CBP-HSF2 structural and functional interplay in Rubinstein-Taybi neurodevelopmental disorder.

Patients carrying autosomal dominant mutations in the histone/lysine acetyl transferases CBP or EP300 develop a neurodevelopmental disorder: Rubinstein-Taybi syndrome (RSTS). The biological pathways underlying these neurodevelopmental defects remain elusive. Here, we unravel the contribution of a stress-responsive pathway to RSTS. We characterize the structural and functional interaction between CBP/EP300 and heat-shock factor 2 (HSF2), a tuner of brain cortical development and major player in prenatal stress responses in the neocortex: CBP/EP300 acetylates HSF2, leading to the stabilization of the HSF2 protein. Consequently, RSTS patient-derived primary cells show decreased levels of HSF2 and HSF2-dependent alteration in their repertoire of molecular chaperones and stress response. Moreover, we unravel a CBP/EP300-HSF2-N-cadherin cascade that is also active in neurodevelopmental contexts, and show that its deregulation disturbs neuroepithelial integrity in 2D and 3D organoid models of cerebral development, generated from RSTS patient-derived iPSC cells, providing a molecular reading key for this complex pathology.

High frequency repetitive Transcranial Magnetic Stimulation promotes long lasting phrenic motoneuron excitability via GABAergic networks.

Repetitive transcranial magnetic stimulation (rTMS) is a promising, innovative, and non-invasive therapy used clinically. Efficacy of rTMS has been demonstrated to ameliorate psychiatric disorders and neuropathic pain through neuromodulation of affected neural circuits. However, little is known about the mechanisms and the specific neural circuits via which rTMS facilitates these functional effects. The aim of this study was to begin revealing the mechanisms by which rTMS may tap into existing neural circuits, by using a well characterized spinal motor circuit - the phrenic circuit. Here we hypothesized that rTMS can be used to enhance phrenic motoneuron excitability in anesthetized Sprague Dawley rats. Multiple acute rTMS protocols were used revealing 10 Hz rTMS protocol induced a robust, long-lasting increase in phrenic motoneuron excitability, functionally evaluated by diaphragm motor evoked potentials (59.1 ± 21.1 % of increase compared to baseline 60 min after 10 Hz protocol against 6.0 ± 5.8 % (p = 0.007) for Time Control, -5.8 ± 7.4 % (p < 0.001) for 3 Hz, and 5.2 ± 12.5 % (p = 0.008) for 30 Hz protocols). A deeper analyze allowed to discriminate "responder" and "non-responder" subgroups among 10 Hz rTMS treated animals. Intravenous injections of GABAA and GABAB receptor agonists prior to 10 Hz rTMS treatment, abolished the enhanced phrenic motoneuron excitability, suggesting GABAergic input plays a mechanistic role in rTMS-induced phrenic excitability. These data demonstrate that a single high frequency rTMS protocol at 10 Hz increases phrenic motoneuron excitability, mediated by a local GABAergic "disinhibition". By understanding how rTMS can be used to affect neural circuits non-invasively we can begin to harness the therapeutic potential of this neuromodulatory strategy to promote recovery after disease or injury to the central nervous system.

Orofacial Myofunctional Therapy in Obstructive Sleep Apnea Syndrome: A Pathophysiological Perspective.

Obstructive sleep apnea (OSA) syndrome is a multi-factorial disorder. Recently identified pathophysiological contributing factors include airway collapsibility, poor pharyngeal muscle responsiveness, a low arousal threshold, and a high loop gain. Understanding the pathophysiology is of pivotal importance to select the most effective treatment option. It is well documented that conventional treatments (continuous positive airway pressure (CPAP), upper airway surgery, and dental appliance) may not always be successful in the presence of non-anatomical traits, especially in mild to moderate OSA. Orofacial myofunctional therapy (OMT) consists of isotonic and isometric exercises targeted to oral and oropharyngeal structures, with the aim of increasing muscle tone, endurance, and coordinated movements of pharyngeal and peripharyngeal muscles. Recent studies have demonstrated the efficacy of OMT in reducing snoring, apnea-hypopnea index, and daytime sleepiness, and improving oxygen saturations and sleep quality. Myofunctional therapy helps to reposition the tongue, improve nasal breathing, and increase muscle tone in pediatric and adult OSA patients. Studies have shown that OMT prevents residual OSA in children after adenotonsillectomy and helps adherence in CPAP-treated OSA patients. Randomized multi-institutional studies will be necessary in the future to determine the effectiveness of OMT in a single or combined modality targeted approach in the treatment of OSA. In this narrative review, we present up-to-date literature data, focusing on the role of OSA pathophysiology concepts concerning pharyngeal anatomical collapsibility and muscle responsiveness, underlying the response to OMT in OSA patients.

