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Lipoxygenase (LOX) activity provides oxidative lipid metabolites, which are involved in inflammatory disorders and tumorigenesis. Activity‐based probes to detect the activity of LOX enzymes in their cellular context provide opportunities to explore LOX biology and LOX inhibition. Here, we developed Labelox B as a potent covalent LOX inhibitor for one‐step activity‐based labeling of proteins with LOX activity. Labelox B was used to establish an ELISA‐based assay for affinity capture and antibody‐based detection of specific LOX isoenzymes. Moreover, Labelox B enabled efficient activity‐based labeling of endogenous LOXs in living cells. LOX proved to localize in the nucleus, which was rationalized by identification of a functional bromodomain‐like consensus motif in 15‐LOX‐1. This indicates that 15‐LOX‐1 is not only involved in oxidative lipid metabolism, but also in chromatin binding, which suggests a potential role in chromatin modifications., Activity‐based labeling of lipoxygenase (LOX) activity using the novel one‐step labeling probe Labelox B enables detection of isoenzyme inhibitory selectivity in an ELISA‐based assay and visualization of the localization of LOX activity in living cells. The observed nuclear localization was rationalized by identification of a bromodomain‐like region in the 15‐LOX‐1 isoenzyme.

Macrophage migration inhibitory factor (MIF) is involved in protein‐protein interactions that play key roles in inflammation and cancer. Current strategies to develop small molecule modulators of MIF functions are mainly restricted to the MIF tautomerase active site. Here, we use this site to develop proteolysis targeting chimera (PROTAC) in order to eliminate MIF from its protein‐protein interaction network. We report the first potent MIF‐directed PROTAC, denoted MD13, which induced almost complete MIF degradation at low micromolar concentrations with a DC50 around 100 nM in A549 cells. MD13 suppresses the proliferation of A549 cells, which can be explained by deactivation of the MAPK pathway and subsequent induction of cell cycle arrest at the G2/M phase. MD13 also exhibits antiproliferative effect in a 3D tumor spheroid model. In conclusion, we describe the first MIF‐directed PROTAC (MD13) as a research tool, which also demonstrates the potential of PROTACs in cancer therapy., Targeting macrophage migration inhibitory factor (MIF) is challenging, as it is involved in diseases via protein‐protein interactions. Here, the first potent MIF‐directed PROTAC MD13 is reported, which eliminates MIF from its protein‐protein interaction network by inducing MIF degradation. MD13 suppresses the proliferation of A549 cells by reducing MIF protein levels and MIF‐related signaling, demonstrating the potential of PROTACs in cancer therapy.

Genotoxic agents are widely used anti-cancer therapies because of their ability to interfere with highly proliferative cells. An important outcome of these interventions is the induction of a state of permanent arrest also known as cellular senescence. However, senescent cancer cells are characterized by genomic instability and are at risk of escaping the growth arrest to eventually facilitate cancer relapse. The tumor necrosis factor related apoptosis inducing ligand (TRAIL) signals extrinsic apoptosis via Death Receptors (DR) 4 and 5, while Decoy Receptors (DcR) 1 and 2, and Osteoprotegerin (OPG) are homologous to death receptors but incapable of transducing an apoptotic signal. The use of recombinant TRAIL as an anti-cancer strategy in combination with chemotherapy is currently in development, and a major question remains whether senescent cancer cells respond to TRAIL. Here, we show variable sensitivity of cancer cells to TRAIL after senescence induction, and upregulation of both pro-apoptotic and anti-apoptotic receptors in therapy-induced senescent cancer cells. A DR5-selective TRAIL variant (DHER), unable to bind to DcR1 or OPG, was more effective in inducing apoptosis of senescent cancer cells compared to wild-type TRAIL. Importantly, no apoptosis induction was observed in non-cancerous cells, even at the highest concentrations tested. Our results suggest that targeting DR5 can serve as a novel therapeutic strategy for the elimination of therapy-induced senescent cancer cells.

Macrophage migration inhibitory factor (MIF) and its homolog MIF2 (also known as D-dopachrome tautomerase or DDT) play key roles in cell growth and immune responses. MIF and MIF2 expression is dysregulated in cancers and neurodegenerative diseases. Accurate and convenient detection of MIF and MIF2 will facilitate research on their roles in cancer and other diseases. Herein, we report the development and application of a 4-iodopyrimidine based probe 8 for the selective labeling of MIF and MIF2. Probe 8 incorporates a fluorophore that allows in situ imaging of these two proteins. This enabled visualization of the translocation of MIF2 from the cytoplasm to the nucleus upon methylnitronitrosoguanidine stimulation of HeLa cells. This observation, combined with literature on nuclease activity for MIF, enabled the identification of nuclease activity for MIF2 on human genomic DNA.

Non-small-cell lung carcinoma (NSCLC) is one of the most common forms of lung cancer, and a leading cause of cancer death among human beings. There is an urgent demand for novel therapeutics for the treatment of NSCLC to enhance the efficacy of the currently applied Tyrosine kinase inhibitors (TKIs) therapy and to overcome therapy-resistance. Here, we report a novel small-molecule inhibitor that simultaneously targets histone deacetylase (HDAC) and macrophage migration inhibitory factor (MIF). The HDAC/MIF dual inhibitor proved to be toxic for EGFR mutated (H1650, TKI-resistant) or knock out (A549 EGFR-/-) NSCLC cell lines. Further experiments showed that HDAC inhibition inhibits cell survival and proliferation, while MIF inhibition downregulates pAKT or AKT expression level, which both interfere with cell survival. Furthermore, the combination treatment of TKI and HDAC/MIF dual inhibitor showed that the dual inhibitor enhanced TKI inhibitory efficacy, highlighting the advantages of HDAC/MIF dual inhibitor for more effective treatment of NSCLC.

