Deconvoluting kinase inhibitor induced cardiotoxicity

Many drugs designed to inhibit kinases have their clinical utility limited by cardiotoxicity-related label warnings or prescribing restrictions. While this liability is widely recognized, designing safer kinase inhibitors (KI) requires knowledge of the causative kinase(s). Efforts to unravel the kinases have encountered pharmacology with nearly prohibitive complexity. At therapeutically relevant concentrations, KIs show promiscuity distributed across the kinome. Here, to overcome this complexity, 65 KIs with known kinome-scale polypharmacology profiles were assessed for effects on cardiomyocyte (CM) beating. Changes in human iPSC-CM beat rate and amplitude were measured using label-free cellular impedance. Correlations between beat effects and kinase inhibition profiles were mined by computation analysis (Matthews Correlation Coefficient) to identify associated kinases. Thirty kinases met criteria of having (1) pharmacological inhibition correlated with CM beat changes, (2) expression in both human-induced pluripotent stem cell-derived cardiomyocytes and adult heart tissue, and (3) effects on CM beating following single gene knockdown. A subset of these 30 kinases were selected for mechanistic follow up. Examples of kinases regulating processes spanning the excitation–contraction cascade were identified, including calcium flux (RPS6KA3, IKBKE) and action potential duration (MAP4K2). Finally, a simple model was created to predict functional cardiotoxicity whereby inactivity at three sentinel kinases (RPS6KB1, FAK, STK35) showed exceptional accuracy in vitro and translated to clinical KI safety data. For drug discovery, identifying causative kinases and introducing a predictive model should transform the ability to design safer KI medicines. For cardiovascular biology, discovering kinases previously unrecognized as influencing cardiovascular biology should stimulate investigation of underappreciated signaling pathways

Listragning i lerbruk

En undersökning rörande listdragning i lerbruk har utförts. Tre olika lerbruk utvärderades på testytor. Resultaten visade att alla tre bruken gav lister med skarpa linjer och släta ytor. Ett av bruken valdes sedan ut till listdragning på en uppmurad rörspis. Följande examensarbete innehåller bakgrund, en beskrivning av arbetet som utförts samt en sektion om resultat, ställningstaganden och diskussion.Uppsats för avläggande av högskoleexamen i Kulturvård, Bygghantverk,7,5 hp, 2011

Structural and functional studies on glutaredoxins and members of the thioredoxin superfamily

The active site of E.coli glutaredoxin 3 was investigated using a combination of experimental and theoretical techniques. Starting from different conformations, molecular dynamics simulations converged to an active site conformation where the Cys11 thiolate was hydrogen bonded to surrounding amide protons and to the thiol proton of Cys14. The Cys14 chi1 and chi2 torsion angles determined by NMR supported the simulation result. The torsion angles of Cys11 could not be determined due to degenerate β-protons. The NMR titration of His15 showed that it was the sidechain was in the ε tautomer form and had a pKa value of 6.0. The pH-induced unfolding of WT and the C14A and K8A mutants were monitored by UV- and CD-spectroscopy. The Cys11 thiolate becomes protonated when the protein unfolds, or the protein unfolds when Cys11 becomes protonated. The removal of the thiol group in the C14A had a large impact on the outcome of the titrations while the K8A mutant only had a marginal effect. Human spermatid-specific thioredoxin-1 is a protein that is expressed only in sperm during maturation. Analysis of crystallization trials showed that only one part of the protein was left. CD spectra of the full length protein and the two parts were collected. It was clear from the spectra of the N-terminal domain that it was largely unstructured. In contrast, the C-terminal domain was that of a folded protein and was found to be quite similar to human Trx1. There are two human dithol glutaredoxins. The redox potentials of these were determined using glutathione redox buffer, direct protein-protein equilibration and thermodynamic linkage. The values were found to be -232 mV and -221 mV for hGrx1 and hGrx2, respectively. Furthermore, a second disulfide bond was discovered. The redox potential was determined to be -317 mV. Hence, it is present as a disulfide in the protein except under very reducing conditions in vivo. Phylogenetic analysis showed that there are three distinct groups of glutaredoxins, Grx1, Grx2 and the monothiol Grx5. A high precision NMR structure of E.coli Grx3 was solved. The RMSD of the backbone atoms was 0.26 Å relative to the mean. The Cys11 residue in active site showed two conformations. In the first, the thiolate was the acceptor in a hydrogen bond network where the donors where the surrounding amide protons and the thiol proton of Cys14. The other conformation displayed a hydrogen bond between the Cys11 and Thr10. Double mutant cycle analyses showed that there is a favorable interaction between the side chains of Cys11 and Cys14 (-1.63 kcal/mol). However, the interaction between Thr10 and Cys11 was unfavorable (+0.68 kcal/mol). The structures of the oxidized form and of the Grx3-glutathione complex were recalculated in order to make a valid comparison to the reduced form. The analysis showed that there are small differences at the backbone level between the redox forms. There are some differences in the active site. The most apparent ones are the conformation of Tyr13 and the position of Val52, both important in substrate binding

Crystal structure of the ATPase domain of the human AAA+ protein paraplegin/SPG7.

