Publications by authors named "Gerrit J Poelarends"

98 Publications

Unlocking Asymmetric Michael Additions in an Archetypical Class I Aldolase by Directed Evolution.

ACS Catal 2021 Nov 15;11(21):13236-13243. Epub 2021 Oct 15.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.

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 , 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acscatal.1c03911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8576802PMC
November 2021

4-Iodopyrimidine Labeling Reveals Nuclear Translocation and Nuclease Activity for Both MIF and MIF2*.

Chemistry 2021 Nov 1. Epub 2021 Nov 1.

Department Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy (GRIP), University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/chem.202103030DOI Listing
November 2021

Biocatalytic Asymmetric Cyclopropanations via Enzyme-Bound Iminium Ion Intermediates.

Angew Chem Int Ed Engl 2021 11 5;60(45):24059-24063. Epub 2021 Oct 5.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.202110719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8596749PMC
November 2021

Biocatalytic enantioselective hydroaminations enabling synthesis of -arylalkyl-substituted L-aspartic acids.

Org Biomol Chem 2021 Jul;19(29):6407-6411

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands. g.j.poelarends.rug.nl.

N-Substituted l-aspartic acids are important chiral building blocks for pharmaceuticals and food additives. Here we report the asymmetric synthesis of various N-arylalkyl-substituted l-aspartic acids using ethylenediamine-N,N'-disuccinic acid lyase (EDDS lyase) as a biocatalyst. This C-N lyase shows a broad non-natural amine substrate scope and outstanding enantioselectivity, allowing the efficient addition of structurally diverse arylalkylamines to fumarate to afford the corresponding N-arylalkyl-substituted l-aspartic acids in good isolated yield (up to 79%) and with excellent enantiopurity (>99% ee). These results further demonstrate that C-N lyases working in reverse constitute an extremely powerful synthetic tool to prepare difficult noncanonical amino acids.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/d1ob00748cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8317194PMC
July 2021

D-dopachrome tautomerase contributes to lung epithelial repair via atypical chemokine receptor 3-dependent Akt signaling.

EBioMedicine 2021 Jun 4;68:103412. Epub 2021 Jun 4.

Groningen Research Institute of Pharmacy, Department of Molecular Pharmacology, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, the Netherlands; University Medical Center Groningen, Groningen Research Institute of Asthma and COPD, University of Groningen, Hanzeplein 1, 9713 GZ Groningen, the Netherlands. Electronic address:

Background: Emphysematous COPD is characterized by aberrant alveolar repair. Macrophage migration inhibitory factor (MIF) contributes to alveolar repair, but for its structural and functional homolog D-dopachrome tautomerase (DDT) this is unknown. MIF mediates its effects through CD74 and/or C-X-C chemokine receptors 2 (CXCR2), 4(CXCR4), and possibly 7 (ACKR3). DDT can also signal through CD74, but interactions with other receptors have not been described yet. We therefore aimed at investigating if and how DDT contributes to epithelial repair in COPD.

Methods: We studied effects of recombinant DDT on cell proliferation and survival by clonogenic assay and annexin V-PI staining respectively. DDT-induced signaling was investigated by Western blot. Effects on epithelial growth and differentiation was studied using lung organoid cultures with primary murine or human epithelial cells and incubating with DDT or an ACKR3-blocking nanobody. DDT-ACKR3 interactions were identified by ELISA and co-immunoprecipitation.

Findings: We found that DDT promoted proliferation of and prevented staurosporine-induced apoptosis in A549 lung epithelial cells. Importantly, DDT also stimulated growth of primary alveolar epithelial cells as DDT treatment resulted in significantly more and larger murine and human alveolar organoids compared to untreated controls. The anti-apoptotic effect of DDT and DDT-induced organoid growth were inhibited in the presence of an ACKR3-blocking nanobody. Furthermore, ELISA assay and co-immunoprecipitation suggested DDT complexes with ACKR3. DDT could activate the PI3K-Akt pathway and this activation was enhanced in ACKR3-overexpressing cells.

Interpretation: In conclusion, DDT contributes to alveolar epithelial repair via ACKR3 and may thus augment lung epithelial repair in COPD.

