ResearchPad - chemical-bonding Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Atco, a yeast mitochondrial complex of Atp9 and Cox6, is an assembly intermediate of the ATP synthase]]> Mitochondrial oxidative phosphorylation (oxphos) is the process by which the ATP synthase conserves the energy released during the oxidation of different nutrients as ATP. The yeast ATP synthase consists of three assembly modules, one of which is a ring consisting of 10 copies of the Atp9 subunit. We previously reported the existence in yeast mitochondria of high molecular weight complexes composed of mitochondrially encoded Atp9 and of Cox6, an imported structural subunit of cytochrome oxidase (COX). Pulse-chase experiments indicated a correlation between the loss of newly translated Atp9 complexed to Cox6 and an increase of newly formed Atp9 ring, but did not exclude the possibility of an alternate source of Atp9 for ring formation. Here we have extended studies on the functions and structure of this complex, referred to as Atco. We show that Atco is the exclusive source of Atp9 for the ATP synthase assembly. Pulse-chase experiments show that newly translated Atp9, present in Atco, is converted to a ring, which is incorporated into the ATP synthase with kinetics characteristic of a precursor-product relationship. Even though Atco does not contain the ring form of Atp9, cross-linking experiments indicate that it is oligomeric and that the inter-subunit interactions are similar to those of the bona fide ring. We propose that, by providing Atp9 for biogenesis of ATP synthase, Atco complexes free Cox6 for assembly of COX. This suggests that Atco complexes may play a role in coordinating assembly and maintaining proper stoichiometry of the two oxphos enzymes

<![CDATA[The role of the C<sub>2</sub>A domain of synaptotagmin 1 in asynchronous neurotransmitter release]]> Following nerve stimulation, there are two distinct phases of Ca2+-dependent neurotransmitter release: a fast, synchronous release phase, and a prolonged, asynchronous release phase. Each of these phases is tightly regulated and mediated by distinct mechanisms. Synaptotagmin 1 is the major Ca2+ sensor that triggers fast, synchronous neurotransmitter release upon Ca2+ binding by its C2A and C2B domains. It has also been implicated in the inhibition of asynchronous neurotransmitter release, as blocking Ca2+ binding by the C2A domain of synaptotagmin 1 results in increased asynchronous release. However, the mutation used to block Ca2+ binding in the previous experiments (aspartate to asparagine mutations, sytD-N) had the unintended side effect of mimicking Ca2+ binding, raising the possibility that the increase in asynchronous release was directly caused by ostensibly constitutive Ca2+ binding. Thus, rather than modulating an asynchronous sensor, sytD-N may be mimicking one. To directly test the C2A inhibition hypothesis, we utilized an alternate C2A mutation that we designed to block Ca2+ binding without mimicking it (an aspartate to glutamate mutation, sytD-E). Analysis of both the original sytD-N mutation and our alternate sytD-E mutation at the Drosophila neuromuscular junction showed differential effects on asynchronous release, as well as on synchronous release and the frequency of spontaneous release. Importantly, we found that asynchronous release is not increased in the sytD-E mutant. Thus, our work provides new mechanistic insight into synaptotagmin 1 function during Ca2+-evoked synaptic transmission and demonstrates that Ca2+ binding by the C2A domain of synaptotagmin 1 does not inhibit asynchronous neurotransmitter release in vivo.

<![CDATA[Assessing the effects of intratendinous genipin injections: Mechanical augmentation and spatial distribution in an ex vivo degenerative tendon model]]>


Tendinopathy is a common musculoskeletal disorder and current treatment options show limited success. Genipin is an effective collagen crosslinker with low cytotoxicity and a promising therapeutic strategy for stabilizing an intratendinous lesion.


This study examined the mechanical effect and delivery of intratendinous genipin injection in healthy and degenerated tendons.

Study design

Controlled laboratory study


Bovine superficial digital flexor tendons were randomized into four groups: Healthy control (N = 25), healthy genipin (N = 25), degenerated control (N = 45) and degenerated genipin (N = 45). Degeneration was induced by Collagenase D injection. After 24h, degenerated tendons were subsequently injected with either 0.2ml of 80mM genipin or buffer only. 24h post-treatment, samples were cyclically loaded for 500 cycles and then ramp loaded to failure. Fluorescence and absorption assays were performed to analyze genipin crosslink distribution and estimate tissue concentration after injection.


