Chemistry and biology of natural product derived protease inhibitors

Im Rahmen dieser Dissertation sollten Naturstoffe und davon abgeleitete Derivate synthetisiert und im Hinblick auf ihre biologische Aktivität als Protease-Inhibitoren untersucht werden. Für die Naturstoffklasse der 3-Amino-6-Hydroxy-2-piperidon(Ahp)-Cyclodepsipeptide, die als nicht-kovalente Serin-Protease-Inhibitoren bekannt sind, konnte eine Festphasensynthese basierend auf einem allgemeinen Ahp-Vorläufermolekül entwickelt werden. Für den Ahp-Vorläufer wurde eine Lösungssynthese entwickelt. Die Festphasenstrategie ermöglicht die Synthese maßgeschneiderter Ahp-Cyclodepsipeptide; sie beinhaltet eine Veresterung an der festen Phase, sowie eine Festphasen-Macrolaktamisierung und etabliert ein neues Protokoll zur Generierung eines Aldehyds bei Abspaltung von der festen Phase. Zur Überprüfung der neu-entwickelten Strategie wurde der Chymotrypsin-Inhibitor Symplocamide A erfolgreich an der festen Phase synthetisiert und im Anschluss auf seine biologische Aktivität hin untersucht. Mit Symplocamide A als der Leitstruktur wurden vereinfachte Analoga von Ahp-Cyclodepsipeptiden synthetisiert, um zu überprüfen ob diese Analoga, wie die Ahp-Cyclodepsipeptide auch, die sogenannte kanonische Konformation einnehmen. Die kanonische Konformation wurde zuerst in proteinogenen Serinprotease-Inhibitoren beobachtet und studiert. Kürzlich wurden auch peptidische Analoga dieser Inhibitoren untersucht und ein Zusammenhang mit den Ahp-Cyclodepsipeptiden erkannt, welche ebenfalls durch die Einnahme der kanonischen Konformation inhibieren. Im Rahmen dieser Dissertation war es möglich zu zeigen, dass die Ahp-Einheit unter Retention der biologischen Aktivität durch kommerziell erhältliche Aminosäuren ersetzt werden kann. Mittels Struktur-Wirkungsuntersuchungen konnten zudem die kritischen Determinanten zur Einnahme der kanonischen Konformation bestimmt, sowie ein Einblick in die molekulare Grundlage einer effizienten Inhibition gewonnen werden. Für den Naturstoff Symplostatin 4 wurde eine konvergente Lösungssynthese entwickelt, die einen flexiblen Zugang zu modifizierten Derivaten ermöglichte. Speziell die Synthese von Sonden für die Untersuchung von Symplostatin 4 mittels aktivitätsbasiertem Proteinprofiling war durch eine 1,3-dipolare Huisgen-Cycloaddition von propargyl-modifizierten Spezies mit Azid-modifizierten Reportermolekülen vereinfacht möglich. Durch aktivitätsbasiertes Profiling konnten mit den Cystein-Proteasen RD21A und RD21B die Zielenzyme von Symplostatin 4 in der Modellpflanze Arabidopsis thaliana identifiziert werden. Desweiteren wurde die anti-Malaria-Aktivität, die für das chemisch identische Gallinamide A publiziert worden war, für Symplostatin 4 und seine Derivate studiert. Im Rahmen von Untersuchungen mit dem Malariaerreger Plasmodium falciparum konnte festgestellt werden, dass Symplostatin 4 ein nanomolarer Inhibitor der Cystein-Proteasen Falcipain 2 und 3 ist, die für den Hemoglobinverdau zuständig sind und als wichtige Zielenzyme einer alternativen Malaria-Chemotherapie betrachtet werden

Conserved Biochemical Defenses Underpin Host Responses to Oomycete Infection in an Early-Divergent Land Plant Lineage.

