Discovery and optimization of cyclohexane-1,4-diamines as allosteric MALT1 inhibitors

Inhibition of mucosa-associated lymphoid tissue lymphoma translocation protein-1 (MALT1) is a promising strategy to modulate NF-κB signaling, with the potential to treat B-cell lymphoma and autoimmune diseases. We describe the discovery and optimization of (1s,4s)-N,N′-diaryl cyclohexane-1,4-diamines, a novel series of allosteric MALT1 inhibitors, resulting in compound 8 with single digit micromolar cell potency. X-ray analysis confirms that this compound binds to an induced allosteric site in MALT1. Compound 8 is highly selective and has an excellent in vivo rat PK profile with low clearance and high oral bioavailability, making it a promising lead for further optimization

Design and synthesis of soluble and cell-permeable PI3Kδ inhibitors for long-acting inhaled administration

PI3Kδ is a lipid kinase that is believed to be important in the migration and activation of cells of the immune system. Inhibition is hypothesised to provide a powerful yet selective immunomodulatory effect that may be beneficial for the treatment of conditions such as asthma or rheumatoid arthritis. In this work we describe the identification of inhibitors based on a thiazolopyridone core structure and their subsequent optimisation for inhalation. The initially identified compound (13) had good potency and isoform selectivity but was not suitable for inhalation. Addition of basic substituents to a region of the molecule pointing to solvent was tolerated (enzyme inhibition pIC50 >9) and by careful manipulation of the pKa and lipophilicity we were able to discover compounds (20b, 20f) with good lung retention and cell potency that could be taken forward to in-vivo studies where significant target engagement could be demonstrated

Structure-function studies of beta-lactam biosynthetic enzymes

Beta-lactam compounds belong to the most important antibiotics in current use. The increasing occurrence of bacterial resistance against antibiotics, which threatens to move us back to pre-antibiotic era, calls for the development of new antibiotics. Beta-lactams are produced by fermentation in the microorganism, therefore knowledge about the biosynthetic enzymes involved is vital for the production of new antibiotics to escape resistance. This thesis presents structure-function studies on enzymes involved in the biosynthesis of the beta-lactam antibiotics cephalosporins and cephamycins DAOCS is a non-heme Fe(II) dioxygenase and catalyses the oxidative ring expansion of the penicillin nucleus into the nucleus of cephalosporins. Structures of DAOCS with substrates, product and cofactors obtained from two different crystal forms are presented here. The structural results suggest a mechanism for cephalosporin formation where 2-oxoglutarate and dioxygen need to react first to produce an oxidizing iron species, followed by reaction with the penicillin substrate. Structural differences in the two crystal forms indicate conformational flexibility in the C terminus and point to a role for the C terminus in catalysis. Biosynthesis of cephamycins (C7-methoxylated cephalosporins) is catalysed by two enzymes, cmcI and cmcJ, but the details of catalysis are largely unknown. The crystal structure of cmcI, presented here, is a hexamer consisting of a C-terminal Rossman domain and a smaller N-terminal domain. The N-terminal domain is involved in oligomerisation and the Rossmann domain binds SAM and SAH in a fashion common for methyltransferases, with a bound magnesium ion in the vicinity of SAM. The expected cephalosporin binding site is occupied by PEG, which ligates the magnesium ion. From docking studies of a cephalosoporin molecule to the cmcI-Mg2+-SAM structure, a model for substrate binding is proposed. Altogether, the results suggest cmcI is a methyltransferase that catalyses the second catalytic step in cephamycin biosynthesis

Gränsdragning vid värdepappershandel : -beskattning av finansiella- och icke-finansiella företag

