Sulfur(VI) Fluoride Exchange Chemistry in Solid-Phase Synthesis of Compound Arrays: Discovery of Histone Deacetylase Inhibitors.

Multistep synthesis performed on solid support is a powerful means to generate small-molecule libraries for the discovery of chemical probes to dissect biological mechanisms as well as for drug discovery. Therefore, expansion of the collection of robust chemical transformations amenable to solid-phase synthesis is desirable for achieving chemically diverse libraries for biological testing. Here, we show that sulfur(VI) fluoride exchange (SuFEx) chemistry, exemplified by pairing phenols with aryl fluorosulfates, can be used for the solid-phase synthesis of biologically active compounds. As a case study, we designed and synthesized a library of 84 hydroxamic acid-containing small molecules, providing a rich source of inhibitors with diverse selectivity profiles across the human histone deacetylase enzyme family. Among other discoveries, we identified a scaffold that furnished inhibitors of HDAC11 with exquisite selectivity in vitro and a selective inhibitor of HDAC6 that was shown to affect the acetylation of α-tubulin over histone sites H3K18, H3K27, as well as SMC3 in cultured cells. Our results encourage the further use of SuFEx chemistry for the synthesis of diverse small-molecule libraries and provide insight for future design of selective HDAC inhibitors.

Contemporary Applications of Thioamides and Methods for Their Synthesis.

Thioamides are naturally occurring isosteres of amide bonds in which the chalcogen atom of the carbonyl is changed from oxygen to sulfur. This substitution gives rise to altered nucleophilicity and hydrogen bonding properties with importance for both chemical reactivity and non-covalent interactions. As such, thioamides have been introduced into biologically active compounds to achieve improved target affinity and/or stability towards hydrolytic enzymes but have also been applied as probes of protein and peptide folding and dynamics. Recently, a series of new methods have been developed for the synthesis of thioamides as well as their utilization in peptide chemistry. Further, novel strategies for the incorporation of thioamides into proteins have been developed, enabling both structural and functional studies to be performed. In this Review, we highlight the recent developments in the preparation of thioamides and their applications for peptide modification and study of protein function.

Emergent Worldsheet for the AdS Virasoro-Shapiro Amplitude.

We construct a representation for the first anti-de Sitter curvature correction to the Virasoro-Shapiro amplitude, as an integral over the Riemann sphere. The integrand is that of the Virasoro-Shapiro amplitude in flat space, with the extra insertion of a linear combination of single-valued multiple polylogarithms of weight three. The integral representation implies an elegant, manifestly single-valued representation for the Wilson coefficients of the low-energy expansion.

Substrates and Cyclic Peptide Inhibitors of the Oligonucleotide-Activated Sirtuin 7.

The sirtuins are NAD+ -dependent lysine deacylases, comprising seven isoforms (SIRT1-7) in humans, which are involved in the regulation of a plethora of biological processes, including gene expression and metabolism. The sirtuins share a common hydrolytic mechanism but display preferences for different ϵ-N-acyllysine substrates. SIRT7 deacetylates targets in nuclei and nucleoli but remains one of the lesser studied of the seven isoforms, in part due to a lack of chemical tools to specifically probe SIRT7 activity. Here we expressed SIRT7 and, using small-angle X-ray scattering, reveal SIRT7 to be a monomeric enzyme with a low degree of globular flexibility in solution. We developed a fluorogenic assay for investigation of the substrate preferences of SIRT7 and to evaluate compounds that modulate its activity. We report several mechanism-based SIRT7 inhibitors as well as de novo cyclic peptide inhibitors selected from mRNA-display library screening that exhibit selectivity for SIRT7 over other sirtuin isoforms, stabilize SIRT7 in cells, and cause an increase in the acetylation of H3 K18.

Sarcopenia and loss of muscle mass in patients with lung cancer undergoing chemotherapy treatment: a systematic review and meta-analysis.

