Extreme thrombocytosis with an aggressive evolution harboring a novel variant of calreticulin (CALR) in exon 3.

We describe the case of a patient with extreme thrombocytosis whose evolution was rapidly fatal. No cause of secondary thrombocytosis was found. There was no sign of myelofibrosis but the megakaryocytes were small and dysplastic. The patient presented a calreticulin (CALR) variant in exon 3 (C105S), as well as concomitant mutations of ASXL1, U2AF1, and EZH2. This variant of CALR has never been described before, and after sorting, all identified mutations were found in myeloid cells but not in lymphoid cells. Therefore, the diagnosis of a frontier case of myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN) was made. A treatment with hydroxycarbamide was started because of a high risk of thrombosis. Upon worsening of the hematological status two new mutations appeared, SETBP1 and ETV6, and the CALR mutation was still detectable, as well as the three other mutations found in the chronic stage. Our results show that this variant could contribute to MDS/MPN pathogenesis in that patient.

Bergofungin D, a peptaibol template for the introduction of chemical modifications, synthesis of analogs and comparative studies of their structures.

Bergofungin D is a helical peptide of the peptaibol family consisting of 14 amino acids, six of which are the helix inducer aminoisobutyric acid (Aib). In the second third of the sequence, a hydroxyproline causes a bending of the helix and a disruption of the hydrogen bond network, and Aib7 is the only amino acid in this region involved in the hydrogen bond network. Therefore, modification of this residue can serve as a probe to monitor the effect of introducing amino acid substitutions on this more fragile helical turn. To validate this approach, we simplified the original bergofungin D by reducing the number of non-classical amino acids, replacing the (R)-isovaleric acid by its enantiomer or an Aib and the hydroxyproline with a proline, respectively, without affecting its secondary structure. Within the modified structure, we replaced Aib7-Aib8 by its 1,2,3-triazolodipeptide equivalent or Aib7 by a serine or a dehydrobutyrine. We have reported and analyzed five crystal structures, three of which are new, demonstrating the usefulness of the modified bergofungin D as a probe for monitoring the introduction of amino acid substitutions within a helical structure.

Planar Chiral p,p'-Terphenyl-Based Cyclophanes with Remarkable Enantiomer Stability: Synthesis, Theoretical Investigations, and Complexation Studies.

Design of conformationally stable compounds with planar chirality is a topic of great interest mainly because of their potential applications as enantioselective ligands or other functional materials. Herein, we present the design and synthesis of novel planar chiral cyclophanes, obtained by ortho, ortho″ anchoring of the p,p'-terphenyl unit, with bridges of different lengths and rigidities, along with their nuclear magnetic resonance, mass spectrometry, and X-ray characterizations. We investigated the influence of the structural particularities of the bridges over the stability of the enantiomers, by means of nuclear magnetic resonance and chiral high-performance liquid chromatography as well as by density functional theory calculations. We also demonstrated the ability of one of the cyclophanes to preferentially bind arginine with Ka > 110 M-1 (ΔG > -11 kJ mol-1) in acetonitrile solutions containig 10 % water, in the presence of other amino acids.

1-Aminobicyclo[2.2.2]octane-2-carboxylic Acid and Derivatives As Chiral Constrained Bridged Scaffolds for Foldamers and Chiral Catalysts.

The improvement of molecular diversity is one of the major concerns of chemists since the continuous development of original synthetic molecules provides unique scaffolds usable in organic and bioorganic chemistry. The challenge is to develop versatile platforms with highly controlled chemical three-dimensional space thanks to controlled chirality and conformational restraints. In this respect, cyclic ß-amino acids are of great interest with applications in various fields of chemistry. In addition to their intrinsic biological properties, they are important precursors for the synthesis of new generations of bioactive compounds such as antibiotics, enzyme inhibitors, and antitumor agents. They have also been involved in asymmetric synthesis as efficient organo-catalysts in their free form and as derivatives. Finally, constrained cyclic ß-amino acids have been incorporated into oligomers to successfully stabilize original structures in foldamer science with recent successes in health, material science, and catalysis. Over the last ∼10 years, we focused on bicyclic ß-amino acids possessing a bicyclo[2.2.2]octane structure. This latter is a structural key element in numerous families of biologically active natural and synthetic products and is an interesting template for asymmetric synthesis. Nonetheless, reported studies on bicyclic carbo-bridged compounds are rather limited compared to those on bicyclic-fused and heterobridged derivatives. In this Account, we particularly focused on the synthesis and applications of the 1-aminobicyclo[2.2.2]octane-2-carboxylic acid, named, ABOC, and its derivatives. This highly constrained bicyclic ß-amino acid, with a sterically hindered bridgehead primary amine and an endocyclic chiral center, displays drastically reduced conformational freedom. In addition, its high bulkiness strongly impacts the spatial orientation of the appended functionalities and the conformation of adjacent building blocks. Thus, we have first expanded a fundamental synthetic work by a wide ranging study in the field of foldamers, in the design of various stable peptide/peptidomimetic helical structures incorporating the ABOC residue (11/9-, 18/16-, 12/14/14-, and 12/10-helices). In addition, such bicyclic residue was fully compatible with and stabilized the canonical oligourea helix, whereas very few cyclic ß-amino acids have been incorporated into oligoureas. In addition, we have pursued with the synthesis of some ABOC derivatives, in particular the 1,2-diaminobicyclo[2.2.2]octane chiral diamine, named DABO, and its investigation in chiral catalytic systems. Covalent organo-catalysis of the aldol reaction using ABOC-containing tripeptide catalysts provided a range of aldol products with high enantioselectivity. Moreover, the double reductive condensation of DABO with various aldehydes allowed the building of new chiral ligands that proved their efficiency in the copper-catalyzed asymmetric Henry reaction.

