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Die enzymkatalysierte Funktionalisierung (engl.: late‐stage functionalization (LSF)), wie z. B. die Methylierung von Arzneimittelmolekülen und Leitstrukturen, ermöglicht den direkten Zugang zu wirksameren pharmazeutischen Wirkstoffen (API). S‐Adenosyl‐l‐Methionin‐abhängige Methyltransferasen (MTs) können eine Schlüsselrolle bei der Entwicklung neuer Wirkstoffe spielen, da sie die chemo‐ und regioselektive Methylierung von O‐, N‐, S‐ und C‐Atomen katalysieren und damit den herkömmlichen chemischen Wegen überlegen sind. Um geeignete MTs zu identifizieren, haben wir einen kontinuierlichen fluoreszenzbasierten Hochdurchsatzassay für SAM‐abhängige Methyltransferasen entwickelt, der das Screening unter Verwendung von E. coli Zelllysaten erleichtert. Dieser Assay besteht aus zwei enzymatischen Schritten für die Umwandlung von S‐Adenosyl‐l‐Homocystein zu H2S, was zu einer selektiven Fluoreszenzmessung durch Reduktion eines Azidocoumarin‐Sulfidsensors führt. Die Untersuchung von zwei O‐MTs und einer N‐MT bestätigte, dass dieser Assay für die Bestimmung der Methyltransferase‐Aktivität in E. coli Zelllysaten geeignet ist. [ABSTRACT FROM AUTHOR]

Die Hauptprotease von SARS‐CoV‐2 (Mpro), dem Auslöser von COVID‐19, ist ein wichtiges Arzneistoff‐Target. Ein neues fluorogenes Substrat, das kinetisch mit einem intern gequenchten fluoreszierenden Peptid verglichen wurde, erwies sich als ideal geeignet für ein Hochdurchsatz‐Screening mit rekombinant exprimierter Mpro. Zwei Klassen von Protease‐Inhibitoren, Azanitrile und Pyridylester, wurden identifiziert, optimiert und biochemisch charakterisiert. Maßgeschneiderte Peptide mit einer reaktiven Azanitril‐Kopfgruppe zeigten eine duale Inhibition von Mpro und Cathepsin L, einer Protease, welche die virale Zellinvasion befördert. Zur Optimierung der Pyridylindolester wurde ein Positions‐Scanning durchgeführt. Unser fokussierter Ansatz zur Entwicklung von Mpro‐Inhibitoren erwies sich dem virtuellen Screening als überlegen. Mit den beiden irreversiblen Inhibitoren Azanitril 8 (kinac/Ki=37 500 m−1 s−1, Ki=24.0 nm) und Pyridylester 17 (kinac/Ki=29 100 m−1 s−1, Ki=10.0 nm) wurden vielversprechende Kandidaten für die zukünftige Arzneistoffentwicklung entdeckt. [ABSTRACT FROM AUTHOR]

Microfluidic droplet sorting enables the high‐throughput screening and selection of water‐in‐oil microreactors at speeds and volumes unparalleled by traditional well‐plate approaches. Most such systems sort using fluorescent reporters on modified substrates or reactions that are rarely industrially relevant. We describe a microfluidic system for high‐throughput sorting of nanoliter droplets based on direct detection using electrospray ionization mass spectrometry (ESI‐MS). Droplets are split, one portion is analyzed by ESI‐MS, and the second portion is sorted based on the MS result. Throughput of 0.7 samples s−1 is achieved with 98 % accuracy using a self‐correcting and adaptive sorting algorithm. We use the system to screen ≈15 000 samples in 6 h and demonstrate its utility by sorting 25 nL droplets containing transaminase expressed in vitro. Label‐free ESI‐MS droplet screening expands the toolbox for droplet detection and recovery, improving the applicability of droplet sorting to protein engineering, drug discovery, and diagnostic workflows. [ABSTRACT FROM AUTHOR]

RNA‐based therapies offer a wide range of therapeutic interventions including the treatment of skin diseases; however, the strategies to efficiently deliver these biomolecules are still limited due to obstacles related to the cellular uptake and cytoplasmic delivery. Herein, we report the synthesis of a triggerable polymeric nanoparticle (NP) library composed of 160 formulations, presenting physico‐chemical diversity and differential responsiveness to light. Six formulations were more efficient (up to 500 %) than commercially available lipofectamine in gene‐knockdown activity. These formulations showed differential internalization by skin cells and the endosomal escape was rapid (minutes range). The NPs were effective in the release of siRNA and miRNA. Acute skin wounds treated with the top hit NP complexed with miRNA‐150‐5p healed faster than wounds treated with scrambled miRNA. Light‐activatable NPs offer a new strategy to topically deliver non‐coding RNAs. [ABSTRACT FROM AUTHOR]

