Publications by authors named "Wim J Quax"

145 Publications

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

Engineering of Multiple Modules to Improve Amorphadiene Production in Using CRISPR-Cas9.

J Agric Food Chem 2021 Apr 20;69(16):4785-4794. Epub 2021 Apr 20.

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

Engineering strategies to improve terpenoids' production in mainly focus on 2-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 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 . Overall, this study provides a universal strategy for further increasing terpenoids' production in by comprehensive and systematic metabolic engineering.
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http://dx.doi.org/10.1021/acs.jafc.1c00498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8154554PMC
April 2021

Current State and Future Directions of Genetics and Genomics of Endophytic Fungi for Bioprospecting Efforts.

Front Bioeng Biotechnol 2021 15;9:649906. Epub 2021 Mar 15.

Groningen Institute of Pharmacy, Chemical and Pharmaceutical Biology, University of Groningen, Groningen, Netherlands.

The bioprospecting of secondary metabolites from endophytic fungi received great attention in the 1990s and 2000s, when the controversy around taxol production from spp. endophytes was at its height. Since then, hundreds of reports have described the isolation and characterization of putative secondary metabolites from endophytic fungi. However, only very few studies also report the genetic basis for these phenotypic observations. With low sequencing cost and fast sample turnaround, genetics- and genomics-based approaches have risen to become comprehensive approaches to study natural products from a wide-range of organisms, especially to elucidate underlying biosynthetic pathways. However, in the field of fungal endophyte biology, elucidation of biosynthetic pathways is still a major challenge. As a relatively poorly investigated group of microorganisms, even in the light of recent efforts to sequence more fungal genomes, such as the 1000 Fungal Genomes Project at the Joint Genome Institute (JGI), the basis for bioprospecting of enzymes and pathways from endophytic fungi is still rather slim. In this review we want to discuss the current approaches and tools used to associate phenotype and genotype to elucidate biosynthetic pathways of secondary metabolites in endophytic fungi through the lens of bioprospecting. This review will point out the reported successes and shortcomings, and discuss future directions in sampling, and genetics and genomics of endophytic fungi. Identifying responsible biosynthetic genes for the numerous secondary metabolites isolated from endophytic fungi opens the opportunity to explore the genetic potential of producer strains to discover novel secondary metabolites and enhance secondary metabolite production by metabolic engineering resulting in novel and more affordable medicines and food additives.
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http://dx.doi.org/10.3389/fbioe.2021.649906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005728PMC
March 2021

Betacyanins, major components in Opuntia red-purple fruits, protect against acetaminophen-induced acute liver failure.

Food Res Int 2020 11 18;137:109461. Epub 2020 Jun 18.

Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, the Netherlands; Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, the Netherlands. Electronic address:

Acetaminophen (APAP) misuse or overdose is the most important cause of drug-induced acute liver failure. Overdoses of acetaminophen induce oxidative stress and liver injury by the electrophilic metabolite N-acetyl-p-benzoquinone imine (NAPQI). Plant-based medicine has been used for centuries against diseases or intoxications due to their biological activities. The aim of this study was to evaluate the therapeutic value of Opuntia robusta and Opuntia streptacantha fruit extracts against acetaminophen-induced liver damage and to identify the major biocomponents on them. Opuntia fruit extracts were obtained by peeling and squeezing each specie, followed by lyophilization. HPLC was used to characterize the extracts. The effect of the extracts against acetaminophen-induced acute liver injury was evaluated both in vivo and in vitro using biochemical, molecular and histological determinations. The results showed that betacyanins are the main components in the analyzed Opuntia fruit extracts, with betanin as the highest concentration. Therapeutic treatments with Opuntia extracts reduced biochemical, molecular and histological markers of liver (in vivo) and hepatocyte (in vitro) injury. Opuntia extracts reduced the APAP-increased expression of the stress-related gene Gadd45b. Furthermore, Opuntia extracts exerted diverse effects on the antioxidant related genes Sod2, Gclc and Hmox1, independent of their ROS-scavenging ability. Therefore, betacyanins as betanin from Opuntia robusta and Opuntia streptacantha fruits are promising nutraceutical compounds against oxidative liver damage.
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http://dx.doi.org/10.1016/j.foodres.2020.109461DOI Listing
November 2020

High level production of amorphadiene using Bacillus subtilis as an optimized terpenoid cell factory.

N Biotechnol 2021 Jan 22;60:159-167. Epub 2020 Oct 22.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, 9713 AV, Groningen, the Netherlands. Electronic address:

The anti-malarial drug artemisinin, produced naturally in the plant Artemisia annua, experiences unstable and insufficient supply as its production relies heavily on the plant source. To meet the massive demand for this compound, metabolic engineering of microbes has been studied extensively. In this study, we focus on improving the production of amorphadiene, a crucial artemisinin precursor, in Bacillus subtilis. The expression level of the plant-derived amorphadiene synthase (ADS) was upregulated by fusion with green fluorescent protein (GFP). Furthermore, a co-expression system of ADS and a synthetic operon carrying the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway genes was established. Subsequently, farnesyl pyrophosphate synthase (FPPS), a key enzyme in formation of the sesquiterpene precursor farnesyl pyrophosphate (FPP), was expressed to supply sufficient substrate for ADS. The consecutive combination of these features yielded a B. subtilis strain expressing chromosomally integrated GFP-ADS followed by FPPS and a plasmid encoded synthetic operon showing a stepwise increased production of amorphadiene. An experimental design-aided systematic medium optimization was used to maximize the production level for the most promising engineered B. subtilis strain, resulting in an amorphadiene yield of 416 ± 15 mg/L, which is 20-fold higher than that previously reported in B. subtilis and more than double the production in Escherichia coli or Saccharomyces cerevisiae on a shake flask fermentation level.
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http://dx.doi.org/10.1016/j.nbt.2020.10.007DOI Listing
January 2021

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

Artemisinin Derivatives Stimulate DR5-Specific TRAIL-Induced Apoptosis by Regulating Wildtype P53.