The Unexpected Helical Supramolecular Assembly of a Simple Achiral Acetamide Tecton Generates Selective Water Channels.

Invited for the cover of this issue is the collaborative research team coordinated by Arie van der Lee at the University of Montpellier. The image depicts chiral channels with highly mobile water molecules resulting from the robust self-organization of a simple achiral acetamide. Fully reversible release and re-uptake of water molecules takes place near ambient conditions, with efficient water transport and a good selectivity against NaCl suggesting it to be an efficient candidate for desalination processes. Read the full text of the article at 10.1002/chem.20200383.

The Unexpected Helical Supramolecular Assembly of a Simple Achiral Acetamide Tecton Generates Selective Water Channels.

Achiral 2-hydroxy-N-(diphenylmethyl)acetamide (HNDPA) crystallizes in the P61 chiral space group as a hydrate, building up permeable chiral crystalline helical water channels. The crystallization-driven chiral self-resolution process is highly robust, with the same air-stable crystalline form readily obtained under a variety of conditions. Interestingly, the HNDPA supramolecular helix inner pore is filled by a helical water wire. The whole edifice is mainly stabilized by robust hydrogen bonds involving the HNDPA amide bonds and CH… π interactions between the HNDPA phenyl groups. The crystalline structure shows breathing behavior, with completely reversible release and re-uptake of water inside the chiral channel under ambient conditions. Importantly, the HNDPA channel is able to transport water very efficiently and selectively under biomimetic conditions. With a permeability per channel of 3.3 million water molecules per second in large unilamellar vesicles (LUV) and total selectivity against NaCl, the HNDPA channel is a very promising functional nanomaterial for future applications.

The C-terminal segment of Leishmania major HslU: Toward potential inhibitors of LmHslVU activity.

It is urgent to develop less toxic and more efficient treatments for leishmaniases and trypanosomiases. We explore the possibility to target the parasite mitochondrial HslVU protease, which is essential for growth and has no analogue in the human host. For this, we develop compounds potentially inhibiting the complex assembly by mimicking the C-terminal (C-ter) segment of the ATPase HslU. We previously showed that a dodecapeptide derived from Leishmania major HslU C-ter segment (LmC12-U2, Cpd 1) was able to bind to and activate the digestion of a fluorogenic substrate by LmHslV. Here, we present the study of its structure-activity relationships. By replacing each essential residue with related non-proteinogenic residues, we obtained more potent analogues. In particular, a cyclohexylglycine residue at position 11 (cpd 24) allowed a more than three-fold gain in potency while reducing the size of compound 24 from twelve to six residues (cpd 50) without significant loss of potency, opening the way toward short HslU C-ter peptidomimetics as potential inhibitors of HslV proteolytic function. Finally, conjugates constituted of LmC6-U2 analogues and a mitochondrial penetrating peptide were found to penetrate into the promastigote form of L. infantum and to inhibit the parasite growth without showing toxicity toward human THP-1 cells at the same concentration (i.e. 30 µM).

Helical γ-Peptide Foldamers as Dual Inhibitors of Amyloid-ß Peptide and Islet Amyloid Polypeptide Oligomerization and Fibrillization.

