Virtual Screening of Histone Lysine Demethylase(JMJD2) identifies new inhibitors

The JmjC domain-containing proteins are hydroxylases that confer posttranslational modifications on histone tails, by removing methylation marks on methylated lysine residues. This serves to either promote or repress gene transcription. The JMJD2A-D family members include the enzyme Jumonji domain 2C (JMJD2C), which specifically demethylates di- and trimethylated histone H3 at Lys 9 or Lys 36.[1] Dysregulation of JMJD2C has been implicated in prostate, colonic, and breast cancer as the demethylase can modify the expression levels of oncogenes.[2] The goal of the present study was to identify potent and selective small-molecule inhibitors of JMJD2C, to be used as chemical biology tools to further investigate the role of JMJD2C in cell proliferation and survival. Using high-resolution crystal structures of the JMJD2 subfamily members as templates, we have performed a small molecule virtual docking screen. From the ~3 million molecules that were docked, this experiment identified 21 compounds as possible leads. These compounds were tested against JMJD2C in enzymatic assays and here we report an overall hit rate of 76%, with 8 compounds demonstrating an IC50 of 176μM to 1.18μM. A molecule containing a salicylate core was selected as a candidate for optimization and thus far we have completed several rounds of iterative target-specific compound docking, hybrid molecule design, compound synthesis and in vitro characterization. Notably, our method demonstrated a substantial increase in potency when we linked two docked fragments together and further derivatized this new scaffold, through which we have successfully derived a 65nM inhibitor of JMJD2C. A compound representing the inhibitor scaffold has been co-crystallized with JMJD2A to a resolution of 2.4 Å. In the crystal structure each asymmetric unit contains two JMJD2A monomers, each bound to a single inhibitor molecule. This complex-structure superposes well with the docked pose for the hybrid series of compounds. We are now focusing our efforts on identifying an inhibitor that is selective for the JMJD2 family over other JmjC domain-containing proteins

Structural studies of lyase mediated resistance to group B streptogramins in «Staphylococcus aureus»