Compared to controls, genipin treatment increased ultimate force by 19% in degenerated tendons (median control 530 N vs. 633 N; p = 0.0078). No significant differences in mechanical properties were observed in healthy tendons, while degenerated tendons showed a significant difference in ultimate stress (+23%, p = 0.049), stiffness (+27%, p = 0.037), work to failure (+42%, p = 0.009), and relative stress relaxation (-11%, p < 0.001) after genipin injection. Fluorescence and absorption were significantly higher in genipin treated tendons compared to control groups. A higher degree of crosslinking (+45%, p < 0.001) and a more localized distribution were observed in the treated healthy compared to degenerated tendons, with higher genipin tissue concentrations in healthy (7.9 mM) than in degenerated tissue (2.3 mM).


Using an ex-vivo tendinopathy model, intratendinous genipin injections recovered mechanical strength to the level of healthy tendons. Measured by genipin tissue distribution, injection is an effective method for local delivery.

Clinical relevance

This study provides a proof of concept for the use of intratendinous genipin injection in the treatment of tendinopathy. The results demonstrate that a degenerated tendon can be mechanically augmented by a clinically viable method of local genipin delivery. This warrants further in vivo studies towards the development of a clinically applicable treatment based on genipin.

<![CDATA[Molecular mechanisms of mesoporous silica formation from colloid solution: Ripening-reactions arrest hollow network structures]]>

The agglomeration of silica nanoparticles in aqueous solution is investigated from molecular simulations. Mimicking destabilization of colloidal solutions by full removal of protective moieties or surface charge, association of SiO2/Si(OH)4 core/shell particles leads to rapid proton transfer reactions that account for local silanole → silica ripening reactions. Yet, such virtually barrier-less binding is only observed within a limited contact zone. Agglomeration hence leads to the formation of oligomers of nanoparticles, whilst full merging into a compact precipitate is hampered by the need for extended structural reorganisation. Implementing sufficiently fast supply from colloidal solution, our simulations show the development of silica networks comprised of covalently bound, yet not fully merged nanoparticles. Within the oligomerized nanoparticle network, coordination numbers range from 2 to 5 –which is far below closest packing. Our simulations hence rationalize the formation of covalently bound network structures hosting extended pores. The resulting interfaces to the solvent show water immobilization only for the immediate contact layers, whilst the inner pores exhibit solvent mobility akin to bulk water.

<![CDATA[The ability of locked nucleic acid oligonucleotides to pre-structure the double helix: A molecular simulation and binding study]]>

Locked nucleic acid (LNA) oligonucleotides bind DNA target sequences forming Watson-Crick and Hoogsteen base pairs, and are therefore of interest for medical applications. To be biologically active, such an oligonucleotide has to efficiently bind the target sequence. Here we used molecular dynamics simulations and electrophoresis mobility shift assays to elucidate the relation between helical structure and affinity for LNA-containing oligonucleotides. In particular, we have studied how LNA substitutions in the polypyrimidine strand of a duplex (thus forming a hetero duplex, i.e. a duplex with a DNA polypurine strand and an LNA/DNA polypyrimidine strand) enhance triplex formation. Based on seven polypyrimidine single strand oligonucleotides, having LNAs in different positions and quantities, we show that alternating LNA with one or more non-modified DNA nucleotides pre-organizes the hetero duplex toward a triple-helical-like conformation. This in turn promotes triplex formation, while consecutive LNAs distort the duplex structure disfavoring triplex formation. The results support the hypothesis that a pre-organization in the hetero duplex structure enhances the binding of triplex forming oligonucleotides. Our findings may serve as a criterion in the design of new tools for efficient oligonucleotide hybridization.