Funding: As stated in the Acknowledgments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ebiom.2021.103412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8185224PMC
June 2021

Proteolysis Targeting Chimera (PROTAC) for Macrophage Migration Inhibitory Factor (MIF) Has Anti-Proliferative Activity in Lung Cancer Cells.

Angew Chem Int Ed Engl 2021 08 26;60(32):17514-17521. Epub 2021 Jun 26.

Department Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy (GRIP), University of Groningen, Antonius Deusinglaan 1, 9713, AV, Groningen, The Netherlands.

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 DC 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.202101864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8362126PMC
August 2021

The broad amine scope of pantothenate synthetase enables the synthesis of pharmaceutically relevant amides.

Org Biomol Chem 2021 May;19(20):4515-4519

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/d1ob00238dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8150671PMC
May 2021

Structural Aspects of Photopharmacology: Insight into the Binding of Photoswitchable and Photocaged Inhibitors to the Glutamate Transporter Homologue.

J Am Chem Soc 2021 01 15;143(3):1513-1520. Epub 2021 Jan 15.

Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.

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 in complex with photoresponsive transport inhibitors-azobenzene derivative of TBOA (both in and 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jacs.0c11336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7844824PMC
January 2021

Genetic regulation of gene expression of MIF family members in lung tissue.

Sci Rep 2020 10 12;10(1):16980. Epub 2020 Oct 12.

Department of Molecular Pharmacology, Groningen Research Institute of Pharmacy (GRIP), University of Groningen, Groningen, The Netherlands.

Macrophage migration inhibitory factor (MIF) is a cytokine found to be associated with chronic obstructive pulmonary disease (COPD). However, there is no consensus on how MIF levels differ in COPD compared to control conditions and there are no reports on MIF expression in lung tissue. Here we studied gene expression of members of the MIF family MIF, D-Dopachrome Tautomerase (DDT) and DDT-like (DDTL) in a lung tissue dataset with 1087 subjects and identified single nucleotide polymorphisms (SNPs) regulating their gene expression. We found higher MIF and DDT expression in COPD patients compared to non-COPD subjects and found 71 SNPs significantly influencing gene expression of MIF and DDTL. Furthermore, the platform used to measure MIF (microarray or RNAseq) was found to influence the splice variants detected and subsequently the direction of the SNP effects on MIF expression. Among the SNPs found to regulate MIF expression, the major LD block identified was linked to rs5844572, a SNP previously found to be associated with lower diffusion capacity in COPD. This suggests that MIF may be contributing to the pathogenesis of COPD, as SNPs that influence MIF expression are also associated with symptoms of COPD. Our study shows that MIF levels are affected not only by disease but also by genetic diversity (i.e. SNPs). Since none of our significant eSNPs for MIF or DDTL have been described in GWAS for COPD or lung function, MIF expression in COPD patients is more likely a consequence of disease-related factors rather than a cause of the disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-74121-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7552402PMC
October 2020

7-Hydroxycoumarins Are Affinity-Based Fluorescent Probes for Competitive Binding Studies of Macrophage Migration Inhibitory Factor.

J Med Chem 2020 10 13;63(20):11920-11933. Epub 2020 Oct 13.

Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy (GRIP), University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.

Macrophage migration inhibitory factor (MIF) is a cytokine with key roles in inflammation and cancer, which qualifies it as a potential drug target. Apart from its cytokine activity, MIF also harbors enzyme activity for keto-enol tautomerization. MIF enzymatic activity has been used for identification of MIF binding molecules that also interfere with its biological activity. However, MIF tautomerase activity assays are troubled by irregularities, thus creating a need for alternative methods. In this study, we identified a 7-hydroxycoumarin fluorophore with high affinity for the MIF tautomerase active site ( = 18 ± 1 nM) that binds with concomitant quenching of its fluorescence. This property enabled development of a novel competition-based assay format to quantify MIF binding. We also demonstrated that the 7-hydroxycoumarin fluorophore interfered with the MIF-CD74 interaction and inhibited proliferation of A549 cells. Thus, we provide a high-affinity MIF binder as a novel tool to advance MIF-oriented research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.0c01160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7586407PMC
October 2020

Using Mutability Landscapes To Guide Enzyme Thermostabilization.

Chembiochem 2021 01 30;22(1):170-175. Epub 2020 Sep 30.