BACKGROUND: In patients with cancer, sarcopenia is associated with treatment related complications, treatment cessation, poor quality of life and reduced overall survival. Despite this, there is limited knowledge about changes in skeletal muscle mass during chemotherapy. The aim of this systematic review and meta-analysis was to investigate the change of skeletal muscle mass and sarcopenia during chemotherapy treatment among patients with lung cancer. METHODS: A systematic literature search was conducted in three databases, PubMed, EMBASE and Web of Science. Observational studies with patients with lung cancer were eligible for inclusion if skeletal muscle mass was measured before and after receiving chemotherapy treatment. RESULTS: Ten cohort studies with a total of 867 participants met the inclusion criteria. During 5.2 ± 2.9 months of chemotherapy treatment, patients with lung cancer experienced a significant loss of skeletal muscle mass with a standardized mean difference (SMD) of: -0.25 (95% CI -0.47 to -0.03). The pretreatment prevalence of sarcopenia varied across studies from 35% to 74%. Only one study reported prevalence of sarcopenia both before and after chemotherapy treatment with an increase from 35% to 59%. CONCLUSION: The present data demonstrate a marked loss of skeletal muscle mass in patients with lung cancer undergoing chemotherapy treatment, as well as a high prevalence of sarcopenia. As sarcopenia is associated with poor clinical outcomes, it seems important to include and use assessments of skeletal muscle mass in clinical practice to identify patients in need for interventions. Moreover, interventional studies to hinder development of sarcopenia are needed.

Aryl Fluorosulfate Based Inhibitors That Covalently Target the SIRT5 Lysine Deacylase.

The sirtuin enzymes are a family of lysine deacylases that regulate gene transcription and metabolism. Sirtuin 5 (SIRT5) hydrolyzes malonyl, succinyl, and glutaryl ϵ-N-carboxyacyllysine posttranslational modifications and has recently emerged as a vulnerability in certain cancers. However, chemical probes to illuminate its potential as a pharmacological target have been lacking. Here we report the harnessing of aryl fluorosulfate-based electrophiles as an avenue to furnish covalent inhibitors that target SIRT5. Alkyne-tagged affinity-labeling agents recognize and capture overexpressed SIRT5 in cultured HEK293T cells and can label SIRT5 in the hearts of mice upon intravenous injection of the compound. This work demonstrates the utility of aryl fluorosulfate electrophiles for targeting of SIRT5 and suggests this as a means for the development of potential covalent drug candidates. It is our hope that these results will serve as inspiration for future studies investigating SIRT5 and general sirtuin biology in the mitochondria.

Investigation of Carboxylic Acid Isosteres and Prodrugs for Inhibition of the Human SIRT5 Lysine Deacylase Enzyme.

Sirtuin 5 (SIRT5) is a protein lysine deacylase enzyme that regulates diverse biology by hydrolyzing ϵ-N-carboxyacyllysine posttranslational modifications in the cell. Inhibition of SIRT5 has been linked to potential treatment of several cancers but potent compounds with activity in cells have been lacking. Here we developed mechanism-based inhibitors that incorporate isosteres of a carboxylic acid residue that is important for high-affinity binding to the enzyme active site. By masking of the tetrazole moiety of the most potent candidate from our initial SAR study, we achieved potent and cytoselective growth inhibition for the treatment of SIRT5-dependent leukemic cancer cell lines in culture. Thus, we provide an efficient, cellularly active small molecule that targets SIRT5, which can help elucidate its function and potential as a future drug target. This work shows that masked isosteres of carboxylic acids are viable chemical motifs for the development of inhibitors that target mitochondrial enzymes, which may have applications beyond the sirtuin field.

Investigating GIPR (ant)agonism: A structural analysis of GIP and its receptor.

The glucose-dependent insulinotropic polypeptide (GIP) is a 42-residue metabolic hormone that is actively being targeted for its regulatory role of glycemia and energy balance. Limited structural data of its receptor has made ligand design tedious. This study investigates the structure and function of the GIP receptor (GIPR), using a homology model based on the GLP-1 receptor. Molecular dynamics combined with in vitro mutational data were used to pinpoint residues involved in ligand binding and/or receptor activation. Significant differences in binding mode were identified for the naturally occurring agonists GIP(1-30)NH2 and GIP(1-42) compared with high potency antagonists GIP(3-30)NH2 and GIP(5-30)NH2. Residues R1832.60, R1902.67, and R3005.40 are shown to be key for activation of the GIPR, and evidence suggests that a disruption of the K293ECL2-E362ECL3 salt bridge by GIPR antagonists strongly reduces GIPR activation. Combinatorial use of these findings can benefit rational design of ligands targeting the GIPR.