Cancers (Basel) 2020 Sep 4;12(9). Epub 2020 Sep 4.

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

Artemisinin derivatives, widely known as commercial anti-malaria drugs, may also have huge potential in treating cancer cells. It has been reported that artemisinin derivatives can overcome resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in liver and cervical cancer cells. In our study, we demonstrated that artesunate (ATS) and dihydroartemisinin (DHA) are more efficient in killing colon cancer cells compared to artemisinin (ART). ATS/DHA induces the expression of DR5 in a P53 dependent manner in HCT116 and DLD-1 cells. Both ATS and DHA overcome the resistance to DHER-induced apoptosis in HCT116, mainly through upregulating death receptor 5 (DR5). We also demonstrate that DHA sensitizes HCT116 cells to DHER-induced apoptosis via P53 regulated DR5 expression in P53 knockdown assays. Nevertheless, a lower effect was observed in DLD-1 cells, which has a single SerPhe mutation in the P53 DNA binding domain. Thus, the status of P53 could be one of the determinants of TRAIL resistance in some cancer cells. Finally, the combination treatment of DHA and the TRAIL variant DHER increases cell death in 3D colon cancer spheroid models, which shows its potential as a novel therapy.
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http://dx.doi.org/10.3390/cancers12092514DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7563660PMC
September 2020

Development of phenylthiourea derivatives as allosteric inhibitors of pyoverdine maturation enzyme PvdP tyrosinase.

Bioorg Med Chem Lett 2020 09 15;30(17):127409. Epub 2020 Jul 15.

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

Infections caused by Pseudomonas aeruginosa become increasingly difficult to treat because these bacteria have acquired various mechanisms for antibiotic resistance, which creates the need for mechanistically novel antibiotics. Such antibiotics might be developed by targeting enzymes involved in the iron uptake mechanism because iron is essential for bacterial survival. For P. aeruginosa, pyoverdine has been described as an important virulence factor that plays a key role in iron uptake. Therefore, inhibition of enzymes involved in the pyoverdine synthesis, such as PvdP tyrosinase, can open a new window for the treatment of P. aeruginosa infections. Previously, we reported phenylthiourea as the first allosteric inhibitor of PvdP tyrosinase with high micromolar potency. In this report, we explored structure-activity relationships (SAR) for PvdP tyrosinase inhibition by phenylthiourea derivatives. This enables identification of a phenylthiourea derivative (3c) with a potency in the submicromolar range (IC = 0.57 + 0.05 µM). Binding could be rationalized by molecular docking simulation and 3c was proved to inhibit the bacterial pyoverdine production and bacterial growth in P. aeruginosa PA01 cultures.
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http://dx.doi.org/10.1016/j.bmcl.2020.127409DOI Listing
September 2020

Sortase mutants with improved protein thermostability and enzymatic activity obtained by consensus design.

Protein Eng Des Sel 2019 12;32(12):555-564

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

Staphylococcus aureus sortase A (SaSrtA) is an enzyme that anchors proteins to the cell surface of Gram-positive bacteria. During the transpeptidation reaction performed by SaSrtA, proteins containing an N-terminal glycine can be covalently linked to another protein with a C-terminal LPXTG motif (X being any amino acid). Since the sortase reaction can be performed in vitro as well, it has found many applications in biotechnology. Although sortase-mediated ligation has many advantages, SaSrtA is limited by its low enzymatic activity and dependence on Ca2+. In our study, we evaluated the thermodynamic stability of the SaSrtA wild type and found the enzyme to be stable. We applied consensus analysis to further improve the enzyme's stability while at the same time enhancing the enzyme's activity. As a result, we found thermodynamically improved, more active and Ca2+-independent mutants. We envision that these new variants can be applied in conjugation reactions in low Ca2+ environments.
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http://dx.doi.org/10.1093/protein/gzaa018DOI Listing
December 2019

Death Receptor 5 Displayed on Extracellular Vesicles Decreases TRAIL Sensitivity of Colon Cancer Cells.

Front Cell Dev Biol 2020 19;8:318. Epub 2020 May 19.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Groningen, Netherlands.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is considered to be a promising antitumor drug because of its selective proapoptotic properties on tumor cells. However, the clinical application of TRAIL is until now limited because of the resistance of several cancer cells, which can occur at various levels in the TRAIL signaling pathway. The role of decoy receptors that can side-track TRAIL, thereby preventing the formation of an activated death receptor, has been extensively studied. In this study, we have focused on extracellular vesicles (EVs) that are known to play a role in cell-to-cell communication and that can be released by donor cells into the medium transferring their components to recipient cells. TRAIL-induced apoptotic signaling is triggered upon the binding of two death receptors, DR4 and DR5. Here, we found that DR5 but not DR4 is present in the conditioned medium (CM)-derived from various cancer cells. Moreover, we observed that DR5 was exposed on EVs and can act as "decoy receptor" for binding to TRAIL. This results in a strongly reduced number of apoptotic cells upon treatment with DR5-specific TRAIL variant DHER in CM. This reduction happened with EVs containing either the long or short isoform of DR5. Taken together, we demonstrated that colon rectal tumor cells can secrete DR5-coated EVs, and this can cause TRAIL resistance. This is to our knowledge a novel finding and provides new insights into understanding TRAIL sensitivity.
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http://dx.doi.org/10.3389/fcell.2020.00318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7248226PMC
May 2020

Engineering the specificity of Streptococcus pyogenes sortase A by loop grafting.