Rieske non-heme iron oxygenases (ROs) are primarily known for their ability to catalyze the stereoselective formation of vicinal cis-diols in a single step, endowing valuable products for pharmaceutical and chemical applications. In addition, ROs can catalyze several other oxidation reactions with high regio- and stereoselectivity and typically broad substrate scope. Owing to their dependence on multicomponent electron transfer, the majority of synthetic applications of ROs relies on recombinant whole-cell catalysts. In this context, important properties of the multicomponent system that determine the catalytic efficiency, including electron transfer via redox partner proteins, stability and uncoupling, have been investigated to a lesser extent in recent years. Here, we show for one of the most prominent ROs, the cumene dioxygenase from Pseudomonas fluorescens IP01 (CDO) that by developing and optimizing an efficient in vitro system, high catalytic activities can be achieved. In addition, we highlight that an efficient and continuous supplementation of electrons to the oxygenase is required to sustain their catalytic activity, while uncoupling can be a major limitation in CDO efficiency and stability.

The recent increase of interest in photocatalysis spread to biocatalysis and triggered a rush for the development of light-dependent enzyme-mediated or enzyme-coupled processes. After several years of intense research on photobiocatalysis, it is time to evaluate the state of the field in a structured manner. In this Perspective, we suggest to group photobiocatalysis into distinct disciplines and provide principal guidelines and standards for the reporting of photobiocatalytic research results as well as advice on performing photobiocatalytic reactions. Overall, we assess that the field contributes to the diversity of biocatalytic reactions while offering the selectivity of enzymes to photocatalysis. We foresee that the ongoing excitement for light-dependent enzymatic processes will lead to the discovery of novel photobiocatalytic mechanisms to complement biocatalysis with new bond-forming reactions and will provide additional innovative strategies to utilize light as a possible benign energy source.

Oxygen-directed methylation is a ubiquitous tailoring reaction in natural product pathways catalysed by 15 O-methyltransferases (OMTs). Promiscuous OMT biocatalysts are thus a valuable asset in the toolkit for 16 sustainable synthesis and optimization of known bioactive scaffolds for drug development. Here, we 17 characterized two bacterial OMTs from Desulforomonas acetoxidans and Streptomyces avermitilis in 18 terms of their enzymatic properties and substrate scope and determined their crystal structures. Both 19 OMTs methylated a wide range of catechol-like substrates, including flavonoids, coumarins, 20 hydroxybenzoic acids and their respective aldehydes, an anthraquinone and an indole. One enzyme also 21 accepted a steroid. The product range included pharmaceutically relevant compounds such as 22 (iso)fraxidin, iso(scopoletin), chrysoeriol, alizarin 1-methyl ether and 2-methoxyestradiol. Interestingly, 23 certain non-catechol flavonoids and hydroxybenzoic acids were also methylated. This study expands the 24 knowledge on substrate preference and structural diversity of bacterial catechol OMTs and paves the way 25 for their use in (combinatorial) pathway engineering.

Various diseases are deeply associated with aberrations in HDAC8 functions. These aberrations can be assigned to either structural functions or catalytic functions of HDAC8. Therefore, development of HDAC8 degradation inducers might be more promising than HDAC8 inhibitors. We employed the proteolysis targeting chimera (PROTAC) strategy to develop a selective and potent HDAC8 degradation inducer CT-4 with single-digit nanomolar DC50 values and over 95% Dmax in both triple-negative breast cancer MDA-MB-231 cells and T-cell leukemia cells. Notably, CT-4 demonstrated potent anti-migration activity and limited anti-proliferative activity in MDA-MB-231 cells. In contrast, CT-4 effectively induced apototic cell death in Jurkat cells, as assessed by a caspase 3/7 activity assay and flow cytometry. Our findings suggest that the development of HDAC8 degradation inducers holds great potential for the treatment of HDAC8-related diseases.

Idiopathic pulmonary fibrosis is a progressive lung disease that causes scarring and loss of lung function. Macrophages play a key role in fibrosis, but their responses to altered morphological and mechanical properties of the extracellular matrix in fibrosis is relatively unexplored. Our previous work showed functional changes in murine fetal liver-derived alveolar macrophages on fibrous or globular collagen morphologies. In this study, we applied differential proteomics to further investigate molecular mechanisms underlying the observed functional changes. Macrophages cultured on uncoated, fibrous, or globular collagen-coated plastic were analyzed by liquid chromatography-mass spectrometry. The presence of collagen affected expression of 77 proteins, while 142 were differentially expressed between macrophages grown on fibrous or globular collagen. Biological process and pathway enrichment analysis revealed that culturing on any type of collagen induced higher expression of enzymes involved in glycolysis. However, this did not lead to a higher rate of glycolysis, probably because of a concomitant decrease in activity of these enzymes. Our data suggest that macrophages sense collagen morphologies and can respond with changes in expression and activity of metabolism-related proteins. These findings suggest intimate interactions between macrophages and their surroundings that may be important in repair or fibrosis of lung tissue.

Biocatalysis has established itself as a vital tool in the chemical and pharmaceutical industries. However, challenged by the sheer number of chemical reactions available to small-molecule catalysts, the quest to harness biocatalysts for novel transformations has become a veritable hotbed of research. Herein, we discuss previous and actual developments in this area ranging from enzyme discovery for novel function to directed evolution, computational enzyme design, and the construction of artificial (metallo)enzymes, as well as exciting new developments in the field of photobiocatalysis for tapping into abiological reaction chemistries in enzyme catalysis.

The invention relates to the field of medicinal chemistry and pharmacology, specifically to novel inhibitors of histone deacetylase (HDAC), and to compositions, methods of making, and using them, for instance in the treatment of chronic obstructive pulmonary disease (COPD). Provided is a compound of the formula A or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein A is H or an in vivo hydrolysable group, and L is a linker group.

