Search

Fast prediction of mode of action for bioactive compounds would immensely foster bioactivity annotation in compound collections and may early on reveal off-targets in chemical biology research and drug discovery. A variety of target-based assays is available for addressing the modulation of druggable proteins. However, they cannot precisely predict how a compound would influence cellular processes due to polypharmacology. Furthermore, non-protein targets are often not considered. Morphological profiling, e.g., using the Cell Painting assay that monitors hundreds of morphological features upon compound perturbation and staining of cellular components, offers a fast, unbiased assessment of compound activity on various targets and cellular processes in one single experiment. However, due to incomplete bioactivity annotation and unknown activities of reference (landmark) compounds, prediction of bioactivity is not straightforward. Here we introduce the concept of subprofile analysis to map the mode of action for both reference and unexplored compounds. We defined mode-of-action clusters for a group of reference compounds and extracted cluster subprofiles that contain only a subset of morphological features (i.e., subprofiles) to represent a consensus profile. Subprofile analysis allows for assignment of compounds to, currently, ten different targets or modes of action in one single assay and bypasses the need of exploring all biosimilar reference compounds for the generation of target hypothesis. This approach will enable rapid bioactivity annotation of compound collections, particularly of uncharacterized small molecules, and will be extended to further bioactivity clusters in future. The data is public accessible viahttps://github.com/mpimp-comas/2022_pahl_ziegler_subprofilesand the web app toolhttp://cpcse.pythonanywhere.com/.

Nature Communications 12(1), 2426 (2021). doi:10.1038/s41467-021-22596-0, To adapt to fluctuating protein folding loads in the endoplasmic reticulum (ER), the Hsp70 chaperone BiP is reversibly modified with adenosine monophosphate (AMP) by the ER-resident Fic-enzyme FICD/HYPE. The structural basis for BiP binding and AMPylation by FICD has remained elusive due to the transient nature of the enzyme-substrate-complex. Here, we use thiol-reactive derivatives of the cosubstrate adenosine triphosphate (ATP) to covalently stabilize the transient FICD:BiP complex and determine its crystal structure. The complex reveals that the TPR-motifs of FICD bind specifically to the conserved hydrophobic linker of BiP and thus mediate specificity for the domain-docked conformation of BiP. Furthermore, we show that both AMPylation and deAMPylation of BiP are not directly regulated by the presence of unfolded proteins. Together, combining chemical biology, crystallography and biochemistry, our study provides structural insights into a key regulatory mechanism that safeguards ER homeostasis., Published by Nature Publishing Group UK, [London]

The macrophage migration inhibitory factor (MIF), a vital cytokine and biomarker, has been suggested to closely associate with the pathogenesis of liver cancer. However, a simple and effective approach for monitoring the change and distribution of cellular MIF is currently lacking and urgently needed, which could be helpful for a better understanding of its role in the progression of cancer. Herein, we report a novel activity-based probe, TPP2, which allows for direct labeling and imaging of endogenous MIF activity within live cells, clinical tissues, and

ATP and GTP are exceptionally important molecules in biology with multiple, and often discrete, functions. Therefore, enzymes that bind to either of them must develop robust mechanisms to selectively utilize one or the other. Here, this specific problem is addressed by molecular studies of the human NMP kinase AK3 which uses GTP to phosphorylate AMP. AK3 plays an important role in the citric acid cycle where it is responsible for GTP/GDP recycling. By combining a structural biology approach with functional experiments, we present a comprehensive structural and mechanistic understanding of the enzyme. We discovered that AK3 functions by recruitment of GTP to the active site, while ATP is rejected and non-productively bound to the AMP binding site. Consequently, ATP acts as an inhibitor with respect to GTP and AMP. The overall features with specific recognition of the correct substrate and non-productive binding by the incorrect substrate bears strong similarity to previous findings for the ATP specific NMP kinase adenylate kinase. Taken together we are now able to provide the fundamental principles for GTP and ATP selectivity in the large NMP kinase family. As a side-result originating from non-linearity of chemical shifts in GTP and ATP titrations, we find that protein surfaces offer a general and weak binding affinity for both GTP and ATP. These non-specific interactions likely act to lower the available intra-cellular GTP and ATP concentrations and may have driven evolution to adapt the Michaelis constants of NMP kinases accordingly.