Proteins 2020 11 21;88(11):1394-1400. Epub 2020 Jun 21.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Groningen, The Netherlands.

Sortases are a group of enzymes displayed on the cell-wall of Gram-positive bacteria. They are responsible for the attachment of virulence factors onto the peptidoglycan in a transpeptidation reaction through recognition of a pentapeptide substrate. Most housekeeping sortases recognize one specific pentapeptide motif; however, Streptococcus pyogenes sortase A (SpSrtA WT) recognizes LPETG, LPETA and LPKLG motifs. Here, we examined SpSrtA's flexible substrate specificity by investigating the role of the β7/β8 loop in determining substrate specificity. We exchanged the β7/β8 loop in SpSrtA with corresponding β7/β8 loops from Staphylococcus aureus (SaSrtA WT) and Bacillus anthracis (BaSrtA WT). While the BaSrtA-derived variant showed no enzymatic activity toward either LPETG or LPETA substrates, the activity of the SaSrtA-derived mutant toward the LPETA substrate was completely abolished. Instead, the mutant had an improved activity toward LPETG, the preferred substrate of SaSrtA WT.
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http://dx.doi.org/10.1002/prot.25958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7586933PMC
November 2020

Production of Squalene in by Squalene Synthase Screening and Metabolic Engineering.

J Agric Food Chem 2020 Apr 3;68(15):4447-4455. Epub 2020 Apr 3.

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

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 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 . Furthermore, we achieved a 29-fold increase of squalene yield (0.26-7.5 mg/L) by expressing SQS from and eliminating bottlenecks within the upstream methylerythritol-phosphate pathway. Moreover, our findings showed that also could positively affect the production of squalene.
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http://dx.doi.org/10.1021/acs.jafc.0c00375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7168599PMC
April 2020

A novel histone acetyltransferase inhibitor A485 improves sensitivity of non-small-cell lung carcinoma cells to TRAIL.

Biochem Pharmacol 2020 05 12;175:113914. Epub 2020 Mar 12.

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

Transcriptional coactivators p300 and CBP catalyze the acetylation of lysine residues in histone proteins. Upregulation of p300 and CBP has been associated with lung, colorectal and hepatocellular cancer, indicating an important role of p300 and CBP in tumorigenesis. Recently, the novel p300 and CBP-selective inhibitor A485 became available, which was shown to inhibit proliferation of 124 different cancer cell lines. Here, we found that downregulation of EP300 or CREBBP enhances apoptosis upon TRAIL stimulation in non-small-cell lung cancer (NSCLC) cells. A485 upregulates pro- and anti-apoptotic genes at the mRNA level, implying an apoptosis-modulating effect in NSCLC cells. However, A485 alone does not induce apoptosis. Interestingly, we observed that the number of apoptotic cells increases upon combined treatment with A485 and TRAIL. Therefore, A485, as a TRAIL-sensitizer, was used in combination with TRAIL in wild type of NSCLC cell lines (HCC827 and H1650) and cells with acquired erlotinib resistance (HCC827-ER and H1650-ER). Our results show that the combination of A485 and TRAIL synergistically increases cell death and inhibits long-term cell proliferation. Furthermore, this combination inhibits the growth of 3D spheroids of EGFR-TKI-resistant cells. Taken together, we demonstrate a successful combination of A485 and TRAIL in EGFR-TKI-sensitive and resistant NSCLC cells.
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http://dx.doi.org/10.1016/j.bcp.2020.113914DOI Listing
May 2020

Immobilized Acylase PvdQ Reduces Biofilm Formation on PDMS Silicone.

Front Chem 2020 5;8:54. Epub 2020 Feb 5.

Chemical and Pharmaceutical Biology Department, University of Groningen, Groningen, Netherlands.

The bacterial biofilm plays a key role in nosocomial infections, especially those related to medical devices in sustained contact with patients. The active dispersion of bacterial cells out of biofilms acts as a reservoir for infectious diseases. The formation of such biofilms is a highly complex process, which is coordinated by many regulatory mechanisms of the pathogen including quorum sensing (QS). Many bacteria coordinate the expression of key virulence factors dependent on their population density through QS. The inhibition of this system is called quorum quenching (QQ). Thus, preventing the development of biofilms is considered a promising approach to prevent the development of hard to treat infections. Enzymatic QQ is the concept of interfering with the QS system of bacteria outside the cell. PvdQ is an acylase with an N-terminal nucleophile (Ntn-hydrolase) that is a part of the pyoverdine gene cluster (). It is able to cleave irreversibly the amide bond of long chain N-acyl homoserine lactones (AHL) rendering them inactive. Long chain AHLs are the main signaling molecule in the QS system of the gram-negative pathogen PA01, which is known for surface-associated biofilms on indwelling catheters and is also the cause of catheter-associated urinary tract infections. Furthermore, PA01 is a well characterized pathogen with respect to QS as well as QQ. In this study, we immobilized the acylase PvdQ on polydimethylsiloxane silicone (PDMS), creating a surface with quorum quenching properties. The goal is to control infections by minimizing the colonization of indwelling medical devices such as urinary catheters or intravascular catheters. The enzyme activity was confirmed by testing the degradation of the main auto-inducer that mediates QS in . In this article we report for the first time a successful immobilization of the quorum quenching acylase PvdQ on PDMS silicone. We could show that immobilized PvdQ retained its activity after the coating procedure and showed a 6-fold reduction of the auto-inducer 3-oxo-C12 in a biosensor setup. Further we report significant reduction of a PA01 biofilm on a coated PDMS surface compared to the same untreated material.
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http://dx.doi.org/10.3389/fchem.2020.00054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012999PMC
February 2020

