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Pseudomonas aeruginosa is an opportunistic pathogen prone to developing drug-resistance and is a major cause of infection for burn patients and patients suffering from cystic fibrosis or are hospitalized in intensive care units. One of the virulence factors of this bacterium is the lipase enzyme that degrades the extracellular matrix of the host tissue and promotes invasion. Bromhexine is a mucolytic drug and has recently been reported to function as a competitive inhibitor of lipase with an IC50 value of 49 µM. In the present study, an attempt was made to identify stronger inhibitors from the ChEMBL database of bioactive compounds, as compared to the reference compound Bromhexine. Following docking and MD simulations, four hit compounds (N1-N4) were selected that showed promising binding modes and low RMSD values indicative of stable protein-ligand complexes. From subsequent binding pose metadynamics (BPMD) simulations, two of these (N2 and N4) stood out as more potent than Bromhexine, displaying stable interactions with residues in the catalytic site of the enzyme. Biological investigations were performed for all four compounds. Among them, the same two hit compounds were found to be the most effective binders with IC50 values of 22.1 and 27.5 µM, respectively; i.e. roughly twice as efficient as the reference Bromhexine. Taken together, our results show that these hits can be promising new candidates to use as leads for the development of drugs targeting the P. aeruginosa lipase enzyme. Communicated by Ramaswamy H. Sarma

The static and dynamic structures of DNA duplexes affected by 5S-Tg (Tg, Thymine glycol) epimers were studied using MD simulations and Markov State Models (MSMs) analysis. The results show that the 5S,6S-Tg base caused little perturbation to the helix, and the base-flipping barrier was determined to be 4.4 kcal mol−1 through the use of enhanced sampling meta-eABF calculations, comparable to 5.4 kcal mol−1 of the corresponding thymine flipping. Two conformations with the different hydrogen bond structures between 5S,6R-Tg and A19 were identified in several independent MD trajectories. The 5S,6R-Tg:O6HO6•••N1:A19 hydrogen bond is present in the high-energy conformation displaying a clear helical distortion, and near barrier-free Tg base flipping. The low-energy conformation always maintains Watson–Crick base pairing between 5S,6R-Tg and A19, and 5S-Tg base flipping is accompanied by a small barrier of ca. 2.0 KBT (T = 298 K). The same conformations are observed in the MSMs analysis. Moreover, the transition path and metastable structures of the damaged base flipping are for the first time verified through MSMs analysis. The data clearly show that the epimers have completely different influence on the stability of the DNA duplex, thus implying different enzymatic mechanisms for DNA repair.

International audience; Inositol-requiring enzyme 1 (IRE1) is a major mediator of the unfolded protein response (UPR), which is activated upon endoplasmic reticulum (ER) stress. Tumor cells experience ER stress due to adverse microenvironmental cues, a stress overcome by relying on IRE1 signaling as an adaptive mechanism. Herein, we report the discovery of structurally new IRE1 inhibitors identified through the structural exploration of its kinase domain. Characterization in in vitro and in cellular models showed that they inhibit IRE1 signaling and sensitize glioblastoma (GB) cells to the standard chemotherapeutic, temozolomide (TMZ). Finally, we demonstrate that one of these inhibitors, Z4P, permeates the blood-brain barrier (BBB), inhibits GB growth, and prevents relapse in vivo when administered together with TMZ. The hit compound disclosed herein satisfies an unmet need for targeted, non-toxic IRE1 inhibitors and our results support the attractiveness of IRE1 as an adjuvant therapeutic target in GB.

