Exploiting the Innate Plasticity of the Programmed Cell Death-1 (PD1) Receptor to Design Pembrolizumab H3 Loop Mimics.

Checkpoint blockade of the immunoreceptor programmed cell death-1 (PD1) with its ligand-1 (PDL1) by monoclonal antibodies such as pembrolizumab provided compelling clinical results in various cancer types, yet the molecular mechanism by which this drug blocks the PD1/PDL1 interface remains unclear. To address this question, we examined the conformational motion of PD1 associated with the binding of pembrolizumab. Our results revealed that the innate plasticity of both C'D and FG loops is crucial to form a deep binding groove (371 Å3 ) across several distant epitopes of PD1. This analysis ultimately provided a rational-design to create pembrolizumab H3 loop mimics [RDYRFDMGFD] into ß-hairpin scaffolds. As a result, a 20-residue long ß-hairpin peptide 1 e was identified as a first-in-class potent PD1-inhibitor (EC50 of 0.29 µM; Ki of 41 nM).

Crystal structure of Mokola virus glycoprotein in its post-fusion conformation.

Mokola virus (MOKV) belongs to the lyssavirus genus. As other genus members-including rabies virus (RABV)-it causes deadly encephalitis in mammals. MOKV entry into host cells is mediated by its transmembrane glycoprotein G. First, G binds cellular receptors, triggering virion endocytosis. Then, in the acidic endosomal environment, G undergoes a conformational change from its pre- toward its post-fusion state that catalyzes the merger of the viral and endosomal membranes. Here, we have determined the crystal structure of a soluble MOKV G ectodomain in which the hydrophobic fusion loops have been replaced by more hydrophilic sequences. The crystal structure corresponds to a monomer that is similar to the protomer of the trimeric post-fusion state of vesicular stomatitis virus (VSV) G. However, by electron microscopy, we show that, at low pH, at the surface of pseudotyped VSV, MOKV spikes adopt the trimeric post-fusion conformation and have a tendency to reorganize into regular arrays. Sequence alignment between MOKV G and RABV G allows a precise location of RABV G antigenic sites. Repositioning MOKV G domains on VSV G pre-fusion structure reveals that antigenic sites are located in the most exposed part of the molecule in its pre-fusion conformation and are therefore very accessible to antibodies. Furthermore, the structure allows the identification of pH-sensitive molecular switches. Specifically, the long helix, which constitutes the core of the post-fusion trimer for class III fusion glycoproteins, contains many acidic residues located at the trimeric interface. Several of them, aligned along the helix, point toward the trimer axis. They have to be protonated for the post-fusion trimer to be stable. At high pH, when they are negatively charged, they destabilize the interface, which explains the conformational change reversibility. Finally, the present structure will be of great help to perform rational mutagenesis on lyssavirus glycoproteins.

Role of Macrocyclic Conformational Steering in a Kinetic Route toward Bielschowskysin.

Macrocyclic furanobutenolide-derived cembranoids (FBCs) are the biosynthetic precursors to a wide variety of highly congested and oxygenated polycyclic (nor)diterpenes (e.g. plumarellide, verrillin, and bielschowskysin). These architecturally complex metabolites are thought to originate from site-selective oxidation of the macrocycle backbone and a series of intricate transannular reactions. Yet the development of a common biomimetic route has been hampered by a lack of synthetic methods for the pivotal furan dearomatization in a regio- and stereoselective manner. To address these shortcomings, a concise strategy of epoxidation followed by a kinetically controlled furan dearomatization is reported. The surprising switch of facial α:ß-discrimination observed in the epoxidation of the most strained E-acerosolide versus E-deoxypukalide and E-bipinnatin J derived macrocycles has been rationalized by the variation of the 3D conformational landscape between macrocyclic scaffolds. A careful conformational analysis of these macrocycles by VT-NMR and NOESY experiments at low temperature was supported by DFT calculations to characterize these equilibrating macrocyclic conformers. The shift in conformational topology associated with a swing of the butenolide ring in E-deoxypukalide is in general agreement with the reversal of ß-selectivity observed in the epoxidation. We also describe the downstream functionalization of FBC-macrocycles and how the C-7 epoxide configuration is retentively translated to the C-3 stereogenicity in dearomatized products under kinetic control to secure the requisite 3S,7S,8S configurations for the bielschowskysin synthesis. Unlike previously speculated, our results suggest that the most strained FBC-macrocycles bearing a E-(Δ7,8)-alkene moiety may stand as the true biosynthetic precursors to bielschowskysin and several other polycyclic natural products of this class.