A Bispecific Inhibitor of the EGFR/ADAM17 Axis Decreases Cell Proliferation and Migration of EGFR-Dependent Cancer Cells.

Cancers (Basel) 2020 02 10;12(2). Epub 2020 Feb 10.

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

Dysregulated epidermal growth factor receptor (EGFR) is an oncogenic driver of many human cancers, promoting aberrant cell proliferation, migration, and survival. Pharmacological targeting of EGFR is often challenged by acquired mechanisms of resistance. Ligand-dependent mechanisms in EGFR wild-type cells rely on ligand or receptor overexpression, allowing cells to outcompete inhibitors and perpetuate signaling in an autocrine manner. Importantly, EGFR ligands are synthesized as membrane-bound precursors that must be solubilized to enable receptor-ligand interactions. The A disintegrin and metalloproteinase 17 (ADAM17) is considered the main sheddase of several EGFR ligands, and a potential pharmacological target. However, its broad substrate range and ubiquitous expression complicate its therapeutic targeting. Here, we present a novel bispecific fusion protein construct consisting of the inhibitory prodomain of ADAM17 (TPD), fused to an EGFR-targeting designed ankyrin repeat protein (DARPin). TPD is a natural inhibitor of ADAM17, maintaining the protease in a zymogen-like form. Meanwhile, the high affinity anti-EGFR DARPin E01 binds to EGFR and inhibits ligand binding. The resulting fusion protein E01-GS-TPD retained binding ability to both molecular targets EGFR and ADAM17. The large difference in affinity for each target resulted in enrichment of the fusion protein in EGFR-positive cells compared to EGFR-negative cells, suggesting a possible application in autocrine signaling inhibition. Accordingly, E01-GS-TPD decreased migration and proliferation of EGFR-dependent cell lines with no significant increase in apoptotic cell death. Finally, inhibition of proliferation was observed through EGFR ligand-dependent mechanisms as growth inhibition was not observed in EGFR mutant or KRAS mutant cell lines. The use of bispecific proteins targeting the EGFR/ADAM17 axis could be an innovative strategy for the treatment of EGFR-dependent cancers.
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http://dx.doi.org/10.3390/cancers12020411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072247PMC
February 2020

A novel mechanism of inhibition by phenylthiourea on PvdP, a tyrosinase synthesizing pyoverdine of Pseudomonas aeruginosa.

Int J Biol Macromol 2020 Mar 30;146:212-221. Epub 2019 Dec 30.

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

The biosynthesis of pyoverdine, the major siderophore of Pseudomonas aeruginosa, is a well-organized process involving a discrete number of enzyme-catalyzed steps. The final step of this process involves the PvdP tyrosinase, which converts ferribactin into pyoverdine. Thus, inhibition of the PvdP tyrosinase activity provides an attractive strategy to interfere with siderophore synthesis to manage P. aeruginosa infections. Here, we report phenylthiourea as a non-competitive inhibitor of PvdP for which we solved a crystal structure in complex with PvdP. The crystal structure indicates that phenylthiourea binds to an allosteric binding site and thereby interferes with its tyrosinase activity. We further provide proofs that PvdP tyrosinase inhibition by phenylthiourea requires the C-terminal lid region. This provides opportunities to develop inhibitors that target the allosteric site, which seems to be confined to fluorescent pseudomonads, and not the tyrosinase active site. Furthermore, increases the chances to identify PvdP inhibitors that selectively interfere with siderophore synthesis.
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http://dx.doi.org/10.1016/j.ijbiomac.2019.12.252DOI Listing
March 2020

A regulated synthetic operon facilitates stable overexpression of multigene terpenoid pathway in Bacillus subtilis.

J Ind Microbiol Biotechnol 2020 Feb 1;47(2):243-249. Epub 2020 Jan 1.

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

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 C 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 C carotenoid production.
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http://dx.doi.org/10.1007/s10295-019-02257-4DOI Listing
February 2020

Antifungal and biofilm inhibitory effect of Cymbopogon citratus (lemongrass) essential oil on biofilm forming by Candida tropicalis isolates; an in vitro study.

J Ethnopharmacol 2020 Jan 27;246:112188. Epub 2019 Aug 27.

Hacettepe University, Faculty of Sciences, Department of Biology (Biotechnology Division) Beytepe, 06800, Ankara, Turkey. Electronic address:

Ethnopharmacological Relevance: Cymbopogon citratus (lemongrass) essential oil has been widely used as a traditional medicine and is well known for antimicrobial properties. Therefore, it might be a potent anti-infective and biofilm inhibitive against Candida tropicalis infections. Until now, no ideal coating or cleaning method based on an essential oil has been described to prevent biofilm formation of Candida strains on silicone rubber maxillofacial prostheses, voice prostheses and medical devices susceptible to C. tropicalis infections.