Tire wear particle and leachate exposures from a pristine and road-worn tire to Hyalella azteca: Comparison of chemical content and biological effects.

Tire emissions have emerged as an environmental contaminant of concern. To fully understand their effects to biota, research is needed from different stages of a tire's lifecycle. In this study we exposed freshwater Hyalella azteca to tire wear particles (TWPs) as particle suspensions or their respective chemical leachates (the chemicals released from tire particles into water) from a pristine (P-TWP) and worn (W-TWP) tire of same make and model. Acute and long-term toxicity experiments on H. azteca showed that P-TWP suspensions were more toxic than W-TWP suspensions with estimated LC50 values of 364 ± 64 particles (0.19 ± 0.03 g L-1) and 3073 ± 211 particles (0.91 ± 0.06 g L-1), respectively. However, leachates from W- and P-TWPs appeared equally toxic, but did not conform to a sigmoidal dose-response pattern and LC50 values could not be derived. In long-term tests (21 d) P-TWP suspensions showed no significant effects on H. azteca mortality (p = 0.970) or reproduction (p = 0.123), but growth was significantly reduced (p = 0.003) at the highest concentration tested (250 particles mL-1 or 0.127 g L-1). Chemical analysis of both particle types and their leachates showed that four compounds, benzothiazole, 1-indanone, aluminum and zinc, consistently leached from TWPs into water. Analysis of the two TWPs showed a difference in the concentration of the various compounds. Specifically, P-TWPs contained significantly more 1-octanethiol, phenanthrene, anthracene and aluminum than W-TWPs, suggesting that they are possible candidates for the increased toxicity observed following P-TWP exposure.

Development of Sarcopenia in Patients With Bladder Cancer: A Systematic Review.

OBJECTIVE: Sarcopenia is known to influence cancer-related complications and overall survival. However, the effect of cancer treatment on the development or progression of sarcopenia is relatively unknown. The primary aim of this systematic review was to determine the prevalence and development of sarcopenia among people with bladder cancer. DATA SOURCES: A systematic search was performed in PubMed, Web of Science, and EMBASE. Studies with ≥2 assessments of sarcopenia were eligible for inclusion. Five retrospective cohorts were included with a total of 438 participants. The baseline prevalence of sarcopenia across studies varied from 25% to 69% and post-treatment prevalence from 50% to 81%. The average loss of muscle mass was 2.2% to 10% during a time course of 3 to 12 months. CONCLUSION: The prevalence of sarcopenia markedly increased during cancer treatment in patients with bladder cancer. Further research into the effect of different treatment regimens on the development of sarcopenia, and how these changes might affect functional capacity and survival is needed. IMPLICATIONS FOR NURSING PRACTICE: The development of sarcopenia is important to understand because of its negative affect on quality of life, complications, and mortality. Further, understanding how sarcopenia develops during treatment could potentially strengthen nurses' future care plans for patients with bladder cancer.

Towards the molecular architecture of the peroxisomal receptor docking complex.

Import of yeast peroxisomal matrix proteins is initiated by cytosolic receptors, which specifically recognize and bind the respective cargo proteins. At the peroxisomal membrane, the cargo-loaded receptor interacts with the docking protein Pex14p that is tightly associated with Pex17p. Previous data suggest that this interaction triggers the formation of an import pore for further translocation of the cargo. The mechanistic principles, however, are unclear, mainly because structures of higher-order assemblies are still lacking. Here, using an integrative approach, we provide the structural characterization of the major components of the peroxisomal docking complex Pex14p/Pex17p, in a native bilayer environment, and reveal its subunit organization. Our data show that three copies of Pex14p and a single copy of Pex17p assemble to form a 20-nm rod-like particle. The different subunits are arranged in a parallel manner, showing interactions along their complete sequences and providing receptor binding sites on both membrane sides. The long rod facing the cytosol is mainly formed by the predicted coiled-coil domains of Pex14p and Pex17p, possibly providing the necessary structural support for the formation of the import pore. Further implications of Pex14p/Pex17p for formation of the peroxisomal translocon are discussed.

A piggybacking mechanism enables peroxisomal localization of the glyoxylate cycle enzyme Mdh2 in yeast.