Structural Impacts of Drug-Resistance Mutations Appearing in HIV-2 Protease.

The use of antiretroviral drugs is accompanied by the emergence of HIV-2 resistances. Thus, it is important to elucidate the mechanisms of resistance to antiretroviral drugs. Here, we propose a structural analysis of 31 drug-resistant mutants of HIV-2 protease (PR2) that is an important target against HIV-2 infection. First, we modeled the structures of each mutant. We then located structural shifts putatively induced by mutations. Finally, we compared wild-type and mutant inhibitor-binding pockets and interfaces to explore the impacts of these induced structural deformations on these two regions. Our results showed that one mutation could induce large structural rearrangements in side-chain and backbone atoms of mutated residue, in its vicinity or further. Structural deformations observed in side-chain atoms are frequent and of greater magnitude, that confirms that to fight drug resistance, interactions with backbone atoms should be favored. We showed that these observed structural deformations modify the conformation, volume, and hydrophobicity of the binding pocket and the composition and size of the PR2 interface. These results suggest that resistance mutations could alter ligand binding by modifying pocket properties and PR2 stability by impacting its interface. Our results reinforce the understanding of the effects of mutations that occurred in PR2 and the different mechanisms of PR2 resistance.

Impacts of drug resistance mutations on the structural asymmetry of the HIV-2 protease.

BACKGROUND: Drug resistance is a severe problem in HIV treatment. HIV protease is a common target for the design of new drugs for treating HIV infection. Previous studies have shown that the crystallographic structures of the HIV-2 protease (PR2) in bound and unbound forms exhibit structural asymmetry that is important for ligand recognition and binding. Here, we investigated the effects of resistance mutations on the structural asymmetry of PR2. Due to the lack of structural data on PR2 mutants, the 3D structures of 30 PR2 mutants of interest have been modeled using an in silico protocol. Structural asymmetry analysis was carried out with an in-house structural-alphabet-based approach. RESULTS: The systematic comparison of the asymmetry of the wild-type structure and a large number of mutants highlighted crucial residues for PR2 structure and function. In addition, our results revealed structural changes induced by PR2 flexibility or resistance mutations. The analysis of the highlighted structural changes showed that some mutations alter protein stability or inhibitor binding. CONCLUSIONS: This work consists of a structural analysis of the impact of a large number of PR2 resistant mutants based on modeled structures. It suggests three possible resistance mechanisms of PR2, in which structural changes induced by resistance mutations lead to modifications in the dimerization interface, ligand recognition or inhibitor binding.

Comment on "Bad Language": Resolving some Ambiguities about Chirality.

In this comment on the Essay entitled "Bad Language" by Dunitz I add new elements which enlighten the discussion. I hope that they contribute to suppress ambiguities and preconceived ideas about chirality.

CoMPARA: Collaborative Modeling Project for Androgen Receptor Activity.