Glucose transporters play an essential role in cancer cell proliferation and survival and have been pursued as promising cancer drug targets. Using microarrays of a library of new macrocycles known as rapafucins, which were inspired by the natural product rapamycin, we screened for new inhibitors of GLUT1. We identified multiple hits from the rapafucin 3D microarray and confirmed one hit as a bona fide GLUT1 ligand, which we named rapaglutin A (RgA). We demonstrate that RgA is a potent inhibitor of GLUT1 as well as GLUT3 and GLUT4, with an IC50 value of low nanomolar for GLUT1. RgA was found to inhibit glucose uptake, leading to a decrease in cellular ATP synthesis, activation of AMP‐dependent kinase, inhibition of mTOR signaling, and induction of cell‐cycle arrest and apoptosis in cancer cells. Moreover, RgA was capable of inhibiting tumor xenografts in vivo without obvious side effects. RgA could thus be a new chemical tool to study GLUT function and a promising lead for developing anticancer drugs. [ABSTRACT FROM AUTHOR]

Natural products (NPs) inspire the design and synthesis of novel biologically relevant chemical matter, for instance through biology‐oriented synthesis (BIOS). However, BIOS is limited by the partial coverage of NP‐like chemical space by the guiding NPs. The design and synthesis of "pseudo NPs" overcomes these limitations by combining NP‐inspired strategies with fragment‐based compound design through de novo combination of NP‐derived fragments to unprecedented compound classes not accessible through biosynthesis. We describe the development and biological evaluation of pyrano‐furo‐pyridone (PFP) pseudo NPs, which combine pyridone‐ and dihydropyran NP fragments in three isomeric arrangements. Cheminformatic analysis indicates that the PFPs reside in an area of NP‐like chemical space not covered by existing NPs but rather by drugs and related compounds. Phenotypic profiling in a target‐agnostic "cell painting" assay revealed that PFPs induce formation of reactive oxygen species and are structurally novel inhibitors of mitochondrial complex I. [ABSTRACT FROM AUTHOR]

Carbohydrates are attached and removed in living systems through the action of carbohydrate‐active enzymes such as glycosyl transferases and glycoside hydrolases. The molecules resulting from these enzymes have many important roles in organisms, such as cellular communication, structural support, and energy metabolism. In general, each carbohydrate transformation requires a separate catalyst, and so these enzyme families are extremely diverse. To make this diversity manageable, high‐throughput approaches look at many enzymes at once. Similarly, high‐throughput approaches can be a powerful way of finding inhibitors that can be used to tune the reactivity of these enzymes, either in an industrial, a laboratory, or a medicinal setting. In this review, we provide an overview of how these enzymes and inhibitors can be sought using techniques such as high‐throughput natural product and combinatorial library screening, phage and mRNA display of (glyco)peptides, fluorescence‐activated cell sorting, and metagenomics. [ABSTRACT FROM AUTHOR]

Assaying for enzymatic activity is a persistent bottleneck in biocatalyst and drug development. Existing high‐throughput assays for enzyme activity tend to be applicable only to a narrow range of biochemical transformations, whereas universal enzyme characterization methods usually require chromatography to determine substrate turnover, greatly diminishing throughput. We present an enzyme activity assay that allows the high‐throughput mass‐spectrometric detection of enzyme activity in complex matrices without the need for a chromatographic step. This technology, which we call probing enzymes with click‐assisted NIMS (PECAN), can detect the activity of medically and biocatalytically significant cytochrome P450s in cell lysate, microsomes, and bacteria. Using this approach, a cytochrome P450BM3 mutant library was successfully screened for the ability to catalyze the oxidation of the sesquiterpene valencene. [ABSTRACT FROM AUTHOR]

Die gelenkte oder gerichtete Evolution ("directed evolution") hat sich zu einem Standardverfahren entwickelt, um natürliche Proteine für eine breite Palette von Anwendungen maßzuschneidern. Es gelang über verschiedene Industriebereiche hinweg, eine hohe Wertschöpfung und einen hohen gesellschaftlichen Nutzen zu erzielen. Beispiele sind die enzymatische Herstellung von Chemikalien und Pharmazeutika sowie Anwendungen in Ernährung, Futtermittel und Waschmittel. Diese nachhaltigen Industrieverfahren sind essenziell für den Übergang von einer Erdöl‐basierten Wirtschaft hin zu einer Bioökonomie, die auf nachwachsenden Rohstoffe beruht. Ein vergleichbarer Durchbruch wurde durch die Technologie des Phagen‐Displays ("phage display") erzielt, die es durch gerichtete Evolution ermöglicht, hochselektive Antikörper durch physikalische Kopplung des Phänotyps und des zugehörigen Genotyps zu finden, die für eine breite Palette von Anwendungen in Diagnostik und Therapie zur Verfügung stehen. Der Chemie‐Nobelpreis 2018 wurde zur Hälfte Frances H. Arnold für die gerichtete Evolution von Enzymen verliehen. Die andere Hälfte teilen sich George P. Smith und Sir Gregory P. Winter für das Phagen‐Display von Peptiden und Antikörpern. Dieses Highlight würdigt die Beiträge der Nobellaureaten und fasst die Historie, den derzeitigen Status, Herausforderungen und Anwendungen der gerichteten Evolution und des Phagen‐Display zusammen. [ABSTRACT FROM AUTHOR]