Eukaryotic cells have evolved organelles that allow the compartmentalization and regulation of metabolic processes. Knowledge of molecular mechanisms that allow temporal and spatial organization of enzymes within organelles is therefore crucial for understanding eukaryotic metabolism. Here, we show that the yeast malate dehydrogenase 2 (Mdh2) is dually localized to the cytosol and to peroxisomes and is targeted to peroxisomes via association with Mdh3 and a Pex5-dependent piggybacking mechanism. This dual localization of Mdh2 contributes to our understanding of the glyoxylate cycle and provides a new perspective on compartmentalization of cellular metabolism, which is critical for the perception of metabolic disorders and aging.

Pex14p Phosphorylation Modulates Import of Citrate Synthase 2 Into Peroxisomes in Saccharomyces cerevisiae.

The peroxisomal biogenesis factor Pex14p is an essential component of the peroxisomal matrix protein import machinery. Together with Pex13p and Pex17p, it is part of the membrane-associated peroxisomal docking complex in yeast, facilitating the binding of cargo-loaded receptor proteins for translocation of cargo proteins into the peroxisome. Furthermore, Pex14p is part of peroxisomal import pores. The central role of Pex14p in peroxisomal matrix protein import processes renders it an obvious target for regulatory mechanisms such as protein phosphorylation. To explore this possibility, we examined the state of Pex14p phosphorylation in Saccharomyces cerevisiae. Phos-tag-SDS-PAGE of Pex14p affinity-purified from solubilized membranes revealed Pex14p as multi-phosphorylated protein. Using mass spectrometry, we identified 16 phosphorylation sites, with phosphorylation hot spots located in the N- and C-terminal regions of Pex14p. Analysis of phosphomimicking and non-phosphorylatable variants of Pex14p revealed a decreased import of GFP carrying a peroxisomal targeting signal type 1, indicating a functional relevance of Pex14p phosphorylation in peroxisomal matrix protein import. We show that this effect can be ascribed to the phosphomimicking mutation at serine 266 of Pex14p (Pex14p-S266D). We further screened the subcellular distribution of 23 native GFP-tagged peroxisomal matrix proteins by high-content fluorescence microscopy. Only Cit2p, the peroxisomal isoform of citrate synthase, was affected in the Pex14p-S266D mutant, showing increased cytosolic localization. Cit2p is part of the glyoxylate cycle, which is required for the production of essential carbohydrates when yeast is grown on non-fermentable carbon sources. Pex14p-S266 phosphosite mutants showed reversed growth phenotypes in oleic acid and ethanol with acetyl-CoA formed in peroxisomes and the cytosol, respectively. Overexpression of Cit2p rescued the growth phenotype of yeast cells expressing Pex14p-S266D in oleic acid. Our data indicate that phosphorylation of Pex14p at S266 provides a mechanism for controlling the peroxisomal import of Cit2p, which helps S. cerevisiae cells to adjust their carbohydrate metabolism according to the nutritional conditions.

New-Onset Atrial Fibrillation Among Patients With Infection in the Emergency Department: A Multicenter Cohort Study of 1-Year Stroke Risk.

BACKGROUND: Patients with new-onset atrial fibrillation in relation to infection are frequent in emergency departments (EDs) and may require antithrombotic therapy because of the increased risk of stroke. Our objective was to describe the 1-year risk of stroke in patients in the ED with infection, new-onset atrial fibrillation, and no antithrombotic therapy. METHODS: This was a population-based cohort study at 4 EDs in Denmark and Sweden. Atrial fibrillation was identified by electrocardiogram (ECG) upon arrival at the ED, and infection was identified by discharge diagnosis. Patient history was followed for 12 months or until initiation of oral anticoagulant therapy, ischemic stroke, or death. Primary outcome was stroke within 12 months compared to patients with infection and no atrial fibrillation. RESULTS: In the analysis, 15,505 patients were included; 48.7% were male and the median age was 71 (IQR, 56-83). Among the included patients, 2107 (13.6%) had atrial fibrillation of any kind and 822 (39.0%) of these had new-onset atrial fibrillation with a median CHA2DS2-VASc score of 3 (IQR 2-4). New-onset atrial fibrillation during infection showed an absolute postdischarge 1-year risk of stroke of 2.7% (95% CI 1.6-4.2), corresponding to a crude hazard ratio (HR) of 1.4 (95% CI 0.9-2.3), a sex and age adjusted HR of 1.0 (95% CI 0.6-1.6), and a CHA2DS2-VASc adjusted HR of 1.1 (95% CI, 0.7-1.8) compared to patients with infection but no atrial fibrillation. CONCLUSIONS: Patients in the ED with infection and new-onset atrial fibrillation without current oral anticoagulant therapy had a 2.7% absolute 1-year risk of stroke. Stroke events were mainly related to sex and age and risk factors identified by the CHA2DS2-VASc score.