BACKGROUND: Endocrine disrupting chemicals (EDCs) are xenobiotics that mimic the interaction of natural hormones and alter synthesis, transport, or metabolic pathways. The prospect of EDCs causing adverse health effects in humans and wildlife has led to the development of scientific and regulatory approaches for evaluating bioactivity. This need is being addressed using high-throughput screening (HTS) in vitro approaches and computational modeling. OBJECTIVES: In support of the Endocrine Disruptor Screening Program, the U.S. Environmental Protection Agency (EPA) led two worldwide consortiums to virtually screen chemicals for their potential estrogenic and androgenic activities. Here, we describe the Collaborative Modeling Project for Androgen Receptor Activity (CoMPARA) efforts, which follows the steps of the Collaborative Estrogen Receptor Activity Prediction Project (CERAPP). METHODS: The CoMPARA list of screened chemicals built on CERAPP's list of 32,464 chemicals to include additional chemicals of interest, as well as simulated ToxCast™ metabolites, totaling 55,450 chemical structures. Computational toxicology scientists from 25 international groups contributed 91 predictive models for binding, agonist, and antagonist activity predictions. Models were underpinned by a common training set of 1,746 chemicals compiled from a combined data set of 11 ToxCast™/Tox21 HTS in vitro assays. RESULTS: The resulting models were evaluated using curated literature data extracted from different sources. To overcome the limitations of single-model approaches, CoMPARA predictions were combined into consensus models that provided averaged predictive accuracy of approximately 80% for the evaluation set. DISCUSSION: The strengths and limitations of the consensus predictions were discussed with example chemicals; then, the models were implemented into the free and open-source OPERA application to enable screening of new chemicals with a defined applicability domain and accuracy assessment. This implementation was used to screen the entire EPA DSSTox database of ∼875,000 chemicals, and their predicted AR activities have been made available on the EPA CompTox Chemicals dashboard and National Toxicology Program's Integrated Chemical Environment. https://doi.org/10.1289/EHP5580.

Molecular Chirality in Classical Spacetime: Solving the Controversy about the Spinning Cone Model of Rotating Molecules.

The spinning cone is a model of rotating molecules used by Barron in 1986 in relation to asymmetric synthesis and to parity violation. He considered that the non-translating cone spinning about its symmetry axis has false chirality (i.e., it is not chiral), whereas Mislow concluded in 1999 that it is indeed chiral and severely criticized the true versus false chirality nomenclature introduced by Barron, who still disagreed in 2013 with the conclusion of Mislow. Here, it is shown that this controversy comes from an ambiguity in the spinning cone model and that in fact both authors were right. Light is thrown on the true chirality versus false chirality controversy with a very recently published result, which was thus unavailable to both authors: this is a new definition of chirality that encompasses the one introduced by Lord Kelvin at the end of the 19th century.

New physiological bench test reproducing nocturnal breathing pattern of patients with sleep disordered breathing.

Previous studies have shown that Automatic Positive Airway Pressure devices display different behaviors when connected to a bench using theoretical respiratory cycle scripts. However, these scripts are limited and do not simulate physiological behavior during the night. Our aim was to develop a physiological bench that is able to simulate patient breathing airflow by integrating polygraph data. We developed an algorithm analyzing polygraph data and transformed this information into digital inputs required by the bench hardware to reproduce a patient breathing profile on bench. The inputs are respectively the simulated respiratory muscular effort pressure input for an artificial lung and the sealed chamber pressure to regulate the Starling resistor. We did simulations on our bench for a total of 8 hours and 59 minutes for a breathing profile from the demonstration recording of a Nox T3 Sleep Monitor. The simulation performance results showed that in terms of relative peak-valley amplitude of each breathing cycle, simulated bench airflow was biased by only 1.48% ± 6.80% compared to estimated polygraph nasal airflow for a total of 6,479 breathing cycles. For total respiratory cycle time, the average bias ± one standard deviation was 0.000 ± 0.288 seconds. For patient apnea events, our bench simulation had a sensitivity of 84.7% and a positive predictive value equal to 90.3%, considering 149 apneas detected both in polygraph nasal simulated bench airflows. Our new physiological bench would allow personalizing APAP device selection to each patient by taking into account individual characteristics of a sleep breathing profile.

High Impact: The Role of Promiscuous Binding Sites in Polypharmacology.

