Peptide-Based Covalent Inhibitors Bearing Mild Electrophiles to Target a Conserved His Residue of the Bacterial Sliding Clamp.

Peptide-based covalent inhibitors targeted to nucleophilic protein residues have recently emerged as new modalities to target protein-protein interactions (PPIs) as they may provide some benefits over more classic competitive inhibitors. Covalent inhibitors are generally targeted to cysteine, the most intrinsically reactive amino acid residue, and to lysine, which is more abundant at the surface of proteins but much less frequently to histidine. Herein, we report the structure-guided design of targeted covalent inhibitors (TCIs) able to bind covalently and selectively to the bacterial sliding clamp (SC), by reacting with a well-conserved histidine residue located on the edge of the peptide-binding pocket. SC is an essential component of the bacterial DNA replication machinery, identified as a promising target for the development of new antibacterial compounds. Thermodynamic and kinetic analyses of ligands bearing different mild electrophilic warheads confirmed the higher efficiency of the chloroacetamide compared to Michael acceptors. Two high-resolution X-ray structures of covalent inhibitor-SC adducts were obtained, revealing the canonical orientation of the ligand and details of covalent bond formation with histidine. Proteomic studies were consistent with a selective SC engagement by the chloroacetamide-based TCI. Finally, the TCI of SC was substantially more active than the parent noncovalent inhibitor in an in vitro SC-dependent DNA synthesis assay, validating the potential of the approach to design covalent inhibitors of protein-protein interactions targeted to histidine.

Oligourea helix bundle binds detergents with diverse polar head groups.

Here we report a series of crystal structures (and accompanying biophysical data) of an array of diverse detergent guests bound to an oligourea foldamer helix bundle. These results significantly increase our structural and chemical understanding of aqueous guest recognition by oligourea foldamers and will aid the design of further functionalised oligourea-based self-assemblies.

Synthesis and Crystallographic Characterization of Helical Hairpin Oligourea Foldamers.

Invited for the cover of this issue is the group of Gilles Guichard at the University of Bordeaux. The image depicts sketches and technical drawing tools to illustrate the creation and precise characterization of foldamer tertiary structures. Read the full text of the article at 10.1002/chem.202300087.

Unexpected binding modes of inhibitors to the histone chaperone ASF1 revealed by a foldamer scanning approach.

In the search for foldamer inhibitors of the histone chaperone ASF1, we explored the possibility of substituting four α-residues (≈one helix turn) by 3-urea segments and scanned the sequence of a short α-helical peptide known to bind ASF1. By analysing the impact of the different foldamer replacements within the peptide chain, we uncovered new binding modes of the peptide-urea chimeras to ASF1.

Synthesis and Crystallographic Characterization of Helical Hairpin Oligourea Foldamers.

Oligomers designed to form a helix-turn-helix super-secondary structure have been prepared by covalently bridging aliphatic oligourea foldamer helices with either rigid aromatic or more flexible aliphatic spacers. The relative helix orientation in these dimers was investigated at high resolution using X-ray diffraction analysis. In several cases, racemic crystallography was used to facilitate crystallization and structure determination. All structures were solved by direct methods. Well-defined parallel helical hairpin motifs were observed in all cases when 4,4'-methylene diphenyl diisocyanate was employed as a dimerizing agent, irrespective of primary sequence and chain length.

PSL Chemical Biology Symposia Third Edition: A Branch of Science in its Explosive Phase.

This symposium is the third PSL (Paris Sciences & Lettres) Chemical Biology meeting (2016, 2019, 2023) held at Institut Curie. This initiative originally started at Institut de Chimie des Substances Naturelles (ICSN) in Gif-sur-Yvette (2013, 2014), under the directorship of Professor Max Malacria, with a strong focus on chemistry. It was then continued at the Institut Curie (2015) covering a larger scope, before becoming the official PSL Chemical Biology meeting. This latest edition was postponed twice for the reasons that we know. This has given us the opportunity to invite additional speakers of great standing. This year, Institut Curie hosted around 300 participants, including 220 on site and over 80 online. The pandemic has had, at least, the virtue of promoting online meetings, which we came to realize is not perfect but has its own merits. In particular, it enables those with restricted time and resources to take part in events and meetings, which can now accommodate unlimited participants. We apologize to all those who could not attend in person this time due to space limitation at Institut Curie.