Synercid® is used to treat infections caused by vancomycin resistant Enterococcus faecium, methicillin resistant Staphylococcus species and some Streptococcus species. It belongs to a class of antimicrobials, known as streptogramins, which is comprised of Group A (e.g. dalfopristin) and Group B (e.g. quinupristin) compounds. Independently, these compounds are bacteriostatic against Gram-positive bacteria, however, in combination they exhibit synergistic bactericidal effects due to permanent inhibition of the bacterial 50S ribosomal subunit. Resistance to the Group B streptogramins is conferred enzymatically by streptogramin B lyases (Vgb), which cleave the ring structure of these peptide drugs.Here we present the first crystal structure of a streptogramin B lyase from Staphylococcus aureus to a resolution of 1.65 Å. High-resolution data sets were collected for both the native and L-selenomethionine substituted Vgb and the structure was solved using a combination of MAD and molecular replacement techniques. The structure of Vgb, is that of a beta-propeller consisting of seven blades made of highly twisted beta-sheets arranged in a circular array. A similar fold has been observed in the WD40, Kelch, YWTD, PQQ and AxSPD families, however, Vgb has no sequence similarity to these proteins. This identifies Vgb as possessing a novel beta-propeller motif. Initial attempts to crystallize Vgb with a catalytically inactive mutant were met with limited success due to problems with crystal packing. Using structure-inspired loop mutagenesis, we were able to crystallize Vgb in a new crystal form conducive to substrate binding. This structure reveals that the antibiotic, quinupristin, binds in a large depression on the top face of Vgb. Guided by the structure, we propose a reaction mechanism in which the initial proton abstraction is facilitated by a Mg2+ linked conjugated system, with His270, His228, Try18, Glu268, and Glu284 playing roles in the catalytic mechanism. Since Mg2+ is only indirectly involved in the opening of the lactone ring, this represents a novel enzymatic mechanism.A comparison of Vgb and the ribosome structures reveals that Mg2+ facilitates the binding of the antibiotic through the 3-hydroxypicolinic acid moiety in both targets, however, the predominant associations are due to van der Waals interactions. In Vgb, interactions between the enzyme and the substrate are predominantly through interactions parallel to the plane of the lactone, while in the ribosome the antibiotic bound is at the periphery of the ring. This allows for extension of the antibiotic at the L-phenylglycine moiety when bound to the ribosome, while such an alteration would preclude efficient binding to Vgb. Lastly, we have characterized an engineered Group B streptogramin that has a variation at the L-phenylglycine position and we find that this new ligand retains antibacterial properties while it is a much weaker substrate for Vgb. This information has laid out the foundation
 for further structure-based drug design of Group B streptogramins that will bind the ribosome with greater affinity and not be susceptible to inactivation by Vgb.Le Synercide® est utilisé pour traiter les infections causées par Enterococcus faecium résistant à la vancomycine, les espèces de Staphylocoques résistantes à la méthicilline, ainsi que certaines espèces de Streptocoques. Il appartient à une catégorie d'antibiotiques dénommée streptogramines. Cette dernière est composée de deux sous-groupes d'antibiotiques, notamment Groupe A (ex. dalfopristine) et Groupe B (e.x. quinopristine). Utilisés indépendamment, ces composés ont un effet bactériostatique contre les bactéries à Gram positif. Cependant, en combinaison, elles exercent un effet synergique bactéricide grâce à l'inhibition permanente de l'unité 50S du ribosome. La résistance aux streptogramines du Groupe B est conférée par des enzymes lyases streptogramine B(Vgb), qui scindent la structure cyclique de ces antibiotique.Dans ce thèse, nous présentons la première structure cristallographique d'une lyase streptogramine B de Staphylococcus aureus à une résolution de 1.65 Å. Des données à haute résolution ont été obtenues pour la protéine Vgb native, ainsi que celle qui contenait des substitutions de L-sélénométhionine. La structure a été résolue en utilisant une combinaison des techniques de MAD et de remplacements moléculaires. La structure de Vgb est celle d'une hélice β composée de sept ailettes de feuillets β tordus et arrangés en formation circulaire. Un pli semblable fut observé dans les familles WD40, Kelch, YWTD, PQQ et AxSPD, cependant Vgb ne possède aucune similarité au niveau de sa séquence avec ces protéines. Ceci indique que Vgb possède une hélice β inédite. Les efforts initiaux pour cristalliser Vgb avec un mutant catalytiquement inactif ont été limités par des problèmes d'empaquetage de cristaux. En utilisant la mutagenèse d'une boucle, nous avons réussi à cristalliser Vgb dans une nouvelle configuration qui permet la liaison de son substrat. Cette structure révèle que la quinupristine se lie dans un large creux situé sur la surface supérieure de Vgb. Guidés par la structure, nous proposons un mécanisme de réaction dans lequel le départ initiale du proton est favorisée par un système conjugué lié au Mg2+, avec His270, His228, Tyr 18, Glu268 et Glu284 jouant des rôles clés dans le mécanisme catalytique. Étant donné que le Mg2+ est impliqué de manière indirecte dans l'ouverture de l'anneau du lactone, ceci représente un nouveau mécanisme enzymatique.En comparant Vgb et le ribosome, on observe que le Mg2+ favorise la liaison de l'antibiotique par le biais de l'acide 3-hydroxypicolinique aux deux sites, cependant, les interactions van der Waals représentent le mécanisme principal de liaison. Au niveau de Vgb, les interactions entre l'enzyme et son substrat sont favorisées par des interactions van der Waals parallèle au plan de la lactone, quant au ribosome, la liaison de l'antibiotique a lieu à la périphérie de l'anneau. Ceci permet l'extension de l'antibiotique sur la moitié L-phénylglycine lorsque lié au ribosome, mais un changement pareil empêcherait la liaison efficace à Vgb. Nous avons caractérisé une streptogramine B modifiée qui possède une variation à cette position et nous avons remarqué que cette nouvelle streptogramine garde son action antibactérienne tout en étant un faible substrat de Vgb. Cette information forme une base pour la création d'autres streptogramines de Groupe B qui se fixeront avec plus d'efficacité au ribosome sans être susceptibles à l'inactivation par Vgb

Dizziness Handicap Inventory in Clinical Evaluation of Dizzy Patients

(1) Objectives: The evaluation of dizzy patients is difficult due to nonspecific symptoms that require a multi-specialist approach. The Dizziness Handicap Inventory (DHI) is widely used in the assessment of dizziness-related disability, but its clinical efficacy needs further expansion. The aim of this study was to identify the subscales of DHI that may correlate with some vestibular or nonvestibular dysfunctions. (2) Material and methods: This observational study included 343 dizzy patients with one of the following clinical conditions: Vestibular impairment noncompensated or compensated, central or bilateral, benign paroxysmal positional vertigo (BPPV), migraine and psychogenic dizziness. Principal component analysis was used to examine the factorial structure of the questionnaire. (3) Results: The DHI questionnaire total scoring and its vestibular subscale distinguished between patients with compensated and uncompensated vestibular dysfunction with positive predictive values of 76% and 79%, respectively. The DHI items composing the F3 (positional) subscale revealed the highest scoring in the BPPV group with 75% sensitivity and 92% negative predictive value (NPV) in reference to Dix–Hallpike tests. The DHI total score and the subscales scores correlated with anxiety-depression, and the highest correlation coefficients were calculated for vestibular (F2 0.56) and anxiety (F5 0.51) subscales. (4) Conclusions: Our analysis revealed that the DHI vestibular subscale distinguishes between patients with compensated and uncompensated vestibular dysfunction. The positional subscale showed the highest scoring in the BPPV group with high sensitivity and low specificity of the test. The DHI is highly correlated with patients’ psychological status