Type 2 diabetes (T2D) and Alzheimer's disease (AD) belong to the 10 deadliest diseases and are sorely lacking in effective treatments. Both pathologies are part of the degenerative disorders named amyloidoses, which involve the misfolding and the aggregation of amyloid peptides, hIAPP for T2D and Aß1-42 for AD. While hIAPP and Aß1-42 inhibitors have been essentially designed to target ß-sheet-rich structures composing the toxic amyloid oligomers and fibrils of these peptides, the strategy aiming at trapping the non-toxic monomers in their helical native conformation has been rarely explored. We report herein the first example of helical foldamers as dual inhibitors of hIAPP and Aß1-42 aggregation and able to preserve the monomeric species of both amyloid peptides. A foldamer composed of 4-amino(methyl)-1,3-thiazole-5-carboxylic acid (ATC) units, adopting a 9-helix structure reminiscent of 310 helix, was remarkable as demonstrated by biophysical assays combining thioflavin-T fluorescence, transmission electronic microscopy, capillary electrophoresis and mass spectrometry.

Prospect of Thiazole-based γ-Peptide Foldamers in Enamine Catalysis: Exploration of the Nitro-Michael Addition.

As three-dimensional folding is prerequisite to biopolymer activity, complex functions may also be achieved through foldamer science. Because of the diversity of sizes, shapes and folding available with synthetic monomers, foldamer frameworks enable a numerous opportunities for designing new generations of catalysts. We herein demonstrate that heterocyclic γ-peptide scaffolds represent a versatile platform for enamine catalysis. One central feature was to determine how the catalytic activity and the transfer of chiral information might be under the control of the conformational behaviours of the oligomer.

The HslV Protease from Leishmania major and Its Activation by C-terminal HslU Peptides.

HslVU is an ATP-dependent proteolytic complex present in certain bacteria and in the mitochondrion of some primordial eukaryotes, including deadly parasites such as Leishmania. It is formed by the dodecameric protease HslV and the hexameric ATPase HslU, which binds via the C-terminal end of its subunits to HslV and activates it by a yet unclear allosteric mechanism. We undertook the characterization of HslV from Leishmania major (LmHslV), a trypanosomatid that expresses two isoforms for HslU, LmHslU1 and LmHslU2. Using a novel and sensitive peptide substrate, we found that LmHslV can be activated by peptides derived from the C-termini of both LmHslU1 and LmHslU2. Truncations, Ala- and D-scans of the C-terminal dodecapeptide of LmHslU2 (LmC12-U2) showed that five out of the six C-terminal residues of LmHslU2 are essential for binding to and activating HslV. Peptide cyclisation with a lactam bridge allowed shortening of the peptide without loss of potency. Finally, we found that dodecapeptides derived from HslU of other parasites and bacteria are able to activate LmHslV with similar or even higher efficiency. Importantly, using electron microscopy approaches, we observed that the activation of LmHslV was accompanied by a large conformational remodeling, which represents a yet unidentified layer of control of HslV activation.

Indoloazepinone-Constrained Oligomers as Cell-Penetrating and Blood-Brain-Barrier-Permeating Compounds.

Non-cationic and amphipathic indoloazepinone-constrained (Aia) oligomers have been synthesized as new vectors for intracellular delivery. The conformational preferences of the [l-Aia-Xxx]n oligomers were investigated by circular dichroism (CD) and NMR spectroscopy. Whereas Boc-[l-Aia-Gly]2,4 -OBn oligomers 12 and 13 and Boc-[l-Aia-ß3 -h-l-Ala]2,4 -OBn oligomers 16 and 17 were totally or partially disordered, Boc-[l-Aia-l-Ala]2 -OBn (14) induced a typical turn stabilized by C5 - and C7 -membered H-bond pseudo-cycles and aromatic interactions. Boc-[l-Aia-l-Ala]4 -OBn (15) exhibited a unique structure with remarkable T-shaped π-stacking interactions involving the indole rings of the four l-Aia residues forming a dense hydrophobic cluster. All of the proposed FITC-6-Ahx-[l-Aia-Xxx]4 -NH2 oligomers 19-23, with the exception of FITC-6-Ahx-[l-Aia-Gly]4 -NH2 (18), were internalized by MDA-MB-231 cells with higher efficiency than the positive references penetratin and Arg8 . In parallel, the compounds of this series were successfully explored in an in vitro blood-brain barrier (BBB) permeation assay. Although no passive diffusion permeability was observed for any of the tested Ac-[l-Aia-Xxx]4 -NH2 oligomers in the PAMPA model, Ac-[l-Aia-l-Arg]4 -NH2 (26) showed significant permeation in the in vitro cell-based human model of the BBB, suggesting an active mechanism of cell penetration.