Oxygenases and peroxygenases incorporate oxygen from the environmentally friendly oxidants O2 and H2O2, respectively, into their organic substrates in a stereo- and regioselective manner. While the majority of oxygenases and peroxygenases apply a cofactor, a few peculiar enzymes have been discovered, which are able to catalyse oxygenation and peroxygenation reactions without making use of a cofactor. However, the cofactor-independent oxygenases and peroxygenases known up to date are unsatisfactorily explored. In the work reported in this thesis, we investigated one tautomerase superfamily member with oxygenase activity, RhCC from Rhodococcus jostii, and one tautomerase superfamily member with promiscuous peroxygenase activity, 4 oxalocrotonate tautomerase (4 OT) from Pseudomonas putida. By screening selected enzymes homologous to RhCC, we discovered Rhop3 from Rhodococcus opacus with three-times enhanced oxygenase activity. However, the oxygenase activity of Rhop3 appears to be dependent on metal ions, disqualifying it as a cofactor-independent system. Furthermore, we present an overview of enzymes with peroxygenase activity available in nature and discuss enzyme engineering strategies, which have been applied to improve their peroxygenase activity. Applying directed evolution, we enhanced the promiscuous peroxygenase activity of a tandem-fused enzyme variant of 4 OT by about 150-times compared to 4-OT wild-type and realized the synthesis of α,β-epoxy-aldehydes in gram-scale with high enantioselectivity. Following, we applied this improved artificial peroxygenase in a one-pot multi-enzymatic cascade to synthesize enantioenriched epoxides and triols from biomass-derived starting materials.

Biocatalysis might effectively address the challenges associated with green and sustainable chemical synthesis. In the work described in this thesis, native and newly engineered enzymes were used for the step-economic and more sustainable biocatalytic synthesis of valuable precursors to pharmaceuticals and nutraceuticals, including enantioenriched epoxides, vicinal triols and amino acids. First, a non-natural aldolase and a non-natural peroxygenase, both evolved in the laboratory from the promiscuous enzyme 4-oxalocrotonate tautomerase, were combined to develop a one-pot two-step enzymatic cascade for the preparation of α,β-epoxy-aldehydes. The addition of a specifically selected natural epoxide hydrolase to the same pot yielded a short pathway to enantioenriched aryl glycerols from simple biomass-derived starting materials. Second, the laboratory evolution of the enzyme ethylenediamine-N,N'-disuccinic acid (EDDS) lyase generated an enzyme variant that showed a large improvement in activity, enabling the effective and enantioselective synthesis of complex L-aspartic acid derivatives, valuable precursors to the artificial dipeptide sweeteners neotame and advantame. These results present new opportunities to develop practical multienzymatic processes for the more sustainable and step-economic synthesis of an important class of food additives. Finally, a study into the structure, mechanism and biocatalytic potential of a newly discovered monooxygenase, named Rhop3, is presented.

With an ever-increasing amount of (meta)genomic data being deposited in sequence databases, (meta)genome mining for natural product biosynthetic pathways occupies a critical role in the discovery of novel pharmaceutical drugs, crop protection agents and biomaterials. The genes that encode these pathways are often organised into biosynthetic gene clusters (BGCs). In 2015, we defined the Minimum Information about a Biosynthetic Gene cluster (MIBiG): a standardised data format that describes the minimally required information to uniquely characterise a BGC. We simultaneously constructed an accompanying online database of BGCs, which has since been widely used by the community as a reference dataset for BGCs and was expanded to 2021 entries in 2019 (MIBiG 2.0). Here, we describe MIBiG 3.0, a database update comprising large-scale validation and re-annotation of existing entries and 661 new entries. Particular attention was paid to the annotation of compound structures and biological activities, as well as protein domain selectivities. Together, these new features keep the database up-to-date, and will provide new opportunities for the scientific community to use its freely available data, e.g. for the training of new machine learning models to predict sequence-structure-function relationships for diverse natural products. MIBiG 3.0 is accessible online at https://mibig.secondarymetabolites.org/.

Lignin is presently regarded as both highly under-utilized and prom-ising feedstock for diverse applications. Enzymes for aromatic O-de-methylations are of particular interest in this context. In this issue ofChem Catalysis, Michener and co-workers expand the enzyme uni-verse with Rieske-type aryl-O-demethylases acting on lignin-derivedsubstrates.

Class I aldolases catalyze asymmetric aldol addition reactions and have found extensive application in the biocatalytic synthesis of chiral β-hydroxy-carbonyl compounds. However, the usefulness of these powerful enzymes for application in other C-C bond-forming reactions remains thus far unexplored. The redesign of class I aldolases to expand their catalytic repertoire to include non-native carboligation reactions therefore continues to be a major challenge. Here, we report the successful redesign of 2-deoxy-d-ribose-5-phosphate aldolase (DERA) from Escherichia coli, an archetypical class I aldolase, to proficiently catalyze enantioselective Michael additions of nitromethane to α,β-unsaturated aldehydes to yield various pharmaceutically relevant chiral synthons. After 11 rounds of directed evolution, the redesigned DERA enzyme (DERA-MA) carried 12 amino-acid substitutions and had an impressive 190-fold enhancement in catalytic activity compared to the wildtype enzyme. The high catalytic efficiency of DERA-MA for this abiological reaction makes it a proficient "Michaelase" with potential for biocatalytic application. Crystallographic analysis provides a structural context for the evolved activity. Whereas an aldolase acts naturally by activating the enzyme-bound substrate as a nucleophile (enamine-based mechanism), DERA-MA instead acts by activating the enzyme-bound substrate as an electrophile (iminium-based mechanism). This work demonstrates the power of directed evolution to expand the reaction scope of natural aldolases to include asymmetric Michael addition reactions and presents opportunities to explore iminium catalysis with DERA-derived catalysts inspired by developments in the organocatalysis field.