<![CDATA[Conformational regulation of Escherichia coli DNA polymerase V by RecA and ATP]]>

Mutagenic translesion DNA polymerase V (UmuD′2C) is induced as part of the DNA damage-induced SOS response in Escherichia coli, and is subjected to multiple levels of regulation. The UmuC subunit is sequestered on the cell membrane (spatial regulation) and enters the cytosol after forming a UmuD′2C complex, ~ 45 min post-SOS induction (temporal regulation). However, DNA binding and synthesis cannot occur until pol V interacts with a RecA nucleoprotein filament (RecA*) and ATP to form a mutasome complex, pol V Mut = UmuD′2C-RecA-ATP. The location of RecA relative to UmuC determines whether pol V Mut is catalytically on or off (conformational regulation). Here, we present three interrelated experiments to address the biochemical basis of conformational regulation. We first investigate dynamic deactivation during DNA synthesis and static deactivation in the absence of DNA synthesis. Single-molecule (sm) TIRF-FRET microscopy is then used to explore multiple aspects of pol V Mut dynamics. Binding of ATP/ATPγS triggers a conformational switch that reorients RecA relative to UmuC to activate pol V Mut. This process is required for polymerase-DNA binding and synthesis. Both dynamic and static deactivation processes are governed by temperature and time, in which onoff switching is “rapid” at 37°C (~ 1 to 1.5 h), “slow” at 30°C (~ 3 to 4 h) and does not require ATP hydrolysis. Pol V Mut retains RecA in activated and deactivated states, but binding to primer-template (p/t) DNA occurs only when activated. Studies are performed with two forms of the polymerase, pol V Mut-RecA wt, and the constitutively induced and hypermutagenic pol V Mut-RecA E38K/ΔC17. We discuss conformational regulation of pol V Mut, determined from biochemical analysis in vitro, in relation to the properties of pol V Mut in RecA wild-type and SOS constitutive genetic backgrounds in vivo.

<![CDATA[DeepDrug3D: Classification of ligand-binding pockets in proteins with a convolutional neural network]]>

Comprehensive characterization of ligand-binding sites is invaluable to infer molecular functions of hypothetical proteins, trace evolutionary relationships between proteins, engineer enzymes to achieve a desired substrate specificity, and develop drugs with improved selectivity profiles. These research efforts pose significant challenges owing to the fact that similar pockets are commonly observed across different folds, leading to the high degree of promiscuity of ligand-protein interactions at the system-level. On that account, novel algorithms to accurately classify binding sites are needed. Deep learning is attracting a significant attention due to its successful applications in a wide range of disciplines. In this communication, we present DeepDrug3D, a new approach to characterize and classify binding pockets in proteins with deep learning. It employs a state-of-the-art convolutional neural network in which biomolecular structures are represented as voxels assigned interaction energy-based attributes. The current implementation of DeepDrug3D, trained to detect and classify nucleotide- and heme-binding sites, not only achieves a high accuracy of 95%, but also has the ability to generalize to unseen data as demonstrated for steroid-binding proteins and peptidase enzymes. Interestingly, the analysis of strongly discriminative regions of binding pockets reveals that this high classification accuracy arises from learning the patterns of specific molecular interactions, such as hydrogen bonds, aromatic and hydrophobic contacts. DeepDrug3D is available as an open-source program at with the accompanying TOUGH-C1 benchmarking dataset accessible from

<![CDATA[Metal based donepezil analogues designed to inhibit human acetylcholinesterase for Alzheimer’s disease]]>

Among neurodegenerative disorders, Alzheimer’s disease (AD) is one of the most common disorders showing slow progressive cognitive decline. Targeting acetylcholinesterase (AChE) is one of the major strategies for AD therapeutics, as cholinergic pathways in the cerebral cortex and basal forebrain are compromised. Herein, we report the design of some copper and other metal based donepezil derivatives, employing density functional theory (DFT). All designed compounds are optimized at the B3LYP/SDD level of theory. Dipole moments, electronic energie, enthalpies, Gibbs free energies, and HOMO-LUMO gaps of these modified compounds are also investigated in the subsequent analysis. The molecules were then subjected to molecular docking analysis with AChE to study the molecular interactions broadly. Ensemble based docking and molecular dynamics (MD) simulations of the best candidates were also performed. Docking and MD simulation reveal that modified drugs are more potent than unmodified donepezil, where Trp86, Tyr337, Phe330 residues play some important roles in drug-receptor interactions. According to ensemble based docking, D9 shows greater binding affinity compared to the parent in most conformations obtained from protein data bank and MD simulation. In addition, it is observed that the π- π stacking with the residues of Trp86, Tyr337, Tyr341, Tyr124 and Trp286 may be required for strong ligand binding. Moreover, ADME/T analysis suggests that modified derivatives are less toxic and have improved pharmacokinetic properties than those of the parent drug. These results further confirm the ability of metal-directed drugs to bind simultaneously to the active sites of AChE and support them as potential candidates for the future treatment of Alzheimer’s disease.