Cell-free synthesis of amyloid fibrils with infectious properties and amenable to sub-milligram magic-angle spinning NMR analysis.

Structural investigations of amyloid fibrils often rely on heterologous bacterial overexpression of the protein of interest. Due to their inherent hydrophobicity and tendency to aggregate as inclusion bodies, many amyloid proteins are challenging to express in bacterial systems. Cell-free protein expression is a promising alternative to classical bacterial expression to produce hydrophobic proteins and introduce NMR-active isotopes that can improve and speed up the NMR analysis. Here we implement the cell-free synthesis of the functional amyloid prion HET-s(218-289). We present an interesting case where HET-s(218-289) directly assembles into infectious fibril in the cell-free expression mixture without the requirement of denaturation procedures and purification. By introducing tailored 13C and 15N isotopes or CF3 and 13CH2F labels at strategic amino-acid positions, we demonstrate that cell-free synthesized amyloid fibrils are readily amenable to high-resolution magic-angle spinning NMR at sub-milligram quantity.

Reaching High Stereoselectivity and Activity in Organocatalyzed Ring-Opening Polymerization of Racemic Lactide by the Combined Use of a Chiral (Thio)Urea and a N-Heterocyclic Carbene.

Stereochemical control during polymerization is a key strategy of polymer chemistry to achieve semicrystalline engineered plastics. The stereoselective ring-opening polymerization (ROP) of racemic lactide (rac-LA), which can lead to highly isotactic polylactide (PLA), is one of the emblematic examples in this area. Surprisingly, stereoselective ROP of rac-LA employing chiral organocatalysts has been under-leveraged. Here we show that a commercially available chiral thiourea (TU1), or its urea homologue (U1), can be used in conjunction with an appropriately selected N-heterocyclic carbene (NHC) to trigger the stereoselective ROP of rac-LA at room temperature in toluene. Both a high organic catalysis activity (>90% monomer conversion in 5-9 h) and a high stereoselectivity (probability of formation of meso dyads, Pm, in the range 0.82-0.93) can be achieved by thus pairing a NHC and a chiral amino(thio)urea. The less sterically hindered and the more basic NHC, that is, a NHC bearing tert-butyl substituents (NHCtBu), provides the highest stereoselectivity when employed in conjunction with the chiral TU1 or U1. This asymmetric organic catalysis strategy, as applied here in polymerization chemistry, further expands the field of possibilities to achieve bioplastics with adapted thermomechanical properties.

Hexafluoroisobutylation of enolates through a tandem elimination/allylic shift/hydrofluorination reaction.

The isobutyl side chain is a highly prevalent hydrophobic group in drugs, and it notably constitutes the side chain of leucine. Its replacement by a hexafluorinated version containing two CF3 groups may endow the target compound with new and advantageous properties, yet this modification remains overlooked due to the absence of a general and practical synthetic methodology. Herein, we report the first general method to introduce the hexafluoroisobutyl group into ketoesters, malonates, 1,3-diketones, Schiff base esters and malononitrile. We demonstrated that the reaction occurs through an elimination/allylic shift/hydrofluorination cascade process which efficiently overcomes the usual fluoride ß-elimination observed with α-CF3-vinyl groups. We showed that with alkali metal bases, a pentafluorinated alkene is obtained predominantly, whereas the use of tetrabutylammonium fluoride (TBAF) allows hydrofluorination to occur. This tandem process represents a conceptually new pathway to synthesize bis-trifluoromethylated compounds. This methodology was applied to the multigram-scale synthesis of enantiopure (S)-5,5,5,5',5',5'-hexafluoroleucine.

Adaptive Binding of Alkyl Glycosides by Nonpeptidic Helix Bundles in Water: Toward Artificial Glycolipid Binding Proteins.

Amphipathic water-soluble helices formed from synthetic peptides or foldamers are promising building blocks for the creation of self-assembled architectures with non-natural shapes and functions. While rationally designed artificial quaternary structures such as helix bundles have been shown to contain preformed cavities suitable for guest binding, there are no examples of adaptive binding of guest molecules by such assemblies in aqueous conditions. We have previously reported a foldamer 6-helix bundle that contains an internal nonpolar cavity able to bind primary alcohols as guest molecules. Here, we show that this 6-helix bundle can also interact with larger, more complex guests such as n-alkyl glycosides. X-ray diffraction analysis of co-crystals using a diverse set of guests together with solution and gas-phase studies reveals an adaptive binding mode whereby the apo form of the 6-helix bundle undergoes substantial conformational change to accommodate the hydrocarbon chain in a manner reminiscent of glycolipid transfer proteins in which the cavity forms upon lipid uptake. The dynamic nature of the self-assembling and molecular recognition processes reported here marks a step forward in the design of functional proteomimetic molecular assemblies.