Cyclopropane rings are an important structural motif frequently found in many natural products and pharmaceuticals. Commonly, biocatalytic methodologies for the asymmetric synthesis of cyclopropanes rely on repurposed or artificial heme enzymes. Here, we engineered an unusual cofactor‐independent cyclopropanation enzyme based on a promiscuous tautomerase for the enantioselective synthesis of various cyclopropanes via the nucleophilic addition of diethyl 2‐chloromalonate to α,β‐unsaturated aldehydes. The engineered enzyme promotes formation of the two new carbon‐carbon bonds with excellent stereocontrol over both stereocenters, affording the desired cyclopropanes with high diastereo‐ and enantiopurity (d.r. up to 25:1; e.r. up to 99:1). Our results highlight the usefulness of promiscuous enzymes for expanding the biocatalytic repertoire for non‐natural reactions., Engineering of a promiscuous 4‐oxalocrotonate tautomerase (4‐OT) resulted in an unusual cofactor‐independent cyclopropanation enzyme that promotes the stereoselective addition of diethyl 2‐chloromalonate to substituted cinnamaldehydes to afford various cyclopropanes with high diastereo‐ and enantiopurity.

Following DNA replication, equal amounts of chromatin proteins are distributed over sister chromatids by re-deposition of parental chromatin proteins and deposition of newly synthesized chromatin proteins. Molecular mechanisms balancing the allocation of new and old chromatin proteins remain largely unknown. Here, we studied the genome-wide distribution of new chromatin proteins relative to parental DNA template strands and replication initiation zones using the double-click-seq. Under control conditions, new chromatin proteins were preferentially found on DNA replicated by the lagging strand machinery. Strikingly, replication stress induced by hydroxyurea or curaxin treatment and inhibition of ataxia telangiectasia and Rad3-related protein (ATR) or p53 inactivation inverted the observed chromatin protein deposition bias to the strand replicated by the leading strand polymerase in line with previously reported effects on replication protein A occupancy. We propose that asymmetric deposition of replication protein occupancy. propose asymmetric deposition newly synthesized chromatin proteins onto sister chromatids reflects differences in the processivity of leading and lagging strand synthesis.

Enzymes have several advantages over conventional catalysts for organic synthesis. Over the last two decades, much effort has been made to further extend the scope of biocatalytic reactions available to synthetic chemists, particularly by expanding the repertoire of enzymes for abiological transformations. In this regard, exciting new developments in the area of photobiocatalysis enable now the introduction of non-natural reactivity in enzymes to solve long-standing synthetic challenges. A recently described example from the Hyster group demonstrates in an unprecedented way how the combination of photochemistry with enzyme catalysis empowers the catalytic asymmetric construction of Csp 3–Csp 3 bonds with high chemo- and enantioselectivity.

The acquisition of iron is a crucial mechanism for the survival of pathogenic bacteria such as Pseudomonas aeruginosa in eukaryotic hosts. The key iron chelator in this organism is the siderophore pyoverdine, which was shown to be crucial for iron homeostasis. Pyoverdine is a non-ribosomal peptide with several maturation steps in the cytoplasm and others in the periplasmatic space. A key enzyme for its maturation is the acylase PvdQ. The inhibition of PvdQ stops the maturation of pyoverdine causing a significant imbalance in the iron homeostasis and hence can negatively influence the survival of P. aeruginosa. In this work, we successfully synthesized chromene-derived inhibitory molecules targeting PvdQ in a low micromolar range. In silico modeling as well as kinetic evaluations of the inhibitors suggest a competitive inhibition of the PvdQ function. Further, we evaluated the inhibitor in vivo on P. aeruginosa cells and report a dose-dependent reduction of pyoverdine formation. The compound also showed a protecting effect in a Galleria mellonella infection model.

Peroxygenases selectively incorporate oxygen into organic molecules making use of the environmentally friendly oxidant H2O2 with water being the sole by-product. These biocatalysts can provide ‘green’ routes for the synthesis of enantioenriched epoxides, which are fundamental intermediates in the production of pharmaceuticals. The peroxyzyme 4‑oxalocrotonate tautomerase (4‑OT), catalysing the epoxidation of a variety of α,β-unsaturated aldehydes with H2O2, is outstanding because of its independence from any cost-intensive cofactor. However, its low-level peroxygenase activity and the decrease in the enantiomeric excess of the corresponding α,β-epoxy-aldehydes under preparative-scale conditions is limiting the potential of 4‑OT. Herein we report the directed evolution of a tandem-fused 4‑OT variant, which showed an ~150-fold enhanced peroxygenase activity compared to 4-OT wild type, enabling the synthesis of α,β-epoxy-aldehydes in milligram- and gram-scale with high enantiopurity (up to 98% ee) and excellent conversions. This engineered cofactor-independent peroxyzyme can provide new opportunities for the eco-friendly and practical synthesis of enantioenriched epoxides at large scale.

The implementation of light-driven catalytic processes in biocatalysis opens a golden window of opportunities. We hereby report the merging of photocatalytic C-C bond formation with enzymatic asymmetric reduction for the direct conversion of simple aldehydes and acrylates or unsaturated carboxylic acids into chiral ?-lactones. Tetrabutylammonium decatungstate (TBADT) is employed as the photocatalyst to trigger the hydroacylation of the starting olefins, yielding the corresponding keto esters/acids. Subsequently, an alcohol dehydrogenase converts the intermediate to the chiral alcohol, which undergoes lactonization to the desired ?-lactone. The photochemoenzymatic synthesis of aliphatic and aromatic ?-lactones was thereby achieved with up to >99 % ee and >99 % yield. This synthesis highlights the power of building molecular complexity by merging photocatalysis with biocatalysis to access high-value added chiral compounds from simple, cheap and largely available starting materials.

Pantothenate synthetase from Escherichia coli (PSE. coli) catalyzes the ATP-dependent condensation of (R)-pantoic acid and β-alanine to yield (R)-pantothenic acid (vitamin B5), the biosynthetic precursor to coenzyme A. Herein we show that besides the natural amine substrate β-alanine, the enzyme accepts a wide range of structurally diverse amines including 3-amino-2-fluoropropionic acid, 4-amino-2-hydroxybutyric acid, 4-amino-3-hydroxybutyric acid, and tryptamine for coupling to the native carboxylic acid substrate (R)-pantoic acid to give amide products with up to >99% conversion. The broad amine scope of PSE. coli enabled the efficient synthesis of pharmaceutically-relevant vitamin B5 antimetabolites with excellent isolated yield (up to 89%). This biocatalytic amide synthesis strategy may prove to be useful in the quest for new antimicrobials that target coenzyme A biosynthesis and utilisation., Pantothenate synthetase from Escherichia coli (PSE. coli) has a broad substrate scope, accepting diverse amines in the amidation of (R)-pantoate, enabling the facile synthesis of pharmaceutically relevant vitamin B5 antimetabolites.

