller in order to extract the best possible performance from such simple controller.

Homeoprotein transcription factors have the property of interacting with membranes through their DNA-binding homeodomain, which is involved in unconventional internalization and secretion. Both processes depend on membrane-translocating events but their detailed molecular mechanisms are still poorly understood. We have previously characterized the conformational properties of Engrailed 2 homeodomain (EnHD) in aqueous solution and in micelles as membrane-mimetic environments. In the present study, we used small isotropic lipid bicelles as a more relevant membrane-mimetic model to characterize the membrane-bound state of EnHD. We show that lipid bicelles, in contrast to micelles, adequately reproduce the requirement of anionic lipids in the membrane binding and conformational transition of EnHD. The fold-unfold transition of EnHD induced by anionic lipids was characterized by NMR using 1H, 13C, 15N chemical shifts, nuclear Overhauser effects, residual dipolar couplings, intramolecular and intermolecular paramagnetic relaxation enhancements induced by site-directed spin-label or paramagnetic lipid probe, respectively. A global unpacking of EnHD helices is observed leading to a loss of the native fold. However, near-native propensities of EnHD backbone conformation are maintained in membrane environment, including not only the three helices but also the turn connecting helices H2 and H3. NMR and coarse-grained molecular dynamics simulations reveal that the EnHD adopts a shallow insertion in the membrane, with the three helices oriented parallel to the membrane. EnHD explores extended conformations and closed U-shaped conformations, which are stabilized by anionic lipid recruitment.

In preclinical models, the development and optimization of protein-drug conjugates require accurate determination of the plasma and tissue profiles of both the protein and its conjugated drug. To this aim, we developed a bioanalytical strategy based on dual radiolabeling and ex vivo digital imaging. By combining enzymatic and chemical reactions, we obtained homogeneous dual-labeled anti-MMP-14 Fabs (antigen-binding fragments) conjugated to monomethyl auristatin E where the protein scaffold was labeled with carbon-14 (14C) and the conjugated drug with tritium (3H). These antibody-drug conjugates with either a noncleavable or a cleavable linker were then evaluated in vivo. By combining liquid scintillation counting and ex vivo dual-isotope radio-imaging, it was possible not only to monitor both components simultaneously during their circulation phase but also to quantify accurately their amount accumulated within the different organs.

Cylindrospermopsin, a major cyanotoxin, is produced by freshwater cyanobacteria. Its biosynthesis starts from arginine and glycine and involves five polyketide synthases and several tailoring enzymes. We report the identification of 7-deoxy-desulfo-argino-cylindrospermopsin in several cylindrospermopsin-producing cyanobacteria using mass spectrometry experiments. We have purified this new metabolite and established its structure by 1D and 2D NMR spectroscopy using scalar-based 1H-1H, 1H-13C, and 1H-15N as well as 2D 1H-1H ROESY correlation experiments. Using labeled arginines in isotopic incorporation experiments, we have shown that arginine is fully incorporated into 7-deoxy-desulfo-argino-cylindrospermopsin and that the uracil ring of cylindrospermopsin originates from the guanidino moiety of arginine, thus solving a long-standing puzzling question. CyrG and CyrH from the cylindrospermopsin-producing Oscillatoria sp. PCC 6506 were overproduced in Escherichia coli and purified to homogeneity. We showed that CyrG is a zinc-dependent hydrolase, homologous to adenosine deaminases, that transforms 7-deoxy-desulfo-argino-cylindrospermopsin into 7-deoxy-desulfo-cylindrospermopsin and ornithine, with the following kinetic parameters: KM = 0.21 ± 0.05 μM and kcat = 0.19 ± 0.02 min-1. CyrG contained 0.55 mol of zinc per mol of monomer but could be activated by FeII or CoII. CyrH contained almost no metal and showed no such activity even in the presence of excess metal. Using structure-based alignments and secondary structure predictions, we propose that the fifth and last polyketide synthase CyrF in cylindrospermopsin biosynthesis contains an unprecedented C-terminal domain homologous to N-acetyltransferases. We suggest that this domain catalyzes the condensation of the CyrF product with arginine to give 7-deoxy-desulfo-argino-cylindrospermopsin. This would be an unprecedented termination step for a polyketide synthase.

Cell-penetrating peptides cross cell membranes through various parallel internalization pathways. Herein, we analyze the role of the negatively charged lipid phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) in the internalization of Penetratin. Contributions of both inner leaflet and outer leaflet pools of PI(4,5)P2 were revealed by quantifying the internalization of Penetratin in cells treated with PI(4,5)P2 binders. Studies on model systems showed that Penetratin has a strong affinity for PI(4,5)P2 and interacts selectively with this lipid, even in the presence of other negatively charged lipids, as demonstrated by affinity photo-crosslinking experiments. Differential scanning calorimetry experiments showed that Penetratin induces lateral segregation in PI(4,5)P2-containing liposomes, which was confirmed by coarse-grained molecular dynamics simulations. NMR experiments indicated that Penetratin adopts a stabilized helical conformation in the presence of PI(4,5)P2-containing membranes, with an orientation parallel to the bilayer plane, which was also confirmed by all-atom simulations. NMR and photo-crosslinking experiments also suggest a rather shallow insertion of the peptide in the membrane. Put together, our findings suggest that PI(4,5)P2 is a privileged interaction partner for Penetratin and that it plays an important role in Penetratin internalization.

Oligomers of α-aminoisobutyric acid (Aib) are achiral peptides that adopt 310 helical structures with equal population of left- and right-handed conformers. The screw-sense preference of the helical chain may be controlled by a single chiral residue located at one terminus. 1 H and 19 F NMR, X-ray crystallography and circular dichroism studies on new Aib oligomers show that the incorporation of a chiral quaternary α-trifluoromethylalanine at their N-terminus induces a reversal of the screw-sense preference of the 310 -helix compared to that of a non-fluorinated analogue having an l-α-methyl valine residue. This work demonstrates that, among the many particular properties of introducing a trifluoromethyl group into foldamers, its stereo-electronic properties are of major interest to control the helical screw sense. Its use as an easy-to-handle 19 F NMR probe to reliably determine both the magnitude of the screw-sense preference and its sign assignment is also of remarkable interest.

Studies on the synthetic methodologies and the structural propensity of peptides containing consecutive aza-amino acids are still in their infancy. Here, details of the synthesis and conformational analysis of tripeptides containing two consecutive aza-amino acids are provided. The demonstration that the type I β-turn folding is induced, even in aqueous media, by the introduction of one or two lateral chains on the diaza-peptide unit is of particular importance for the design of peptidomimetics of biological interest.

In light of the recent accumulated knowledge on SARS-CoV-2 and its mode of human cells invasion, the binding of viral spike glycoprotein to human Angiotensin Converting Enzyme 2 (hACE2) receptor plays a central role in cell entry. We designed a series of peptides mimicking the N-terminal helix of hACE2 protein which contains most of the contacting residues at the binding site, exhibiting a high helical folding propensity in aqueous solution. Our best peptide-mimics are able to block SARS-CoV-2 human pulmonary cell infection with an inhibitory concentration (IC50) in the nanomolar range upon binding to the virus spike protein with high affinity. These first-in-class blocking peptide mimics represent powerful tools that might be used in prophylactic and therapeutic approaches to fight the coronavirus disease 2019 (COVID-19).