<![CDATA[Interaction between the transmembrane domains of Sho1 and Opy2 enhances the signaling efficiency of the Hog1 MAP kinase cascade in Saccharomyces cerevisiae]]>

To cope with increased extracellular osmolarity, the budding yeast Saccharomyces cerevisiae activates the Hog1 mitogen-activated protein kinase (MAPK), which controls a variety of adaptive responses. Hog1 is activated through the high-osmolarity glycerol (HOG) pathway, which consists of a core MAPK cascade and two independent upstream branches (SHO1 and SLN1 branches) containing distinct osmosensing machineries. In the SHO1 branch, a homo-oligomer of Sho1, the four-transmembrane (TM) osmosensor, interacts with the transmembrane co-osmosensors, Hkr1 and Msb2, and the membrane anchor protein Opy2, through their TM domains, and activates the Ste20-Ste11-Pbs2-Hog1 kinase cascade. In this study, we isolated and analyzed hyperactive mutants of Sho1 and Opy2 that harbor mutations within their TM domains. Several hyperactive mutations enhanced the interaction between Sho1 and Opy2, indicating the importance of the TM-mediated interaction between Sho1 and Opy2 for facilitating effective signaling. The interaction between the TM domains of Sho1 and Opy2 will place their respective cytoplasmic binding partners Pbs2 and Ste11 in close proximity. Indeed, genetic analyses of the mutants showed that the Sho1-Opy2 interaction enhances the activation of Pbs2 by Ste11, but not Hog1 by Pbs2. Some of the hyperactive mutants had mutations at the extracellular ends of either Sho1 TM4 or Opy2 TM, and defined the Sho1-Opy2 binding site 1 (BS1). Chemical crosslinking and mutational analyses revealed that the cytoplasmic ends of Sho1 TM1 and Opy2 TM also interact with each other, defining the Sho1-Opy2 binding site 2 (BS2). A geometric consideration constrains that one Opy2 molecule must interact with two adjacent Sho1 molecules in Sho1 oligomer. These results raise a possibility that an alteration of the conformation of the Sho1-Opy2 complex might contributes to the osmotic activation of the Hog1 MAPK cascade.

<![CDATA[Thioguanine-based DENV-2 NS2B/NS3 protease inhibitors: Virtual screening, synthesis, biological evaluation and molecular modelling]]>

Dengue virus Type 2 (DENV-2) is predominant serotype causing major dengue epidemics. There are a number of studies carried out to find its effective antiviral, however to date, there is still no molecule either from peptide or small molecules released as a drug. The present study aims to identify small molecules inhibitor from National Cancer Institute database through virtual screening. One of the hits, D0713 (IC50 = 62 μM) bearing thioguanine scaffold was derivatised into 21 compounds and evaluated for DENV-2 NS2B/NS3 protease inhibitory activity. Compounds 18 and 21 demonstrated the most potent activity with IC50 of 0.38 μM and 16 μM, respectively. Molecular dynamics and MM/PBSA free energy of binding calculation were conducted to study the interaction mechanism of these compounds with the protease. The free energy of binding of 18 calculated by MM/PBSA is -16.10 kcal/mol compared to the known inhibitor, panduratin A (-11.27 kcal/mol), which corroborates well with the experimental observation. Results from molecular dynamics simulations also showed that both 18 and 21 bind in the active site and stabilised by the formation of hydrogen bonds with Asn174.

<![CDATA[Effect of Sec61 interaction with Mpd1 on endoplasmic reticulum-associated degradation]]>

Proteins that misfold in the endoplasmic reticulum (ER) are transported back to the cytosol for ER-associated degradation (ERAD). The Sec61 channel is one of the candidates for the retrograde transport conduit. Channel opening from the ER lumen must be triggered by ERAD factors and substrates. Here we aimed to identify new lumenal interaction partners of the Sec61 channel by chemical crosslinking and mass spectrometry. In addition to known Sec61 interactors we detected ERAD factors including Cue1, Ubc6, Ubc7, Asi3, and Mpd1. We show that the CPY* ERAD factor Mpd1 binds to the lumenal Sec61 hinge region. Deletion of the Mpd1 binding site reduced the interaction between both proteins and caused an ERAD defect specific for CPY* without affecting protein import into the ER or ERAD of other substrates. Our data suggest that Mpd1 binding to Sec61 is a prerequisite for CPY* ERAD and confirm a role of Sec61 in ERAD of misfolded secretory proteins.