Aim Of The Study: To investigate the antifungal and biofilm inhibitory effects of Cymbopogon citratus oil. Clinical isolates of C. tropicalis biofilms on different biomaterials were used to study the inhibitory effect.

Materials And Methods: The efficacy of Cymbopogon citratus, Cuminum cyminum, Citrus limon and Cinnamomum verum essential oils were compared on biofilm formation of three C. tropicalis isolates on 24 well polystyrene plates. C. citratus oil coated silicone rubber surfaces were prepared using hypromellose ointment as a vehicle. The antifungal tests to determine minimum inhibitory and minimum fungicidal concentrations were assessed by a microbroth dilution method and biofilm formation was determined by a crystal violet binding assay.

Results: C. tropicalis strains formed more biofilm on hydrophobic materials than on hydrophilic glass. C. citratus oil showed a high antifungal effect against all C. tropicalis strains. For comparison, C. limon oil and C. cyminum oil showed minor to no killing effect against the C. tropicalis strains. C. citratus oil had the lowest minimal inhibitory concentration of all essential oils tested and inhibited biofilm formation of all C. tropicalis strains. C. citratus oil coating on silicone rubber resulted in a 45-76% reduction in biofilm formation of all C. tropicalis strains.

Conclusion: Cymbopogon citratus oil has good potential to be used as an antifungal and antibiofilm agent on silicone rubber prostheses and medical devices on which C. tropicalis biofilms pose a serious risk for skin infections and may cause a shorter lifespan of the prosthesis.
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http://dx.doi.org/10.1016/j.jep.2019.112188DOI Listing
January 2020

Inhibitory selectivity among class I HDACs has a major impact on inflammatory gene expression in macrophages.

Eur J Med Chem 2019 Sep 18;177:457-466. Epub 2019 May 18.

Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy (GRIP), University of Groningen, Groningen, 9713 AV, the Netherlands. Electronic address:

Histone deacetylases (HDACs) play an important role in cancer, degenerative diseases and inflammation. The currently applied HDAC inhibitors in the clinic lack selectivity among HDAC isoforms, which limits their application for novel indications such as inflammatory diseases. Recent, literature indicates that HDAC 3 plays an important role among class I HDACs in gene expression in inflammation. In this perspective, the development and understanding of inhibitory selectivity among HDACs 1, 2 and 3 and their respective influence on gene expression need to be characterized to facilitate drug discovery. Towards this aim, we synthesized nine structural analogues of the class I HDAC inhibitor Entinostat and investigated their selectivity profile among HDACs 1, 2 and 3. We found that we can explain the observed structure activity relationships by small structural and conformational differences between HDAC 1 and HDAC 3 in the 'lid' interacting region. Cell-based studies indicated, however, that application of inhibitors with improved HDAC 3 selectivity did not provide an anti-inflammatory response in contrast to expectations from biochemical evidence in literature. Altogether, in this study, we identified structure activity relationships among class I HDACs and we connected isoform selectivity among class I HDACs with pro- and anti-inflammatory gene transcription in macrophages.
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http://dx.doi.org/10.1016/j.ejmech.2019.05.038DOI Listing
September 2019

Regulation of Survival Networks in Senescent Cells: From Mechanisms to Interventions.

J Mol Biol 2019 07 31;431(15):2629-2643. Epub 2019 May 31.

European Institute for the Biology of Aging (ERIBA), University Medical Center Groningen (UMCG), University of Groningen, Groningen, the Netherlands. Electronic address:

Cellular senescence is a state of stable cell cycle arrest arising in response to DNA and mitochondrial damages. Senescent cells undergo morphological, structural and functional changes that are influenced by a number of variables, including time, stress, tissue, and cell type. The heterogeneity of the senescent phenotype is exemplified by the many biological properties that senescent cells can cover. The advent of innovative model organisms has demonstrated a functional role of senescent cells during embryogenesis, tissue remodeling, tumorigenesis and aging. Importantly, prolonged and aberrant persistence of senescent cells is often associated with tissue dysfunction and pathology, and is partially the consequence of mechanisms that enhance survival and resistance to cell death. Here, we describe the main molecular players involved in promoting survival of senescent cells, with particular emphasis on the regulation of senescence-associated anti-apoptotic pathways. We discuss the consequences these pathways have in providing resistance to intrinsic and extrinsic pro-apoptotic signals. Finally, we highlight the importance of these pathways in the development of targets for senolytic interventions.
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http://dx.doi.org/10.1016/j.jmb.2019.05.036DOI Listing
July 2019

Exoproteome Heterogeneity among Closely Related Staphylococcus aureus t437 Isolates and Possible Implications for Virulence.

J Proteome Res 2019 07 5;18(7):2859-2874. Epub 2019 Jun 5.

University of Groningen , University Medical Center Groningen, Department of Medical Microbiology , Hanzeplein 1 , P.O. Box 30001, 9700 RB Groningen , The Netherlands.