Department of Chemical and Pharmaceutical Biology Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen (The, Netherlands.

Thermostabilizing enzymes while retaining their activity and enantioselectivity for applied biocatalysis is an important topic in protein engineering. Rational and computational design strategies as well as directed evolution have been used successfully to thermostabilize enzymes. Herein, we describe an alternative mutability-landscape approach that identified three single mutations (R11Y, R11I and A33D) within the enzyme 4-oxalocrotonate tautomerase (4-OT), which has potential as a biocatalyst for pharmaceutical synthesis, that gave rise to significant increases in apparent melting temperature T (up to 20 °C) and in half-life at 80 °C (up to 111-fold). Introduction of these beneficial mutations in an enantioselective but thermolabile 4-OT variant (M45Y/F50A) afforded improved triple-mutant enzyme variants showing an up to 39 °C increase in T value, with no reduction in catalytic activity or enantioselectivity. This study illustrates the power of mutability-landscape-guided protein engineering for thermostabilizing enzymes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cbic.202000442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7821111PMC
January 2021

Recent Applications of Carbon-Nitrogen Lyases in Asymmetric Synthesis of Noncanonical Amino Acids and Heterocyclic Compounds.

Chembiochem 2020 10 8;21(19):2733-2742. Epub 2020 Jun 8.

Department of Chemical and Pharmaceutical Biology Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands.

Carbon-nitrogen (C-N) lyases are enzymes that normally catalyze the cleavage of C-N bonds. Reversing this reaction towards carbon-nitrogen bond formation can be a powerful approach to prepare valuable compounds that could find applications in everyday life. This review focuses on recent (last five years) applications of native and engineered C-N lyases, either as stand-alone biocatalysts or as part of multienzymatic and chemoenzymatic cascades, in enantioselective synthesis of noncanonical amino acids and dinitrogen-fused heterocycles, which are useful tools for neurobiological research and important synthetic precursors to pharmaceuticals and food additives.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cbic.202000214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7586795PMC
October 2020

The role of MIF in chronic lung diseases: looking beyond inflammation.

Am J Physiol Lung Cell Mol Physiol 2020 06 25;318(6):L1183-L1197. Epub 2020 Mar 25.

Department of Molecular Pharmacology, Groningen Research Institute of Pharmacy, University of Groningen, Groningen, The Netherlands.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been associated with many diseases. Most studies found in literature describe MIF as a proinflammatory cytokine involved in chronic inflammatory conditions, but evidence from last years suggests that many of its key effects are not directly related to inflammation. In fact, MIF is constitutively expressed in most human tissues and in some cases in high levels, which does not reflect the pattern of expression of a classic proinflammatory cytokine. Moreover, MIF is highly expressed during embryonic development and decreases during adulthood, which point toward a more likely role as growth factor. Accordingly, MIF knockout mice develop age-related spontaneous emphysema, suggesting that MIF presence (e.g., in younger individuals and wild-type animals) is part of a healthy lung. In view of this new line of evidence, we aimed to review data on the role of MIF in the pathogenesis of chronic lung diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajplung.00521.2019DOI Listing
June 2020

Enantiocomplementary Epoxidation Reactions Catalyzed by an Engineered Cofactor-Independent Non-natural Peroxygenase.

Angew Chem Int Ed Engl 2020 06 14;59(26):10374-10378. Epub 2020 Apr 14.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713, AV, Groningen, The Netherlands.

Peroxygenases are heme-dependent enzymes that use peroxide-borne oxygen to catalyze a wide range of oxyfunctionalization reactions. Herein, we report the engineering of an unusual cofactor-independent peroxygenase based on a promiscuous tautomerase that accepts different hydroperoxides (t-BuOOH and H O ) to accomplish enantiocomplementary epoxidations of various α,β-unsaturated aldehydes (citral and substituted cinnamaldehydes), providing access to both enantiomers of the corresponding α,β-epoxy-aldehydes. High conversions (up to 98 %), high enantioselectivity (up to 98 % ee), and good product yields (50-80 %) were achieved. The reactions likely proceed via a reactive enzyme-bound iminium ion intermediate, allowing tweaking of the enzyme's activity and selectivity by protein engineering. Our results underscore the potential of catalytic promiscuity for the engineering of new cofactor-independent oxidative enzymes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.202001373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7317984PMC
June 2020

Enantioselective Aldol Addition of Acetaldehyde to Aromatic Aldehydes Catalyzed by Proline-Based Carboligases.