<![CDATA[Potent, multi-target serine protease inhibition achieved by a simplified β-sheet motif]]>

Engagement of an extended β-sheet is a common substrate/inhibitor interaction at the active site of serine proteases and is an important feature of Laskowski mechanism inhibitors that present a substrate-like loop to a target protease. This loop is cleaved but subsequently relegated forming a stable inhibitor/protease complex. Laskowski inhibitors are ubiquitous in nature and are used extensively in serine protease inhibitor design. However, most studies concentrate on introducing new sidechain interactions rather than the direct contributions of the substrate-like β-sheet to enzyme inhibition. Here we report the crystal structure of an simplified β-sheet inhibitory motif within the Sunflower Trypsin Inhibitor (SFTI) in complex with trypsin. We show that the intramolecular hydrogen bond network of this SFTI variant (SFTI-TCTR) engages the inhibitor sidechains that would normally interact with a target protease, giving mainchain interactions a more prominent role in complex formation. Despite having reduced sidechain interactions, this SFTI variant is remarkably potent and inhibits a diverse range of serine proteases. Crystal structural analysis and molecular modelling of SFTI-TCTR complexes again indicates an interface dominated by β–sheet interactions, highlighting the importance of this motif and the adaptability of SFTI as a scaffold for inhibitor design.

<![CDATA[The Vaccinia virion: Filling the gap between atomic and ultrastructure]]>

We have investigated the molecular-level structure of the Vaccinia virion in situ by protein-protein chemical crosslinking, identifying 4609 unique-mass crosslink ions at an effective FDR of 0.33%, covering 2534 unique pairs of crosslinked protein positions, 625 of which were inter-protein. The data were statistically non-random and rational in the context of known structures, and showed biological rationality. Crosslink density strongly tracked the individual proteolytic maturation products of p4a and p4b, the two major virion structural proteins, and supported the prediction of transmembrane domains within membrane proteins. A clear sub-network of four virion structural proteins provided structural insights into the virion core wall, and proteins VP8 and A12 formed a strongly-detected crosslinked pair with an apparent structural role. A strongly-detected sub-network of membrane proteins A17, H3, A27 and A26 represented an apparent interface of the early-forming virion envelope with structures added later during virion morphogenesis. Protein H3 seemed to be the central hub not only for this sub-network but also for an ‘attachment protein’ sub-network comprising membrane proteins H3, ATI, CAHH(D8), A26, A27 and G9. Crosslinking data lent support to a number of known interactions and interactions within known complexes. Evidence is provided for the membrane targeting of genome telomeres. In covering several orders of magnitude in protein abundance, this study may have come close to the bottom of the protein-protein crosslinkome of an intact organism, namely a complex animal virus.

<![CDATA[The integration of pharmacophore-based 3D QSAR modeling and virtual screening in safety profiling: A case study to identify antagonistic activities against adenosine receptor, A2A, using 1,897 known drugs]]>

Safety pharmacology screening against a wide range of unintended vital targets using in vitro assays is crucial to understand off-target interactions with drug candidates. With the increasing demand for in vitro assays, ligand- and structure-based virtual screening approaches have been evaluated for potential utilization in safety profiling. Although ligand based approaches have been actively applied in retrospective analysis or prospectively within well-defined chemical space during the early discovery stage (i.e., HTS screening and lead optimization), virtual screening is rarely implemented in later stage of drug discovery (i.e., safety). Here we present a case study to evaluate ligand-based 3D QSAR models built based on in vitro antagonistic activity data against adenosine receptor 2A (A2A). The resulting models, obtained from 268 chemically diverse compounds, were used to test a set of 1,897 chemically distinct drugs, simulating the real-world challenge of safety screening when presented with novel chemistry and a limited training set. Due to the unique requirements of safety screening versus discovery screening, the limitations of 3D QSAR methods (i.e., chemotypes, dependence on large training set, and prone to false positives) are less critical than early discovery screen. We demonstrated that 3D QSAR modeling can be effectively applied in safety assessment prior to in vitro assays, even with chemotypes that are drastically different from training compounds. It is also worth noting that our model is able to adequately make the mechanistic distinction between agonists and antagonists, which is important to inform subsequent in vivo studies. Overall, we present an in-depth analysis of the appropriate utilization and interpretation of pharmacophore-based 3D QSAR models for safety screening.