Abstract: Functional metagenomics has opened new opportunities for enzyme discovery. To exploit the full potential of this new tool, the design of selective screens is essential, especially when searching for rare enzymes. To identify novel glycosidases that employ cleavage strategies other than the conventional Koshland mechanisms, a suitable screen was needed. Focusing on the unsaturated glucuronidases (UGLs), it was found that use of simple aryl glycoside substrates did not allow sufficient discrimination against β‐glucuronidases, which are widespread in bacteria. While conventional glycosidases cannot generally hydrolyze thioglycosides efficiently, UGLs follow a distinct mechanism that allows them to do so. Thus, fluorogenic thioglycoside substrates featuring thiol‐based self‐immolative linkers were synthesized and assessed as selective substrates. The generality of the approach was validated with another family of unconventional glycosidases, the GH4 enzymes. Finally, the utility of these substrates was tested by screening a small metagenomic library. [ABSTRACT FROM AUTHOR]

Abstract: Amyotrophic lateral sclerosis, or Lou Gehrig's disease, is characterized by motor neuron death, with average survival times of two to five years. One cause of this disease is the misfolding of superoxide dismutase 1 (SOD1), a phenomenon influenced by point mutations spanning the protein. Herein, we used an epitope‐specific high‐throughput screen to identify a peptide ligand that stabilizes the SOD1 native conformation and accelerates its folding by a factor of 2.5. This strategy may be useful for fundamental studies of protein energy landscapes as well as designing new classes of therapeutics. [ABSTRACT FROM AUTHOR]

Die ClpXP‐Protease von Staphylococcus aureus ist ein wichtiger Regulator der Zellhomöostase und Virulenz. Mithilfe eines Hochdurchsatz‐Screenings gegen den ClpXP‐Komplex wurde ein spezifischer Inhibitor des ClpX‐Chaperons identifiziert, der den oligomeren Zustand zerstört. Die Synthese von 34 Derivaten offenbarte, dass das molekulare Grundgerüst nur eingeschränkte Veränderungen ermöglicht und nur geringe Änderungen toleriert werden. Eine Analyse der aktivsten Verbindungen zeigte eine starke Verringerung der Toxinproduktion von S. aureus, die durch eine maßgeschneiderte, MS‐basierte Assayplattform quantifiziert wurde. Transkriptom‐ und Gesamtproteomstudien bestätigten die globale Virulenzreduktion und enthüllten eine charakteristische Signatur der Proteinexpression in mit Substanz behandelten Zellen. Obwohl diese Signatur teilweise mit dem Muster der ClpX‐Deletionszellen übereinstimmte, wurde eine weitere Verringerung der Toxine beobachtet, was zu der vielversprechenden Möglichkeit führt, dass weitere Virulenzpfade direkt oder indirekt von der niedermolekularen Verbindung adressiert werden könnten. Ein Hochdurchsatz‐Screening gegen die ClpXP‐Protease enthüllte die ersten nicht‐kovalenten Inhibitoren des ClpX‐Chaperons. Bindung des Inhibitors induziert die Zerstörung des ClpX‐Hexamers und sogar des kompletten proteolytischen ClpXP‐Komplexes. Behandlung von Staphylococcus aureus mit den Verbindungen führt zu einer globalen Herunterregulation einer Vielzahl sekretierter Virulenzfaktoren. [ABSTRACT FROM AUTHOR]

We present an automated microfluidic platform for in-flow studies of visible-light photoredox catalysis in liquid or gas-liquid reactions at the 15 μL scale. An oscillatory flow strategy enables a flexible residence time while preserving the mixing and heat transfer advantages of flow systems. The adjustable photon flux made possible with the platform is characterized using actinometry. Case studies of oxidative hydroxylation of phenylboronic acids and dimerization of thiophenol demonstrate the capabilities and advantages of the system. Reaction conditions identified through droplet screening translate directly to continuous synthesis with minor platform modifications. [ABSTRACT FROM AUTHOR]