Various pathogenic bacteria use post-translational modifications to manipulate the central components of host cell functions. Many of the enzymes released by these bacteria belong to the large Fic family, which modify targets with nucleotide monophosphates. The lack of a generic method for identifying the cellular targets of Fic family enzymes hinders investigation of their role and the effect of the post-translational modification. Here, we establish an approach that uses reactive co-substrate-linked enzymes for proteome profiling. We combine synthetic thiol-reactive nucleotide derivatives with recombinantly produced Fic enzymes containing strategically placed cysteines in their active sites to yield reactive binary probes for covalent substrate capture. The binary complexes capture their targets from cell lysates and permit subsequent identification. Furthermore, we determined the structures of low-affinity ternary enzyme-nucleotide-substrate complexes by applying a covalent-linking strategy. This approach thus allows target identification of the Fic enzymes from both bacteria and eukarya.

Summary AMPylation is a post-translational modification that modifies amino acid side chains with adenosine monophosphate (AMP). Recently, a role of AMPylation as a universal regulatory mechanism in infection and cellular homeostasis has emerged, driving the demand for universal tools to study this modification. Here, we describe three monoclonal anti-AMP antibodies (mAbs) from mouse that are capable of protein backbone-independent recognition of AMPylation, in denatured (western blot) as well as native (ELISA, IP) applications, thereby outperforming previously reported tools. These antibodies are highly sensitive and specific for AMP modifications, highlighting their potential as tools for new target identification, as well as for validation of known targets. Interestingly, applying the anti-AMP mAbs to various cancer cell lines reveals a previously undescribed broad and diverse AMPylation pattern. In conclusion, these anti-AMP mABs will further advance the current understanding of AMPylation and the spectrum of modified targets., Graphical Abstract, Highlights • Generation of murine monoclonal anti-AMP antibodies via synthetic AMPylated peptide • Characterization in the applications western blot, ELISA, and immunoprecipitation • Sensitive and specific recognition of AMPylation independent of protein backbone • Expansion of toolbox for the detection and enrichment of AMPylation, Biochemistry; Biomolecules; Molecular Biology; Biotechnology

Colibactin is a small molecule produced by certain bacterial species of the human microbiota that harbour the pks genomic island. Pks(+) bacteria induce a genotoxic phenotype in eukaryotic cells an ...

Legionella pneumophila infects eukaryotic cells by forming a replicative organelle – the Legionella containing vacuole. During this process, the bacterial protein DrrA/SidM is secreted and manipulates the activity and post-translational modification (PTM) states of the vesicular trafficking regulator Rab1. As a result, Rab1 is modified with an adenosine monophosphate (AMP), and this process is referred to as AMPylation. Here, we use a chemical approach to stabilise low-affinity Rab:DrrA complexes in a site-specific manner to gain insight into the molecular basis of the interaction between the Rab protein and the AMPylation domain of DrrA. The crystal structure of the Rab:DrrA complex reveals a previously unknown non-conventional Rab-binding site (NC-RBS). Biochemical characterisation demonstrates allosteric stimulation of the AMPylation activity of DrrA via Rab binding to the NC-RBS. We speculate that allosteric control of DrrA could in principle prevent random and potentially cytotoxic AMPylation in the host, thereby perhaps ensuring efficient infection by Legionella., Nature Communications, 12 (1), ISSN:2041-1723

Small-molecule flux in tissue-microdomains is essential for organ function, but knowledge of this process is scant due to the lack of suitable methods. We developed two independent techniques that allow the quantification of advection (flow) and diffusion in individual bile canaliculi and in interlobular bile ducts of intact livers in living mice, namely Fluorescence Loss After Photoactivation (FLAP) and Intravital Arbitrary Region Image Correlation Spectroscopy (IVARICS). The results challenge the prevailing 'mechano-osmotic' theory of canalicular bile flow. After active transport across hepatocyte membranes bile acids are transported in the canaliculi primarily by diffusion. Only in the interlobular ducts, diffusion is augmented by regulatable advection. Photoactivation of fluorescein bis-(5-carboxymethoxy-2-nitrobenzyl)-ether (CMNB-caged fluorescein) in entire lobules demonstrated the establishment of diffusive gradients in the bile canalicular network and the sink function of interlobular ducts. In contrast to the bile canalicular network, vectorial transport was detected and quantified in the mesh of interlobular bile ducts. In conclusion, the liver consists of a diffusion dominated canalicular domain, where hepatocytes secrete small molecules and generate a concentration gradient and a flow-augmented ductular domain, where regulated water influx creates unidirectional advection that augments the diffusive flux.