International audience; CD95 is a member of the TNF receptor superfamily that is ubiquitously expressed in healthy and pathological tissues. Stimulation of CD95 by its physiological ligand CD95L induces its oligomerization leading in turn to the transduction of either apoptotic or non-apoptotic signals. CD95L can exist as both membrane-anchored and soluble forms (sCD95L), the latter resulting from the proteolytic cleavage of the former. Candidate proteases able to achieve CD95L cleavage were identified as matrix metalloproteases (MMP) due to their demonstrated ability to cleave other TNF superfamily ligands. The main goal of this study was to systematically identify the MMP family members capable of cleaving CD95L and subsequently determine the corresponding cleavage sites. By using different orthogonal biochemical approaches and combining them with molecular modeling, we confirmed data from the literature regarding CD95L cleavage by MMP-3 and MMP-7. Moreover, we found that MMP-2 and MMP-12 can cleave CD95L and characterized their resulting cleavage sites. This study provides a systematic approach to analyze the cleavage of CD95L, which until now had only been poorly described.

International audience; Ganglioside-monosialic acid (GM1) gangliosidosis, a rare autosomal recessive disorder, is frequently caused by deleterious single nucleotide variants (SNVs) in GLB1 gene. These variants result in reduced β-galactosidase (β-gal) activity, leading to neurodegeneration associated with premature death. Currently, no effective therapy for GM1 gangliosidosis is available. Three ongoing clinical trials aim to deliver a functional copy of the GLB1 gene to stop disease progression. In this study, we show that 41% of GLB1 pathogenic SNVs can be replaced by adenine base editors (ABEs). Our results demonstrate that ABE efficiently corrects the pathogenic allele in patient-derived fibroblasts, restoring therapeutic levels of β-gal activity. Off-target DNA analysis did not detect off-target editing activity in treated patient’s cells, except a bystander edit without consequences on β-gal activity based on 3D structure bioinformatics predictions. Altogether, our results suggest that gene editing might be an alternative strategy to cure GM1 gangliosidosis.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that is growing rapidly among the elderly population around the world. Studies show that a lack of acetylcholine and butyrylcholine due to the overexpression of enzymes Acetylcholinesterase (AChE) and Butyrylcholinesterase (BChE) may lead to reduced communication between neuron cells. As a result, seeking novel inhibitors targeting these enzymes might be vital for future treatment of AD. Ondansetron is used to prevent nausea and vomiting caused by chemotherapy or radiation treatments, and is herein shown to be a potent inhibitor of cholinesterase. Comparison is made between Ondansetron and FDA-approved cholinesterase inhibitors Rivastigmine and Tacrine. Molecular docking demonstrates that interactions between the studied ligand and aromatic residues in the peripheral region of the active site are important in binding. Molecular dynamics simulations and binding pose metadynamics show that Ondansetron is highly potent against both enzymes, and far better than Rivastigmine. Inhibitor activities evaluated by in vitro studies confirm that the drug inhibits AChE and BChE by non-competitive and mixed inhibition, respectively, with IC50 values 33 µM (AChE) and 2.5 µM (BChE). Based on the findings, we propose that Ondansetron may have therapeutic applications in inhibiting cholinesterase, especially for BChE.

Diphthamide is a post-translationally modified histidine essential for messenger RNA translation and ribosomal protein synthesis. We present evidence for DPH5 as a novel cause of embryonic lethality and profound neurodevelopmental delays (NDDs).Molecular testing was performed using exome or genome sequencing. A targeted Dph5 knockin mouse (C57BL/6Ncrl-Dph5DPH5 variants p.His260Arg (homozygous), p.Asn110Ser and p.Arg207Ter (heterozygous), and p.Asn174LysfsTer10 (homozygous) were identified in 3 unrelated families with distinct overlapping craniofacial features, profound NDDs, multisystem abnormalities, and miscarriages. Dph5 p.His260Arg homozygous knockin was embryonically lethal with only 1 subviable mouse exhibiting impaired growth, craniofacial dysmorphology, and multisystem dysfunction recapitulating the human phenotype. Adenosine diphosphate-ribosylation assays showed absent to decreased function in DPH5-knockout human and yeast cells. In silico modeling of the variants showed altered DPH5 structure and disruption of its interaction with eEF2.We provide strong clinical, biochemical, and functional evidence for DPH5 as a novel cause of embryonic lethality or profound NDDs with multisystem involvement and expand diphthamide-deficiency syndromes and ribosomopathies.