Proteintemplat ‐ gesteuerte Fragmentligationen sind ein neuartiges Konzept zur Unterstützung der Wirkstofffindung und können dazu beitragen, die Wirksamkeit von Proteinliganden zu verbessern. Es handelt sich dabei um chemische Reaktionen zwischen niedermolekularen Verbindungen (“Fragmenten”), die die Oberfläche eines Proteins als Reaktionsgefäß verwenden, um die Bildung eines Proteinliganden mit erhöhter Bindungsaffinität zu katalysieren. Die Methode nutzt die molekulare Erkennung kleiner reaktiver Fragmente durch die Proteine sowohl zur Assemblierung der Liganden als auch zur Identifizierung bioaktiver Fragmentkombinationen. Chemische Synthese und Bioassay werden dabei in einem Schritt vereint. Dieser Aufsatz diskutiert die biophysikalischen Grundlagen der reversiblen und irreversiblen Fragmentligationen und gibt einen Überblick über die Methoden, mit denen die durch das Proteintemplat gebildeten Ligationsprodukte detektiert werden können. Der chemische Reaktionsraum und aktuelle Anwendungen wie auch die Bedeutung dieses Konzeptes für die Wirkstofffindung werden erörtert. Können Wirkstoffe durch Proteine als Reaktionsgefäße entdeckt werden? Proteine können reversible und irreversible Verknüpfungen von Protein ‐ bindenden Fragmenten induzieren. In diesem Aufsatz werden die Chemie und die Biophysik derartiger Reaktionen dargestellt. Das Potenzial von Templat ‐ katalysierten Reaktionen für die Wirkstoffforschung und als ein alternativer Wirkmechanismus von Medikamenten wird diskutiert. [ABSTRACT FROM AUTHOR]

Efficient delivery of biomacromolecules (e.g., proteins, nucleic acids) into cell cytosol remains a critical challenge for the development of macromolecular therapeutics or diagnostics. To date, most common approaches to assess cytosolic delivery rely on fluorescent labeling of macromolecules with an 'always on' reporter and subcellular imaging of endolysosomal escape by confocal microscopy. This strategy is limited by poor signal-to-noise ratio and only offers low throughput, qualitative information. Herein we describe a quantitative redox-activatable sensor (qRAS) for the real-time monitoring of cytosolic delivery of macromolecules. qRAS-labeled macromolecules are silent (off) inside the intact endocytic organelles, but can be turned on by redox activation after endolysosomal disruption and delivery into the cytosol, thereby greatly improving the detection accuracy. In addition to confocal microscopy, this quantitative sensing technology allowed for a high-throughput screening of a panel of polymer carriers toward efficient cytosolic delivery of model proteins on a plate reader. The simple and versatile qRAS design offers a useful tool for the investigation of new strategies for endolysosomal escape of biomacromolecules to facilitate the development of macromolecular therapeutics for a variety of disease indications. [ABSTRACT FROM AUTHOR]

Hydrogenases, ferredoxins, and ferredoxin-NADP+ reductases (FNR) are redox proteins that mediate electron metabolism in vivo, and are also potential components for biological H2 production technologies. A high-throughput H2 production assay device (H2PAD) is presented that enables simultaneous evaluation of 96 individual H2 production reactions to identify components that improve performance. Using a CCD camera and image analysis software, H2PAD senses the chemo-optical response of Pd/WO3 thin films to the H2 produced. H2PAD-enabled discovery of hydrogenase and FNR mutants that enhance biological H2 production is reported. From a library of 10 080 randomly mutated Clostridium pasteurianum [FeFe] hydrogenases, we found a mutant with nearly 3-fold higher H2 production specific activity. From a library of 400 semi-randomly mutated Oryza sativa FNR, the top hit enabled a 60 % increase in NADPH-driven H2 production rates. H2PAD can also facilitate elucidation of fundamental biochemical mechanisms within these systems. [ABSTRACT FROM AUTHOR]

Aptamers are a promising class of affinity reagents because they are chemically synthesized, thus making them highly reproducible and distributable as sequence information rather than a physical entity. Although many high-quality aptamers have been previously reported, it is difficult to routinely generate aptamers that possess both high affinity and specificity. One of the reasons is that conventional aptamer selection can only be performed either for affinity (positive selection) or for specificity (negative selection), but not both simultaneously. In this work, we harness the capacity of fluorescence activated cell sorting (FACS) for multicolor sorting to simultaneously screen for affinity and specificity at a throughput of 107 aptamers per hour. As a proof of principle, we generated DNA aptamers that exhibit picomolar to low nanomolar affinity in human serum for three diverse proteins, and show that these aptamers are capable of outperforming high-quality monoclonal antibodies in a standard ELISA detection assay. [ABSTRACT FROM AUTHOR]

Poly(ADP-ribose) polymerases (PARPs) are key enzymes in a variety of cellular processes. Most small-molecule PARP inhibitors developed to date have been against PARP1, and suffer from poor selectivity. PARP14 has recently emerged as a potential therapeutic target, but its inhibitor development has trailed behind. Herein, we describe a small molecule microarray-based strategy for high-throughput synthesis, screening of >1000 potential bidentate inhibitors of PARPs, and the successful discovery of a potent PARP14 inhibitor H10 with >20-fold selectivity over PARP1. Co-crystallization of the PARP14/ H10 complex indicated H10 bound to both the nicotinamide and the adenine subsites. Further structure-activity relationship studies identified important binding elements in the adenine subsite. In tumor cells, H10 was able to chemically knockdown endogenous PARP14 activities. [ABSTRACT FROM AUTHOR]