The Cover Feature illustrates how dual catalysis between a photocatalyst and an enzyme accomplishes the synthesis of chiral γ-lactones from simple starting materials. In their Research Article, D. Ravelli, S. Schmidt and co-workers explored a decatungstate photocatalyst that, upon near-UV light irradiation, promotes the radical hydroacylation of α,β-unsaturated esters or acids with aldehydes. Subsequently, an alcohol dehydrogenase reduces the intermediate keto esters/acids to the corresponding chiral alcohols, which undergo lactonization to the desired chiral γ-lactones. This study highlights the powerfulness of merging photo- with biocatalysis for building molecular complexity to access high-value added chiral compounds from simple, cheap, and largely available starting materials. More information can be found in the Research Article by D. Ravelli, S. Schmidt and co-workers.

Biaryl sulfonamides are excellent candidates for the azologization approach that yields photoswitchable drugs more active in their metastable cis state, compared to the stable trans state. Here we present the scope and limitations of this strategy for rational design in photopharmacology.

Engineering strategies to improve terpenoids production in Bacillus subtilis mainly focus on 2C-methyl-D-erythritol-4-phosphate (MEP) pathway overexpression. To systematically engineer the chassis strain for higher amorphadiene (precursor of artemisinin) production, a clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) system was established in B. subtilis to facilitate precise and efficient genome editing. Then, this system was employed to engineer three more modules to improve amorphadiene production, including the terpene synthase module, the branch pathway module, and the central metabolic pathway module. Finally, our combination of all of the useful strategies within one strain significantly increased extracellular amorphadiene production from 81 to 116 mg/L after 48 h flask fermentation without medium optimization. For the first time, we attenuated the FPP-derived competing pathway to improve amorphadiene biosynthesis and investigated how the TCA cycle affects amorphadiene production in B. subtilis. Overall, this study provides a universal strategy for further increasing terpenoids' production in B. subtilis by comprehensive and systematic metabolic engineering.

doi:10.1021/jacs.0c11336, Photopharmacology addresses the challenge of drug selectivity and side effects through creation of photoresponsive molecules activated with light with high spatiotemporal precision. This is achieved through incorporation of molecular photoswitches and photocages into the pharmacophore. However, the structural basis for the light-induced modulation of inhibitory potency in general is still missing, which poses a major design challenge for this emerging field of research. Here we solved crystal structures of the glutamate transporter homologue Glt$_{Tk}$ in complex with photoresponsive transport inhibitors���azobenzene derivative of TBOA (both in trans and cis configuration) and with the photocaged compound ONB-hydroxyaspartate. The essential role of glutamate transporters in the functioning of the central nervous system renders them potential therapeutic targets in the treatment of neurodegenerative diseases. The obtained structures provide a clear structural insight into the origins of photocontrol in photopharmacology and lay the foundation for application of photocontrolled ligands to study the transporter dynamics by using time-resolved X-ray crystallography.

The study was designed to determine the potential of collagen hydrolysate produced from the skin of Kacang goat through chymotrypsin hydrolysis to be used as an inhibitor of angiotensin converting enzyme (ACE). This research was conducted in three replications, with the measured parameters include ACE inhibitory potential and collagen hydrolysate fractionation. The results showed that collagen extraction of Kacang goat skin by chymotrypsin hydrolysis yielded 9.74% (dry matter, v/v) collagen, with pH at 6.6. The extracted collagen contained α1, α2, and β collagen chains with molecular weights of 151 kDa, 141 kDa, and 240 kDa, respectively. Furthermore, the collagen hydrolysis produced protein peptides confirmed at molecular weights of 43 to 107 kDa. The hydrolysate fractionation at molecular weights of 5 kDa showed proteins concentrations of 2.33 mg/mL, 3.81 mg/mL, and 3.93 mg/mL, respectively. The hydrolysate fractionation with molecular weight

Chiral dihydrobenzoxazinones and dihydroquinoxalinones serve as essential building blocks for pharmaceuticals and agrochemicals. Here, we report short chemoenzymatic synthesis routes for the facile preparation of these complex heterocycles in an optically pure form. These synthetic routes involve a highly stereoselective hydroamination step catalyzed by ethylenediamine-N,N′-disuccinic acid lyase (EDDS lyase). This enzyme is capable of catalyzing the asymmetric addition of various substituted 2-aminophenols to fumarate to give a broad range of substituted N-(2-hydroxyphenyl)-l-aspartic acids with excellent enantiomeric excess (ee up to >99%). This biocatalytic hydroamination step was combined with an acid-catalyzed esterification–cyclization sequence to convert the enzymatically generated noncanonical amino acids into the desired dihydrobenzoxazinones in good overall yield (up to 63%) and high optical purity (ee up to >99%). By means of a similar one-pot, two-step chemoenzymatic approach, enantioenriched dihydroquinoxalinones (ee up to >99%) were prepared in good overall yield (up to 78%) using water as solvent for both steps. These chemoenzymatic methodologies offer attractive alternative routes to challenging dihydrobenzoxazinones and dihydroquinoxalinones, starting from simple and commercially available achiral building blocks.