Folding in Place: Design of ß-Strap Motifs to Stabilize the Folding of Hairpins with Long Loops.

Despite their pivotal role in defining antibody affinity and protein function, ß-hairpins harboring long noncanonical loops remain synthetically challenging because of the large entropic penalty associated with their conformational folding. Little is known about the contribution and impact of stabilizing motifs on the folding of ß-hairpins with loops of variable length and plasticity. Here, we report a design of minimalist ß-straps (strap = strand + cap) that offset the entropic cost of long-loop folding. The judicious positioning of noncovalent interactions (hydrophobic cluster and salt-bridge) within the novel 8-mer ß-strap design RW(V/H)W···WVWE stabilizes hairpins with up to 10-residue loops of varying degrees of plasticity (Tm up to 52 °C; 88 ± 1% folded at 18 °C). This "hyper" thermostable ß-strap outperforms the previous gold-standard technology of ß-strand-ß-cap (16-mer) and provides a foundation for producing new classes of long hairpins as a viable and practical alternative to macrocyclic peptides.

SMCHD1 is involved in de novo methylation of the DUX4-encoding D4Z4 macrosatellite.

The DNA methylation epigenetic signature is a key determinant during development. Rules governing its establishment and maintenance remain elusive especially at repetitive sequences, which account for the majority of methylated CGs. DNA methylation is altered in a number of diseases including those linked to mutations in factors that modify chromatin. Among them, SMCHD1 (Structural Maintenance of Chromosomes Hinge Domain Containing 1) has been of major interest following identification of germline mutations in Facio-Scapulo-Humeral Dystrophy (FSHD) and in an unrelated developmental disorder, Bosma Arhinia Microphthalmia Syndrome (BAMS). By investigating why germline SMCHD1 mutations lead to these two different diseases, we uncovered a role for this factor in de novo methylation at the pluripotent stage. SMCHD1 is required for the dynamic methylation of the D4Z4 macrosatellite upon reprogramming but seems dispensable for methylation maintenance. We find that FSHD and BAMS patient's cells carrying SMCHD1 mutations are both permissive for DUX4 expression, a transcription factor whose regulation has been proposed as the main trigger for FSHD. These findings open new questions as to what is the true aetiology for FSHD, the epigenetic events associated with the disease thus calling the current model into question and opening new perspectives for understanding repetitive DNA sequences regulation.

Structure-Property Relationship Study of N-(Hydroxy)Peptides for the Design of Self-Assembled Parallel ß-Sheets.

The design of novel and functional biomimetic foldamers remains a major challenge in creating mimics of native protein structures. Herein, we report the stabilization of a remarkably short ß-sheet by incorporating N-(hydroxy)glycine (Hyg) residues into the backbone of peptides. These peptide-peptoid hybrids form unique parallel ß-sheet structures by self-assembly upon hydrogenation. Our spectroscopic and crystallographic data suggest that the local conformational perturbations induced by N-(hydroxy)amides are outweighed by a network of strong interstrand hydrogen bonds.

Deciphering the complexity of the 4q and 10q subtelomeres by molecular combing in healthy individuals and patients with facioscapulohumeral dystrophy.