ACS Catal 2020 Feb 28;10(4):2522-2527. Epub 2020 Jan 28.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.

Aromatic β-hydroxyaldehydes, 1,3-diols, and α,β-unsaturated aldehydes are valuable precursors to biologically active natural products and drug molecules. Herein we report the biocatalytic aldol condensation of acetaldehyde with various aromatic aldehydes to give a number of aromatic α,β-unsaturated aldehydes using a previously engineered variant of 4-oxalocrotonate tautomerase [4-OT(M45T/F50A)] as carboligase. Moreover, an efficient one-pot two-step chemoenzymatic route toward chiral aromatic 1,3-diols has been developed. This one-pot chemoenzymatic strategy successfully combined a highly enantioselective aldol addition step catalyzed by a proline-based carboligase [4-OT(M45T/F50A) or TAUT015] with a chemical reduction step to convert enzymatically prepared aromatic β-hydroxyaldehydes into the corresponding 1,3-diols with high optical purity (e.r. up to >99:1) and in good isolated yield (51-92%). These developed (chemo)enzymatic methodologies offer alternative synthetic choices to prepare a variety of important drug precursors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acscatal.0c00039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7045556PMC
February 2020

Selective Colorimetric "Turn-On" Probe for Efficient Engineering of Iminium Biocatalysis.

ACS Omega 2020 Feb 28;5(5):2397-2405. Epub 2020 Jan 28.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, Groningen 9713 AV, The Netherlands.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsomega.9b03849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017405PMC
February 2020

Aminocarboxylic acids related to aspergillomarasmine A (AMA) and ethylenediamine-N,N'-disuccinic acid (EDDS) are strong zinc-binders and inhibitors of the metallo-beta-lactamase NDM-1.

Chem Commun (Camb) 2020 Mar;56(20):3047-3049

Biological Chemistry Group, Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands.

A series of aminocarboxylic acid analogues of aspergillomarasmine A (AMA) and ethylenediamine-N,N'-disuccinic acid (EDDS) were chemoenzymatically synthesized via the addition of various mono- and diamine substrates to fumaric acid catalyzed by the enzyme EDDS lyase. Many of these novel AMA and EDDS analogues demonstrate potent inhibition of the bacterial metallo-β-lactamase NDM-1. Isothermal titration calorimetry assays revealed a strong correlation between the inhibitory potency of the compounds and their ability to bind zinc. Compounds 1a (AMA), 1b (AMB), 5 (EDDS), followed by 1d and 8a, demonstrate the highest synergy with meropenem resensitizing an NDM-1 producing strain of E. coli to this important carbapenem of last resort.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/d0cc00356eDOI Listing
March 2020

Tuning Enzyme Activity for Nonaqueous Solvents: Engineering an Enantioselective "Michaelase" for Catalysis in High Concentrations of Ethanol.

Chembiochem 2020 05 18;21(10):1499-1504. Epub 2020 Feb 18.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands.

Enzymes have evolved to function under aqueous conditions and may not exhibit features essential for biocatalytic application, such as the ability to function in high concentrations of an organic solvent. Consequently, protein engineering is often required to tune an enzyme for catalysis in non-aqueous solvents. In this study, we have used a collection of nearly all single mutants of 4-oxalocrotonate tautomerase, which promiscuously catalyzes synthetically useful Michael-type additions of acetaldehyde to various nitroolefins, to investigate the effect of each mutation on the ability of this enzyme to retain its "Michaelase" activity in elevated concentrations of ethanol. Examination of this mutability landscape allowed the identification of two hotspot positions, Ser30 and Ala33, at which mutations are beneficial for catalysis in high ethanol concentrations. The "hotspot" position Ala33 was then randomized in a highly enantioselective, but ethanol-sensitive 4-OT variant (L8F/M45Y/F50A) to generate an improved enzyme variant (L8F/A33I/M45Y/F50A) that showed great ethanol stability and efficiently catalyzes the enantioselective addition of acetaldehyde to nitrostyrene in 40 % ethanol (permitting high substrate loading) to give the desired γ-nitroaldehyde product in excellent isolated yield (89 %) and enantiopurity (ee=98 %). The presented work demonstrates the power of mutability-landscape-guided enzyme engineering for efficient biocatalysis in non-aqueous solvents.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cbic.201900721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7317446PMC
May 2020

In Situ Acetaldehyde Synthesis for Carboligation Reactions.