Paraplegin is an m-AAA protease of the mitochondrial inner membrane that is linked to hereditary spastic paraplegias. The gene encodes an FtsH-homology protease domain in tandem with an AAA+ homology ATPase domain. The protein is believed to form a hexamer that uses ATPase-driven conformational changes in its AAA-domain to deliver substrate peptides to its protease domain. We present the crystal structure of the AAA-domain of human paraplegin bound to ADP at 2.2 A. This enables assignment of the roles of specific side chains within the catalytic cycle, and provides the structural basis for understanding the mechanism of disease mutations.This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1

Novel globoside-like oligosaccharide expression patterns in nontypeable Haemophilus influenzae lipopolysaccharide: FEBS J.

We report the novel pattern of lipopolysaccharide (LPS) expressed by two disease-associated nontypeable Haemophilus influenzae strains, 1268 and 1200. The strains express the common structural motifs of H. influenzae; globotetraose [beta-d-GalpNAc-(1-->3)-alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Gl cp] and its truncated versions globoside [alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glcp] and lactose [beta-d-Galp-(1-->4)-beta-d-Glcp] linked to the terminal heptose (HepIII) and the corresponding structures with an alpha-d-Glcp as the reducing sugar linked to the middle heptose (HepII) in the same LPS molecule. Previously these motifs had been found linked only to either the proximal heptose (HepI) or HepIII of the triheptosyl inner-core moiety l-alpha-d-Hepp-(1-->2)-[PEtn-->6]-l-alpha-d-Hepp-(1-->3)-l-alpha-d-Hepp-(1 -->5)-[PPEtn-->4]-alpha-Kdo-(2-->6)-lipid A. This novel finding was obtained by structural studies of LPS using NMR techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material, as well as electrospray ionization-multiple-step tandem mass spectrometry on permethylated dephosphorylated oligosaccharide material. A lpsA mutant of strain 1268 expressed LPS of reduced complexity that facilitated unambiguous structural determination. Using capillary electrophoresis-ESI-MS/MS we identified sialylated glycoforms that included sialyllactose as an extension from HepII, this is a further novel finding for H. influenzae LPS. In addition, each LPS was found to carry phosphocholine and O-linked glycine. Nontypeable H. influenzae strain 1200 expressed identical LPS structures to 1268 with the difference that strain 1200 LPS had acetates substituting HepIII, whereas strain 1268 LPS has glycine at the same positionNRC publication: Ye

Human spermatid-specific thioredoxin-1 (Sptrx-1) is a two-domain protein with oxidizing activity

24 páginas, 5 figuras.Spermatid-specific thioredoxin-1 (Sptrx-1) is the first member of the thioredoxin family of proteins with a tissue-specific expression pattern, found exclusively in the tail of elongating spermatids and spermatozoa. We describe here further biochemical characterization of human Sptrx-1 protein structure and enzymatic activity. In gel filtration chromatography human Sptrx-1 eluates as a 400 kDa protein consistent with either an oligomeric form, not maintained by intermolecular disulfide bonding, and/or a highly asymmetrical structure. Analysis of circular dichroism spectra of fragments 1–360 and 361–469 and comparison to spectra of full-length Sptrx-1 supports a two-domain organization with a largely unstructured N-terminal domain and a folded thioredoxin-like C-terminal domain. Functionally, Sptrx-1 behaves as an oxidant in vitro when using selenite, but not oxidized glutathione, as electron acceptor. This oxidizing enzymatic activity suggests that Sptrx-1 might govern the stabilization (by disulfide cross-linking) of the different structures in the developing tail of spermatids and spermatozoa.This work was supported by grants from the Swedish Medical Research Council (Projects 03P-14096-01A, 03X-14041-01A), the Åke Wibergs Stiftelse and the Karolinska Institutet to A.M.-V. and the Fundación Margit y Folke Pehrzon to A.J.Peer reviewe

Deconvoluting Kinase Inhibitor Induced Cardiotoxicity

Many drugs designed to inhibit kinases have their clinical utility limited by cardiotoxicity-related label warnings or prescribing restrictions. While this liability is widely recognized, designing safer kinase inhibitors (KI) requires knowledge of the causative kinase(s). Efforts to unravel the kinases have encountered pharmacology with nearly prohibitive complexity. At therapeutically relevant concentrations, KIs show promiscuity distributed across the kinome. Here, to overcome this complexity, 65 KIs with known kinome-scale polypharmacology profiles were assessed for effects on cardiomyocyte (CM) beating. Changes in human iPSC-CM beat rate and amplitude were measured using label-free cellular impedance. Correlations between beat effects and kinase inhibition profiles were mined by computation analysis (Matthews Correlation Coefficient) to identify associated kinases. Thirty kinases met criteria of having (1) pharmacological inhibition correlated with CM beat changes, (2) expression in both human-induced pluripotent stem cell-derived cardiomyocytes and adult heart tissue, and (3) effects on CM beating following single gene knockdown. A subset of these 30 kinases were selected for mechanistic follow up. Examples of kinases regulating processes spanning the excitation–contraction cascade were identified, including calcium flux (RPS6KA3, IKBKE) and action potential duration (MAP4K2). Finally, a simple model was created to predict functional cardiotoxicity whereby inactivity at three sentinel kinases (RPS6KB1, FAK, STK35) showed exceptional accuracy in vitro and translated to clinical KI safety data. For drug discovery, identifying causative kinases and introducing a predictive model should transform the ability to design safer KI medicines. For cardiovascular biology, discovering kinases previously unrecognized as influencing cardiovascular biology should stimulate investigation of underappreciated signaling pathways