BACKGROUND: Subtelomeres are variable regions between telomeres and chromosomal-specific regions. One of the most studied pathologies linked to subtelomeric imbalance is facioscapulohumeral dystrophy (FSHD). In most cases, this disease involves shortening of an array of D4Z4 macrosatellite elements at the 4q35 locus. The disease also segregates with a specific A-type haplotype containing a degenerated polyadenylation signal distal to the last repeat followed by a repetitive array of ß-satellite elements. This classification applies to most patients with FSHD. A subset of patients called FSHD2 escapes this definition and carries a mutation in the SMCHD1 gene. We also recently described patients carrying a complex rearrangement consisting of a cis-duplication of the distal 4q35 locus identified by molecular combing. METHODS: Using this high-resolution technology, we further investigated the organisation of the 4q35 region linked to the disease and the 10q26 locus presenting with 98% of homology in controls and patients. RESULTS: Our analyses reveal a broad variability in size of the different elements composing these loci highlighting the complexity of these subtelomeres and the difficulty for genomic assembly. Out of the 1029 DNA samples analysed in our centre in the last 7 years, we also identified 54 cases clinically diagnosed with FSHD carrying complex genotypes. This includes mosaic patients, patients with deletions of the proximal 4q region and 23 cases with an atypical chromosome 10 pattern, infrequently found in the control population and never reported before. CONCLUSION: Overall, this work underlines the complexity of these loci challenging the diagnosis and genetic counselling for this disease.

Type 1 FSHD with 6-10 Repeated Units: Factors Underlying Severity in Index Cases and Disease Penetrance in Their Relatives Attention.

Molecular defects in type 1 facioscapulohumeral muscular dystrophy (FSHD) are caused by a heterozygous contraction of the D4Z4 repeat array from 1 to 10 repeat units (RUs) on 4q35. This study compared (1) the phenotype and severity of FSHD1 between patients carrying 6-8 vs. 9-10 RUs, (2) the amount of methylation in different D4Z4 regions between patients with FSHD1 with different clinical severity scores (CSS). This cross-sectional multicenter study was conducted to measure functional scales and for genetic analysis. Patients were classified into two categories according to RUs: Group 1, 6-8; Group 2, 9-10. Methylation analysis was performed in 27 patients. A total of 99 carriers of a contracted D4Z4 array were examined. No significant correlations between RUs and CSS (r = 0.04, p = 0.73) and any of the clinical outcome scales were observed between the two groups. Hypomethylation was significantly more pronounced in patients with high CSS (>3.5) than those with low CSS (<1.5) (in DR1 and 5P), indicating that the extent of hypomethylation might modulate disease severity. In Group 1, the disease severity is not strongly correlated with the allele size and is mostly correlated with the methylation of D4Z4 regions.

Enantioselective Synthesis of α-Allyl Amino Esters via Hydrogen-Bond-Donor Catalysis.

We report a chiral-squaramide-catalyzed enantio- and diastereoselective synthesis of α-allyl amino esters. The optimized protocol provides access to N-carbamoyl-protected amino esters via nucleophilic allylation of readily accessible α-chloro glycinates. A variety of useful α-allyl amino esters were prepared, including crotylated products bearing vicinal stereocenters that are inaccessible through enolate alkylation, with high enantioselectivity (up to 97% ee) and diastereoselectivity (>10:1). The reactions display first-order kinetic dependence on both the α-chloro glycinate and the nucleophile, consistent with rate-limiting C-C bond formation. Computational analysis of the uncatalyzed reaction predicts an energetically inaccessible iminium intermediate, and a lower energy concerted SN2 mechanism.

Total synthesis of (±)-fumimycin and analogues for biological evaluation as peptide deformylase inhibitors.

A concise 7-step total synthesis of (±)-fumimycin in 11.6 % overall yield is reported. An acid-catalyzed intramolecular aza-Friedel-Crafts cyclization was developed to construct the benzofuranone skeleton of the natural product bearing an α,α-disubstituted amino acid moiety in a single step. Regioselective chlorination followed by a Suzuki-Miyaura cross-coupling rapidly enabled the preparation of a library of analogues which were evaluated against peptide deformylase for antibacterial activity.

Intercepted-Knoevenagel condensation for the synthesis of unsymmetrical fused-tricyclic 4H-pyrans.