The expansion of plants onto land necessitated the evolution of robust defense strategies to protect against a wide array of microbial invaders. Whereas host responses to microbial colonization are extensively explored in evolutionarily young land plant lineages such as angiosperms, we know relatively little about plant-pathogen interactions in early-diverging land plants thought to better represent the ancestral state. Here, we define the transcriptional and proteomic response of the early-divergent liverwort Marchantia polymorpha to infection with the oomycete pathogen Phytophthora palmivora. We uncover a robust molecular response to oomycete colonization in Marchantia that consists of conserved land plant gene families. Direct macroevolutionary comparisons of host infection responses in Marchantia and the model angiosperm Nicotiana benthamiana further reveal a shared set of orthologous microbe-responsive genes that include members of the phenylpropanoid metabolic pathway. In addition, we identify a role for the Marchantia R2R3-MYB transcription factor MpMyb14 in activating phenylpropanoid (flavonoid) biosynthesis during oomycete infection. Mpmyb14 mutants infected with P. palmivora fail to activate phenylpropanoid biosynthesis gene expression and display enhanced disease susceptibility compared to wild-type plants. Conversely, the ectopic induction of MpMyb14 led to the accumulation of anthocyanin-like pigments and dramatically enhanced liverwort resistance to P. palmivora infection. Collectively, our results demonstrate that the Marchantia response to oomycete infection displays evolutionarily conserved features indicative of an ancestral pathogen deterrence strategy centered on phenylpropanoid-mediated biochemical defenses.Gatsby Charitable Foundation (GAT3395/GLD) Royal Society (UF160413, RGF\EA\180002) BBSRC OpenPlant initiative (BB/L014130/1) Natural Environment Research Council (NERC; NE/N00941X/1), Natural Sciences and Engineering Research Council of Canada (NSERC) postdoctoral fellowship, Max-Planck-Gesellschaft

Gene modification by fast-track recombineering for cellular localization and isolation of components of plant protein complexes.

To accelerate the isolation of plant protein complexes and study cellular localization and interaction of their components, an improved recombineering protocol is described for simple and fast site-directed modification of plant genes in bacterial artificial chromosomes (BACs). Coding sequences of fluorescent and affinity tags were inserted into genes and transferred together with flanking genomic sequences of desired size by recombination into Agrobacterium plant transformation vectors using three steps of E. coli transformation with PCR-amplified DNA fragments. Application of fast-track recombineering is illustrated by the simultaneous labelling of CYCLINDEPENDENT KINASE D (CDKD) and CYCLIN H (CYCH) subunits of kinase module of TFIIH general transcription factor and the CDKD-activating CDKF;1 kinase with green fluorescent protein (GFP) and mCherry (green and red fluorescent protein) tags, and a PIPL (His18-StrepII-HA) epitope. Functionality of modified CDKF;1 gene constructs is verified by complementation of corresponding T-DNA insertion mutation. Interaction of CYCH with all three known CDKD homologues is confirmed by their co-localization and co-immunoprecipitation. Affinity purification and mass spectrometry analyses of CDKD;2, CYCH, and DNA-replication-coupled HISTONE H3.1 validate their association with conserved TFIIH subunits and components of CHROMATIN ASSEMBLY FACTOR 1, respectively. The results document that simple modification of plant gene products with suitable tags by fast-track recombineering is well suited to promote a wide range of protein interaction and proteomics studies

Pathogen effector recognition-dependent association of NRG1 with EDS1 and SAG101 in TNL receptor immunity

Plants utilise intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors to detect pathogen effectors and activate local and systemic defence. NRG1 and ADR1 “helper” NLRs (RNLs) cooperate with enhanced disease susceptibility 1 (EDS1), senescence-associated gene 101 (SAG101) and phytoalexin-deficient 4 (PAD4) lipase-like proteins to mediate signalling from TIR domain NLR receptors (TNLs). The mechanism of RNL/EDS1 family protein cooperation is not understood. Here, we present genetic and molecular evidence for exclusive EDS1/SAG101/NRG1 and EDS1/PAD4/ADR1 co-functions in TNL immunity. Using immunoprecipitation and mass spectrometry, we show effector recognition-dependent interaction of NRG1 with EDS1 and SAG101, but not PAD4. An EDS1-SAG101 complex interacts with NRG1, and EDS1-PAD4 with ADR1, in an immune-activated state. NRG1 requires an intact nucleotide-binding P-loop motif, and EDS1 a functional EP domain and its partner SAG101, for induced association and immunity. Thus, two distinct modules (NRG1/EDS1/SAG101 and ADR1/EDS1/PAD4) mediate TNL receptor defence signalling

