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1.
Pharmacol Res ; 185: 106510, 2022 11.
Article in English | MEDLINE | ID: mdl-36252775

ABSTRACT

Glioblastoma multiforme (GBM) is the most common malignant brain tumor with limited therapeutic options. Besides surgery, chemotherapy using temozolomide, carmustine or lomustine is the main pillar of therapy. However, therapy success is limited and prognosis still is very poor. One restraining factor is drug resistance caused by drug transporters of the ATP-binding cassette family, e.g. ABCB1 and ABCG2, located at the blood-brain barrier and on tumor cells. The active efflux of xenobiotics including drugs, e.g. temozolomide, leads to low intracellular drug concentrations and subsequently insufficient anti-tumor effects. Nevertheless, the role of efflux transporters in GBM is controversially discussed. In the present study, we analyzed the role of ABCB1 and ABCG2 in GBM cells showing that ABCB1, but marginally ABCG2, is relevant. Applying a CRISPR/Cas9-derived ABCB1 knockout, the response to temozolomide was significantly augmented demonstrated by decreased cell number (p < 0.001) and proliferation rate (p = 0.04), while apoptosis was increased (p = 0.04). For carmustine, a decrease of cells in G1-phase was detected pointing to cell cycle arrest in the ABCB1 knockout (p = 0.006). For lomustine, however, loss of ABCB1 did not alter the response to the treatment. Overall, this study shows that ABCB1 is involved in the active transport of temozolomide out of the tumor cells diminishing the response to temozolomide. Interestingly, loss of ABCB1 also affected the response to the lipophilic drug carmustine. These findings show that ABCB1 is not only relevant at the blood-brain barrier, but also in the tumor cells diminishing success of chemotherapy.


Subject(s)
Glioblastoma , Humans , Temozolomide/pharmacology , Temozolomide/therapeutic use , Glioblastoma/drug therapy , Glioblastoma/genetics , Glioblastoma/pathology , Carmustine/pharmacology , Carmustine/therapeutic use , ATP Binding Cassette Transporter, Subfamily G, Member 2/metabolism , Lomustine/therapeutic use , Lomustine/pharmacology , CRISPR-Cas Systems , ATP-Binding Cassette Transporters/metabolism , Neoplasm Proteins/metabolism , Cell Line, Tumor , Drug Resistance, Neoplasm , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/metabolism
2.
Org Lett ; 23(6): 2024-2028, 2021 03 19.
Article in English | MEDLINE | ID: mdl-33656898

ABSTRACT

Oxepinamides are fungal oxepine-pyrimidinone-ketopiperazine derivatives. In this study, we elucidated the biosynthetic pathway of oxepinamide D in Aspergillus ustus by gene deletion, heterologous expression, feeding experiments, and enzyme assays. We demonstrated that the cytochrome P450 enzymes catalyzed highly specific and stereoselective oxepin ring formation.


Subject(s)
Aspergillus/metabolism , Cytochrome P-450 Enzyme System/chemistry , Fungi/chemistry , Oxepins/chemistry , Aspergillus/chemistry , Biosynthetic Pathways , Catalysis , Cytochrome P-450 Enzyme System/metabolism , Enzyme Assays , Fungi/metabolism , Molecular Structure
3.
Org Biomol Chem ; 18(26): 4946-4948, 2020 07 08.
Article in English | MEDLINE | ID: mdl-32588866

ABSTRACT

Heterologous expression has been proven to be a successful strategy for the identification of metabolites encoded by cryptic/silent genes. Expression of a nonreducing polyketide synthase (NR-PKS) gene from Penicillium crustosum in Aspergillus nidulans led to the accumulation of three isocoumarins 1-3. Feeding experiments revealed that the PKS product 1 can be converted by the host enzymes to its hydroxylated (2) and methylated (3) derivatives. These results provided one additional example that unexpected further modifications of an enzyme product can take place in a heterologous host.