Insight into Substrate Recognition by Urea-Based Helical Foldamer Catalysts Using a DFT Global Optimization Approach.

Peptides and foldamers have recently gained increasing attention as chiral catalysts to achieve challenging (asymmetric) transformations. We previously reported that short helically folded aliphatic oligoureas in combination with achiral Brønsted bases are effective H-bonding catalysts for C-C bond-forming reactions─i.e., the conjugate addition of 1,3-dicarbonyl pronucleophiles to nitroalkenes─with high reactivity and selectivity and at remarkably low chiral catalyst/substrate molar ratios. This theoretical investigation at the density functional theory level of theory, aims to both analyze how the substrates of the reaction interact with the foldamer catalyst and rationalize a chain-length dependence effect on the catalytic properties. We confirm that the first two ureas are the only H-bond donors available to interact with external molecules. Moreover, each urea site interacts with one of the two reactants allowing a short distance between the two reacting carbons, thus facilitating the conjugated addition. Additionally, it was observed that the molecular recognition and catalyst-substrate interactions are mainly governed by electrostatic interactions but not orbital interactions (see from NBO if this is finally true). On these grounds, an electrostatic potential (ESP) analysis showed an important internal charge separation in the catalyst, the positive ESP region being concentrated around the first two ureas, with its area extending as the number of residues increases.

Block Copolymer Synthesis by a Sequential Addition Strategy from the Organocatalytic Group Transfer Polymerization of Methyl Methacrylate to the Ring-Opening Polymerization of Lactide.

Sequential block copolymerization involving comonomers belonging to different classes, e.g., a vinyl-type monomer and a heterocycle, is a challenging task in macromolecular chemistry, as corresponding propagating species do not interconvert easily from one to the other by crossover reactions. Here, it is first evidenced that 1-methoxy 2-methyl 1-trimethylsilyloxypropene (MTS), i.e., a silyl ketene acetal (SKA)-containing initiator, can be used in presence of the P4 -t-Bu phosphazene organic base to control the ring-opening polymerization (ROP) of racemic lactide (rac-LA). The elementary reaction, which rapidly transforms SKA groups into propagating alkoxides, can be leveraged to directly synthesize well-defined poly(methyl methacrylate)-b-polylactide block copolymers. This is achieved using P4 -t-Bu as the single organic catalyst and MTS as the initiator for the group transfer polymerization of methyl methacrylate, followed by the ROP of rac-LA. Both polymerization methods are implemented under selective and controlled/living conditions at room temperature in THF. This sequential addition strategy further expands the scope of organic catalysis of polymerizations for macromolecular engineering of block copolymers involving propagating species of disparate reactivity.

Design of Oligourea-Based Foldamers with Antibacterial and Antifungal Activities.

There is an urgent need to develop new therapeutic strategies to fight the emergence of multidrug resistant bacteria. Many antimicrobial peptides (AMPs) have been identified and characterized, but clinical translation has been limited partly due to their structural instability and degradability in physiological environments. The use of unnatural backbones leading to foldamers can generate peptidomimetics with improved properties and conformational stability. We recently reported the successful design of urea-based eukaryotic cell-penetrating foldamers (CPFs). Since cell-penetrating peptides and AMPs generally share many common features, we prepared new sequences derived from CPFs by varying the distribution of histidine- and arginine-type residues at the surface of the oligourea helix, and evaluated their activity on both Gram-positive and Gram-negative bacteria as well as on fungi. In addition, we prepared and tested new amphiphilic block cofoldamers consisting of an oligourea and a peptide segment whereby polar and charged residues are located in the peptide segment and more hydrophobic residues in the oligourea segment. Several foldamer sequences were found to display potent antibacterial activities even in the presence of 50% serum. Importantly, we show that these urea-based foldamers also possess promising antifungal properties.

Iterative Structure-Based Optimization of Short Peptides Targeting the Bacterial Sliding Clamp.