Staphylococcus aureus with spa-type t437 has been identified as a predominant community-associated methicillin-resistant S. aureus clone from Asia, which is also encountered in Europe. Molecular typing has previously shown that t437 isolates are highly similar regardless of geographical regions or host environments. The present study was aimed at assessing to what extent this high similarity is actually reflected in the production of secreted virulence factors. We therefore profiled the extracellular proteome, representing the main reservoir of virulence factors, of 20 representative clinical isolates by mass spectrometry. The results show that these isolates can be divided into three groups and nine subgroups based on exoproteome abundance signatures. This implies that S. aureus t437 isolates show substantial exoproteome heterogeneity. Nonetheless, 30 highly conserved extracellular proteins, of which about 50% have a predicted role in pathogenesis, were dominantly identified. To approximate the virulence of the 20 investigated isolates, we employed infection models based on Galleria mellonella and HeLa cells. The results show that the grouping of clinical isolates based on their exoproteome profile can be related to virulence. We consider this outcome important as our approach provides a tool to pinpoint differences in virulence among seemingly highly similar clinical isolates of S. aureus.
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http://dx.doi.org/10.1021/acs.jproteome.9b00179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6617432PMC
July 2019

Histone Deacetylase Inhibitors Sensitize TRAIL-Induced Apoptosis in Colon Cancer Cells.

Cancers (Basel) 2019 May 10;11(5). Epub 2019 May 10.

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

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered as a promising anti-cancer therapeutic. However, many cancers have been found to be or to become inherently resistant to TRAIL. A combination of epigenetic modifiers, such as histone deacetylase inhibitors (HDACi's), with TRAIL was effective to overcome TRAIL resistance in some cancers. Broad spectrum HDACi's, however, show considerable toxicity constraining clinical use. Since overexpression of class I histone deacetylase (HDAC) has been found in colon tumors relative to normal mucosa, we have focused on small spectrum HDACi's. We have now tested agonistic receptor-specific TRAIL variants rhTRAIL 4C7 and DHER in combination with several class I specific HDACi's on TRAIL-resistant colon cancer cells DLD-1 and WiDr. Our data show that TRAIL-mediated apoptosis is largely improved in WiDr cells by pre-incubation with Entinostat-a HDAC1, 2, and 3 inhibitor- and in DLD-1 cells by RGFP966-a HDAC3-specific inhibitor- or PCI34051-a HDAC8-specific inhibitor. We are the first to report that using RGFP966 or PCI34051 in combination with rhTRAIL 4C7 or DHER represents an effective cancer therapy. The intricate relation of HDACs and TRAIL-induced apoptosis was confirmed in cells by knockdown of , , or gene expression, which showed more early apoptotic cells upon adding rhTRAIL 4C7 or DHER. We observed that RGFP966 and PCI34051 increased DR4 expression after incubation on DLD-1 cells, while RGFP966 induced more DR5 expression on WiDr cells, indicating a different role for DR4 or DR5 in these combinations. At last, we show that combined treatment of RGFP966 with TRAIL variants (rhTRAIL 4C7/DHER) increases apoptosis on 3D tumor spheroid models.
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http://dx.doi.org/10.3390/cancers11050645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6562715PMC
May 2019

Creation of RANKL mutants with low affinity for decoy receptor OPG and their potential anti-fibrosis activity.

FEBS J 2019 09 25;286(18):3582-3593. Epub 2019 May 25.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, The Netherlands.

Fibrosis is characterized by the progressive alteration of the tissue structure due to the excessive production of extracellular matrix (ECM). The signaling system encompassing Receptor Activator of Nuclear factor NF-κB Ligand (RANKL)/RANK/Osteoprotegerin (OPG) was discovered to play an important role in the regulation of ECM formation and degradation in bone tissue. However, whether and how this signaling pathway plays a role in liver or pulmonary ECM degradation is unclear up to now. Interestingly, increased decoy receptor OPG levels are found in fibrotic tissues. We hypothesize that RANKL can stimulate RANK on macrophages and initiate the process of ECM degradation. This process may be inhibited by highly expressed OPG in fibrotic conditions. In this case, RANKL mutants that can bind to RANK without binding to OPG might become promising therapeutic candidates. In this study, we built a structure-based library containing 44 RANKL mutants and found that the Q236 residue of RANKL is important for OPG binding. We show that RANKL_Q236D can activate RAW cells to initiate the process of ECM degradation and is able to escape from the obstruction by exogenous OPG. We propose that the generation of RANKL mutants with reduced affinity for OPG is a promising strategy for the exploration of new therapeutics against fibrosis.
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http://dx.doi.org/10.1111/febs.14925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6852375PMC
September 2019

Novel 15-Lipoxygenase-1 Inhibitor Protects Macrophages from Lipopolysaccharide-Induced Cytotoxicity.

J Med Chem 2019 05 19;62(9):4624-4637. Epub 2019 Apr 19.

Department of Chemical Biology 2, Stratingh Institute for Chemistry , University of Groningen , Nijenborgh 7 , 9747 AG Groningen , The Netherlands.

Various mechanisms for regulated cell death include the formation of oxidative mediators such as lipid peroxides and nitric oxide (NO). In this respect, 15-lipoxygenase-1 (15-LOX-1) is a key enzyme that catalyzes the formation of lipid peroxides. The actions of these peroxides are interconnected with nuclear factor-κB signaling and NO production. Inhibition of 15-LOX-1 holds promise to interfere with regulated cell death in inflammatory conditions. In this study, a novel potent 15-LOX-1 inhibitor, 9c (i472), was developed and structure-activity relationships were explored. In vitro, this inhibitor protected cells from lipopolysaccharide-induced cell death, inhibiting NO formation and lipid peroxidation. Thus, we provide a novel 15-LOX-1 inhibitor that inhibits cellular NO production and lipid peroxidation, which set the stage for further exploration of these mechanisms.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6511944PMC
May 2019

Metabolic Engineering of Toward Taxadiene Biosynthesis as the First Committed Step for Taxol Production.

