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Amylase/trypsin-inhibitors (ATIs) comprise about 2-4% of the total wheat grain proteins and may contribute to natural defense against pests and pathogens. However, they are currently among the most widely studied wheat components because of their proposed role in adverse reactions to wheat consumption in humans. ATIs have long been known to contribute to IgE-mediated allergy (notably Bakers' asthma), but interest has increased since 2012 when they were shown to be able to trigger the innate immune system, with attention focused on their role in coeliac disease which affects about 1% of the population and, more recently, in non-coeliac wheat sensitivity which may affect up to 10% of the population. This has led to studies of their structure, inhibitory properties, genetics, control of expression, behavior during processing, effects on human adverse reactions to wheat and, most recently, strategies to modify their expression in the plant using gene editing. We therefore present an integrated account of this range of research, identifying inconsistencies, and gaps in our knowledge and identifying future research needs.NoteThis paper is the outcome of an invited international ATI expert meeting held in Amsterdam, February 3-5 2020

Amylase/trypsin-inhibitors (ATIs) comprise about 2–4% of the total wheat grain proteins and may contribute to natural defense against pests and pathogens. However, they are currently among the most widely studied wheat components because of their proposed role in adverse reactions to wheat consumption in humans. ATIs have long been known to contribute to IgE-mediated allergy (notably Bakers' asthma), but interest has increased since 2012 when they were shown to be able to trigger the innate immune system, with attention focused on their role in coeliac disease which affects about 1% of the population and, more recently, in non-coeliac wheat sensitivity which may affect up to 10% of the population. This has led to studies of their structure, inhibitory properties, genetics, control of expression, behavior during processing, effects on human adverse reactions to wheat and, most recently, strategies to modify their expression in the plant using gene editing. We therefore present an integrated account of this range of research, identifying inconsistencies, and gaps in our knowledge and identifying future research needs.Note This paper is the outcome of an invited international ATI expert meeting held in Amsterdam, February 3-5 2020

Die Nicotinamidadenindinukleotidphosphat-Oxidase Isoform 2 ist ein Enzymsystem, welches reaktive Sauerstoffspezies herstellt und aufgrund dessen im Kontext zahlreicher pathologischer Zustände eine gewichtige Rolle spielt. Die beiden Untereinheiten p47phox und p22phox stellen mit ihrer Interaktion einen entscheidenden Schritt in der Aktivierung des Enzymkomplexes dar und sind somit geeignete Zielmoleküle für die therapeutische Behandlung von oxidativem Stress. Dementsprechend wurden „small-molecule“-Inhibitoren entwickelt, die das Potential besitzen, an die beiden SH3 Domänen des p47phox-Proteins zu binden. Dieser Ansatz nutzte die Fragment-basierteWirkstoffforschung und eine Verknüpfung monomerer Fragmente zu einem dimeren Molekül. Zehn verschiedene Varianten dieser dimeren Moleküle, die sich einzig in ihrer Linker- Struktur unterschieden, wurden mittels Oberflächen-Plasmon-Resonanz sowie Fluoreszenz- Polarisation auf ihr Bindungsvermögen zu den beiden SH3 Domänen von p47phox getestet. Die beiden wirksamsten dimeren Moleküle wiesen submikromolekulare Dissoziationskonstanten auf und wurden für thermodynamische Studien mit Hilfe von isothermer Titrationskalorimetrie ausgewählt. Hierbei offenbarten sich unterschiedliche Enthalpie- sowie Entropie-Beiträge der beiden Moleküle in ihrer Bindung zu p47phox. Beide hatten jedoch die erwartete Bindungs-Stöchiometrie von 1:1 zwischen einem dimeren Molekül und dem p47phox-Protein gemein. Dem gegenüber stand eine 2:1 Bindungs-Stöchiometrie zwischen dem einzelnen monomeren Fragment und p47phox. Analog zu dem Fragment führte die Bindung der dimeren Moleküle zu keiner kompakten Struktur der SH3 Domänen. Solch eine kompakte Struktur war hingegen bei der Bindung eines Peptids des physiologischen Bindungspartners, p22phox, mittels Kleinwinkelröntgenstreuung nachweisbar. Zusammenfassend lässt sich feststellen, dass die Bindung ausgewählter dimerer Moleküle zu den SH3 Domänen von p47phox validiert und anhand der gewählten Verknüpfung der monomeren Fragmente klassifiziert werden konnte. Die isotherme Titrationskalorimetrie konnte überdies das vermutete 1:1-Bindungsverhalten bestätigen und lieferte wichtige Ansatzpunkte für die nachfolgende Phase der Leitstruktur-Optimierung. The nicotinamide adenine dinucleotide phosphate oxidase isoform 2 (NOX2) is an enzyme complex, which produces reactive oxygen species and is linked to several pathological conditions. The interaction between its subunits p47phox and p22phox is essential for the activation of the enzyme complex, and thus suggested as potential target for therapeutic approaches towards oxidative stress. Dimeric small-molecule inhibitors binding to the tandem SH3 domain of p47phox were designed, arising from a fragment-based screen and subsequent fragment linking. Ten dimeric compounds with various linker structures were tested in their binding affinity to the tandem SH3 domain of p47phox by surface plasmon resonance and fluorescence polarization. The two most potent compounds showed submicromolar dissociation constants and were selected for thermodynamic studies using isothermal titration calorimetry. The two molecules differed in their enthalpic and entropic profile, but their supposed binding stoichiometry of 1:1 between a dimeric compound and the tandem SH3 domain was confirmed. This was consistent with a 2:1 binding stoichiometry of the original monomeric fragment. However, just as the fragment, the dimeric compounds did not induce a more compact structure of the tandem SH3 domain. Such a compact structure was seen for a peptide deriving from the physiological binding partner, p22phox, with the aid of small-angle X-ray scattering. In conclusion, binding of selected dimeric compounds to the tandem SH3 domain of p47phox could be validated and affinities determined as a function of the linking type. Isothermal titration calorimetry studies confirmed the expected binding mode and provided possible starting points for the prospective lead optimization phase. submitted by: Felix Peters Auch als Printexemplar in der Bibliothek verfügbar Masterarbeit Wien, FH Campus Wien 2021