Die Anwendung von Tryptophan ‐ Halogenasen für die enzymatische Halogenierung hat noch gravierende Limitierungen. Diese könnten mittels gerichteter Evolution überwunden werden, jedoch bedarf die Durchmusterung großer Mutantenbibliotheken eines robusten Hochdurchsatz ‐ fähigen Testsystems. Im Hinblick darauf wurde eine Suzuki ‐ Miyaura ‐ Kreuzkupplung als quantitativer Halogenase ‐ Assay auf Basis der Bildung eines fluoreszierenden Aryltryptophans entwickelt. Die Methode wurde für die Anwendung im E. ‐ coli ‐ Lysat ohne intermediäre Aufarbeitung optimiert und ermöglicht es, halogenierte Tryptophanderivate mit hoher Spezifität in der Mikrotiterplatte quantitativ nachzuweisen. Unter Verwendung dieses Verfahrens wurde eine thermostabile Tryptophan ‐ 6 ‐ Halogenase gefunden, indem mittels fehlerbehafteter PCR eine Mutantenbibliothek erzeugt wurde, die mithilfe der fluorogenen Kreuzkupplung auf erhöhte Thermostabilität durchmustert wurde. Diese Methode lieferte eine verbesserte Enzymvariante mit deutlich gesteigerter Stabilität sowie 2.5 ‐ fach verbesserter Aktivität. Nadel im Heuhaufen: Die Synthese eines fluoreszierenden Aryltryptohans mittels Suzuki ‐ Miyaura ‐ Kupplung (siehe Schema) ermöglicht die spezifische Detektion von C5 ‐ , C6 ‐ und C7 ‐ bromiertem Tryptophan im E. ‐ coli ‐ Lysat. Dies bietet eine Basis für einen Hochdurchsatz ‐ Assay zum Aktivitätsnachweis von Tryptophan ‐ Halogenasen im Protein ‐ Engineering. Die Anwendung dieses Screenings in der zufallsbasierten Mutagenese lieferte eine thermostabile Tryptophan ‐ 6 ‐ Halogenase ‐ Variante. [ABSTRACT FROM AUTHOR]

The re-emergence of influenza raises a global concern that viral pandemics can unpredictably occur. However, effective approaches that can probe the infection risk of influenza viruses for humans are rare. In this work, we develop a glycofoldamer that can rapidly identify the glycan-receptor specificity of influenza viruses in a high-throughput manner. The coupling of glycan receptors that can be recognized by hemagglutinin (a surface protein on the virion capsid of influenza) to a fluorogenic-dye foldamer produces the glycofoldamers with minimal fluorescence in aqueous solution. After interaction with human-infecting virus strains for only five minutes, the fluorescence intensity of the glycofoldamer is remarkably enhanced with a blue-shifted emission peak. The probes have also proven effective for the rapid identification of 1) the human- or bird-infecting properties of influenza viruses in a high-throughput manner and 2) the receptor-specificity switch of a virus strain by mutations. [ABSTRACT FROM AUTHOR]

The reactivity of a representative set of 17 organozinc pivalates with 18 polyfunctional druglike electrophiles (informers) in Negishi cross-coupling reactions was evaluated by high-throughput experimentation protocols. The high-fidelity scaleup of successful reactions in parallel enabled the isolation of sufficient material for biological testing, thus demonstrating the high value of these new solid zinc reagents in a drug-discovery setting and potentially for many other applications in chemistry. Principal component analysis (PCA) clearly defined the independent roles of the zincates and the informers toward druggable-space coverage. [ABSTRACT FROM AUTHOR]

The synthesis of well-defined polymers in a low-volume, combinatorial fashion has long been a goal in polymer chemistry. Here, we report the preparation of a wide range of highly controlled homo and block co-polymers by Enz-RAFT (enzyme-assisted reversible addition-fragmentation chain transfer) polymerization in microtiter plates in the open atmosphere. The addition of 1 μ m glucose oxidase (GOx) to water/solvent mixtures enables polymerization reactions to proceed in extremely low volumes (40 μL) and low radical concentrations. This procedure provides excellent control and high conversions across a range of monomer families and molecular weights, thus avoiding the need to purify for screening applications. This simple technique enables combinatorial polymer synthesis in microtiter plates on the benchtop without the need of highly specialized synthesizers and at much lower volumes than is currently possible by any other technique. [ABSTRACT FROM AUTHOR]

There is great demand for high-throughput methods to characterize ligand affinity. By combining mRNA display with next-generation sequencing, we determined the kinetic on- and off-rates for over twenty thousand ligands without the need for synthesis or purification of individual members. Our results are reproducible and as accurate as those obtained with other methods of affinity measurement. [ABSTRACT FROM AUTHOR]