Subject(s)
Gene Expression Regulation, Enzymologic/genetics , Isocoumarins/metabolism , Polyketide Synthases/genetics , Polyketide Synthases/metabolism , Aspergillus nidulans/enzymology , Isocoumarins/chemistry , Penicillium/enzymology
4.
Org Lett ; 22(1): 88-92, 2020 01 03.
Article in English | MEDLINE | ID: mdl-31833773

ABSTRACT

Crustosic acid (1) differs from terrestric acid (2) by a 5ß-carboxylmethyl at the tetronate ring instead of a 5α-methyl group in Penicillium crustosum. The formation of 1 via carboxylcrustic and viridicatic acid was confirmed by gene deletion and heterologous expression. The conversion of 1 to 2 requires a decarboxylation-mediated olefination by TraH and subsequent reduction by TraD. The redox-assisted decarboxylation and stereoisomerization proved the biosynthetic relationships of fungal acyltetronates with different stereochemistry.

5.
J Org Chem ; 85(2): 1298-1307, 2020 01 17.
Article in English | MEDLINE | ID: mdl-31860310

ABSTRACT

The active form of clavatol, ortho-quinone methide, can be generated from hydroxyclavatol in an aqueous system and used as a highly reactive intermediate for coupling with diverse natural products under very mild conditions. These include flavonoids, hydroxynaphthalenes, coumarins, xanthones, anthraquinones, phloroglucinols, phenolic acids, indole derivatives, tyrosine analogues, and quinolines. The clavatol moiety was mainly attached via C-C bonds to the ortho- or para-positions of phenolic hydroxyl/amino groups and the C2-position of the indole ring.

6.
J Am Chem Soc ; 141(10): 4225-4229, 2019 03 13.
Article in English | MEDLINE | ID: mdl-30811183

ABSTRACT

Penilactones A and B consist of a γ-butyrolactone and two clavatol moieties. We identified two separate gene clusters for the biosynthesis of these key building blocks in Penicillium crustosum. Gene deletion, feeding experiments, and biochemical investigations proved that a nonreducing PKS ClaF is responsible for the formation of clavatol and the PKS-NRPS hybrid TraA is involved in the formation of crustosic acid, which undergoes decarboxylation and isomerization to the predominant terrestric acid. Both acids are proposed to be converted to γ-butyrolactones with involvement of a cytochrome P450 ClaJ. Oxidation of clavatol to hydroxyclavatol by a nonheme FeII/2-oxoglutarate-dependent oxygenase ClaD and its spontaneous dehydration to an ortho-quinone methide initiate the two nonenzymatic 1,4-Michael addition steps. Spontaneous addition of the methide to the γ-butyrolactones led to peniphenone D and penilactone D, which undergo again stereospecific attacking by methide to give penilactones A/B.

7.
ACS Synth Biol ; 6(6): 1056-1064, 2017 06 16.
Article in English | MEDLINE | ID: mdl-28221769

ABSTRACT

The tryptophan derivative 1-methyl-1,2,3,4-tetrahydro-ß-carboline-3-carboxylic acid (MTCA) is present in many plants and foods including fermentation products of the baker's yeast Saccharomyces cerevisiae. MTCA is formed from tryptophan and acetaldehyde via a Pictet-Spengler reaction. In this study, up to 9 mg/L of MTCA were detected as a mixture of (1S,3S) and (1R,3S) isomers in a ratio of 2.2:1 in Saccharomyces cerevisiae cultures. To the best of our knowledge, this is the first report on the presence of MTCA in laboratory baker's yeast cultures. Expression of three fungal tryptophan prenyltransferase genes, fgaPT2, 5-dmats, and 7-dmats in S. cerevisiae resulted in the formation of MTCA derivatives with prenyl moieties at different positions of the indole ring. Expression of these genes in dimethylallyl diphosphate and tryptophan overproducing strains led to generation of up to 400 mg/L of prenylated MTCAs as mixtures of (1S,3S) and (1R,3S) diastereomers in ratios similar to that of unprenylated MTCA. The structures of the described substances including their stereochemistry were unequivocally elucidated by mass spectrometry as well as one- and two-dimensional NMR spectroscopy. The results of this study provide a convenient system for the production of high amounts of designed prenylated MTCAs in S. cerevisiae. Furthermore, our work can be considered as an excellent example for the construction of more complex molecules by introducing just one key gene.


Subject(s)
Carbolines/metabolism , Dimethylallyltranstransferase/metabolism , Saccharomyces cerevisiae/metabolism , Carbolines/analysis , Cell Culture Techniques , Metabolic Engineering , Prenylation , Tryptophan/metabolism
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