4H-Pyrans (4H-Pys) and 1,4-dihydropyridines (1,4-DHPs) are important classes of heterocyclic scaffolds in medicinal chemistry. Herein, an indium(III)-catalyzed one-pot domino reaction for the synthesis of highly functionalized 4H-Pys, and a model of 1,4-DHP is reported. This alternative approach to the challenging Hantzsch 4-component reaction enables the synthesis of fused-tricyclic heterocycles, and the mechanistic studies underline the importance of an intercepted-Knoevenagel adduct to achieve higher chemoselectivity towards these types of unsymmetrical heterocycles.

Synthesis and Reactivity of α-Haloglycine Esters: Hyperconjugation in Action.

A general and efficient synthesis of α-haloglycine esters from commercially available feedstock chemicals, in a single step, is reported. The reactivity of these α-haloglycine esters with various nucleophiles was studied as surrogates of α-iminoesters upon activation with hydrogen-bond donor catalysts. DFT calculations on the α-haloglycine structures (X = F, Cl, Br) accompanied by an X-ray characterization of the α-bromoglycine ester support the existence of a "generalized" anomeric effect created by hyperconjugation. This peculiar hyperconjugative effect is proposed to be responsible for the enhanced halogen nucleofugality leading to a facile halogen abstraction by hydrogen-bond donor catalysts. This reactivity was exploited with thiourea catalysts on several catalytic transformations (aza-Friedel-Crafts and Mannich reactions) for the synthesis of several types of non-proteinogenic α-amino esters.

Synthesis of α-Arylated Cycloalkanones from Congested Trisubstituted Spiro-epoxides: Application of the House-Meinwald Rearrangement for Ring Expansion.

A three-step sequence for the synthesis of α-arylated cyclohexanones and the most challenging cycloheptanones is reported. First, an efficient one-pot synthesis of ß,ß'-disubstituted benzylidene cycloalkanes (styrenes) using the palladium-catalyzed Barluenga reaction from readily available feedstock chemicals is described. Furthermore, an epoxidation followed by the House-Meinwald rearrangement (HMR) of spiro-epoxides is reported to produce a number of α-arylated cycloalkanones upon ring expansion. Reactions catalyzed by bismuth triflate underwent quasi-exclusively ring expansion for all substrates (electronically poor and rich), with yields ranging from 15% to 95%, thus demonstrating the difficulty of achieving ring enlargement for electron-deficient spiro-epoxides. On the other hand, by means of catalysis with aluminum trichloride, the rearrangement of spiro-epoxides proceeded typically in high yields and with remarkable regioselectivity on a broader substrate scope. In this case, a switch of regioselectivity was achieved for spiro-epoxides with electron-withdrawing substituents which enable the method to be successfully extended to some chemospecific arene shifts and the synthesis of aldehydes bearing a α-quaternary carbon. While the HMR has been extensively studied for smaller ring enlargement, we are pleased to report herein that larger cyclohexanones and cycloheptanones can be obtained efficiently from more sterically demanding trisubstituted spiro-epoxides bearing electron-releasing and electron-neutral arene substituents.

A Kinetic Dearomatization Strategy for an Expedient Biomimetic Route to the Bielschowskysin Skeleton.

Bielschowskysin (1), the flagship of the furanocembranoid diterpene family, has attracted attention from chemists owing to its intriguing and daunting polycyclic architecture and medicinal potential against lung cancer. The high level of functionalization of 1 poses a considerable challenge to synthesis. Herein, a stereoselective furan dearomatization strategy of furanocembranoids was achieved via the intermediacy of chlorohydrins. The stereochemical course of the kinetic dearomatization was established, and the C3 configuration of the resulting exo-enol ether intermediates proved to be essential to complete the late-stage transannular [2+2] photocycloaddition. Overall, this biomimetic strategy starting from the natural product acerosolide (9) featured an unprecedented regio- and highly stereoselective furan dearomatization, which provided rapid access to the pivotal exo-enol ethers en route to the intricate bielschowskyane skeleton.