Gangliosides are important signaling molecules in the cell membrane and are processed by several enzymes. Deficiencies in these enzymes can cause human lysosomal storage diseases. Building an understanding of the pathways of glycosphingolipid catabolism requires methods for the analysis of these enzymatic activities A GM3-derived FRET probe was synthesized chemoenzymatically for the detection and quantitation of a range of ganglioside-degrading enzymes, both in cell lysates and in living cells. This is the first substrate that enables the ratiometric fluorogenic assay of sphingolipid ceramide N-deacylase and endoglycoceramidase and can detect and localize neuraminidase activity in living cells. It is therefore a valuable tool for building a better understanding of membrane-confined enzymology. It also enables the robust and reliable assay of ganglioside-degrading enzymes in a microtiter plate, thus opening the door to screening for novel or engineered biocatalysts or for new inhibitors. [ABSTRACT FROM AUTHOR]

Methods for the rapid and inexpensive discovery of hit compounds are essential for pharmaceutical research and DNA-encoded chemical libraries represent promising tools for this purpose. We here report on the design and synthesis of DAL-100K, a DNA-encoded chemical library containing 103 200 structurally compact compounds. Affinity screening experiments and DNA-sequencing analysis provided ligands with nanomolar affinities to several proteins, including prostate-specific membrane antigen and tankyrase 1. Correlations of sequence counts with binding affinities and potencies of enzyme inhibition were observed and enabled the identification of structural features critical for activity. These results indicate that libraries of this type represent a useful source of small-molecule binders for target proteins of pharmaceutical interest and information on structural features important for binding. [ABSTRACT FROM AUTHOR]

The widespread application of ω-transaminases as biocatalysts for chiral amine synthesis has been hampered by fundamental challenges, including unfavorable equilibrium positions and product inhibition. Herein, an efficient process that allows reactions to proceed in high conversion in the absence of by-product removal using only one equivalent of a diamine donor ( ortho-xylylenediamine) is reported. This operationally simple method is compatible with the most widely used ( R)- and ( S)-selective ω-TAs and is particularly suitable for the conversion of substrates with unfavorable equilibrium positions (e.g., 1-indanone). Significantly, spontaneous polymerization of the isoindole by-product generates colored derivatives, providing a high-throughput screening platform to identify desired ω-TA activity. [ABSTRACT FROM AUTHOR]

Reported herein are two imidazole-based small molecules, termed neurodazine (Nz) and neurodazole (Nzl), which induce neuronal differentiation of pluripotent P19 cells. Their ability to induce neurogenesis of P19 cells is comparable to that of retinoic acid. However, Nz and Nzl were found to be more selective neurogenesis inducers than retinoic acid owing to their unique ability to suppress astrocyte differentiation of P19 cells. Our results also show that Nz and Nzl promote production of physiologically active neurons because P19-cell-derived neurons induced by these substances have functional glutamate responsiveness. The present study suggests that Nz and Nzl could serve as important chemical tools to induce formation of specific populations of neuronal cell types from pluripotent cells. [ABSTRACT FROM AUTHOR]

Biomolekulare Interaktionen, die von zentraler Bedeutung für Pharmakologie und Diagnostik sind, können durch Thermophorese quantifiziert werden. Diese reagiert empfindlich auf bindungsinduzierte Größen-, Ladungs- oder Konformationsänderungen. Etablierte Kapillarmessungen verbrauchen mindestens 0.5 μL pro Probe. Wir reduzierten den Probenverbrauch um den Faktor 50, indem wir 10-nL-Tropfen verwendeten, die mit akustischer Flüssigkeitsübertragung (Labcyte) hergestellt wurden. Die Tropfen wurden in einem Öl-Tensid-Gemisch stabilisiert und mit einem Infrarotlaser lokal erwärmt. Temperaturerhöhung, Marangoni-Fluss und Konzentrationsverteilung wurden mit Fluoreszenzmikroskopie und numerischer Simulation analysiert. In 10-nL-Tropfen quantifizierten wir Affinität, Kooperativität und Pufferabhängigkeit des AMP-Aptamers. Die Miniaturisierung und das 1536-Well-Platten-Format machen die Methode hochdurchsatzfähig und automatisierbar. Dies forciert innovative Anwendungen in der serologischen Diagnostik und markierungsfreien Wirkstoffsuche. [ABSTRACT FROM AUTHOR]

The utilization of information technology in gas chromatography is demonstrated. Multiplexing gas chromatography enhances the overall duty cycle, signal-to-noise ratio and enables achieving extraordinarily high sample throughputs. Parameters such as sequence length, modulation interval and number of repetitive injections in the high-throughput mode determine overall performance of such systems. The portrayed technique opens new perspectives in process analytics by allowing continuous monitoring of chemical processes while analyzing the complete spectrum of analytes. [ABSTRACT FROM AUTHOR]