Selectivity Modulation and Structure of α/aza-ß3 Cyclic Antimicrobial Peptides.

Potent and selective antimicrobial cyclic pseudopeptides (ACPPs) mixing α- and aza-ß3 -amino acids were developed. Cyclopseudopeptide sequences were designed to investigate the impact of some intrinsic molecular parameters on their biological activities. Fine changes in the nature of the side chains strongly modulated the selectivity of the ACPPs with regard to hemolysis versus antimicrobial activity. The conformational preference of such compounds in various media was extensively studied, and the typical structure of cyclic α/aza-ß3 -pseudopeptides is described for the first time. Interestingly, such scaffolds are stabilized by successive inverse γ- and N-N turns (hydrazino turns), a unique feature due to the aza-ß3 residues. The α-amino acid side chains form a cluster on one face of the ring, while the aza-ß3 -amino acid side chains are projected around the ring in the equatorial orientation. Such structural data are particularly valuable to fine-tune the bioactivity of these ACPPs by a structure-based approach.

12/10-Helix in Mixed ß-Peptides Alternating Bicyclic and Acyclic ß-Amino Acids: Probing the Relationship between Bicyclic Side Chain and Helix Stability.

12/10-Helices constitute suitable templates that can be used to design original structures. Nevertheless, they often suffer from a weak stability in polar solvents because they exhibit a mixed hydrogen-bond network resulting in a small macrodipole. In this work, stable and functionalizable 12/10-helices were developed by alternating a highly constrained ß2, 3, 3 -trisubstituted bicyclic amino acid (S)-1-aminobicyclo[2.2.2]octane-2-carboxylic acid ((S)-ABOC) and an acyclic substituted ß-homologated proteinogenic amino acid (l-ß3 -hAA). Based on NMR spectroscopic analysis, it was shown that such mixed ß-peptides display well-defined right-handed 12/10-helices in polar, apolar, and chaotropic solvents; that are, CD3 OH, CDCl3 , and [D6 ]DMSO, respectively. The stability of the hydrogen bonds forming the C10 and C12 pseudocycles as well as the benefit provided by the use of the constrained bicyclic ABOC versus typical acyclic ß-amino acids sequences when designing 12/10-helix were investigated using NH/ND NMR exchange experiments and DFT calculations in various solvents. These studies showed that the ß3 -hAA/(S)-ABOC helix displayed a more stable hydrogen-bond network through specific stabilization of the C10 pseudocycles involving the bridgehead NH of the ABOC bicyclic scaffold.

How are 1,2,3-triazoles accommodated in helical secondary structures?

1,4-Disubstituted-1,2,3-triazole (Tz) is widely used in peptides as a trans-amide bond mimic, despite having hazardous effects on the native peptide activity. The impact of amide bond substitution by Tz on peptide secondary structures is scarcely documented. We performed a Tz scan, by systematically replacing peptide bonds following the Aib residues with Tz on two model peptaibols: alamethicin F50/5 and bergofungin D, which adopt stable α- and 310 helices, respectively. We observed that the Tz insertion, whatever its position in the peptide sequences, abolished their antimicrobial activity. The structural consequences of this insertion were further investigated using CD, NMR and X-ray diffraction. Importantly, five crystal structures that were incorporated with Tz were solved, showing various degrees of alteration of the helical structures, from minor structural perturbation of the helix to partial disorder. Together, these results showed that Tz insertions impair helical secondary structures.

Ribbon-like Foldamers for Cellular Uptake and Drug Delivery.

Different intracellular delivery systems of bioactive compounds have been developed, including cell-penetrating peptides. Although usually nontoxic and biocompatible, these vectors share some of the general drawbacks of peptides, notably low bioavailability and susceptibility to protease degradation, that limit their use. Herein, the conversion of short peptide sequences into poly-α-amino-γ-lactam foldamers that adopt a ribbon-like structure is investigated. This template is used to distribute critical cationic and/or hydrophobic groups on both sides of the backbone, leading to potent short, cell-permeable foldamers with a low positive-charge content. The lead compound showed dramatically improved protease resistance and was able to efficiently deliver a biologically relevant cargo inside cells. This study provided a simple strategy to convert short peptide sequences into efficient protease-resistant cell-penetrating foldamers.

Enhancing the Antimicrobial Activity of Alamethicin F50/5 by Incorporating N-terminal Hydrophobic Triazole Substituents.