International audience; A range of methodologies were herein employed to study the unconventional XBP1 mRNA cleavage mechanism performed by the unfolded protein response (UPR) mediator Inositol Requiring Enzyme 1α (IRE1). Using Protein-RNA molecular docking along with a series of extensive restrained/unrestrained atomistic molecular dynamics (MD) simulations, the dynamical behavior of the system was evaluated and a reliable model of the IRE1/XBP1 mRNA complex was constructed. From a series of well-converged quantum mechanics molecular mechanics well-tempered metadynamics (QM/MM WT-MetaD) simulations using the Grimme dispersion interaction corrected semiempirical parametrization method 6 level of theory (PM6-D3) and the AMBER14SB-OL3 force field, the free energy profile of the cleavage mechanism was determined, along with intermediates and transition state structures. The results show two distinct reaction paths based on general acid-general base type mechanisms, with different activation energies that perfectly match observations from experimental mutagenesis data. The study brings unique atomistic insights into the cleavage mechanism of XBP1 mRNA by IRE1 and clarifies the roles of the catalytic residues H910 and Y892. Increased understanding of the details in UPR signaling can assist in the development of new therapeutic agents for its modulation.

Stress‐induced apoptosis is mediated primarily through the intrinsic pathway that involves caspase‐9. We previously reported that in caspase‐9‐deficient cells, a protein complex containing ATG5 and Fas‐associated death domain (FADD) facilitated caspase‐8 activation and cell death in response to endoplasmic reticulum (ER) stress. Here, we investigated whether this complex could be activated by other forms of cell stress. We show that diverse stress stimuli, including etoposide, brefeldin A and paclitaxel, as well as heat stress and gamma‐irradiation, caused formation of a complex containing ATG5‐ATG12, FADD and caspase‐8 leading to activation of downstream caspases in caspase‐9‐deficient cells. We termed this complex the ‘stressosome’. However, in these cells, only ER stress and heat shock led to stressosome‐dependent cell death. Using in silico molecular modelling, we propose the structure of the stressosome complex, with FADD acting as an adaptor protein, interacting with pro‐caspase‐8 through their respective death effector domains (DEDs) and interacting with ATG5‐ATG12 through its death domain (DD). This suggests that the complex could be regulated by cellular FADD‐like interleukin‐1β‐converting enzyme–inhibitory protein (cFLIPL), which was confirmed experimentally. This study provides strong evidence for an alternative mechanism of caspase‐8 activation involving the stressosome complex.

Inositol Requiring Enzyme 1 (IRE1) is a bifunctional serine/threonine kinase and endoribonuclease. It is a major mediator of the Unfolded Protein Response (UPR), which is activated during endoplasmic reticulum (ER) stress. Tumor cells experience ER stress due to adverse microenvironmental cues such as hypoxia or nutrient shortage and high metabolic/protein folding demand. To cope with those stresses, cancer cells utilize IRE1 signaling as an adaptive mechanism. Here we report the discovery of novel IRE1 inhibitors identified through a structural exploration of the IRE1 kinase domain. We first characterized the candidates in vitro and in cellular models. We showed that all molecules inhibit IRE1 signaling and sensitize glioblastoma cells to the standard chemotherapeutic temozolomide (TMZ). From these inhibitors, we retained a Blood-Brain Barrier (BBB) permeable molecule (Z4P) and demonstrated its ability to inhibit Glioblastoma (GB) growth and to prevent relapse in vivo when administered together with TMZ. These results support the attractiveness of IRE1 as an adjuvant therapeutic target in GB. We thus satisfy an unmet need for targeted, non-toxic, IRE1 inhibitors as adjuvant therapeutic agents against GB.