Molecular combing reveals complex 4q35 rearrangements in Facioscapulohumeral dystrophy.

Facioscapulohumeral dystrophy (FSHD), one of the most common hereditary neuromuscular disorders, is associated with a complex combination of genetic variations at the subtelomeric 4q35 locus. As molecular diagnosis relying on Southern blot (SB) might be challenging in some cases, molecular combing (MC) was recently developed as an additional technique for FSHD diagnosis and exploration of the genomic organization of the 4q35 and 10q26 regions. In complement to the usual SB, we applied MC in a large cohort of 586 individuals with clinical FSHD. In 332 subjects, the two 4q alleles were normal in size, allowing exclusion of FSHD1 while we confirmed FSHD1 in 230 patients. In 14 patients from 10 families, we identified a recurrent complex heterozygous rearrangement at 4q35 consisting of a duplication of the D4Z4 array and a 4qA haplotype, irresolvable by the SB technique. In five families, we further identified variations in the SMCHD1 gene. Impact of the different mutations was tested using a minigene assay and we analyzed DNA methylation after sodium bisulfite modification and NGS sequencing. We discuss the involvement of this rearrangement in FSHD since all mutations in SMCHD1 are not associated with D4Z4 hypomethylation and do not always segregate with the disease.

In Situ-Generated Glycinyl Chloroaminals for a One-Pot Synthesis of Non-proteinogenic α-Amino Esters.

An acetyl chloride-mediated cascade transformation involving a primary carbamate, ethyl glyoxylate, and various types of nucleophiles is reported for the synthesis of orthogonally protected α-amino esters. These reactions proceeded rapidly to afford the pivotal α-chloroglycine intermediate in excellent yields, which can be directly functionalized in situ with various types of nucleophiles. A mild and unique AcOH(cat.)/AcCl system was found to promote an autocatalytic-like condensation and facilitate the multicomponent assembly of non-proteinogenic α-amino esters. To better understand this one-pot transformation and the orchestration of the components' condensations, the investigation of a broader scope of nucleophiles and some kinetic studies are presented. Our findings suggest that the halogenation step toward the formation of α-chloroglycine is the rate-determining step likely proceeding through the formation of N-carbamoyl iminium. Also, the initial kinetic profiling for the nucleophilic substitution supports an SN1-like (SN2C+) mechanism in which nucleophiles add to the iminium-chloride tight ionic pair. These results lead ultimately to the design of a new protocol in which an achiral hydrogen bond donor thiourea catalyst was utilized to enhance the reaction scope and enable silylated nucleophiles to be efficiently exploited to synthesize novel non-proteinogenic α-amino esters.

Insights into herpesvirus tegument organization from structural analyses of the 970 central residues of HSV-1 UL36 protein.

The tegument of all herpesviruses contains a capsid-bound large protein that is essential for multiple viral processes, including capsid transport, decapsidation at the nuclear pore complex, particle assembly, and secondary envelopment, through mechanisms that are still incompletely understood. We report here a structural characterization of the central 970 residues of this protein for herpes simplex virus type 1 (HSV-1 UL36, 3164 residues). This large fragment is essentially a 34-nm-long monomeric fiber. The crystal structure of its C terminus shows an elongated domain-swapped dimer. Modeling and molecular dynamics simulations give a likely molecular organization for the monomeric form and extend our findings to alphaherpesvirinae. Hence, we propose that an essential feature of UL36 is the existence in its central region of a stalk capable of connecting capsid and membrane across the tegument and that the ability to switch between monomeric and dimeric forms may help UL36 fulfill its multiple functions.