Antibiotic crisis – Back to the startA famous quote of Charles Darwin reads: “It is not the strongest of the species that survives, not the most intelligent that survives. It is the one that is the most adaptable to change.” Every living creature is evolving, is adapting to changes of the environment, all the time. With the discovery of antibiotics physicians found, adapted and refined powerful compounds to efficiently kill bacteria. The overuse of antibiotics will eventually render the same useless, as we can observe already right now. Against all logic and warnings antibiotics are still being used carelessly and are expelled to the environment in large quantities. Quorum quenching (QQ) strategies with the acylase PvdQ could be an alternative or addition to conventional antibiotics. The goal of this treatment is to attenuate virulence factors and hinder the formation of biofilms. The clearance of the infection will be performed by the immune system of the host or with the help of antibacterial compounds. Applications of QQ enzymes could be the functionalization of various medical relevant surfaces to mediate anti biofilm properties. We demonstrated in this work that the QQ enzyme PvdQ has indeed the potential to be used against A. baumannii, and P. aeruginosa infections in vitro and in vivo. The potential of QQEs justifies to put more effort in research and refinement of this technique and eventually QQ treatment approaches could offer powerful tools to fight bacterial infections in the future.

Despite the success of drug discovery over the past decades, many potential drug targets still remain intractable for small molecule modulation. The development of proteolysis targeting chimeras (PROTACs) that trigger degradation of the target proteins provides a conceptually novel approach to address drug targets that remained previously elusive. Currently, the main challenge of PROTAC development is the identification of efficient, tissue- and cell-selective PROTAC molecules with good drug-likeness and favorable safety profiles. This review focuses on strategies to enhance the effectiveness and selectivity of PROTACs. We provide a comprehensive summary of recently reported PROTAC design strategies and discuss the advantages and disadvantages of these strategies. Future perspectives for PROTAC design will also be discussed.

A practical, efficient, and selective electrochemicalN-demethylation method of tropane alkaloids to their nortropane derivatives is described. Nortropanes, such as noratropine and norscopolamine, are important intermediates for the semi-synthesis of the medicines ipratropium or oxitropium bromide, respectively. Synthesis was performed in a simple home-made electrochemical batch cell using a porous glassy carbon electrode. The reaction proceeds at room temperature in one step in a mixture of ethanol or methanol and water. The method avoids hazardous oxidizing agents such as H(2)O(2)orm-chloroperbenzoic acid (m-CPBA), toxic solvents such as chloroform, as well as metal-based catalysts. Various key parameters were investigated in electrochemical batch or flow cells, and the optimized conditions were used in batch and flow-cells at gram scale to synthesize noratropine in high yield and purity using a convenient liquid-liquid extraction method without any need for chromatographic purification. Mechanistic studies showed that the electrochemicalN-demethylation proceeds by the formation of an iminium intermediate which is converted by water as the nucleophile. The optimized method was further applied to scopolamine, cocaine, benzatropine, homatropine and tropacocaine, showing that this is a generic way ofN-demethylating tropane alkaloids to synthesize valuable precursors for pharmaceutical products.

The number of genomic aberrations known to be relevant in making therapeutic decisions for non-small cell lung cancer patients has increased in the past decade. Multiple molecular tests are required to reliably establish the presence of these aberrations, which is challenging because available tissue specimens are generally small. To optimize diagnostic testing, we developed a transcriptome-based next-generation sequencing (NGS) assay based on single primed enrichment technology. We interrogated 11 cell lines, two patient-derived frozen biopsies, nine pleural effusion, and 29 formalin-fixed paraffin-embedded (FFPE) samples. All clinical samples were selected based on previously identified mutations at the DNA level in EGFR, KRAS, ALK, PIK3CA, BRAF, AKT1, MET, NRAS, or ROS1 at the DNA level, or fusion genes at the chromosome level, or by aberrant protein expression of ALK, ROS1, RET, and NTRK1. A successful analysis is dependent on the number of unique reads and the RNA quality, as indicated by the DV200 value. In 27 out of 51 samples with &gt, 50 K unique reads and a DV200 &gt, 30, all 19 single nucleotide variants (SNVs)/small insertions and deletions (INDELs), three MET exon 14 skipping events, and 13 fusion gene transcripts were detected at the RNA level, giving a test accuracy of 100%. In summary, this lung-cancer-specific all-in-one transcriptome-based assay for the simultaneous detection of mutations and fusion genes is highly sensitive.

Squalene synthase (SQS) catalyzes the conversion of two farnesyl pyrophosphates to squalene, an important intermediate in between isoprene and valuable triterpenoids. In this study, we have constructed a novel biosynthesis pathway for squalene in Bacillus subtilis and performed metabolic engineering aiming at facilitating further exploitation and production of squalene-derived triterpenoids. Therefore, systematic studies and analysis were performed including selection of multiple SQS candidates from various organisms, comparison of expression vectors, optimization of cultivation temperatures, and examination of rate-limiting factors within the synthetic pathway. We were, for the first time, able to obtain squalene synthesis in B. subtilis. Furthermore, we achieved a 29-fold increase of squalene yield (0.26-7.5 mg/L) by expressing SQS from Bacillus megaterium and eliminating bottlenecks within the upstream methylerythritol-phosphate pathway. Moreover, our findings showed that also ispA could positively affect the production of squalene.

The creation of microbial cell factories for sustainable production of natural products is important for medical and industrial applications. This requires stable expression of biosynthetic pathways in a host organism with favorable fermentation properties such as Bacillus subtilis. The aim of this study is to construct B. subtilis strains that produce valuable terpenoid compounds by overexpressing the innate methylerythritol phosphate (MEP) pathway. A synthetic operon allowing the concerted and regulated expression of multiple genes was developed. Up to 8 genes have been combined in this operon and a stably inherited plasmid-based vector was constructed resulting in a high production of C30 carotenoids. For this, two vectors were examined, one with rolling circle replication and another with theta replication. Theta-replication constructs were clearly superior in structural and segregational stability compared to rolling circle constructs. A strain overexpressing all eight genes of the MEP pathway on a theta-replicating plasmid clearly produced the highest level of carotenoids. The level of transcription for each gene in the operon was similar as RT-qPCR analysis indicated. Hence, that corresponding strain can be used as a stable cell factory for production of terpenoids. This is the first report of merging and stably expressing this large-size operon (eight genes) from a plasmid-based system in B. subtilis enabling high C30 carotenoid production.