Historical chemical annotations of Cinchona bark collections are comparable to results from current day high-pressure liquid chromatography technologies.

ETHNOPHARMACOLOGICAL RELEVANCE: Species of the genus Cinchona (Rubiaceae) have been used in traditional medicine, and as a source for quinine since its discovery as an effective medicine against malaria in the 17th century. Despite being the sole cure of malaria for almost 350 years, little is known about the chemical diversity between and within species of the antimalarial alkaloids found in the bark. Extensive historical Cinchona bark collections housed at the Royal Botanic Gardens, Kew, UK, and in other museums may shed new light on the alkaloid chemistry of the Cinchona genus and the history of the quest for the most effective Cinchona barks. AIM OF THE STUDY: We used High-Pressure Liquid Chromatography (HPLC) coupled with fluorescence detection (FLD) to reanalyze a set of Cinchona barks originally annotated for the four major quinine alkaloids by John Eliot Howard and others more than 150 years ago. MATERIALS AND METHODS: We performed an archival search on the Cinchona bark collections in the Economic Botany Collection housed in Kew, focusing on those with historical alkaloid content information. Then, we performed HPLC analysis of the bark samples to separate and quantify the four major quinine alkaloids and the total alkaloid content using fluorescence detection. Correlations between historic and current annotations were calculated using Spearman's rank correlation coefficient, before paired comparisons were performed using Wilcox rank sum tests. The effects of source were explored using generalized linear modelling (GLM), before the significance of each parameter in predicting alkaloid concentrations were assessed using chi-square tests as likelihood ratio testing (LRT) models. RESULTS: The total alkaloid content estimation obtained by our HPLC analysis was comparatively similar to the historical chemical annotations made by Howard. Additionally, the quantity of two of the major alkaloids, quinine and cinchonine, and the total content of the four alkaloids obtained were significantly similar between the historical and current day analysis using linear regression. CONCLUSIONS: This study demonstrates that the historical chemical analysis by Howard and current day HPLC alkaloid content estimations are comparable. Current day HPLC analysis thus provide a realistic estimate of the alkaloid contents in the historical bark samples at the time of sampling more than 150 years ago. Museum collections provide a powerful but underused source of material for understanding early use and collecting history as well as for comparative analyses with current day samples.

Cleavage of peptidic inhibitors by target protease is caused by peptide conformational transition.

Some peptide sequences can behave as either substrates or inhibitors of serine proteases. Working with a cyclic peptidic inhibitor of the serine protease urokinase-type plasminogen activator (uPA), we have now demonstrated a new mechanism for an inhibitor-to-substrate switch. The peptide, CSWRGLENHAAC (upain-2), is a competitive inhibitor of human uPA, but is also slowly converted to a substrate in which the bond between Arg4 and Gly5 (the P1-P1' bond) is cleaved. Substituting the P2 residue Trp3 to an Ala or substituting the P1 Arg4 residue with 4-guanidino-phenylalanine strongly increased the substrate cleavage rate. We studied the structural basis for the inhibitor-to-substrate switch by determining the crystal structures of the various peptide variants in complex with the catalytic domain of uPA. While the slowly cleaved peptides bound clearly in inhibitory mode, with the oxyanion hole blocked by the side chain of the P3' residue Glu7, peptides behaving essentially as substrates with a much accelerated rate of cleavage was observed to be bound to the enzyme in substrate mode. Our analysis reveals that the inhibitor-to-substrate switch was associated with a 7 Štranslocation of the P2 residue, and we conclude that the inhibitor-to-substrate switch of upain-2 is a result of a major conformational change in the enzyme-bound state of the peptide. This conclusion is in contrast to findings with so-called standard mechanism inhibitors in which the inhibitor-to-substrate switch is linked to minor conformational changes in the backbone of the inhibitory peptide stretch.