Handel med värdepapper förekommer både i finansiella- och icke-finansiella företag. Det är främst finansiella företag såsom kreditinstitut och värdepappersbolag som handlar med värdepapper. Dessa företag presumeras bedriva värdepappersrörelse då de yrkesmässigt bedriver kapitalförvaltning utåtriktat. Handel med värdepapper förekommer också i icke-finansiella företag, exempelvis handels- och produktionsbolag. Dessa företag handlar med värdepapper i syfte att förvalta och hantera kapital för egen räkning. Förvaltningen kan, då den uppnår viss omfattning, klassificeras som värdepappersrörelse. Uppsatsen behandlar gränsdragningsfrågan när förvaltning övergår till yrkesmässig värdepappersrörelse. Frågan är av vikt för företag då det leder till tillämpning av skilda resultatberäkningsregler och därmed skilda skattekonsekvenser. Värdepapper i ett företag som anses bedriva värdepappersförvaltning klassificeras skatterättsligt som kapitaltillgångar och beskattningen av dessa sker enligt kapitalreglerna. För ett företag som anses bedriva värdepappersrörelse klassificeras istället värdepapperen skatterättsligt som lagertillgångar och beskattningen sker då enligt rörelsereglerna. Gränsdragningsfrågan vid värdepappershandel har utvecklats genom rättspraxis i avsaknad av klara rättsregler på området. Det har i doktrin diskuterats att kvantitativa kriterier utvecklats i rättspraxis och att dessa torde vara vägledande vid gränsdragningen. Kriterierna har sin utgångspunkt i rekvisitet yrkesmässighet i 13 kapitlet (kap). 1 § 1 st. Inkomstskattelag (1999:1229) (IL). Med yrkesmässighet menas att det ska företas en utåtriktad handel, i viss omfattning, vara varaktigt och regelbundet. I doktrin framförs ytterligare kriterier såsom bolagsordningens och redovisningens betydelse. Av rättspraxis kan vi utläsa att det inte finns några tydligt uppställda kriterier för företag att uppfylla för att anses utgöra värdepappersrörelse, istället sker en bedömning i det enskilda fallet. Vi är av åsikten att det är av vikt för företag att på förhand kunna förutse sin beskattning och att gränsdragningen leder till en minskad förutsebarhet urett företagsperspektiv. Detta är inte en önskvärd situation och föranleder att en tydligare gränsdragning på området måste ske. Vi har därför utformat två förslag: en lösning baserad på lagstiftning om krav på hur stor omsättningen bör vara, och en lösning med redovisningen som utgångspunkt för den skatterättsliga klassificeringen. Slutligen har vi funnit att den lösningen som är baserad på lagstiftning är mest fördelaktigt ur både ur ett nationellt- och ett företagsperspektiv.  Both financial and non-financial corporations are trading in securities. It is mainly financial companies such as credit and securities companies dealing in securities. These companies are presumed to conduct securities business as they are offering clients professional asset management. Non-financial companies, such as trade and production companies also conduct trading in securities. These companies deal in securities in order to manage their own financial assets. The management can be classified as a securities business if it reaches a certain level. The thesis deals with the demarcation issue when regular asset management exceeds a certain level and turns into a securities business. The issue is of importance for companies as it leads to different tax consequences. When conducting asset management the securities will be classified as capital assets. The company will then be taxed according to certain tax rules applicable for capital assets. When conducting securities business the securities will be classified as stock assets and the company will then be taxed according to certain tax rules applicable for those assets. The demarcation issue has been developed through case law in the absence of clear rules of law in the area. It has been discussed in the doctrine that quantitative criteria were developed in case law and that these can serve as guidelines when solving the issue of demarcation. The criteria are based on the requirement of professionalism in the Swedish Income Tax Act Chapter 13 paragraph 1. Professionalism means that there should be an outward trade, to a certain extent, be stable and on a regular basis. The doctrine states that additional criteria such as the corporate charter and the companies accounting also can be of importance.

Does Gender Matter? : Nurses' communications with children during blood test procedures

Equal opportunities for children are in general regarded as crucial; nevertheless, children are still often treated differently due to their sex. This could limit a child's inherent way of expressing him/herself. Nurses need to be aware of how gender constructions influence their interactions with children. The aim of this study was to illuminate interpretative repertoires that a group of nurses use when communicating with children during blood test procedures in two children's hospitals in Sweden. Data was collected by semi-structured observations of nurses conducting blood test procedures on children, and the observations were analyzed using discourse psychology. Two main groups of interpretative repertoires were found. In one group the repertoires were supporting gender stereotyping and in the other group the repertoires were weakening gender stereotyping. In conclusion, nurses' interactions with children during procedures offer the children different socially and culturally constructed interpretative repertoires about gender. Increased consciousness of gender issues is needed among nurses to enable children to be and act freely, without being forced into limited gendered expectations

Patients’ health and quality of life after complex endovascular aortic repair : a prospective cohort study