<![CDATA[Trajectory-based training enables protein simulations with accurate folding and Boltzmann ensembles in cpu-hours]]>

An ongoing challenge in protein chemistry is to identify the underlying interaction energies that capture protein dynamics. The traditional trade-off in biomolecular simulation between accuracy and computational efficiency is predicated on the assumption that detailed force fields are typically well-parameterized, obtaining a significant fraction of possible accuracy. We re-examine this trade-off in the more realistic regime in which parameterization is a greater source of error than the level of detail in the force field. To address parameterization of coarse-grained force fields, we use the contrastive divergence technique from machine learning to train from simulations of 450 proteins. In our procedure, the computational efficiency of the model enables high accuracy through the precise tuning of the Boltzmann ensemble. This method is applied to our recently developed Upside model, where the free energy for side chains is rapidly calculated at every time-step, allowing for a smooth energy landscape without steric rattling of the side chains. After this contrastive divergence training, the model is able to de novo fold proteins up to 100 residues on a single core in days. This improved Upside model provides a starting point both for investigation of folding dynamics and as an inexpensive Bayesian prior for protein physics that can be integrated with additional experimental or bioinformatic data.

<![CDATA[Molecular basis for the increased affinity of an RNA recognition motif with re-engineered specificity: A molecular dynamics and enhanced sampling simulations study]]>

The RNA recognition motif (RRM) is the most common RNA binding domain across eukaryotic proteins. It is therefore of great value to engineer its specificity to target RNAs of arbitrary sequence. This was recently achieved for the RRM in Rbfox protein, where four mutations R118D, E147R, N151S, and E152T were designed to target the precursor to the oncogenic miRNA 21. Here, we used a variety of molecular dynamics-based approaches to predict specific interactions at the binding interface. Overall, we have run approximately 50 microseconds of enhanced sampling and plain molecular dynamics simulations on the engineered complex as well as on the wild-type Rbfox·pre-miRNA 20b from which the mutated systems were designed. Comparison with the available NMR data on the wild type molecules (protein, RNA, and their complex) served to establish the accuracy of the calculations.

Free energy calculations suggest that further improvements in affinity and selectivity are achieved by the S151T replacement.

<![CDATA[Feasibility of the annulus fibrosus repair with in situ gelating hydrogels – A biomechanical study]]>

The surgical standard of care for lumbar discectomy leaves the annulus fibrosus (AF) defect unrepaired, despite considerable risk for a recurrent herniation. Identification of a viable defect repair strategy has until now been elusive. The scope of this ex vivo biomechanical study was to evaluate crosslinking hydrogels as potentially promising AF defect sealants, and provide a baseline for their use in combination with collagen scaffolds that restore disc volume. This study directly compared genipin crosslinked fibrin hydrogel (FibGen) as a promising preclinical candidate against a clinically available adhesive composed of glutaraldehyde and albumin (BioGlue). Forty-two bovine coccygeal functional spine units (FSU) were randomly allocated into four groups, namely untreated (control, n = 12), repaired with either one of the tested hydrogels (BioGlue, n = 12; FibGen, n = 12), or FibGen used in combination with a collagen hydrogel scaffold (FibGen+Scaffold, n = 6). All specimens underwent a moderate mechanical testing protocol in intact, injured and repaired states. After completion of the moderate testing protocol, the samples underwent a ramp-to-failure test. Lumbar discectomy destabilized the FSU as quantified by increased torsional range of motion (28.0° (19.1, 45.1) vs. 41.39° (27.3, 84.9), p<0.001), torsional neutral zone (3.1° (1.2, 7.7) vs. 4.8° (2.1, 12.1), Z = -3.49, p < 0.001), hysteresis(24.4 J (12.8, 76.0) vs. 27.6 J (16.4, 54.4), Z = -2.61, p = 0.009), with loss of both disc height (7.0 mm (5.0, 10.5) vs 6.1 mm (4.0, 9.3), Z = -5.16, p < 0.001) and torsional stiffness (0.76 Nmdeg-1 (0.38, 1.07) vs. 0.66 Nmdeg-1 (0.38, 0.97), Z = -3.98, p < 0.001). Most FibGen repaired AF endured the entire testing procedure whereas only a minority of BioGlue repaired AF and all FibGen+Scaffold repaired AF failed (6/10 vs. 3/12 vs. 0/6 respectively, p = 0.041). Both BioGlue and FibGen+Scaffold repaired AF partially restored disc height (0.47 mm (0.07, 2.41), p = 0.048 and 1.52 mm (0.41, 2.57), p = 0.021 respectively) compared to sham treatment (0.08 mm (-0.63, 0.88)) whereas FibGen-only repaired AF had no such effect (0.04 mm (-0.73, 1.13), U = 48.0, p = 1). The AF injury model demonstrated considerable change of FSU mechanics that could be partially restored by use of an AF sealant. While inclusion of a volumetric collagen scaffold led to repair failure, use of FibGen alone demonstrated clinically relevant promise for prevention of mechanical reherniation, outperforming an FDA approved sealant in this ex vivo test series.