AMPylation is a post-translational modification that modifies amino acid side chains with adenosine monophosphate (AMP). Recent progress in the field reveals an emerging role of AMPylation as a universal regulatory mechanism in infection and cellular homeostasis, however, generic tools to study AMPylation are required. Here, we describe three monoclonal anti-AMP antibodies (mAbs) from mouse which are capable of protein backbone independent recognition of AMPylation, in denatured (Western Blot) as well as native (ELISA, IP) applications, thereby outperforming previously reported tools. These antibodies are highly sensitive and specific for AMP modifications, highlighting their potential as tools for new target identification, as well as for validation of known targets. Interestingly, applying the anti-AMP mAbs to various cancer cell lines reveals a previously undescribed broad and diverse AMPylation pattern. In conclusion, the anti-AMP mABs will aid the advancement of understanding AMPylation and the spectrum of modified targets.

Using a chemical approach, the crystal structure of Legionella AnkX in complex with human Rab1b is obtained and characterized., The causative agent of Legionnaires disease, Legionella pneumophila, translocates the phosphocholine transferase AnkX during infection and thereby posttranslationally modifies the small guanosine triphosphatase (GTPase) Rab1 with a phosphocholine moiety at S76 using cytidine diphosphate (CDP)–choline as a cosubstrate. The molecular basis for Rab1 binding and enzymatic modification have remained elusive because of lack of structural information of the low-affinity complex with AnkX. We combined thiol-reactive CDP-choline derivatives with recombinantly introduced cysteines in the AnkX active site to covalently capture the heterocomplex. The resulting crystal structure revealed that AnkX induces displacement of important regulatory elements of Rab1 by placing a β sheet into a conserved hydrophobic pocket, thereby permitting phosphocholine transfer to the active and inactive states of the GTPase. Together, the combination of chemical biology and structural analysis reveals the enzymatic mechanism of AnkX and the family of filamentation induced by cyclic adenosine monophosphate (FIC) proteins.

Incorporation of the fluorogenic l-(7-hydroxycoumarin-4-yl)ethylglycine into proteins is a valuable biological tool. Coumarins are typically accessed via the Pechmann reaction, which requires acidic conditions and lacks substrate flexibility. A Pd-mediated coupling is described between o-methoxyboronic acids and a glutamic acid derived (Z)-vinyl triflate, forming latent coumarins. Global deprotection with BBr3 forms the coumarin scaffold in a single step. This mild and scalable route yielded five analogues, including a probe suitable for use at lower pH.

Neutrophils are short-lived leukocytes that migrate to sites of infection as part of the acute immune response, where they phagocytose, degranulate, and form neutrophil extracellular traps (NETs). During NET formation, the nuclear lobules of neutrophils disappear and the chromatin expands and, accessorized with neutrophilic granule proteins, is expelled. NETs can be pathogenic in, for example, sepsis, cancer, and autoimmune and cardiovascular diseases. Therefore, the identification of inhibitors of NET formation is of great interest. Screening of a focused library of natural-product-inspired compounds by using a previously validated phenotypic NET assay identified a group of tetrahydroisoquinolines as new NET formation inhibitors. This compound class opens up new avenues for the study of cellular death through NET formation (NETosis) at different stages, and might inspire new medicinal chemistry programs aimed at NET-dependent diseases.

Small-molecule flux in tissue-microdomains is essential for organ function, but knowledge of this process is scant due to the lack of suitable methods. We developed two independent techniques that allow the quantification of advection (flow) and diffusion in individual bile canaliculi and in interlobular bile ducts of intact livers in living mice, namely Fluorescence Loss After Photoactivation (FLAP) and Intravital Arbitrary Region Image Correlation Spectroscopy (IVARICS). The results challenge the prevailing ‘mechano-osmotic’ theory of canalicular bile flow. After active transport across hepatocyte membranes bile acids are transported in the canaliculi primarily by diffusion. Only in the interlobular ducts, diffusion is augmented by regulatable advection. Photoactivation of fluorescein bis-(5-carboxymethoxy-2-nitrobenzyl)-ether (CMNB-caged fluorescein) in entire lobules demonstrated the establishment of diffusive gradients in the bile canalicular network and the sink function of interlobular ducts. In contrast to the bile canalicular network, vectorial transport was detected and quantified in the mesh of interlobular bile ducts. In conclusion, the liver consists of a diffusion dominated canalicular domain, where hepatocytes secrete small molecules and generate a concentration gradient and a flow-augmented ductular domain, where regulated water influx creates unidirectional advection that augments the diffusive flux.One Sentence Summary/KeywordsBile flux proceeds by diffusion in canaliculi, augmented by advection in ducts.