A simple and efficient strategy is proposed to significantly improve the antibacterial activity of peptaibols and other antimicrobial peptides by N-terminal capping with 1,2,3-triazole bearing various hydrophobic substituents on C-4. Such N-terminal insertions on alamethicin F50/5 could enhance its antimicrobial activity on Gram-positive bacteria without modification of its overall three-dimensional structure. Although the native peptide and its analogues shared comparable helical contents, the crystal structure of one of the most active derivative showed a local slight distortion of the N-terminal extremity, which was also observed in solution using NMR spectroscopy. Importantly, fluorescence studies showed that the N-capped derivatives had increased affinity for liposomes, which may indicate they interacted more strongly with the bacterial membrane than alamethicin F50/5.

C9/12 Ribbon-Like Structures in Hybrid Peptides Alternating α- and Thiazole-Based γ-Amino Acids.

According to their restricted conformational freedom, heterocyclic γ-amino acids are usually considered to be related to Z-vinylogous γ-amino acids. In this context, oligomers alternating α-amino acids and thiazole-based γ-amino acids (ATCs) were expected to fold into a canonical 12-helical shape as described for α/γ-hybrid peptides composed of cis-α/ß-unsaturated γ-amino acids. However, through a combination of X-ray crystallography, NMR spectroscopy, FTIR experiments, and DFT calculations, it was determined that the folding behavior of ATC-containing hybrid peptides is much more complex. The homochiral α/(S)-ATC sequences were unable to adopt a stable conformation, whereas the heterochiral α/(R)-ATC peptides displayed novel ribbon structures stabilized by unusual C9/12 -bifurcated hydrogen bonds. These ribbon structures could be considered as a succession of pre-organized γ/α dipeptides and may provide the basis for designing original α-helix mimics.

LEON-BIS: multiple alignment evaluation of sequence neighbours using a Bayesian inference system.

BACKGROUND: A standard procedure in many areas of bioinformatics is to use a multiple sequence alignment (MSA) as the basis for various types of homology-based inference. Applications include 3D structure modelling, protein functional annotation, prediction of molecular interactions, etc. These applications, however sophisticated, are generally highly sensitive to the alignment used, and neglecting non-homologous or uncertain regions in the alignment can lead to significant bias in the subsequent inferences. RESULTS: Here, we present a new method, LEON-BIS, which uses a robust Bayesian framework to estimate the homologous relations between sequences in a protein multiple alignment. Sequences are clustered into sub-families and relations are predicted at different levels, including 'core blocks', 'regions' and full-length proteins. The accuracy and reliability of the predictions are demonstrated in large-scale comparisons using well annotated alignment databases, where the homologous sequence segments are detected with very high sensitivity and specificity. CONCLUSIONS: LEON-BIS uses robust Bayesian statistics to distinguish the portions of multiple sequence alignments that are conserved either across the whole family or within subfamilies. LEON-BIS should thus be useful for automatic, high-throughput genome annotations, 2D/3D structure predictions, protein-protein interaction predictions etc.

12/14/14-Helix Formation in 2:1 α/ß-Hybrid Peptides Containing Bicyclo[2.2.2]octane Ring Constraints.

The highly constrained ß-amino acid ABOC induces different types of helices in ß urea and 1:1 α/ß amide oligomers. The latter can adopt 11/9- and 18/16-helical folds depending on the chain length in solution. Short peptides alternating proteinogenic α-amino acids and ABOC in a 2:1 α/ß repeat pattern adopted an unprecedented and stable 12/14/14-helix. The structure was established through extensive NMR, molecular dynamics, and IR studies. While the 1:1 α-AA/ABOC helices diverged from the canonical α-helix, the helix formed by the 9-mer 2:1 α/ß-peptide allowed the projection of the α-amino acid side chains in a spatial arrangement according to the α-helix. Such a finding constitutes an important step toward the conception of functional tools that use the ABOC residue as a potent helix inducer for biological applications.

Conformationally Constrained Peptidomimetics as Inhibitors of the Protein Arginine Methyl Transferases.

Protein arginine N-methyl transferases (PRMTs) belong to a family of enzymes that modulate the epigenetic code through modifications of histones. In the present study, peptides emerging from a phage display screening were modified in the search for PRMT inhibitors through substitution with non-proteinogenic amino acids, N-alkylation of the peptide backbone, and incorporation of constrained dipeptide mimics. One of the modified peptides (23) showed an increased inhibitory activity towards several PRMTs in the low µm range and the conformational preference of this peptide was investigated and compared with the original hit using circular dichroism and NMR spectroscopy. Introducing two constrained tryptophan residue mimics (l-Aia) spaced by a single amino acid was found to induce a unique turn structure stabilized by a hydrogen bond and aromatic π-stacking interaction between the two l-Aia residues.