<![CDATA[Iron chelating properties of Eltrombopag: Investigating its role in thalassemia-induced osteoporosis]]>

Chronic blood transfusions are responsible to cause iron overload, which leads to several complications to end organs and osteoporosis. Iron chelation is needed to remove iron excess and to contain bone-mass loss. Deferasirox is the most recent oral iron chelator that prevents transfusion related iron overload complications. Recently Eltrombopag (ELT) iron chelating properties are emerging. ELT is an agonist at Thrombopoietin receptor, used in treatment of thrombocytopenia. We tested ELT and Deferasirox in iron overloaded osteoclasts from thalassemic patients and donors measuring intracellular iron, TRAP expression and osteoclast activity. We confirmed ELT iron chelation capacity also in bone tissue and a synergic effect when used with Deferasirox. Moreover, having demonstrated its effects on osteoclast activity, we suggest for the first time that ELT could ameliorate bone tissue’s health reducing bone mass loss.

<![CDATA[Construction of an integrated database for hERG blocking small molecules]]>

The inhibition of the hERG potassium channel is closely related to the prolonged QT interval, and thus assessing this risk could greatly facilitate the development of therapeutic compounds and the withdrawal of hazardous marketed drugs. The recent increase in SAR information about hERG inhibitors in public databases has led to many successful applications of machine learning techniques to predict hERG inhibition. However, most of these reports constructed their prediction models based on only one SAR database because the differences in the data format and ontology hindered the integration of the databases. In this study, we curated the hERG-related data in ChEMBL, PubChem, GOSTAR, and hERGCentral, and integrated them into the largest database about hERG inhibition by small molecules. Assessment of structural diversity using Murcko frameworks revealed that the integrated database contains more than twice as many chemical scaffolds for hERG inhibitors than any of the individual databases, and covers 18.2% of the Murcko framework-based chemical space occupied by the compounds in ChEMBL. The database provides the most comprehensive information about hERG inhibitors and will be useful to design safer compounds for drug discovery. The database is freely available at

<![CDATA[Allosteric binding sites in Rab11 for potential drug candidates]]>

Rab11 is an important protein subfamily in the RabGTPase family. These proteins physiologically function as key regulators of intracellular membrane trafficking processes. Pathologically, Rab11 proteins are implicated in many diseases including cancers, neurodegenerative diseases and type 2 diabetes. Although they are medically important, no previous study has found Rab11 allosteric binding sites where potential drug candidates can bind to. In this study, by employing multiple clustering approaches integrating principal component analysis, independent component analysis and locally linear embedding, we performed structural analyses of Rab11 and identified eight representative structures. Using these representatives to perform binding site mapping and virtual screening, we identified two novel binding sites in Rab11 and small molecules that can preferentially bind to different conformations of these sites with high affinities. After identifying the binding sites and the residue interaction networks in the representatives, we computationally showed that these binding sites may allosterically regulate Rab11, as these sites communicate with switch 2 region that binds to GTP/GDP. These two allosteric binding sites in Rab11 are also similar to two allosteric pockets in Ras that we discovered previously.