The intracellular pathogen Legionella pneumophila infects lung macrophages and injects numerous effector proteins into the host cell to establish a vacuole for proliferation. The necessary interference with vesicular trafficking of the host is achieved by modulation of the function of Rab GTPases. The effector protein AnkX chemically modifies Rab1b and Rab35 by covalent phosphocholination of serine or threonine residues using CDP-choline as a donor. So far, the phosphoryl transfer mechanism and the relevance of observed autophosphocholination of AnkX remained disputable. We designed tailored caged compounds to make this type of enzymatic reaction accessible for time-resolved Fourier transform infrared difference spectroscopy. By combining spectroscopic and biochemical methods, we determined that full length AnkX is autophosphocholinated at Ser521, Thr620, and Thr943. However, autophosphocholination loses specificity for these sites in shortened constructs and does not appear to be relevant for the catalysis of the phosphoryl transfer. In contrast, transient phosphocholination of His229 in the conserved catalytic motif might exist as a short-lived reaction intermediate. Upon substrate binding, His229 is deprotonated and locked in this state, being rendered capable of a nucleophilic attack on the pyrophosphate moiety of the substrate. The proton that originated from His229 is transferred to a nearby carboxylic acid residue. Thus, our combined findings support a ping-pong mechanism involving phosphocholination of His229 and subsequent transfer of phosphocholine to the Rab GTPase. Our approach can be extended to the investigation of further nucleotidyl transfer reactions, which are currently of reemerging interest in regulatory pathways of host-pathogen interactions.

Woodward's reagent K (WRK) is a reactive heterocyclic compound that has been employed in protein chemistry to covalently and unspecifically label proteins at nucleophilic amino acids, notably at histidine and cysteine. We have developed a panel of WRK-derived activity-based probes and show that surprisingly and unexpectedly, these probes are fairly selective for a few proteins in the human proteome. The WRK-derived probes show unique reactivity towards the catalytic N-terminal proline in the macrophage migration inhibitory factor (MIF) and can be used to label and, if equipped with a fluorophore, to image MIF activities in living cells.

The present study describes the synthesis and biological studies of a small series of head-to-tail cyclic tetrapeptides of the general structure c(Lys-β

The site-selective incorporation of fluorescent amino acids into proteins has emerged as a valuable alternative to expressible protein reporters. For successful application, a robust and scalable, yet flexible, route to non-natural amino acids is required. This work describes an improved synthesis of coumarin-conjugated lysine derivatives where fluorinated variants are accessed. These analogues can be utilized at low pH and should find application probing biological processes that operate under acidic conditions.

Enzymatic substrate selectivity is critical for the precise control of metabolic pathways. In cases where chemically related substrates are present inside cells, robust mechanisms of substrate selectivity are required. Here, we report the mechanism utilized for catalytic ATP versus GTP selectivity during adenylate kinase (Adk) -mediated phosphorylation of AMP. Using NMR spectroscopy we found that while Adk adopts a catalytically competent and closed structural state in complex with ATP, the enzyme is arrested in a catalytically inhibited and open state in complex with GTP. X-ray crystallography experiments revealed that the interaction interfaces supporting ATP and GTP recognition, in part, are mediated by coinciding residues. The mechanism provides an atomic view on how the cellular GTP pool is protected from Adk turnover, which is important because GTP has many specialized cellular functions. In further support of this mechanism, a structure–function analysis enabled by synthesis of ATP analogs suggests that a hydrogen bond between the adenine moiety and the backbone of the enzyme is vital for ATP selectivity. The importance of the hydrogen bond for substrate selectivity is likely general given the conservation of its location and orientation across the family of eukaryotic protein kinases.

Wir prasentieren eine neue Methode zur Proteinmarkierung, die auf der kovalenten enzymatischen Phosphocholinierung einer spezifischen Octapeptidsequenz intakter Proteine beruht. Das Enzym AnkX aus Legionellen wurde etabliert, um funktionalisierte Phosphocholingruppen aus synthetischen CDP-Cholin-Derivaten auf N- und C-Termini, sowie interne Schleifenregionen zu ubertragen. Zudem kann die kovalente Modifikation durch das Legionellen-Enzym Lem3 hydrolytisch entfernt werden. Lediglich eine 8 Aminosauren kurze Peptidsequenz und eine kleine Verbindungsgruppe (PEG-Phosphocholin) sind fur die effiziente Proteinmarkierung erforderlich.