Computational quantum chemistry within the density functional theory (DFT) and time-dependent density functional theory (TD-DFT) framework is used to investigate the photodegradation mechanism as well as the photochemical and photophysical properties of benoxaprofen (BP), a non steroid anti-inflammatory molecule (2-[2-(4-chlorophenyl)-1,3-benzoxazol-5-yl] propanoic acid). BP is a highly phototoxic agent that causes cutaneous phototoxicity shortly after its administration. On the grounds of concern about serious side effects, especially hepatotoxicity, it was withdrawn from the world market after only 2 years of its release. Our study shows that the drug has the capability to absorb radiation in the UV region, mainly between 300 and 340 nm, and undergoes spontaneous photoinduced decarboxylation from the triplet state. It shows very similar photochemical properties to the highly photolabile non-steroidal anti-inflammatory drugs (NSAIDs) ketoprofen, suprofen, and tiaprofenic acid. Like ketoprofen, BP can also decarboxylate from excited singlet states by overcoming low energy barriers. The differences in molecular orbital (highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO)) distributions between the neutral and deprotonated BP, their absorption spectra, and the energetics and fate of various photoproducts produced throughout the photodegradation are discussed. Initiation and termination of decarboxylated BP radical species and initiation of propagating lipid peroxidation reactions due to the addition of molecular oxygen giving rise to the corresponding peroxyl radical are also explored in detail.

Peptides are an important modality in drug discovery. While current peptide optimization focuses predominantly on the small number of natural and commercially available non-natural amino acids, the chemical spaces available for small molecule drug discovery are in the billions of molecules. In the present study, we describe the development of a large virtual library of readily synthesizable non-natural amino acids that can power the virtual screening protocols and aid in peptide optimization. To that end, we enumerated nearly 380 thousand amino acids and demonstrated their vast chemical diversity compared to the 20 natural and commercial residues. Furthermore, we selected a diverse ten thousand amino acid subset to validate our virtual screening workflow on the Keap1-Neh2 complex model system. Through in silico mutations of Neh2 peptide residues to those from the virtual library, our docking-based protocol identified a number of possible solutions with a significantly higher predicted affinity toward the Keap1 protein. This protocol demonstrates that the non-natural amino acid chemical space can be massively extended and virtually screened with a reasonable computational cost.

Split luciferase complementary assay has been used to investigate the effect of WD domain deletion on Apaf-1 oligomerization. Apaf-1 is an adaptor molecule in formation of apoptosome that activates caspase-9, an activation that is a key event in the mitochondrial cell death pathway. Structural studies suggest that normally Apaf-1 is held in an inactive conformation by intramolecular interactions between Apaf-1's nucleotide binding domain and one of its WD40 domains (WD1). In the prevailing model of Apaf-1 activation, cytochrome c binds to sites in WD1 and in Apaf-1's second WD40 domain (WD2), moving WD1 and WD2 closer together and rotating WD1 away from the nucleotide binding domain. This allows Apaf-1 to bind dATP or ATP and to form the apoptosome, which activates caspase-9. This model predicts that cytochrome c binding to both WD domains is necessary for apoptosome formation and that an Apaf-1 with only WD1 will be locked in an inactive conformation that cannot be activated by cytochrome c. Here we investigated the effect of removing one WD domain (Apaf-1 1-921) on Apaf-1 interactions and caspase activation. Apaf-1 1-921 could not activate caspase-9, even in the presence of cytochrome c. These data show that a single WD domain is sufficient to lock Apaf-1 in an inactive state and this state cannot be altered by cytochrome c.

International audience; IRE1α (inositol requiring enzyme 1 alpha) is one of the main transducers of the unfolded protein response (UPR). IRE1α plays instrumental protumoral roles in several cancers, and high IRE1α activity has been associated with poorer prognoses. In this context, IRE1α has been identified as a potentially relevant therapeutic target. Pharmacological inhibition of IRE1α activity can be achieved by targeting either the kinase domain or the RNase domain. Herein, the recent advances in IRE1α pharmacological targeting is summarized. We describe the identification and optimization of IRE1α inhibitors as well as their mode of action and limitations as anticancer drugs. The potential pitfalls and challenges that could be faced in the clinic, and the opportunities that IRE1α modulating strategies may present are discussed.