Inhibition of postsynaptic density protein-95 (PSD-95) decouples N-methyl-d-aspartate (NMDA) receptor downstream signaling and results in neuroprotection after focal cerebral ischemia. We have previously developed UCCB01-144, a potent dimeric PSD-95 inhibitor, which binds PSD-95 with high affinity and is neuroprotective in experimental stroke. Here, we investigate the selectivity, efficacy and toxicity of UCCB01-144 and compare with the monomeric drug candidate Tat-NR2B9c. Fluorescence polarization using purified proteins and pull-downs of mouse brain lysates showed that UCCB01-144 potently binds all four PSD-95-like membrane-associated guanylate kinases (MAGUKs). In addition, UCCB01-144 affected NMDA receptor signaling pathways in ischemic brain tissue. UCCB01-144 reduced infarct size in young and aged male mice at various doses when administered 30 min after permanent middle cerebral artery occlusion, but UCCB01-144 was not effective in young male mice when administered 1 h post-ischemia or in female mice. Furthermore, UCCB01-144 was neuroprotective in a transient stroke model in rats, and in contrast to Tat-NR2B9c, high dose of UCCB01-144 did not lead to significant changes in mean arterial blood pressure or heart rate. Overall, UCCB01-144 is a potent MAGUK inhibitor that reduces neurotoxic PSD-95-mediated signaling and improves neuronal survival following focal brain ischemia in rodents under various conditions and without causing cardiovascular side effects, which encourages further studies towards clinical stroke trials.

The proteins belonging to the cereal trypsin/alpha-amylase inhibitor family are abundant water/salt-soluble flour proteins active against alpha-amylases from several seed parasites and pests and inactive against endogenous alpha-amylases. Three alpha-amylase inhibitor families have been described in cereals that vary in size and are differently expressed among Triticeae seeds. The present work investigates the presence of human salivary alpha-amylase inhibitors in emmer (Triticum dicoccon Schrank) flour. The isolation was obtained by a series of chromatography steps, and the purification progress was monitored through the inhibition of human salivary alpha-amylase activity. The purified fraction was subjected to protein sequencing by tandem mass spectrometry (MSMS) of the tryptic digests obtained after the sample separation on 2-DE. MSMS data indicated that the emmer alpha-amylase inhibitory fraction was composed of two newly identified proteins [emmer dimeric inhibitor 1 (EDI-1) and emmer dimeric inhibitor 2 (EDI-2)] sharing very high identity levels with related proteins from Triticum aestivum.