The first total synthesis of breitfussin A and B is described. The approach features two palladium-catalyzed cross-couplings installing the indole and pyrrole onto the oxazole core and selective lithiation/iodination of a common indole-oxazole fragment providing 2,4-diiodinated or 2-iodinated oxazoles as potential precursors for breitfussin A and B, respectively. An unexpected acid promoted deiodination was utilized in the synthesis of breitfussin B. Comparison of the synthetic material with previously reported spectral data of isolated breitfussin A and B verified the structure of the breitfussin framework.

The localization and signaling of S-palmitoylated peripheral membrane proteins is sustained by an acylation cycle in which acyl protein thioesterases (APTs) depalmitoylate mislocalized palmitoylated proteins on endomembranes. However, the APTs are themselves reversibly S-palmitoylated, which localizes thioesterase activity to the site of the antagonistc palmitoylation activity on the Golgi. Here, we resolve this conundrum by showing that palmitoylation of APTs is labile due to autodepalmitoylation, creating two interconverting thioesterase pools: palmitoylated APT on the Golgi and depalmitoylated APT in the cytoplasm, with distinct functionality. By imaging APT-substrate catalytic intermediates, we show that it is the depalmitoylated soluble APT pool that depalmitoylates substrates on all membranes in the cell, thereby establishing its function as release factor of mislocalized palmitoylated proteins in the acylation cycle. The autodepalmitoylating activity on the Golgi constitutes a homeostatic regulation mechanism of APT levels at the Golgi that ensures robust partitioning of APT substrates between the plasma membrane and the Golgi. Fil: Vartak, Nachiket. Institut Max Planck Fur Molekulare Physiologie; Alemania Fil: Papke, Bjoern. Institut Max Planck Fur Molekulare Physiologie; Alemania Fil: Grecco, Hernan Edgardo. Institut Max Planck Fur Molekulare Physiologie; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Rossmannek, Lisaweta. Institut Max Planck Fur Molekulare Physiologie; Alemania Fil: Waldmann, Herbert. Institut Max Planck Fur Molekulare Physiologie; Alemania Fil: Hedberg, Christian. Institut Max Planck Fur Molekulare Physiologie; Alemania Fil: Bastiaens, Philippe I. H.. Institut Max Planck Fur Molekulare Physiologie; Alemania

Despite their structural similarity, the natural products omuralide and vibralactone have different biological targets. While omuralide blocks the chymotryptic activity of the proteasome with an IC50 value of 47 nM, vibralactone does not have any effect at this protease up to a concentration of 1 mM. Activity-based protein profiling in HeLa cells revealed that the major targets of vibralactone are APT1 and APT2.

Editing the translations: Adenylylation and phosphocholination have recently been found as important post-translational modifications used by pathogenic bacteria during the infection process. This review discusses the combined use of chemical handles and specific antibodies for the identification of previously unknown substrates of these post-translational modifications in infected host cells.

Although the addition of a 5'-adenosine phosphodiester group to proteins, called adenylylation, has been known for decades, the possibility that adenylylation could be a molecular switch in cellular signaling pathways has emerged recently. The distinct mass shift upon adenylation of threonine or tyrosine residues renders it a good target for MS detection and identification; however, the fragmentation of adenylylated peptides derived from proteolytic digestion of adenylylated proteins has not yet been systematically investigated. Here, we demonstrate that adenylylated peptides show loss of parts of the adenosine monophosphate (AMP) upon different fragmentation techniques. As expected, causing the least fragmentation of the AMP group, electron transfer dissociation yields less complicated spectra. In contrast, CID and higher energy collision (HCD) fragmentation caused AMP to fragment, generating characteristic ions that could be utilized in the specific identification of adenylylated peptides. The characteristic ions and losses upon CID and higher energy collision fragmentation from the AMP group turned out to be highly dependent on which amino acid was adenylylated, with different reporter ions for adenylylated threonine and tyrosine. We also investigated how adenylylation is best incorporated into search engines, exemplified by Mascot and showed that it is possible to identify adenylylation by search engines.