Chembiochem 2020 05 12;21(10):1505-1509. Epub 2020 Feb 12.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands.

The enzyme 4-oxalocrotonate tautomerase (4-OT) can promiscuously catalyze various carboligation reactions using acetaldehyde as a nucleophile. However, the highly reactive nature of acetaldehyde requires intricate handling, which can impede its usage in practical synthesis. Therefore, we investigated three enzymatic routes to synthesize acetaldehyde in situ in one-pot cascade reactions with 4-OT. Two routes afforded practical acetaldehyde concentrations, using an environmental pollutant, trans-3-chloroacrylic acid, or a bio-renewable, ethanol, as starting substrate. These routes can be combined with 4-OT catalyzed Michael-type additions and aldol condensations in one pot. This modular systems biocatalysis methodology provides a stepping stone towards the development of larger artificial metabolic networks for the practical synthesis of important chemical synthons.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cbic.201900666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7318290PMC
May 2020

Structure-activity relationships for binding of 4-substituted triazole-phenols to macrophage migration inhibitory factor (MIF).

Eur J Med Chem 2020 Jan 11;186:111849. Epub 2019 Nov 11.

Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy (GRIP), University of Groningen, Groningen, the Netherlands. Electronic address:

Macrophage migration inhibitory factor (MIF) is a versatile protein that plays a role in inflammation, autoimmune diseases and cancers. Development of novel inhibitors will enable further exploration of MIF as a drug target. In this study, we investigated structure-activity relationships of MIF inhibitors using a MIF tautomerase activity assay to measure binding. Importantly, we notified that transition metals such as copper (II) and zinc (II) interfere with the MIF tautomerase activity under the assay conditions applied. EDTA was added to the assay buffer to avoid interference of residual heavy metals with tautomerase activity measurements. Using these assay conditions the structure-activity relationships for MIF binding of a series of triazole-phenols was explored. The most potent inhibitors in this series provided activities in the low micromolar range. Enzyme kinetic analysis indicates competitive binding that proved reversible. Binding to the enzyme was confirmed using a microscale thermophoresis (MST) assay. Molecular modelling was used to rationalize the observed structure-activity relationships. The most potent inhibitor 2d inhibited proliferation of A549 cells in a clonogenic assay. In addition, 2d attenuated MIF induced ERK phosphorylation in A549 cells. Altogether, this study provides insights in the structure-activity relationships for MIF binding of triazole-phenols and further validates this class of compounds as MIF binding agents in cell-based studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejmech.2019.111849DOI Listing
January 2020

Engineered C-N Lyase: Enantioselective Synthesis of Chiral Synthons for Artificial Dipeptide Sweeteners.

Angew Chem Int Ed Engl 2020 01 19;59(1):429-435. Epub 2019 Nov 19.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713, AV, Groningen, The Netherlands.

Aspartic acid derivatives with branched N-alkyl or N-arylalkyl substituents are valuable precursors to artificial dipeptide sweeteners such as neotame and advantame. The development of a biocatalyst to synthesize these compounds in a single asymmetric step is an as yet unmet challenge. Reported here is an enantioselective biocatalytic synthesis of various difficult N-substituted aspartic acids, including N-(3,3-dimethylbutyl)-l-aspartic acid and N-[3-(3-hydroxy-4-methoxyphenyl)propyl]-l-aspartic acid, precursors to neotame and advantame, respectively, using an engineered variant of ethylenediamine-N,N'-disuccinic acid (EDDS) lyase from Chelativorans sp. BNC1. This engineered C-N lyase (mutant D290M/Y320M) displayed a remarkable 1140-fold increase in activity for the selective hydroamination of fumarate compared to that of the wild-type enzyme. 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.201910704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6973171PMC
January 2020

SLC1A3 contributes to L-asparaginase resistance in solid tumors.

EMBO J 2019 10 16;38(21):e102147. Epub 2019 Sep 16.