The efficient engineering of iminium biocatalysis has drawn considerable attention, with many applications in pharmaceutical synthesis. Here, we report a tailor-made iminium-activated colorimetric "turn-on" probe, specifically designed as a prescreening tool to facilitate engineering of iminium biocatalysis. Upon complexation of the probe with the catalytic Pro-1 residue of the model enzyme 4-oxalocrotonate tautomerase (4-OT), a brightly colored merocyanine-dye-type structure is formed. 4-OT mutants that formed this brightly colored species upon incubation with the probe proved to have a substantial activity for the iminium-based Michael-type addition of nitromethane to cinnamaldehyde, whereas mutants that showed no staining by the probe exhibited no or very low-level "Michaelase" activity. This system was exploited in a solid-phase prescreening assay termed as activated iminium colony staining (AICS) to enrich libraries for active mutants. AICS prescreening reduced the screening effort up to 20-fold. After two rounds of directed evolution, two artificial Michaelases were identified with up to 39-fold improvement in the activity for the addition of nitromethane to cinnamaldehyde, yielding the target γ-nitroaldehyde product with excellent isolated yield (up to 95%) and enantiopurity (up to >99% ee). The colorimetric activation of the turn-on probe could be extended to the class I aldolase 2-deoxy-d-ribose 5-phosphate aldolase, implicating a broader application of AICS in engineering iminium biocatalysis.

Flavoenzymes are oxidoreductases that catalyze an extensive range of different types of reactions. An advanced and powerful approach to achieving transformations that are normally outside the realm of flavoenzymes is the synergistic combination of photocatalysis and biocatalysis. Here we report the identification of a promiscuous flavin-dependent nitroreductase, BaNTR1, that is able to promote enantioselective photobiocatalytic reductions of a broad range of structurally diverse ketones to yield the corresponding alcohols with high conversion (up to >99%) and outstanding enantiopurity (up to >99:1 e.r). Noteworthy, BaNTR1 was able to promote the photoenzymatic reduction of various α,ß-unsaturated ketones to give the corresponding optically pure alcohols without reducing the C=C or C≡C bond, illustrating its remarkably high chemoselectivity. Our results highlight the usefulness of photocatalysis for expanding the catalytic repertoire of nitroreductases to include highly enantio- and chemoselective reductions of non-native ketone substrates to produce optically pure alcohols. This includes difficult to prepare allyl alcohols that are not accessible via photoenzymatic conversions using ene-reductases.

Gene duplication and fusion are among the primary natural processes that generate new proteins from simpler ancestors. Here we adopted this strategy to evolve a promiscuous homohexameric 4-oxalocrotonate tautomerase (4-OT) into an efficient biocatalyst for enantioselective Michael reactions. We first designed a tandem-fused 4-OT to allow independent sequence diversification of adjacent subunits by directed evolution. This fused 4-OT was then subjected to eleven rounds of directed evolution to give variant 4-OT(F11), which showed an up to 320-fold enhanced activity for the Michael addition of nitromethane to cinnamaldehydes. Crystallographic analysis revealed that 4-OT(F11) has an unusual asymmetric trimeric architecture in which one of the monomers is flipped 180 degrees relative to the others. This gene duplication and fusion strategy to break structural symmetry is likely to become an indispensable asset of the enzyme engineering toolbox, finding wide use in engineering oligomeric proteins.

The opportunistic pathogen Pseudomonas aeruginosa employs quorum sensing to govern the production of many virulence factors. Interference with quorum sensing signaling has therefore been put forward as an attractive approach to disarm this pathogen. Here, we analyzed the quorum quenching properties of natural and engineered (2-alkyl-)3-hydroxy-4(1H)-quinolone 2,4-dioxygenases (HQDs) that inactivate the P. aeruginosa signal molecule PQS (Pseudomonas quinolone signal; 2-heptyl-3-hydroxy-4(1H)-quinolone). When added exogenously to P. aeruginosa cultures, all HQDs tested significantly reduced the levels of PQS and other alkylquinolone-type secondary metabolites deriving from the biosynthetic pathway, such as the respiratory inhibitor 2-heptyl-4-hydroxyquinoline N-oxide. HQDs from Nocardia farcinica and Streptomyces bingchenggensis, which combine low KM values for PQS with thermal stability and resilience in the presence of P. aeruginosa exoproducts, respectively, attenuated production of the virulence factors pyocyanin and pyoverdine. A delay in mortality was observed when Galleria mellonella larvae were infected with P. aeruginosa suspensions treated with the S. bingchenggensis HQD or with inhibitors of alkylquinolone biosynthesis. Our data indicate that quenching of PQS signaling has potential as an anti-virulence strategy; however, an efficient anti-virulence therapy against P. aeruginosa likely requires a combination of agents addressing multiple targets.

The application of biocatalysis in conquering challenging synthesis requires the constant input of new enzymes. This thesis used directed evolution guided by catalytic promiscuity to develop a series of enzymes to perform iminium catalysis, a powerful catalysis mode originating from organocatalysis. Three such iminium biocatalysts have been created: a cofactor-independent peroxygenase achieving enantiocomplementary epoxidation reactions, and two efficient Michaelases, one based on a class I aldolase DERA and the other one based on an artificially tandem-fused tautomerase enzyme. These developments illustrate the power of combining chemomimetic enzyme design and directed evolution to create synthetically useful new-to-natural enzymes.