Rationale: Complex endovascular aortic repair often involves multiple major procedures over time with a high risk of complications and little time for recovery. This exposes patients to great stress, both physically and mentally, with potentially long-lasting effects. There is limited knowledge about these effects and who is most at risk – information on this could help vascular nurses and other healthcare professionals anticipate and meet care needs. Aim: To investigate the health and quality of life effects of complex endovascular aortic repair, in relation to patients’ demographic and health characteristics. Design: A prospective cohort study. Methods: Patients undergoing elective complex endovascular aortic repair were consecutively recruited from one university hospital during one year (n=25). Self-report questionnaires on health disability (WHODAS 2.0), quality of life (WHOQoL-BREF) and symptoms of anxiety and depression (HADS) were filled out preoperatively and repeated one and six months postoperatively. Prospective changes in health and quality of life, and associations with patient demographics and preoperative health characteristics, were assessed. Ethical approval was obtained prior to study performance. Results: Overall, patients had significantly greater health disability at one month (WHODAS 2.0 score median 31.5, range 1.1–63.0) than preoperatively (median 13.6, range 0.0–41.3) (n=22, p=.017); the majority had recovered at six months (median 11.4, range 3.3–58.7) (n=18, p=.042). No significant effects were seen in quality of life and symptoms of anxiety and depression (p>.05). However, the participants showed heterogeneity, with certain individuals not recovered at six months (n=8). Factors associated with worse six-month outcomes were being female, age < 70 years, postoperative complications, and history of anxiety or depression. Conclusions: Complex endovascular aortic repair have limited long-term negative effects on patients’ health and quality of life. However, some patients are not recovered at six months postoperatively, which could be explained by individual characteristics. To improve recovery outcomes, vascular nurses and other health care professionals should be aware of the possible recovery trajectories and factors associated with impaired recovery, and use them to anticipate and meet the patients’ individual care needs

Conformational Flexibility of the C Terminus with Implications for Substrate Binding and Catalysis Revealed in a New Crystal Form of Deacetoxycephalosporin C Synthase

Deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus catalyses the oxidative ring expansion of the penicillin nucleus into the nucleus of cephalosporins. The reaction requires dioxygen and 2-oxoglutarate as co-substrates to create a reactive iron–oxygen intermediate from a ferrous iron in the active site. The active enzyme is monomeric in solution. The structure of DAOCS was determined earlier from merohedrally twinned crystals where the last four C-terminal residues (308–311) of one molecule penetrate the active site of a neighbouring molecule, creating a cyclic trimeric structure in the crystal. Shortening the polypeptide chain from the C terminus by more than four residues diminishes activity. Here, we describe a new crystal form of DAOCS in which trimer formation is broken and the C-terminal arm is free. These crystals show no signs of twinning, and were obtained from DAOCS labelled with an N-terminal His-tag. The modified DAOCS is catalytically active. The free C-terminal arm protrudes into the solvent, and the C-terminal domain (residues 268–299) is rotated by about 16° towards the active site. The last 12 residues (300–311) are disordered. Structures for various enzyme–substrate and enzyme–product complexes in the new crystal form confirm overlapping binding sites for penicillin and 2-oxoglutarate. The results support the notion that 2-oxoglutarate and dioxygen need to react first to produce an oxidizing iron species, followed by reaction with the penicillin substrate. The position of the penicillin nucleus is topologically similar in the two crystal forms, but the penicillin side-chain in the new non-twinned crystals overlaps with the position of residues 304–306 of the C-terminal arm in the twinned crystals. An analysis of the interactions between the C-terminal region and residues in the active site indicates that DAOCS could also accept polypeptide chains as ligands, and these could bind near the iron.

Insights into Cephamycin Biosynthesis: the Crystal Structure of CmcI from Streptomyces clavuligerus

Cephamycin C-producing microorganisms use two enzymes to convert cephalosporins to their 7α-methoxy derivatives. Here we report the X-ray structure of one of these enzymes, CmcI, from Streptomyces clavuligerus. The polypeptide chain of the enzyme folds into a C-terminal Rossmann domain and a smaller N-terminal domain, and the molecule packs as a hexamer in the crystal. The Rossmann domain binds S-adenosyl-L-methionine (SAM) and the demethylated product, S-adenosyl-L-homocysteine, in a fashion similar to the common binding mode of this cofactor in SAM-dependent methyltransferases. There is a magnesium-binding site in the vicinity of the SAM site with a bound magnesium ion ligated by residues Asp160, Glu186 and Asp187. The expected cephalosporin binding site near the magnesium ion is occupied by polyethyleneglycol (PEG) from the crystallisation medium. The geometry of the SAM and the magnesium binding sites is similar to that found in cathechol O-methyltransferase. The results suggest CmcI is a methyltransferase, and its most likely function is to catalyse the transfer of a methyl group from SAM to the 7α-hydroxy cephalosporin in the second catalytic reaction of cephamycin formation. Based on the docking of the putative substrate, 7α-hydroxy-O-carbamoyldeacetylcephalosporin C, to the structure of the ternary CmcI–Mg2C–SAM complex, we propose a model for substrate binding and catalysis. In this model, the 7-hydroxy group of the β-lactam ring ligates the Mg2C with its α-side facing the methyl group of SAM at a distance that would allow methylation of the hydroxyl-group.