The literature focuses on drug promiscuity, which is a drug's ability to bind to several targets, because it plays an essential role in polypharmacology. However, little work has been completed regarding binding site promiscuity, even though its properties are now recognized among the key factors that impact drug promiscuity. Here, we quantified and characterized the promiscuity of druggable binding sites from protein-ligand complexes in the high quality Mother Of All Databases while using statistical methods. Most of the sites (80%) exhibited promiscuity, irrespective of the protein class. Nearly half were highly promiscuous and able to interact with various types of ligands. The corresponding pockets were rather large and hydrophobic, with high sulfur atom and aliphatic residue frequencies, but few side chain atoms. Consequently, their interacting ligands can be large, rigid, and weakly hydrophilic. The selective sites that interacted with one ligand type presented less favorable pocket properties for establishing ligand contacts. Thus, their ligands were highly adaptable, small, and hydrophilic. In the dataset, the promiscuity of the site rather than the drug mainly explains the multiple interactions between the drug and target, as most ligand types are dedicated to one site. This underlines the essential contribution of binding site promiscuity to drug promiscuity between different protein classes.

Gait Monitoring and Walk Distance Estimation With an Accelerometer During 6-Minute Walk Test.

BACKGROUND: The 6-min walk test (6MWT) encompasses potential and untapped information related to exercise capacity. However, this test does not yield any information about gait pattern. Recently, we used a ventilatory polygraph to reveal respiratory adaptation during the 6MWT with subjects having high or low body mass index (BMI). In this study, we aimed to determine gait parameters with the same device, which integrates an accelerometer. METHODS: Using a 30-m corridor, steps and U-turns were detected with a custom-made algorithm, compared to video recordings as a reference method, and analyzed offline. From the vertical acceleration signal, we were able to determine cadence and step length, and we could calculate the total distance covered in 6 min (6MWD). We then compared these variables between subjects with low BMI (n = 13 subjects) or high BMI (n = 29 subjects). RESULTS: Steps and U-turn detection correlated with video results (r = 0.99, P < .001 for both). The 6MWD calculation was also in line with classical measurements (r = 0.99, P < .001). High BMI subjects had a significantly lower 6MWD, cadence, and step length than controls (P < .001 for each). Walking speed was more closely correlated with step length (r = 0.92) than with cadence (r = 0.64) for both groups. CONCLUSION: Our results demonstrated that a ventilatory polygraph with an embedded accelerometer can be used to detect steps and U-turns, and to calculate 6MWD. This method is sufficiently sensitive to characterize significant BMI-dependent differences in gait pattern during a 6MWT and appears to be a promising tool for routine clinical use.

Four Major Channels Detected in the Cytochrome P450 3A4: A Step toward Understanding Its Multispecificity.

We computed the network of channels of the 3A4 isoform of the cytochrome P450 (CYP) on the basis of 16 crystal structures extracted from the Protein Data Bank (PDB). The calculations were performed with version 2 of the CCCPP software that we developed for this research project. We identified the minimal cost paths (MCPs) output by CCCPP as probable ways to access to the buried active site. The algorithm of calculation of the MCPs is presented in this paper, with its original method of visualization of the channels. We found that these MCPs constitute four major channels in CYP3A4. Among the many channels proposed by Cojocaru et al. in 2007, we found that only four of them open in 3A4. We provide a refined description of these channels together with associated quantitative data.

In Silico Design of New Inhibitors Against Hemagglutinin of Influenza.

The RNA virus influenza A is a serious public health problem, with epidemics resulting in more than 250 000 deaths every year. A protein cavity was identified on the HA2 subunit of the hemagglutinin responsible for the entry of the virus into the host cell by endocytosis. The binding of a ligand in this zone rich in invariant residues and synthetic lethal couples could prevent therapeutic escape and inhibit the conformational change at pH = 5 which is necessary to initiate the membrane fusion in the endosome. Two pentapeptides, a linear peptide (EQRRS) and a cyclic peptide (DQRRD), have been proposed as potential ligands. Complex stability and the interactions between the ligand and the protein have been studied with the help of molecular dynamics and quantum chemistry methods. A high stability of the interactions has been obtained for these two ligands at both pH = 7 and pH = 5. Indeed, these two peptides present two cooperative modes of action that should prevent the conformational change at the origin of the spring-loaded mechanism at pH = 5, (1) mechanical because they are docked on HA2 and (2) electronic because they modify the protonation states of key residues in the loop. This study thus paves the way toward the development of peptide ligands that can inhibit the membrane fusion process.

Statistical Profiling of One Promiscuous Protein Binding Site: Illustrated by Urokinase Catalytic Domain.