Ligand binding modulates the structural dynamics and activity of urokinase-type plasminogen activator: A possible mechanism of plasminogen activation.

The catalytic activity of trypsin-like serine proteases is in many cases regulated by conformational changes initiated by binding of physiological modulators to exosites located distantly from the active site. A trypsin-like serine protease of particular interest is urokinase-type plasminogen activator (uPA), which is involved in extracellular tissue remodeling processes. Herein, we used hydrogen/deuterium exchange mass spectrometry (HDXMS) to study regulation of activity in the catalytic domain of the murine version of uPA (muPA) by two muPA specific monoclonal antibodies. Using a truncated muPA variant (muPA16-243), containing the catalytic domain only, we show that the two monoclonal antibodies, despite binding to an overlapping epitope in the 37s and 70s loops of muPA16-243, stabilize distinct muPA16-243 conformations. Whereas the inhibitory antibody, mU1 was found to increase the conformational flexibility of muPA16-243, the stimulatory antibody, mU3, decreased muPA16-243 conformational flexibility. Furthermore, the HDXMS data unveil the existence of a pathway connecting the 70s loop to the active site region. Using alanine scanning mutagenesis, we further identify the 70s loop as an important exosite for the activation of the physiological uPA substrate plasminogen. Thus, the data presented here reveal important information about dynamics in uPA by demonstrating how various ligands can modulate uPA activity by mediating long-range conformational changes. Moreover, the results provide a possible mechanism of plasminogen activation.

Cracking the code for maintaining quality training in Olympic distance triathlon: lessons learnt from a squad of elite Scandinavian athletes.

OBJECTIVE: High-quality training is a key determinant of performance in the Olympic distance triathlon and is potentially influenced by a unique array of context-specific biopsychosocial factors. Our objective was to explore and describe these factors among squad members of a university-based, elite Olympic distance triathlete developmental programme. METHOD: A qualitative investigation using a visual communication tool-assisted focus group and longitudinal semistructured individual interviews was conducted. Responses were solicited from the University of Southern Denmark's elite triathlon team (n=8), and inductive coding from the focus group formed the basis of questions for the two rounds of individual interviews 11 months apart. All interviews were transcribed verbatim and then analysed thematically. RESULTS: Seventeen context-relevant factors were identified and 10 themes emerged, these being 'the cold weather ritual', 'digestive system conditioning', 'the curse of the night owl', 'the strings attached to sponsorship', 'my coach-my rock', 'mood maintenance', 'the asynchronous training rhythm', 'psychological slavery', 'the legacy of the asphalt tattoo' and 'the tension of family and friends'. CONCLUSIONS: By reflecting on their personal training vortex, elite triathletes were able to provide context-relevant insights into the maintenance of training quality over the course of a competitive season. Further research is required to elucidate whether and how biopsycholosocial factors can be modified to optimise the achievement of training goals.

Discovery of a novel conformational equilibrium in urokinase-type plasminogen activator.

Although trypsin-like serine proteases have flexible surface-exposed loops and are known to adopt higher and lower activity conformations, structural determinants for the different conformations have remained largely obscure. The trypsin-like serine protease, urokinase-type plasminogen activator (uPA), is central in tissue remodeling processes and also strongly implicated in tumor metastasis. We solved five X-ray crystal structures of murine uPA (muPA) in the absence and presence of allosteric molecules and/or substrate-like molecules. The structure of unbound muPA revealed an unsuspected non-chymotrypsin-like protease conformation in which two ß-strands in the core of the protease domain undergoes a major antiparallel-to-parallel conformational transition. We next isolated two anti-muPA nanobodies; an active-site binding nanobody and an allosteric nanobody. Crystal structures of the muPA:nanobody complexes and hydrogen-deuterium exchange mass spectrometry revealed molecular insights about molecular factors controlling the antiparallel-to-parallel equilibrium in muPA. Together with muPA activity assays, the data provide valuable insights into regulatory mechanisms and conformational flexibility of uPA and trypsin-like serine proteases in general.