Case Studies of the Synthesis of Bioactive Cyclodepsipeptide Natural Products

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Solid phase total synthesis of the 3-amino-6-hydroxy-2-piperidone (Ahp) cyclodepsipeptide and protease inhibitor Symplocamide A

The solid phase total synthesis of the marine cyanobacterial Ahp-cyclodepsipeptide Symplocamide A is reported as a model for a general route for the synthesis of tailor-made non-covalent serine protease inhibitors

Ahp cyclodepsipeptides: the Impact of the Ahp residue on the "Canonical Inhibition" of S1 serine proteases

S1 serine proteases are by far the largest and most diverse family of proteases encoded in the human genome. Although recent decades have seen an enormous increase in our knowledge, the biological functions of most of these proteases remain to be elucidated. Chemical inhibitors have proven to be versatile tools for studying the functions of proteases, but this approach is hampered by the limited availability of inhibitor scaffold structures with the potential to allow rapid discovery of selective, noncovalent small-molecule protease inhibitors. The natural product class of Ahp cyclodepsipeptides is an unusual class of small-molecule canonical inhibitors; the incorporation of protease cleavage sequences into their molecular scaffolds enables the design of specific small-molecule inhibitors that simultaneously target the S and S′ subsites of the protease through noncovalent mechanisms. Their synthesis is tedious, however, so in this study we have investigated the relevance of the Ahp moiety for achieving potent inhibition. We found that although the Ahp residue plays an important role in inhibition potency, appropriate replacement with β-hydroxy amino acids results in structurally less complex derivatives that inhibit serine proteases in the low micromolar range

Development of a solid-phase approach to the natural product class of Ahp-containing cyclodepsipeptides

The 3-amino-6-hydroxy-2-piperidone (Ahp) containing cyclodepsipeptides are an interesting class of natural products that inhibit S1 (trypsin and chymotrypsin-like) serine protease in a reversible, noncovalent manner, turning them into potential chemical tools for protease research. Their systematic use in chemical biology is however hampered by their tedious solution-phase chemical synthesis. To overcome this limitation, we report a solid-phase approach to Ahp cyclodepsipeptides that is based on the use of a maskedglutamic aldehyde moiety as a general Ahp precursor molecule. As a proof-of-concept, we therefore recently reported the solid-phase synthesis of Symplocamide A. Here, we want to give a full account on the development and application of the masked glutamic aldehyde moiety as well as the optimization of the solid-phase synthesis, which allowed the successful synthesis of the natural product Symplocamide A

The Adaptation of <i>Botrytis cinerea</i> Extracellular Vesicles Proteome to Surrounding Conditions: Revealing New Tools for Its Infection Process

Extracellular vesicles (EVs) are membranous particles released by different organisms. EVs carry several sets of macromolecules implicated in cell communication. EVs have become a relevant topic in the study of pathogenic fungi due to their relationship with fungal–host interactions. One of the essential research areas in this field is the characterization protein profile of EVs since plant fungal pathogens rely heavily on secreted proteins to invade their hosts. However, EVs of Botrytis cinerea are little known, which is one of the most devastating phytopathogenic fungi. The present study has two main objectives: the characterization of B. cinerea EVs proteome changes under two pathogenic conditions and the description of their potential role during the infective process. All the experimental procedure was conducted in B. cinerea growing in a minimal salt medium supplemented with glucose as a constitutive stage and deproteinized tomato cell walls (TCW) as a virulence inductor. The isolation of EVs was performed by differential centrifugation, filtration, ultrafiltration, and sucrose cushion ultracentrifugation. EVs fractions were visualised by TEM using negative staining. Proteomic analysis of EVs cargo was addressed by LC-MS/MS. The methodology used allowed the correct isolation of B. cinerea EVs and the identification of a high number of EV proteins, including potential EV markers. The isolated EVs displayed differences in morphology under both assayed conditions. GO analysis of EV proteins showed enrichment in cell wall metabolism and proteolysis under TCW. KEGG analysis also showed the difference in EVs function under both conditions, highlighting the presence of potential virulence/pathogenic factors implicated in cell wall metabolism, among others. This work describes the first evidence of EVs protein cargo adaptation in B. cinerea, which seems to play an essential role in its infection process, sharing crucial functions with the conventional secretion pathways