Division of Oncogenomics, Oncode Institute, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

L-asparaginase (ASNase) serves as an effective drug for adolescent acute lymphoblastic leukemia. However, many clinical trials indicated severe ASNase toxicity in patients with solid tumors, with resistant mechanisms not well understood. Here, we took a functional genetic approach and identified SLC1A3 as a novel contributor to ASNase resistance in cancer cells. In combination with ASNase, SLC1A3 inhibition caused cell cycle arrest or apoptosis, and myriads of metabolic vulnerabilities in tricarboxylic acid (TCA) cycle, urea cycle, nucleotides biosynthesis, energy production, redox homeostasis, and lipid biosynthesis. SLC1A3 is an aspartate and glutamate transporter, mainly expressed in brain tissues, but high expression levels were also observed in some tumor types. Here, we demonstrate that ASNase stimulates aspartate and glutamate consumptions, and their refilling through SLC1A3 promotes cancer cell proliferation. Lastly, in vivo experiments indicated that SLC1A3 expression promoted tumor development and metastasis while negating the suppressive effects of ASNase by fueling aspartate, glutamate, and glutamine metabolisms despite of asparagine shortage. Altogether, our findings identify a novel role for SLC1A3 in ASNase resistance and suggest that restrictive aspartate and glutamate uptake might improve ASNase efficacy with solid tumors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.2019102147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6826201PMC
October 2019

Biocatalytic Asymmetric Michael Additions of Nitromethane to α,β-Unsaturated Aldehydes via Enzyme-bound Iminium Ion Intermediates.

ACS Catal 2019 May 10;9(5):4369-4373. Epub 2019 Apr 10.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.

The enzyme 4-oxalocrotonate tautomerase (4-OT) exploits an N-terminal proline as main catalytic residue to facilitate several promiscuous C-C bond-forming reactions via enzyme-bound enamine intermediates. Here we show that the active site of this enzyme can give rise to further synthetically useful catalytic promiscuity. Specifically, the F50A mutant of 4-OT was found to efficiently promote asymmetric Michael additions of nitromethane to various α,β-unsaturated aldehydes to give γ-nitroaldehydes, important precursors to biologically active γ-aminobutyric acids. High conversions, high enantiocontrol, and good isolated product yields were achieved. The reactions likely proceed via iminium ion intermediates formed between the catalytic Pro-1 residue and the α,β-unsaturated aldehydes. In addition, a cascade of three 4-OT(F50A)-catalyzed reactions followed by an enzymatic oxidation step enables assembly of γ-nitrocarboxylic acids from three simple building blocks in one pot. Our results bridge organo- and biocatalysis, and they emphasize the potential of enzyme promiscuity for the preparation of important chiral synthons.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acscatal.9b00780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6503466PMC
May 2019

Enantioselective Synthesis of Pharmaceutically Active γ-Aminobutyric Acids Using a Tailor-Made Artificial Michaelase in One-Pot Cascade Reactions.

ACS Catal 2019 Feb 7;9(2):1503-1513. Epub 2019 Jan 7.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.

Chiral γ-aminobutyric acid (GABA) analogues represent abundantly prescribed drugs, which are broadly applied as anticonvulsants, as antidepressants, and for the treatment of neuropathic pain. Here we report a one-pot two-step biocatalytic cascade route for synthesis of the pharmaceutically relevant enantiomers of γ-nitrobutyric acids, starting from simple precursors (acetaldehyde and nitroalkenes), using a tailor-made highly enantioselective artificial "Michaelase" (4-oxalocrotonate tautomerase mutant L8Y/M45Y/F50A), an aldehyde dehydrogenase with a broad non-natural substrate scope, and a cofactor recycling system. We also report a three-step chemoenzymatic cascade route for the efficient chemical reduction of enzymatically prepared γ-nitrobutyric acids into GABA analogues in one pot, achieving high enantiopurity (e.r. up to 99:1) and high overall yields (up to 70%). This chemoenzymatic methodology offers a step-economic alternative route to important pharmaceutically active GABA analogues, and highlights the exciting opportunities available for combining chemocatalysts, natural enzymes, and designed artificial biocatalysts in multistep syntheses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acscatal.8b04299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366683PMC
February 2019

Modular Enzymatic Cascade Synthesis of Vitamin B and Its Derivatives.