The bacterial DNA sliding clamp (SC), or replication processivity factor, is a promising target for the development of novel antibiotics. We report a structure-activity relationship study of a new series of peptides interacting within the Escherichia coli SC (EcSC) binding pocket. Various modifications were explored including N-alkylation of the peptide bonds, extension of the N-terminal moiety, and introduction of hydrophobic and constrained residues at the C-terminus. In each category, single modifications were identified that increased affinity to EcSC. A combination of such modifications yielded in several cases to a substantially increased affinity compared to the parent peptides with Kd in the range of 30-80 nM. X-ray structure analysis of 11 peptide/EcSC co-crystals revealed new interactions at the peptide-protein interface (i.e., stacking interactions, hydrogen bonds, and hydrophobic contacts) that can account for the improved binding. Several compounds among the best binders were also found to be more effective in inhibiting SC-dependent DNA synthesis.

Crystal structures capture multiple stoichiometric states of an aqueous self-assembling oligourea foldamer.

We report here an oligourea foldamer able to self-assemble in aqueous conditions into helix bundles of multiple stoichiometries. Importantly, we report crystal structures of several of these stoichiometries, providing a series of high-resolution snap-shots of the structural polymorphism of this foldamer and uncovering a novel self-assembly.

Correction: Antibacterial activity of a dual peptide targeting the Escherichia coli sliding clamp and the ribosome.

[This corrects the article DOI: 10.1039/D0CB00060D.].

Urea based foldamers.

N,N'-linked oligoureas are a class of enantiopure, sequence-defined peptidomimetic oligomers without amino acids that form well-defined and predictable helical structures akin to the peptide α-helix. Oligourea-based foldamers combine a number of features-such as synthetic accessibility, sequence modularity, and folding fidelity-that bode well for their use in a range of applications from medicinal chemistry to catalysis. Moreover, it was recently recognized that this synthetic helical backbone can be combined with regular peptides to generate helically folded peptide-oligourea hybrids that display additional features in terms of helix mimicry and protein-surface recognition properties. Here we provide detailed protocols for the preparation of requested monomers and for the synthesis and purification of homo-oligoureas and peptide-oligourea hybrids.

Optimal anchoring of a foldamer inhibitor of ASF1 histone chaperone through backbone plasticity.

Sequence-specific oligomers with predictable folding patterns, i.e., foldamers, provide new opportunities to mimic α-helical peptides and design inhibitors of protein-protein interactions. One major hurdle of this strategy is to retain the correct orientation of key side chains involved in protein surface recognition. Here, we show that the structural plasticity of a foldamer backbone may notably contribute to the required spatial adjustment for optimal interaction with the protein surface. By using oligoureas as α helix mimics, we designed a foldamer/peptide hybrid inhibitor of histone chaperone ASF1, a key regulator of chromatin dynamics. The crystal structure of its complex with ASF1 reveals a notable plasticity of the urea backbone, which adapts to the ASF1 surface to maintain the same binding interface. One additional benefit of generating ASF1 ligands with nonpeptide oligourea segments is the resistance to proteolysis in human plasma, which was highly improved compared to the cognate α-helical peptide.

A chiral thiourea and a phosphazene for fast and stereoselective organocatalytic ring-opening-polymerization of racemic lactide.

Control of stereoregularity is inherent to precision polymerization chemistry for the development of functional materials. A prototypal example of this strategy is the ring-opening polymerization (ROP) of racemic lactide (rac-LA), a bio-sourced monomer. Despite significant advances in organocatalysis, stereoselective ROP of rac-LA employing chiral organocatalysts remains unexplored. Here we tackle that challenge by resorting to Takemoto's catalyst, a chiral aminothiourea, in the presence of a phosphazene base. This chiral binary organocatalytic system allows for fast, chemo- and stereoselective ROP of rac-LA at room temperature, yielding highly isotactic, semi-crystalline and metal-free polylactide, with a melting temperature as high as 187 °C.

Delivery of siRNA by tailored cell-penetrating urea-based foldamers.

Cell-penetrating foldamers (CPFs) have recently shown promise as efficient and safe nucleic acid delivery systems. However, the application of CPFs to siRNA transport remains scarce. Here, we report helical CPFs tailored with specific end-groups (pyridylthio- or n-octyl-ureas) as effective molecular systems in combination with helper lipids to intracellularly deliver biologically-relevant siRNA.