Die Identifizierung und Validierung von Zielmolekulen biologisch aktiver niedermolekularer Verbindungen ist ein auserst wichtiger, oft masgebender Schritt in der akademischen und pharmazeutischen Forschung. Dank der Entwicklung und der Verfugbarkeit diverser neuer experimenteller Verfahren ist die Identifizierung von Zielmolekulen prinzipiell realisierbar, und die Anzahl der erfolgreichen Beispiele wachst stetig. Allerdings ist bislang keine generische, breit anwendbare Methodik etabliert worden. Dieser Aufsatz fasst die gangigen Methoden zur Identifizierung der Zielmolekule niedermolekularer Substanzen in erster Linie fur Chemiker, aber auch fur Biologen zusammen, die z. B. den molekularen Angriffspunkt (Target) einer bestimmten biologisch aktiven Substanz finden wollen. Wir beschreiben die am haufigsten eingesetzten experimentellen Ansatze zur Zielprotein-Identifizierung und geben reprasentative Beispiele, die den neuesten Stand der Forschung demonstrieren. Von den derzeit verfugbaren Methoden werden die Affinitatsisolierung von Proteinen mittels geeigneter Affinitatssonden (pull down) und die anschliesende massenspektrometrische Analyse der angereicherten Proteine am haufigsten eingesetzt. Um eine Anleitung fur einen schnellen Einstieg in dieses Feld zu geben, beschreiben wir basierend auf unseren eigenen Erfahrungen einen typischen Ablauf der Zielprotein-Identifizierung, der auf dem Einsatz der chemischen Proteomik als Schlusselschritt fur die Formulierung einer Hypothese fur potentielle Zielproteine beruht.

Phosphocholination of eukaryotic host cell proteins has recently been identified as a novel post-translational modification important for bacterial pathogenesis. Here, we describe the first straightforward synthetic strategy for peptides containing phosphocholinated serine, threonine, or tyrosine residues using preformed functional amino acid building blocks, fully compatible with standard Fmoc solid-phase peptide synthesis.

Many signaling proteins such as the members of the Ras superfamily of GTPases are posttranslationally modified by covalent attachment of lipid groups, which is crucial for the correct localization and function of these proteins. Numerous lipidated proteins are oncogens often found mutated in several human cancers. Therefore, several therapeutic strategies have been developed based on the inhibition of the enzymes involved in these lipidation steps. Here, we will summarize the results on protein lipidation inhibition, mainly focusing on the small molecules targeting the isoprenylation and acylation of proteins.

Modulation of protein-protein interactions (PPIs) is a highly demanding, but also a very promising approach in chemical biology and targeted drug discovery. In contrast to inhibiting PPIs with small, chemically tractable molecules, stabilisation of these interactions can only be achieved with complex natural products, like rapamycin, FK506, taxol, forskolin, brefeldin and fusicoccin. Fusicoccin stabilises the activatory complex of the plant H(+)-ATPase PMA2 and 14-3-3 proteins. Recently, we have shown that the stabilising effect of fusicoccin could be mimicked by a trisubstituted pyrrolinone (pyrrolidone1, 1). Here, we report the synthesis, functional activity and crystal structure of derivatives of 1 that stabilise the 14-3-3-PMA2 complex. With a limited compound collection three modifications that are important for activity enhancement could be determined: 1) conversion of the pyrrolinone scaffold into a pyrazole, 2) introduction of a tetrazole moiety to the phenyl ring that contacts PMA2, and 3) addition of a bromine to the phenyl ring that exclusively contacts the 14-3-3 protein. The crystal structure of a pyrazole derivative of 1 in complex with 14-3-3 and PMA2 revealed that the more rigid core of this molecule positions the stabiliser deeper into the rim of the interface, enlarging especially the contact surface to PMA2. Combination of the aforementioned features gave rise to a molecule (37) that displays a threefold increase in stabilising the 14-3-3-PMA2 complex over 1. Compound 37 and the other active derivatives show no effect on two other important 14-3-3 protein-protein interactions, that is, with CRaf and p53. This is the first study that describes the successful optimisation of a PPI stabiliser identified by screening.

2-α-Keto oxazoles containing polar head groups in their C5-side chains were designed as fatty acid amide hydrolase (FAAH) inhibitors. Variation in the spacer length resulted in submicromolar α-keto-oxazole FAAH inhibitor (IC50 = 436 nM) presenting electrostatic stabilizing interactions between its polar head group contained in the C5-side chain and the hydrophilic pocket of the enzyme.