Front Microbiol 2019 20;10:218. Epub 2019 Feb 20.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Groningen, Netherlands.

Terpenoids are natural products known for their medicinal and commercial applications. Metabolic engineering of microbial hosts for the production of valuable compounds, such as artemisinin and Taxol, has gained vast interest in the last few decades. The Generally Regarded As Safe (GRAS) 168 with its broad metabolic potential is considered one of these interesting microbial hosts. In the effort toward engineering as a cell factory for the production of the chemotherapeutic Taxol, we expressed the plant-derived taxadiene synthase (TXS) enzyme. TXS is responsible for the conversion of the precursor geranylgeranyl pyrophosphate (GGPP) to taxa-4,11-diene, which is the first committed intermediate in Taxol biosynthesis. Furthermore, overexpression of eight enzymes in the biosynthesis pathway was performed to increase the flux of the GGPP precursor. This was achieved by creating a synthetic operon harboring the genes encoding the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway (, , , , , , ) together with (encoding geranyl and farnesyl pyrophosphate synthases) responsible for providing farnesyl pyrophosphate (FPP). In addition, a vector harboring the gene (encoding geranylgeranyl pyrophosphate synthase, GGPPS, of ) to increase the supply of GGPP was introduced. The overexpression of the MEP pathway enzymes along with IspA and GGPPS caused an 83-fold increase in the amount of taxadiene produced compared to the strain only expressing TXS and relying on the innate pathway of . The total amount of taxadiene produced by that strain was 17.8 mg/l. This is the first account of the successful expression of taxadiene synthase in . We determined that the expression of GGPPS through the gene is essential for the formation of sufficient precursor, GGPP, in as its innate metabolism is not efficient in producing it. Finally, the extracellular localization of taxadiene production by overexpressing the complete MEP pathway along with IspA and GGPPS presents the prospect for further engineering aiming for semisynthesis of Taxol.
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http://dx.doi.org/10.3389/fmicb.2019.00218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391936PMC
February 2019

Death receptor 5 is activated by fucosylation in colon cancer cells.

FEBS J 2019 02 14;286(3):555-571. Epub 2019 Jan 14.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, The Netherlands.

The remarkable pro-apoptotic properties of tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) have led to considerable interest in this protein as a potential anticancer therapeutic. However, TRAIL is largely ineffective in inducing apoptosis in certain cancer cells, and the mechanisms underlying this selectivity are unknown. In colon adenocarcinomas, posttranslational modifications including O- and N- glycosylation of death receptors were found to correlate with TRAIL-induced apoptosis. Additionally, mRNA levels of fucosyltransferase 3 (FUT3) and 6 (FUT6) were found to be high in the TRAIL-sensitive colon adenocarcinoma cell line COLO 205. In this study, we use agonistic receptor-specific TRAIL variants to dissect the contribution of FUT3 and FUT6-mediated fucosylation to TRAIL-induced apoptosis via its two death receptors, DR4 and DR5. Triggering of apoptosis by TRAIL revealed that the low FUT3/6-expressing cells DLD-1 and HCT 116 are insensitive to DR5 but not to DR4-mediated apoptosis. By contrast, efficient apoptosis is mediated via both receptors in high FUT3/6-expressing COLO 205 cells. The reconstitution of FUT3/6 expression in DR5-resistant cells completely restored TRAIL sensitivity via this receptor, while only marginally enhancing apoptosis via DR4 at lower TRAIL concentrations. Interestingly, we observed that induction of the salvage pathway by external administration of l-fucose restores DR5-mediated apoptosis in both DLD-1 and HCT 116 cells. Finally, we show that fucosylation influences the ligand-independent receptor association that leads to increased death inducing signalling complex (DISC) formation and caspase-8 activation. Taken together, these results provide evidence for the differential impact of fucosylation on signalling via DR4 or DR5. These findings provide novel opportunities to enhance TRAIL sensitivity in colon adenocarcinoma cells that are highly resistant to DR5-mediated apoptosis.
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http://dx.doi.org/10.1111/febs.14742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6849799PMC
February 2019

Catalysis of amorpha-4,11-diene synthase unraveled and improved by mutability landscape guided engineering.

Sci Rep 2018 07 2;8(1):9961. Epub 2018 Jul 2.

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

Amorpha-4,11-diene synthase (ADS) cyclizes the substrate farnesyl pyrophosphate to produce amorpha-4,11-diene as a major product. This is considered the first committed and rate-limiting step in the biosynthesis of the antimalarial artemisinin. Here, we utilize a reported 3D model of ADS to perform mutability landscape guided enzyme engineering. A mutant library of 258 variants along sixteen active site residues was created then screened for catalytic activity and product profile. This allowed for identification of the role of some of these residues in the mechanism. R262 constrains the released pyrophosphate group along with magnesium ions. The aromatic residues (W271, Y519 and F525) stabilize the intermediate carbocations while T296, G400, G439 and L515 help with the 1,6- and 1,10-ring closures. Finally, W271 is suggested to act as active site base along with T399, which ensures regioselective deprotonation. The mutability landscape also helped determine variants with improved catalytic activity. H448A showed ~4 fold increase in catalytic efficiency and the double mutation T399S/H448A improved k by 5 times. This variant can be used to enhance amorphadiene production and in turn artemisinin biosynthesis. Our findings provide the basis for the first step in improving industrial production of artemisinin and they open up possibilities for further engineering and understanding of ADS.
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http://dx.doi.org/10.1038/s41598-018-28177-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028579PMC
July 2018

PvdQ Quorum Quenching Acylase Attenuates Virulence in a Mouse Model of Pulmonary Infection.