Chemistry 2018 Nov 30;24(66):17434-17438. Epub 2018 Oct 30.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713, AV, Groningen, The Netherlands.

Access to vitamin B [(R)-pantothenic acid] and both diastereoisomers of α-methyl-substituted vitamin B [(R)- and (S)-3-((R)-2,4-dihydroxy-3,3-dimethylbutanamido)-2-methylpropanoic acid] was achieved using a modular three-step biocatalytic cascade involving 3-methylaspartate ammonia lyase (MAL), aspartate-α-decarboxylase (ADC), β-methylaspartate-α-decarboxylase (CrpG) or glutamate decarboxylase (GAD), and pantothenate synthetase (PS) enzymes. Starting from simple non-chiral dicarboxylic acids (either fumaric acid or mesaconic acid), vitamin B and both diastereoisomers of α-methyl-substituted vitamin B , which are valuable precursors for promising antimicrobials against Plasmodium falciparum and multidrug-resistant Staphylococcus aureus, can be generated in good yields (up to 70 %) and excellent enantiopurity (>99 % ee). This newly developed cascade process may be tailored and used for the biocatalytic production of various vitamin B derivatives by modifying the pantoyl or β-alanine moiety.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/chem.201804151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6471175PMC
November 2018

Chemoenzymatic Synthesis and Pharmacological Characterization of Functionalized Aspartate Analogues As Novel Excitatory Amino Acid Transporter Inhibitors.

J Med Chem 2018 09 15;61(17):7741-7753. Epub 2018 Aug 15.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy , University of Groningen , Antonius Deusinglaan 1 , 9713 AV Groningen , The Netherlands.

Aspartate (Asp) derivatives are privileged compounds for investigating the roles governed by excitatory amino acid transporters (EAATs) in glutamatergic neurotransmission. Here, we report the synthesis of various Asp derivatives with (cyclo)alkyloxy and (hetero)aryloxy substituents at C-3. Their pharmacological properties were characterized at the EAAT1-4 subtypes. The l- threo-3-substituted Asp derivatives 13a-e and 13g-k were nonsubstrate inhibitors, exhibiting pan activity at EAAT1-4 with IC values ranging from 0.49 to 15 μM. Comparisons between (dl- threo)-19a-c and (dl- erythro)-19a-c Asp analogues confirmed that the threo configuration is crucial for the EAAT1-4 inhibitory activities. Analogues (3b-e) of l-TFB-TBOA (3a) were shown to be potent EAAT1-4 inhibitors, with IC values ranging from 5 to 530 nM. Hybridization of the nonselective EAAT inhibitor l-TBOA with EAAT2-selective inhibitor WAY-213613 or EAAT3-preferring inhibitor NBI-59159 yielded compounds 8 and 9, respectively, which were nonselective EAAT inhibitors displaying considerably lower IC values at EAAT1-4 (11-140 nM) than those displayed by the respective parent molecules.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.8b00700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6139576PMC
September 2018

Small-molecule inhibitors of macrophage migration inhibitory factor (MIF) as an emerging class of therapeutics for immune disorders.

Drug Discov Today 2018 11 21;23(11):1910-1918. Epub 2018 Jun 21.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy (GRIP), University of Groningen, Groningen, The Netherlands. Electronic address:

Macrophage migration inhibitory factor (MIF) is an important cytokine for which an increasing number of functions is being described in the pathogenesis of inflammation and cancer. Nevertheless, the availability of potent and druglike MIF inhibitors that are well-characterized in relevant disease models remains limited. Development of highly potent and selective small-molecule MIF inhibitors and validation of their use in relevant disease models will advance drug discovery. In this review, we provide an overview of recent advances in the identification of MIF as a pharmacological target in the pathogenesis of inflammatory diseases and cancer. We also give an overview of the current developments in the discovery and design of small-molecule MIF inhibitors and define future aims in this field.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.drudis.2018.06.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6453109PMC
November 2018

Structural Basis for the Catalytic Mechanism of Ethylenediamine- N, N'-disuccinic Acid Lyase, a Carbon-Nitrogen Bond-Forming Enzyme with a Broad Substrate Scope.