Metastasis from primary tumors remains a major problem for tumor therapy. In the search for markers of metastasis and more effective therapies, the tumor metabolome is relevant because of its importance to the malignant phenotype and metastatic capacity of tumor cells. Altered choline metabolism is a hallmark of cancer. More specifically, a decreased glycerophosphocholine (GPC) to phosphocholine (PC) ratio was reported in breast, ovarian, and prostate cancers. Improved strategies to exploit this altered choline metabolism are therefore required. However, the critical enzyme cleaving GPC to produce choline, the initial step in the pathway controlling the GPC/PC ratio, remained unknown. In the present work, we have identified the enzyme, here named EDI3 (endometrial differential 3). Purified recombinant EDI3 protein cleaves GPC to form glycerol-3-phosphate and choline. Silencing EDI3 in MCF-7 cells decreased this enzymatic activity, increased the intracellular GPC/PC ratio, and decreased downstream lipid metabolites. Downregulating EDI3 activity inhibited cell migration via disruption of the PKCα signaling pathway, with stable overexpression of EDI3 showing the opposite effect. EDI3 was originally identified in our screening study comparing mRNA levels in metastasizing and nonmetastasizing endometrial carcinomas. Both Kaplan–Meier and multivariate analyses revealed a negative association between high EDI3 expression and relapse-free survival time in both endometrial ( P < 0.001) and ovarian ( P = 0.029) cancers. Overall, we have identified EDI3, a key enzyme controlling GPC and choline metabolism. Because inhibition of EDI3 activity corrects the GPC/PC ratio and decreases the migration capacity of tumor cells, it represents a possible target for therapeutic intervention.

Small molecule signaling promotes the communication between bacteria as well as between bacteria and eukaryotes. The opportunistic pathogenic bacterium Legionella pneumophila employs LAI-1 (3-hydroxypentadecane-4-one) for bacterial cell-cell communication. LAI-1 is produced and detected by the Lqs (Legionella quorum sensing) system, which regulates a variety of processes including natural competence for DNA uptake and pathogen-host cell interactions. In this study, we analyze the role of LAI-1 in inter-kingdom signaling. L. pneumophila lacking the autoinducer synthase LqsA no longer impeded the migration of infected cells, and the defect was complemented by plasmid-borne lqsA. Synthetic LAI-1 dose-dependently inhibited cell migration, without affecting bacterial uptake or cytotoxicity. The forward migration index but not the velocity of LAI-1-treated cells was reduced, and the cell cytoskeleton appeared destabilized. LAI-1-dependent inhibition of cell migration involved the scaffold protein IQGAP1, the small GTPase Cdc42 as well as the Cdc42-specific guanine nucleotide exchange factor ARHGEF9, but not other modulators of Cdc42, or RhoA, Rac1 or Ran GTPase. Upon treatment with LAI-1, Cdc42 was inactivated and IQGAP1 redistributed to the cell cortex regardless of whether Cdc42 was present or not. Furthermore, LAI-1 reversed the inhibition of cell migration by L. pneumophila, suggesting that the compound and the bacteria antagonistically target host signaling pathway(s). Collectively, the results indicate that the L. pneumophila quorum sensing compound LAI-1 modulates migration of eukaryotic cells through a signaling pathway involving IQGAP1, Cdc42 and ARHGEF9., Author Summary Legionella pneumophila is a ubiquitous environmental bacterium, which upon inhalation causes a severe pneumonia termed Legionnaires’ disease. The opportunistic pathogen employs the small molecule LAI-1 (Legionella autoinducer-1) for bacterial cell-cell communication. LAI-1 is produced and detected by the Lqs (Legionella quorum sensing) system, which regulates a variety of processes including pathogen-host cell interactions. In this study, we analyzed whether LAI-1 not only plays a role for bacterial signaling but also modulates gene regulation and cellular responses of eukaryotic cells (amoebae or macrophages). We discovered that the gene encoding the LAI-1 autoinducer synthase, lqsA, indeed promotes the inhibition of cell migration by L. pneumophila, and synthetic LAI-1 dose-dependently inhibits cell migration. LAI-1-dependent inhibition of cell migration required the scaffold protein IQGAP1 and the small GTPase Cdc42, as well as the Cdc42 activator ARHGEF9, but not other modulators of Cdc42 or small GTPases. Treatment with LAI-1 led to inactivation of Cdc42 and redistribution of IQGAP1. In summary, our results reveal that the L. pneumophila signaling compound LAI-1 inhibits the migration of eukaryotic cells through a host signaling pathway comprising IQGAP1, Cdc42 and ARHGEF9.