Front Cell Infect Microbiol 2018 26;8:119. Epub 2018 Apr 26.

Department of Chemical and Pharmaceutical Biology, University of Groningen, Groningen, Netherlands.

is the predominant pathogen in pulmonary infections associated with cystic fibrosis. Quorum sensing (QS) systems regulate the production of virulence factors and play an important role in the establishment of successful infections. Inhibition of the QS system (termed quorum quenching) renders the bacteria avirulent thus serving as an alternative approach in the development of novel antibiotics. Quorum quenching in Gram negative bacteria can be achieved by preventing the accumulation of -acyl homoserine lactone (AHL) signaling molecule via enzymatic degradation. Previous work by us has shown that PvdQ acylase hydrolyzes AHL signaling molecules irreversibly, thereby inhibiting QS in and in a model of infection. The aim of the present study is to assess the therapeutic efficacy of intranasally instilled PvdQ acylase in a mouse model of pulmonary infection. First, we evaluated the deposition pattern of intranasally administered fluorochrome-tagged PvdQ (PvdQ-VT) in mice at different stages of pulmonary infection by imaging studies. Following intranasal instillation, PvdQ-VT could be traced in all lung lobes with 42 ± 7.5% of the delivered dose being deposited at 0 h post-bacterial-infection, and 34 ± 5.2% at 72 h post bacterial-infection. We then treated mice with PvdQ during lethal pulmonary infection and that resulted in a 5-fold reduction of lung bacterial load and a prolonged survival of the infected animals with the median survival time of 57 hin comparison to 42 h for the PBS-treated group. In a sublethal pulmonary infection, PvdQ treatment resulted in less lung inflammation as well as decrease of CXCL2 and TNF-α levels at 24 h post-bacterial-infection by 15 and 20%, respectively. In conclusion, our study has shown therapeutic efficacy of PvdQ acylase as a quorum quenching agent during infection.
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http://dx.doi.org/10.3389/fcimb.2018.00119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5932173PMC
April 2019

A nonalcoholic fatty liver disease cirrhosis model in gerbil: the dynamic relationship between hepatic lipid metabolism and cirrhosis.

Int J Clin Exp Pathol 2018 1;11(1):146-157. Epub 2018 Jan 1.

Laboratory Animal Center, Zhejiang Academy of Medical Sciences Hangzhou, Zhejiang, China.

Nonalcoholic fatty liver disease (NAFLD) usually takes decades to develop into cirrhosis, which limits the longitudinal study of NAFLD. This work aims at developing a NAFLD-caused cirrhosis model in gerbil and examining the dynamic relationship between hepatic lipid metabolism and cirrhosis. We fed gerbil a high-fat and high-cholesterol diet (HFHCD) for 24 weeks, and recorded the gerbil's phenotype at 3, 6, 9, 12, 15, 18, 21, 24 weeks. The model's pathological process, lipid metabolism, oxidative stress, liver collagen deposition and presence of relevant cytokines were tested and evaluated during the full-time frame of disease onset. The gerbil model can induce non-alcoholic steatohepatitis (NASH) within 9 weeks, and can develop cirrhosis after 21 weeks induction. The model's lipids metabolism disorder is accompanied with the liver damage development. During the NAFLD progression, triglycerides (TG) and free fatty acids (FFA) have presented distinct rise and fall tendency, and the turning points are at the fibrosis stage. Besides that, the ratios of total cholesterol (CHO) to high-density lipoprotein cholesterol (HDL-C) exhibited constant growth tendency, and have a good linear relationship with hepatic stellate cells (HSC) (R = 0.802, < 0.001). The gerbil NAFLD cirrhosis model has been developed and possesses positive correlation between lipids metabolism and cirrhosis. The compelling rise and fall tendency of TG and FFA indicated that the fibrosis progression can lead to impairment in lipoprotein synthesis and engender decreased TG level. CHO/HDL-C ratios can imply the fibrosis progress and be used as a blood indicator for disease prediction and prevention.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6957964PMC
January 2018

Cytotoxic Deoxypodophyllotoxin Can Be Extracted in High Purity from Anthriscus sylvestris Roots by Supercritical Carbon Dioxide.

Planta Med 2018 May 18;84(8):544-550. Epub 2017 Dec 18.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Groningen, Netherlands.

Deoxypodophyllotoxin is present in the roots of . This compound is cytotoxic on its own, but it can also be converted into podophyllotoxin, which is in high demand as a precursor for the important anticancer drugs etoposide and teniposide. In this study, deoxypodophyllotoxin is extracted from roots by supercritical carbon dioxide extraction. The process is simple and scalable. The supercritical carbon dioxide method extracts 75 - 80% of the total deoxypodophyllotoxin content, which is comparable to a single extraction by traditional Soxhlet. However, less polar components are extracted. The activity of the supercritical carbon dioxide extract containing deoxypodophyllotoxin was assessed by demonstrating that the extract arrests A549 and HeLa cells in the G/M phase of the cell cycle. We conclude that biologically active deoxypodophyllotoxin can be extracted from by supercritical carbon dioxide extraction. The method is solvent free and more sustainable compared to traditional methods.
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http://dx.doi.org/10.1055/s-0043-123938DOI Listing
May 2018
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