DUX4 and DUX4 downstream target genes are expressed in fetal FSHD muscles.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent adult muscular dystrophies. The common clinical signs usually appear during the second decade of life but when the first molecular dysregulations occur is still unknown. Our aim was to determine whether molecular dysregulations can be identified during FSHD fetal muscle development. We compared muscle biopsies derived from FSHD1 fetuses and the cells derived from some of these biopsies with biopsies and cells derived from control fetuses. We mainly focus on DUX4 isoform expression because the expression of DUX4 has been confirmed in both FSHD cells and biopsies by several laboratories. We measured DUX4 isoform expression by using qRT-PCR in fetal FSHD1 myotubes treated or not with an shRNA directed against DUX4 mRNA. We also analyzed DUX4 downstream target gene expression in myotubes and fetal or adult FSHD1 and control quadriceps biopsies. We show that both DUX4-FL isoforms are already expressed in FSHD1 myotubes. Interestingly, DUX4-FL expression level is much lower in trapezius than in quadriceps myotubes, which is confirmed by the level of expression of DUX4 downstream genes. We observed that TRIM43 and MBD3L2 are already overexpressed in FSHD1 fetal quadriceps biopsies, at similar levels to those observed in adult FSHD1 quadriceps biopsies. These results indicate that molecular markers of the disease are already expressed during fetal life, thus opening a new field of investigation for mechanisms leading to FSHD.

Segregation between SMCHD1 mutation, D4Z4 hypomethylation and Facio-Scapulo-Humeral Dystrophy: a case report.

BACKGROUND: The main form of Facio-Scapulo-Humeral muscular Dystrophy is linked to copy number reduction of the 4q D4Z4 macrosatellite (FSHD1). In 5 % of cases, FSHD phenotype appears in the absence of D4Z4 reduction (FSHD2). In 70-80 % of these patients, variants of the SMCHD1 gene segregate with 4qA haplotypes and D4Z4 hypomethylation. CASE PRESENTATION: We report a family presenting with neuromuscular symptoms reminiscent of FSHD but without D4Z4 copy reduction. We characterized the 4q35 region using molecular combing, searched for mutation in the SMCHD1 gene and determined D4Z4 methylation level by sodium bisulfite sequencing. We further investigated the impact of the SMCHD1 mutation at the protein level and on the NMD-dependent degradation of transcript. In muscle, we observe moderate but significant reduction in D4Z4 methylation, not correlated with DUX4-fl expression. Exome sequencing revealed a heterozygous insertion of 7 bp in exon 37 of the SMCHD1 gene producing a loss of frame with premature stop codon 4 amino acids after the insertion (c.4614-4615insTATAATA). Both wild-type and mutated transcripts are detected. CONCLUSION: The truncated protein is absent and the full-length protein level is similar in patients and controls indicating that in this family, FSHD is not associated with SMCHD1 haploinsufficiency.

Correlation between low FAT1 expression and early affected muscle in facioscapulohumeral muscular dystrophy.

OBJECTIVE: Facioscapulohumeral muscular dystrophy (FSHD) is linked to either contraction of D4Z4 repeats on chromosome 4 or to mutations in the SMCHD1 gene, both of which result in the aberrant expression of the transcription factor DUX4. However, it is still difficult to correlate these genotypes with the phenotypes observed in patients. Because we have recently shown that mice with disrupted Fat1 functions exhibit FSHD-like phenotypes, we have investigated the expression of the human FAT1 gene in FSHD. METHODS: We first analyzed FAT1 expression in FSHD adult muscles and determined whether FAT1 expression was driven by DUX4. We next determined FAT1 expression levels in 64 muscles isolated from 16 control fetuses. These data were further complemented with analysis of Fat1 expression in developing mouse embryos. RESULTS: We demonstrated that FAT1 expression is independent of DUX4. Moreover, we observed that (1) in control fetal human biopsies or in developing mouse embryos, FAT1 is expressed at lower levels in muscles that are affected at early stages of FSHD progression than in muscles that are affected later or are nonaffected; and (2) in adult muscle biopsies, FAT1 expression is lower in FSHD muscles compared to control muscles. INTERPRETATION: We propose a revised model for FSHD in which FAT1 levels might play a role in determining which muscles will exhibit early and late disease onset, whereas DUX4 may worsen the muscle phenotype.