Utility of the Novel MediPost Mobile Posturography Device in the Assessment of Patients with a Unilateral Vestibular Disorder

Balance disorders are a growing problem worldwide. Thus, there is an increasing need to provide an inexpensive and feasible alternative to standard posturographic platforms (SP) used for the assessment of balance and to provide a possible solution for telemonitoring of patients. A novel mobile posturography (MP) MediPost device was developed to address these issues. This prospective study used a Modified Clinical Test of Sensory Interaction on Balance to evaluate healthy individuals and patients with a unilateral vestibular disorder through SP and MP simultaneously. The control group included 65 healthy volunteers, while the study group included 38 patients diagnosed with a unilateral vestibular deficit. The angular velocity values obtained from both methods were compared by intraclass correlation coefficients (ICC) and Bland–Altman plot analysis. Diagnostic capabilities were measured in terms of sensitivity and specificity. The ICC between the two methods for conditions 2–4 was indicative of excellent reliability, with the ICC > 0.9 (p < 0.001), except for Condition 1 (standing stance, eyes open) ICC = 0.685, p < 0.001, which is indicative of moderate reliability. ROC curve analysis of angular velocity for condition 4 represents the most accurate differentiating factor with AUC values of 0.939 for SP and 0.953 for MP. This condition also reported the highest sensitivity, specificity, PPV, and NPV values with 86.4%, 87.7%, 80%, and 90.5% for SP, and 92.1%, 84.6%, 77.8%, and 94.8% for MP, respectively. The newly developed MediPost device has high sensitivity and specificity in distinguishing between healthy individuals and patients with a unilateral vestibular deficit

The Recognition of Unrelated Ligands by Identical Proteins

Unrelated ligands, often found in drug discovery campaigns, can bind to the same receptor, even with the same protein residues. To investigate how this might occur, and whether it might be typically possible to find unrelated ligands for the same drug target, we sought examples of topologically unrelated ligands that bound to the same protein in the same site. Seventy-six pairs of ligands, each bound to the same protein (152 complexes total), were considered, classified into three groups. In the first (31 pairs of complexes), unrelated ligands interacted largely with the same pocket residues through different functional groups. In the second group (39 pairs), the unrelated ligand in each pair engaged different residues, though still within the same pocket. The smallest group (6 pairs) contained ligands with different scaffolds but with shared functional groups interacting with the same residues. We found that there are multiple chemically unrelated but physically similar functional groups that can complement any given local protein pocket; when these functional group substitutions are combined within a single molecule, they lead to topologically unrelated ligands that can each well-complement a site. It may be that many active and orthosteric sites can recognize topologically unrelated ligands

Virtual Reality—A Supplement to Posturography or a Novel Balance Assessment Tool?

Virtual reality (VR) is a well-established technology in medicine. Head-mounted displays (HMDs) have made VR more accessible in many branches of medical research. However, its application in balance evaluation has been vague, and comprehensive literature on possible applications of VR in posture measurement is scarce. The aim of this review is to conduct a literature search on the application of immersive VR delivered using a head-mounted display in posturographic measurements. A systematic search of two databases, PubMed and Scopus, using the keywords “virtual reality” and “posturography,” was performed following PRISMA guidelines for systematic reviews. Initial search results returned 89 non-duplicate records. Two reviewers independently screened the abstracts. Sixteen papers fulfilled the inclusion criteria and none of the exclusion criteria and were selected for complete text retrieval. An additional 16 records were identified from citation searching. Ultimately, 21 studies were included in this review. virtual reality is often used as additional visual stimuli in static and dynamic posturography evaluation. Only one study has attempted to evaluate a VR environment in a head-mounted display as an independent method in the assessment of posture. Further research should be conducted to assess HMD VR as a standalone posturography replacement

Classification of Subjects With Balance Disorders Using 1D-CNN and Inertial Sensors

The article presents the concept of detecting subjects with balance disorders by the use of machine learning techniques. The proposed solution has been developed and tested based on a group of 40 subjects, the group included both patients with uncompensated dysfunction in the vestibular system and healthy volunteers. Presence of dysfunction was verified prior to the study by detailed clinical examination. The data for the study were collected with the use of miniature micromachine sensors, mounted on the body at selected locations. The task performed by the subjects consisted of free gait over a distance of three meters; the task was selected to make it easy to perform in any surroundings and not requiring additional equipment. The collected data was used as an input to an artificial neural network based on a one-dimensional convolution kernel. The proposed solution allows to classify subjects into healthy and non-healthy with an accuracy of 87.5%