Biochemistry 2018 07 18;57(26):3752-3763. Epub 2018 May 18.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy , University of Groningen , Antonius Deusinglaan 1 , 9713 AV Groningen , The Netherlands.

The natural aminocarboxylic acid product ethylenediamine- N, N'-disuccinic acid [( S, S)-EDDS] is able to form a stable complex with metal ions, making it an attractive biodegradable alternative for the synthetic metal chelator ethylenediaminetetraacetic acid (EDTA), which is currently used on a large scale in numerous applications. Previous studies have demonstrated that biodegradation of ( S, S)-EDDS may be initiated by an EDDS lyase, converting ( S, S)-EDDS via the intermediate N-(2-aminoethyl)aspartic acid (AEAA) into ethylenediamine and two molecules of fumarate. However, current knowledge of this enzyme is limited because of the absence of structural data. Here, we describe the identification and characterization of an EDDS lyase from Chelativorans sp. BNC1, which has a broad substrate scope, accepting various mono- and diamines for addition to fumarate. We report crystal structures of the enzyme in an unliganded state and in complex with formate, succinate, fumarate, AEAA, and ( S, S)-EDDS. The structures reveal a tertiary and quaternary fold that is characteristic of the aspartase/fumarase superfamily and support a mechanism that involves general base-catalyzed, sequential two-step deamination of ( S, S)-EDDS. This work broadens our understanding of mechanistic diversity within the aspartase/fumarase superfamily and will aid in the optimization of EDDS lyase for asymmetric synthesis of valuable (metal-chelating) aminocarboxylic acids.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.biochem.8b00406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6034166PMC
July 2018

High yield production of human invariant chain CD74 constructs fused to solubility-enhancing peptides and characterization of their MIF-binding capacities.

Protein Expr Purif 2018 08 27;148:46-53. Epub 2018 Mar 27.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands. Electronic address:

The HLA class II histocompatibility antigen gamma chain, also known as HLA-DR antigen-associated invariant chain or CD74, has been shown to be involved in many biological processes amongst which antigen loading and transport of MHC class II molecules from the endoplasmic reticulum to the Golgi complex. It is also part of a receptor complex for macrophage migration inhibitory factor (MIF), and participates in inflammatory signaling. The inhibition of MIF-CD74 complex formation is regarded as a potentially attractive therapeutic target in inflammation, cancer and immune diseases. In order to be able to produce large quantities of the extracellular moiety of human CD74, which has been reported to be unstable and protease-sensitive, different constructs were made as fusions with two solubility enhancers: the well-known maltose-binding domain and Fh8, a small protein secreted by the parasite Fasciola hepatica. The fusion proteins could be purified with high yields from Escherichia coli and were demonstrated to be active in binding to MIF. Moreover, our results strongly suggest that the MIF binding site is located in the sequence between the transmembrane and the membrane-distal trimerisation domain of CD74, and comprises at least amino acids 113-125 of CD74.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.pep.2018.03.008DOI Listing
August 2018

Discovery of chromenes as inhibitors of macrophage migration inhibitory factor.

Bioorg Med Chem 2018 03 24;26(5):999-1005. Epub 2017 Dec 24.

Department of Chemical and Pharmaceutical Biology, University of Groningen, Groningen, The Netherlands. Electronic address:

Macrophage migration inhibitory factor (MIF) is an essential signaling cytokine with a key role in the immune system. Binding of MIF to its molecular targets such as, among others, the cluster of differentiation 74 (CD74) receptor plays a key role in inflammatory diseases and cancer. Therefore, the identification of MIF binding compounds gained importance in drug discovery. In this study, we aimed to discover novel MIF binding compounds by screening of a focused compound collection for inhibition of its tautomerase enzyme activity. Inspired by the known chromen-4-one inhibitor Orita-13, a focused collection of compounds with a chromene scaffold was screened for MIF binding. The library was synthesized using versatile cyanoacetamide chemistry to provide diversely substituted chromenes. The screening provided inhibitors with IC's in the low micromolar range. Kinetic evaluation suggested that the inhibitors were reversible and did not bind in the binding pocket of the substrate. Thus, we discovered novel inhibitors of the MIF tautomerase activity, which may ultimately support the development of novel therapeutic agents against diseases in which MIF is involved.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmc.2017.12.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6323139PMC
March 2018
-->