We identify a role for the GDI-like solubilizing factor (GSF) PDEδ in modulating signalling through Ras family G proteins by sustaining their dynamic distribution in cellular membranes. We show that the GDI-like pocket of PDEδ binds and solubilizes farnesylated Ras proteins, thereby enhancing their diffusion in the cytoplasm. This mechanism allows more effective trapping of depalmitoylated Ras proteins at the Golgi and polycationic Ras proteins at the plasma membrane to counter the entropic tendency to distribute these proteins over all intracellular membranes. Thus, PDEδ activity augments K/Hras signalling by enriching Ras at the plasma membrane; conversely, PDEδ down-modulation randomizes Ras distributions to all membranes in the cell and suppresses regulated signalling through wild-type Ras and also constitutive oncogenic Ras signalling in cancer cells. Our findings link the activity of PDEδ in determining Ras protein topography to Ras-dependent signalling.

The first straightforward building block based (non-interassembly) synthesis of peptides containing adenylylated serine and threonine residues is described. Key features include final global acidolytic protective group removal as well as full compatibility with standard Fmoc solid-phase peptide synthesis (SPPS). The described Thr-AMP SPPS-building block has been employed in the synthesis of the Thr-adenylylated sequence of human GTPase CDC42 (Ac-SEYVP-T(AMP)-VFDNYGC-NH(2)). Further, we demonstrate proof-of-concept for the synthesis of an Ser-adenylylated peptide (Ac-GSGA-S(AMP)-AGSGC-NH(2)) from the corresponding adenylylated serine building block.

Finding the target: activity-based proteomic profiling probes based on the depalmitoylation inhibitors palmostatin B and M have been synthesized and were found to target acyl protein thioesterase 1 (APT1) and 2 (APT2) in cells.

A matter of common sense: a common recognition motif consisting of a negatively charged group five to six bonds away (red) from the (thio)ester functionality (green) and a positively charged tail group ten to twelve bonds away (blue) was identified in two native acyl protein thioesterase 1 (APT1) substrates. This similarity led to the design of potent inhibitors of the Ras-depalmitoylating enzyme APT1.

During infections, bacterial microorganisms initiate profound interactions with mammalian host cells. Usually defense mechanisms of the host destroy intruding bacteria in rapid manner. However, many bacterial pathogens have evolved in a way to avoid these mechanisms. By use of effector molecules, which can be small organic molecules or proteins with enzymatic activity, the host is manipulated on a molecular level. Effectors mediating post-translational modifications (PTMs) are employed by many pathogens to influence the biological activity of host proteins. In the presented thesis, two related PTMs are investigated in detail: Adenylylation, the covalent transfer of an adenosine monophosphate group from adenosine triphosphate onto proteins, and phosphocholination, the covalent transfer of a phosphocholine moiety onto proteins. Over the past years, enzymes mediating these modifications have been discovered in several pathogens, especially as a mechanism to influence the signaling of eukaryotic cells by adenylylating or phosphocholinating small GTPases. However, the development of reliable methods for the isolation and identification of adenylylated and phosphocholinated proteins remains a vehement challenge in this field of research. This thesis presents general procedures for the synthesis of peptides carrying adenylylated or phosphocholinated tyrosine, threonine and serine residues. From the resulting peptides, mono-selective polyclonal antibodies against adenylylated tyrosine and threonine have been raised. The antibodies were used as tools for proteomic research to isolate unknown substrates of adenylyl transferases from eukaryotic cells. Mass spectrometric fragmentation techniques have been investigated to ease the identification of adenylylated proteins. Furthermore, this work presents a new strategy to identify adenylylated proteins. Additionally, small effector molecules are involved in the regulation of infection mechanisms. In this work, the small molecule LAI-1 (Legionella autoinducer 1) from the pathogen Legionella pneumophila, the causative agent of the Legionnaire’s disease, was synthesised together with its amino-derivatives. LAI-1 showed are a clear pharmacological effect on the regulation of the life cycle of L. pneumophila, initiating transmissive traits like motility and virulence. Furthermore, LAI-1 was shown to have an effect on eukaryotic cells as well. Directed motility of the eukaryotic cells was significantly reduced and the cytoskeletal architecture was reorganised, probably by interfering with the small GTPase Cdc42.