Artemisinin, the most famous anti‐malaria drug initially extracted from Artemisia annua L., also exhibits anti‐tumor properties in vivo and in vitro. To improve its solubility and bioavaila‐bility, multiple derivatives have been synthesized. However, to reveal the anti‐tumor mechanism and improve the efficacy of these artemisinin‐type drugs, studies have been conducted in recent years. In this review, we first provide an overview of the effect of artemisinin‐type drugs on the regulated cell death pathways, which may uncover novel therapeutic approaches. Then, to over‐come the shortcomings of artemisinin‐type drugs, we summarize the recent advances in two differ‐ent therapeutic approaches, namely the combination therapy with biologics influencing regulated cell death, and the use of nanocarriers as drug delivery systems. For the former approach, we dis‐cuss the superiority of combination treatments compared to monotherapy in tumor cells based on their effects on regulated cell death. For the latter approach, we give a systematic overview of nanocarrier design principles used to deliver artemisinin‐type drugs, including inorganic‐based na‐noparticles, liposomes, micelles, polymer‐based nanoparticles, carbon‐based nanoparticles, nanostructured lipid carriers and niosomes. Both approaches have yielded promising findings in vitro and in vivo, providing a strong scientific basis for further study and upcoming clinical trials.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising candidate for cancer treatment due to its tumor specificity. TRAIL binds with death receptor (DR) 4 or 5 to initiate apoptotic signal transduction. In this thesis, we first introduced the protein engineering strategies of TNF ligands to target specific receptors and we reviewed the changes in the biological activity of some key ligands. In the experimental part, we worked with DR4-specific TRAIL variant 4C7 and DR5-specific TRAIL variant DHER to study combination treatments to overcome resistance. Artemisinin is an approved anti-malarial drug extracted from Artemisia annua, which has also been studied as an anti-tumor drug for decades. We showed that artemisinin-type drugs stimulate DR5-specific TRAIL-induced apoptosis by regulating wildtype P53 in colon cancer cells. Then, we investigated the possibility of performing this combination treatment in P53 mutated triple-negative breast cancer (TNBC) cells. The effectiveness of dihydroartemisinin (DHA) was improved by forming adducts with transferrin (TF). These DHA-TF adducts induce apoptosis and ferroptosis in TNBC cells. Meanwhile, they enhance TRAIL-induced apoptosis mainly through DR5 upregulation in a P53-independent and ROS-dependent manner. Based on our findings and literature research, we summarized the effects of artemisinin-type drugs on regulated cell death pathways, the combination strategies with biologics, and the nanocarriers for artemisinin delivery. Finally, the expression pattern of DR4 and DR5 and the influence of ionizing radiation on receptor-specific TRAIL in 2D and 3D spheroids were explored. The whole thesis provided new strategies to enhance receptor-specific TRAIL-induced apoptosis for cancer treatment.

Building complex compounds such as fine chemicals and pharmaceuticals by linking carbon-carbon bonds is at the core of organic synthesis. However, current methods to construct carbon-carbon bonds often have significant shortcomings, such as the use of hazardous substances or the generation of toxic waste. Biocatalysis, the use of enzymes in organic synthesis, potentially offers a more eco-friendly and sustainable approach for carbon-carbon bond formation. However, many powerful transformations known in chemistry are not catalyzed by natural enzymes.The work described in the PhD thesis of Andreas Kunzendorf focuses on the discovery and engineering of new enzymes for several highly interesting carbon-carbon bond-forming reactions. The research yielded new biocatalysts for the synthesis of important cyclopropanes and efficient enzymes for the production of building blocks for pharmaceutically active γ-aminobutyric acids via extensive enzyme optimisation using directed evolution. The optimized enzyme variants could not only perform the reactions much faster but also provided the products with high selectivity. The results of this work are a stepping-stone towards more sustainable synthesis of pharmaceuticals and provide exciting opportunities to develop new enzymatic reactions not known in nature.

Pseudomonas aeruginosa still becomes a bacteria of concern to be investigated due to its resistance to several antibiotics. Many efforts have been done to find a novel drug against the infections caused by P. aeruginosa. Recently, the iron uptake mechanism facilitated by siderophores attracts the attention to be explored as a novel target to find a new treatment against the infections. The biosynthesis of pyoverdine (a major siderophore) involves many enzymes where these enzymes could potentially be exploited as drug targets. In this thesis, Joko Priyanto Wibowo demonstrates that the iron uptake mechanism can be exploited to be a novel target to develop new treatments against P. aeruginosa infections by inhibiting PvdP and PvdQ, both are responsible for the biosynthesis of pyoverdine. The PvdP knock-out mutant demonstrated the importance of the enzyme in the virulence of P. aeruginosa. The solvation of the crystal structure of PvdP especially the one that binds to the inhibitor leads to the development of a novel and highly potent inhibitor of PvdP. In addition, the finding of a new inhibitor of PvdQ also opens more alternative ways to obtain new treatments against Pseudomonas infections. The application of PvdP and PvdQ inhibitor in the Galleria mellonella infection model successfully interferes with the iron uptake into the bacterial cell, as a result, reducing the virulence of P. aeruginosa in vivo.

Affinity ligands such as antibodies are widely used in (bio)medical research for purifying proteins from complex biological samples. These ligands are generally immobilized onto solid supports which facilitate the separation of a captured protein from the sample matrix. Adsorptive microtiter plates are commonly used as solid supports prior to immunochemical detection (e.g., immunoassays) but hardly ever prior to liquid chromatography-mass spectrometry (LC-MS-)-based detection. Here, we describe the use of adsorptive microtiter plates for protein enrichment prior to LC-MS detection, and we discuss opportunities and challenges of corresponding workflows, based on examples of targeted (i.e., soluble receptor for advanced glycation end-products (sRAGE) in human serum) and discovery-based workflows (i.e., transcription factor p65 (NF-κB) in lysed murine RAW 264.7 macrophages and peptidyl-prolyl cis-trans isomerase FKBP5 (FKBP5) in lysed human A549 alveolar basal epithelial cells). Thereby, we aim to highlight the potential usefulness of adsorptive microtiter plates in affinity purification workflows prior to LC-MS detection, which could increase their usage in mass spectrometry-based protein research.

The present invention relates to the use of compounds of formula (I) as inhibitors of RNA guided endonucleases, e.g., Cas9 and Cpfl, and methods for the inhibition of the function of such RNA guided endonucleases, including their use in the treatment and prevention of conditions or diseases.