A simple method is described for the patterned deposition of Teflon on paper to create an integrated platform for parallel organic synthesis and cell-based assays. Solvent-repelling barriers made of Teflon-impregnated paper confine organic solvents to specific zones of the patterned array and allow for 96 parallel flow-through syntheses on paper. The confinement and flow-through mixing significantly improves the peptide yield and simplifies the automation of this synthesis. The synthesis of 100 peptides ranging from 7 to 14 amino acids in length gave over 60 % purity for the majority of the peptides (>95 % yield per coupling/deprotection cycle). The resulting peptide arrays were used in cell-based screening to identify 14 potent bioactive peptides that support the adhesion or proliferation of breast cancer cells in a 3D environment. In the future, this technology could be used for the screening of more complex phenotypic responses, such as cell migration or differentiation. [ABSTRACT FROM AUTHOR]

We report an aptamer discovery technology that reproducibly yields higher affinity aptamers in fewer rounds compared to conventional selection. Our method (termed particle display) transforms libraries of solution-phase aptamers into 'aptamer particles', each displaying many copies of a single sequence on its surface. We then use fluorescence-activated cell sorting (FACS) to individually measure the relative affinities of >108 aptamer particles and sort them in a high-throughput manner. Through mathematical analysis, we identified experimental parameters that enable optimal screening, and demonstrate enrichment performance that exceeds the theoretical maximum achievable with conventional selection by many orders of magnitude. We used particle display to obtain high-affinity DNA aptamers for four different protein targets in three rounds, including proteins for which previous DNA aptamer selection efforts have been unsuccessful. We believe particle display offers an extraordinarily efficient mechanism for generating high-quality aptamers in a rapid and economic manner, towards accelerated exploration of the human proteome. [ABSTRACT FROM AUTHOR]

Die Stabilisierung von Protein ‐ Protein ‐ Interaktionen durch kleine Moleküle ist ein vergleichsweise neuer Ansatz, für den bis dato nur wenige Beispiele erbracht wurden. Hier beschreiben wir die Identifizierung von IBE ‐ 667, einem kleinen Molekül, dass die Affinität von LFA ‐ 1 für ICAM ‐ 1 erhöht, sowie die mittels Röntgencokristallisation erhaltene Struktur des IBE ‐ 667/LFA ‐ 1 ‐ Komplexes. Das Design von IBE ‐ 667 basierte auf der Struktur ‐ Aktivitäts ‐ Beziehung, welche aus einem vorherigen On ‐ Bead ‐ Screen von markierten kombinatorischen One ‐ Bead ‐ One ‐ Compound(OBOC) ‐ Substanzbibliotheken durch konfokales Nanoscanning und Bead Picking (CONA) abgeleitet wurde. Mithilfe von zellulären Analysen an aktivierten Immunzellen konnte gezeigt werden, dass IBE ‐ 667 die Bindung von LFA ‐ 1 an ICAM ‐ 1 verstärkt. Aktivieren statt inhibieren: Die Wechselwirkung zwischen dem Antigen LFA ‐ 1 und dem Adhäsionsmolekül ICAM ‐ 1 auf aktivierten T ‐ Zellen ist ein entscheidender Schritt bei der Immunantwort. Ein niedermolekularer Aktivator des Antigens LFA ‐ 1 wurde durch konfokales On ‐ Bead ‐ Screening identifiziert. Kristallstudien belegen, dass der Aktivator an die I ‐ Domäne von LFA ‐ 1 bindet und dessen Affinität zu ICAM ‐ 1 verstärkt. [ABSTRACT FROM AUTHOR]

Terminal-selective cytochrome P450pyr has been successfully engineered through directed evolution for the subterminal hydroxylation of alkanes with excellent regio- and enantioselectivity. A sensitive colorimetric high-throughput screening (HTS) assay was developed for the measurement of both the regioselectivity and the enantioselectivity of a hydroxylation reaction. By using the HTS assay and iterative saturation mutagenesis, sextuple-mutant P450pyrSM1 was created for the hydroxylation of n-octane ( 1) to give ( S)-2-octanol ( 2) with 98 % ee and >99 % subterminal selectivity. The engineered P450 is the first enzyme for this type of highly selective alkane hydroxylation, being useful for the CH activation and functionalization of alkanes and the preparation of enantiopure alcohols. Molecular modeling provided structure-based understanding of the fully altered regioselectivity and the excellent enantioselectivity. Another sextuple-mutant P450pyrSM2 catalyzed the hydroxylation of propylbenzene ( 3) to afford ( S)-1-phenyl-2-propanol ( 4) with 95 % ee and 98 % subterminal selectivity. [ABSTRACT FROM AUTHOR]