Background Expectant parents may have positive and negative emotions during pregnancy and receive support from different sources. Studies on life situation and support among couples have rarely been conducted. This study aims to explore life situation and support during pregnancy among expectant mothers with depressive symptoms and their partners. Methods Twenty-seven expectant mothers, in the last trimester of pregnancy with depressive symptoms (Edinburgh Postnatal Depression Scale score ≥ 7) from seven public antenatal care clinics in Sakonnakhon, a north-eastern province of Thailand, and their partners were interviewed. In total, 54 semi-structured interviews were subjected to content analysis. Results Four categories emerged: (1) Having obstacles in life, (2) Facing life transition, (3) Enhancing confidence, and (4) Dissatisfaction with support. The informants described obstacles regarding economy, fear of health problems, getting an abnormal child and partners’ behaviours. They received support from family members and social networks, but some were dissatisfied with the support from the healthcare. For example, expectant fathers wished to receive more health information and be more involved. Conclusions Healthcare professionals should be aware of the influence of cultural and contextual factors when providing antenatal care to expectant parents. Male involvement in the care must not be neglected.

The reaction of HO• radical with DNA is intensively studied both mechanistically and analytically for lesions formation. Several aspects related to the reaction paths of purine moieties with the formation of 5',8-cyclopurines (cPu), 8-oxopurines (8-oxo-Pu), and their relationship are not well understood. In this study, we investigated the reaction of HO• radical with a 21-mer double-stranded oligodeoxynucleotide (ds-ODNs) in γ-irradiated aqueous solutions under various oxygen concentrations and accurately quantified the six purine lesions (i.e., four cPu and two 8-oxo-Pu) by LC-MS/MS analysis using isotopomeric internal standards. In the absence of oxygen, 8-oxo-Pu lesions are only ∼4 times more than cPu lesions. By increasing oxygen concentration, the 8-oxo-Pu and the cPu gradually increase and decrease, respectively, reaching a gap of ∼130 times at 2.01 × 10-4 M of O2. Kinetic treatment of the data allows to estimate the C5' radical competition between cyclization and oxygen trapping in ds-ODNs, and lastly the rate constants of the four cyclization steps. Tailored computational studies by means of dispersion-corrected DFT calculations were performed on the CGC and TAT in their double-strand models for each cPu diastereoisomer along with the complete reaction pathways of the cyclization steps. Our findings reveal unheralded reaction mechanisms that resolve the long-standing issues with C5' radical cyclization in purine moieties of DNA sequences.

International audience; ER stress is mediated by three sensors and the most evolutionary conserved IRE1α signals through its cytosolic kinase and endoribonuclease (RNase) activities. IRE1α RNase activity can either catalyze the initial step of XBP1 mRNA unconventional splicing or degrade a number of RNAs through regulated IRE1-dependent decay. Until now, the biochemical and biological outputs of IRE1α RNase activity have been well documented; however, the precise mechanisms controlling whether IRE1α signaling is adaptive or pro-death (terminal) remain unclear. We investigated those mechanisms and hypothesized that XBP1 mRNA splicing and regulated IRE1-dependent decay activity could be co-regulated by the IRE1α RNase regulatory network. We identified that RtcB, the tRNA ligase responsible for XBP1 mRNA splicing, is tyrosine-phosphorylated by c-Abl and dephosphorylated by PTP1B. Moreover, we show that the phosphorylation of RtcB at Y306 perturbs RtcB interaction with IRE1α, thereby attenuating XBP1 mRNA splicing. Our results demonstrate that the IRE1α RNase regulatory network is dynamically fine-tuned by tyrosine kinases and phosphatases upon various stresses and that the extent of RtcB tyrosine phosphorylation determines cell adaptive or death outputs.