While recent literature focuses on drug promiscuity, the characterization of promiscuous binding sites (ability to bind several ligands) remains to be explored. Here, we present a proteochemometric modeling approach to analyze diverse ligands and corresponding multiple binding sub-pockets associated with one promiscuous binding site to characterize protein-ligand recognition. We analyze both geometrical and physicochemical profile correspondences. This approach was applied to examine the well-studied druggable urokinase catalytic domain inhibitor binding site, which results in a large number of complex structures bound to various ligands. This approach emphasizes the importance of jointly characterizing pocket and ligand spaces to explore the impact of ligand diversity on sub-pocket properties and to establish their main profile correspondences. This work supports an interest in mining available 3D holo structures associated with a promiscuous binding site to explore its main protein-ligand recognition tendency.

The Cytochrome P450 3A4 has three Major Conformations: New Clues to Drug Recognition by this Promiscuous Enzyme.

We computed the channels of the 3A4 isoform of the cytochrome P450 3A4 (CYP) on the basis of 24 crystal structures extracted from the Protein Data Bank (PDB). We identified three major conformations (denoted C, O1 and O2) using an enhanced version of the CCCPP software that we developed for the present work, while only two conformations (C and O2 ) are considered in the literature. We established the flowchart of definition of these three conformations in function of the structural and physicochemical parameters of the ligand. The channels are characterized with qualitative and quantitative parameters, and not only with their surrounding secondary structures as it is usually done in the literature.

Cavity Versus Ligand Shape Descriptors: Application to Urokinase Binding Pockets.

We analyzed 78 binding pockets of the human urokinase plasminogen activator (uPA) catalytic domain extracted from a data set of crystallized uPA-ligand complexes. These binding pockets were computed with an original geometric method that does NOT involve any arbitrary parameter, such as cutoff distances, angles, and so on. We measured the deviation from convexity of each pocket shape with the pocket convexity index (PCI). We defined a new pocket descriptor called distributional sphericity coefficient (DISC), which indicates to which extent the protein atoms of a given pocket lie on the surface of a sphere. The DISC values were computed with the freeware PCI. The pocket descriptors and their high correspondences with ligand descriptors are crucial for polypharmacology prediction. We found that the protein heavy atoms lining the urokinases binding pockets are either located on the surface of their convex hull or lie close to this surface. We also found that the radii of the urokinases binding pockets and the radii of their ligands are highly correlated (r = 0.9).

A Fast Algorithm to Compute Conical Pockets in Proteins. Application to the Structural Characterization of γ-Carbonic Anhydrases.

Some major proteins families, such as carbonic anhydrases (CAs), have a conical cavity at the active site. No algorithm was available to compute conical cavities, so we needed to design one. The fast algorithm we designed let us show on a set of 717 CAs extracted from the PDB database that γ-CAs are characterized by active site cavity cone angles significantly larger than those of α-CAs and ß-CAs: the generatrix-axis angles are greater than 60° for the γ-CAs while they are smaller than 50° for the other CAs. Free binaries of the CONICA software implementing the algorithm are available through a software repository at http://petitjeanmichel.free.fr/itoweb.petitjean.freeware.html.

Validity of thoracic respiratory inductive plethysmography in high body mass index subjects.

We aim to evaluate thoracic respiratory inductive plethysmography (RIP) in high body mass index (BMI) subjects with a pneumotachometer (PT) as a reference. We simultaneously evaluated spontaneous breathing by RIP and PT in 10 low and 10 high BMI subjects at rest and in moderate exercise. We then recorded RIP amplitude with different excursions mimicking respiratory thoracic deformation, with different sizes of RIP belts surrounding cylinders of different perimeters with or without deformable foam simulating adipose tissue. RIP responses correlated with PT values in low and high BMI groups for inspiratory time (r=0.86 and r=0.91, respectively), expiratory time (r=0.96 and r=0.91, respectively) and amplitude (r=0.82 for both) but with a bias (-0.23±0.25L) for high BMI subjects. ANOVA revealed the effects of perimeter and simulated adiposity (p<0.001 for both). We concluded that thoracic perimeter and deformity of adipose tissue are responsible for biases in RIP response in high BMI subjects.