Increasing evidence shows that activated mesenchymal migration is a key process of the metastatic cascade. Cancer cells usually gain such migratory capability through an epithelial-to-mesenchymal transition. Herein we present a high-throughput microfluidic device with 3120 microchambers to specifically monitor mesenchymal migration. Through imaging of the whole chip and statistical analysis, we can evaluate the two key factors of velocity and percentage related to cell migratory capacity at different cell densities in culture. We also used the device to screen antimetastatic drugs for their inhibition of mesenchymal migration and prevention of metastatic malignancy. This device will provide an excellent platform for biologists to gain a better understanding of cancer metastasis. [ABSTRACT FROM AUTHOR]

Many compound screening collections are populated by members that possess a low degree of structural complexity. In an effort to generate compounds that are both complex and diverse, we have developed a strategy that uses natural products as a starting point for complex molecule synthesis. Herein we apply this complexity-to-diversity approach to abietic acid, an abundant natural product used commercially in paints, varnishes, and lacquers. From abietic acid we synthesize a collection of complex (as assessed by fraction of sp3-hybridized carbons and number of stereogenic centers) and diverse (as assessed by Tanimoto analysis) small molecules. The 84 compounds constructed herein, and those created through similar efforts, should find utility in a variety of biological screens. [ABSTRACT FROM AUTHOR]

Adenosindeaminase (ADA), ein Enzym, das am Purinmetabolismus beteiligt ist, setzt ein fluoreszierendes Adenosin ‐ Analogon (thA) zu einem Inosin ‐ Analogon (thI) mit anderen spektralen Eigenschaften um, sodass die Enzym ‐ katalysierte Reaktion und deren Inhibierung in Echtzeit verfolgt werden können. Diese empfindliche, fluoreszenzspektroskopisch verfolgbare Transformation wurde zur Analyse von ADA ‐ Inhibitoren im Hochdurchsatz ‐ Verfahren eingesetzt. [ABSTRACT FROM AUTHOR]

Entspannender Ansatz: Anhand des NMR ‐ Relaxationsverhaltens von absorbiertem Lösungsmittel wurden die Oberflächen verschiedener mikroporöser Metall ‐ organischer Gerüste und Zeolithe bestimmt. Die NMR ‐ Technik lässt sich leicht automatisieren und kann die Charakterisierung mikroporöser Materialien beschleunigen, indem sie nichtporöse Strukturen oder Gerüste mit kleinen Oberflächen identifiziert. [ABSTRACT FROM AUTHOR]

Eine Immunassay ‐ basierte Methode dient zum Screening zahlreicher Kombinationen von Dipolen und Dipolarophilen bezüglich ihrer Fähigkeit, chemoselektive und biokompatible [3+2] ‐ Cycloadditionen einzugehen. Dieser Ansatz erfüllt die meisten Bedingungen des Klick ‐ Konzepts und führte zur Entdeckung einer Kupfer ‐ katalysierten Reaktion, die Pyrazole aus Sydnonen und Alkinreagentien aufbaut. [ABSTRACT FROM AUTHOR]

G ‐ Quadruplexe, leuchtet! Ein schnelles Hochdurchsatz ‐ Screening zur Entdeckung von neuen G ‐ Quadruplex ‐ bildenden Oligonucleotiden (GFOs) wurde entwickelt. Durch die Verwendung eines fluoreszierenden G ‐ Quadruplex ‐ Liganden und eines DNA ‐ Mikroarrays wurden 1998 neue mögliche GFOs in CpG ‐ Inseln gefunden (CpG=Cytosin ‐ Phosphat ‐ Guanosin). [ABSTRACT FROM AUTHOR]

Ein Substrat für ein colorimetrisches Screening der Cyclisierungsaktivität von Terpensynthasen wurde entwickelt, um das Enzym ‐ Engineering zu erleichtern. Gerichtete Evolution führte zu einer Sesquiterpen ‐ Synthase mit verbesserter Thermostabilität unter Erhaltung der anderen Eigenschaften. Mithilfe der Screening ‐ Methode konnten für eine andere Terpen ‐ Synthase die Expressionsbedingungen und die Stabilisierung im Lysat schnell optimiert werden. PPO=Diphosphat. [ABSTRACT FROM AUTHOR]

Ein einfaches Testsystem auf der Basis von Graphenoxid (GO) ermöglicht das Screening selektiver Inhibitoren einer Helicase des Hepatitis ‐ C ‐ Virus (HCV) zusammen mit Inhibitoren eines SARS ‐ Coronavirus (SARS ‐ CoV) (siehe Schema). In einem einzelnen Screen wurden fünf hochselektive Inhibitoren der HCV ‐ Helicase gefunden, die ortholog zur SARS ‐ CoV ‐ Helicase sind. Einige dieser Treffer wurden mit dem gleichen GO ‐ Assay validiert. [ABSTRACT FROM AUTHOR]