Ganglioside-monosialic acid (GM1) gangliosidosis, a rare autosomal recessive disorder, is frequently caused by deleterious single nucleotide variants (SNVs) in GLB1 gene. These variants result in reduced β-galactosidase (β-gal) activity, leading to neurodegeneration associated with premature death. Currently, no effective therapy for GM1 gangliosidosis is available. Three ongoing clinical trials aim to deliver a functional copy of the GLB1 gene to stop disease progression. Here, we show that 41% of GLB1 pathogenic SNVs might be cured by adenine base editors (ABEs). Our results demonstrate that ABE efficiently corrects the pathogenic allele in patient-derived fibroblasts, restoring a therapeutic level of β-gal activity. Unbiased off-target DNA analysis did not detect off-target editing activity in treated patient’s cells except a bystander edit without consequences on β-gal activity. Altogether our results suggest that gene editing is an alternative strategy to cure GM1 gangliosidosis, by correcting the root cause of disease and avoiding repetitive adeno-associated virus injections.

Inositol Requiring Enzyme 1 (IRE1) is a bifunctional serine/threonine kinase and endoribonuclease. It is a major mediator of the Unfolded Protein Response (UPR), which is activated during endoplasmic reticulum (ER) stress. Tumor cells experience ER stress due to adverse microenvironmental cues such as hypoxia or nutrient shortage and high metabolic/protein folding demand. To cope with those stresses, cancer cells utilize IRE1 signaling as an adaptive mechanism. Here we report the discovery of novel IRE1 inhibitors identified through a structural exploration of the IRE1 kinase domain. We first characterized these candidates in vitro and in cellular models. We showed that all molecules inhibit IRE1 signaling and sensitize glioblastoma cells to the standard chemotherapeutic temozolomide (TMZ). From these inhibitors, we retained a Blood-Brain Barrier (BBB) permeable molecule (Z4P) and demonstrated its ability to inhibit Glioblastoma (GB) growth and to prevent relapse in vivo when administered together with TMZ. These results support the attractiveness of IRE1 as an adjuvant therapeutic target in GB. We thus satisfy an unmet need for targeted, non-toxic, IRE1 inhibitors as adjuvant therapeutic agents against GB.

AIM: To investigate both quantitatively and qualitatively the differences between participation in team-based visits (TBVs) and perceived needs for TBVs from the perspectives of healthcare professionals, in the context of the Swedish three-tier national Child Healthcare program. METHODS: A study-specific questionnaire, including multiple-choice questions with fixed and free-text response options, was developed, and used. To capture healthcare professionals' experiences and find explanations for the quantitative results in qualitative data, a convergent parallel mixed-methods study design was used. Descriptive statistics and McNemar's test were used to analyze the quantitative data, and content analysis was used to analyze the qualitative data. RESULTS: Healthcare professionals perceived the need for TBVs in the Swedish Child Healthcare Services (CHS) to a high extent. The largest difference between the perceived need for TBVs and experienced TBVs was for indications associated with psychosocial problems. The quantitative findings were explored by the qualitative findings. Both individual and organizational factors influenced TBVs. CONCLUSION: Perceived needs for TBVs in Swedish CHS exceed its existence. Healthcare professionals require TBVs delivered by interprofessional teams, in line with proportionate universalism. Accordingly, organizational structures (e.g., co-location and clear instructions on how to distribute TBVs) and human resources (e.g., psychologists and social worker) are needed.

International audience; IRE1α (inositol-requiring enzyme 1 alpha, referred to IRE1 hereafter) is an Endoplasmic Reticulum (ER) resident transmembrane enzyme with cytosolic kinase/RNAse activities. Upon ER stress IRE1 is activated through trans-autophosphorylation and oligomerization, resulting in a conformational change of the RNase domain, thereby promoting two signaling pathways: i) the non-conventional splicing of XBP1 mRNA and ii) the regulated IRE1-dependent decay of RNA (RIDD). IRE1 RNase activity has been linked to diverse pathologies such as cancer or inflammatory, metabolic, and degenerative diseases and the modulation of IRE1 activity is emerging as an appealing therapeutic strategy against these diseases. Several modulators of IRE1 activity have been reported in the past, but none have successfully translated into the clinics as yet. Based on our expertise in the field, we describe in this chapter the approaches and protocols we used to discover novel IRE1 modulators and characterize their effect on IRE1 activity.