| 1. |
- Costeira-Paulo, Joana, et al.
(författare)
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Lipids Shape the Electron Acceptor-Binding Site of the Peripheral Membrane Protein Dihydroorotate Dehydrogenase
- 2018
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Ingår i: Cell Chemical Biology. - : Elsevier BV. - 2451-9456 .- 2451-9448. ; 25:3, s. 309-317
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Tidskriftsartikel (refereegranskat)abstract
- The interactions between proteins and biological membranes are important for drug development, but remain notoriously refractory to structural investigation. We combine non-denaturing mass spectrometry (MS) with molecular dynamics (MD) simulations to unravel the connections among co-factor, lipid, and inhibitor binding in the peripheral membrane protein dihydroorotate dehydrogenase (DHODH), a key anticancer target. Interrogation of intact DHODH complexes by MS reveals that phospholipids bind via their charged head groups at a limited number of sites, while binding of the inhibitor brequinar involves simultaneous association with detergent molecules. MD simulations show that lipids support flexible segments in the membrane-binding domain and position the inhibitor and electron acceptor-binding site away from the membrane surface, similar to the electron acceptor-binding site in respiratory chain complex I. By complementing MS with MD simulations, we demonstrate how a peripheral membrane protein uses lipids to modulate its structure in a similar manner as integral membrane proteins.
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| 2. |
- Ladds, Marcus J. G. W., et al.
(författare)
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A DHODH inhibitor increases p53 synthesis and enhances tumor cell killing by p53 degradation blockage
- 2018
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- The development of non-genotoxic therapies that activate wild-type p53 in tumors is of great interest since the discovery of p53 as a tumor suppressor. Here we report the identification of over 100 small-molecules activating p53 in cells. We elucidate the mechanism of action of a chiral tetrahydroindazole (HZ00), and through target deconvolution, we deduce that its active enantiomer (R)-HZ00, inhibits dihydroorotate dehydrogenase (DHODH). The chiral specificity of HZ05, a more potent analog, is revealed by the crystal structure of the (R)-HZ05/DHODH complex. Twelve other DHODH inhibitor chemotypes are detailed among the p53 activators, which identifies DHODH as a frequent target for structurally diverse compounds. We observe that HZ compounds accumulate cancer cells in S-phase, increase p53 synthesis, and synergize with an inhibitor of p53 degradation to reduce tumor growth in vivo. We, therefore, propose a strategy to promote cancer cell killing by p53 instead of its reversible cell cycle arresting effect.
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| 3. |
- Ladds, Marcus J. G. W., et al.
(författare)
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Autophagic flux blockage by accumulation of weakly basic tenovins leads to elimination of B-Raf mutant tumour cells that survive vemurafenib
- 2018
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 13:4
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Tidskriftsartikel (refereegranskat)abstract
- Tenovin-6 is the most studied member of a family of small molecules with antitumour activity in vivo. Previously, it has been determined that part of the effects of tenovin-6 associate with its ability to inhibit SirT1 and activate p53. However, tenovin-6 has also been shown to modulate autophagic flux. Here we show that blockage of autophagic flux occurs in a variety of cell lines in response to certain tenovins, that autophagy blockage occurs regardless of the effect of tenovins on SirT1 or p53, and that this blockage is dependent on the aliphatic tertiary amine side chain of these molecules. Additionally, we evaluate the contribution of this tertiary amine to the elimination of proliferating melanoma cells in culture. We also demonstrate that the presence of the tertiary amine is sufficient to lead to death of tumour cells arrested in G1 phase following vemurafenib treatment. We conclude that blockage of autophagic flux by tenovins is necessary to eliminate melanoma cells that survive B-Raf inhibition and achieve total tumour cell kill and that autophagy blockage can be achieved at a lower concentration than by chloroquine. This observation is of great relevance as relapse and resistance are frequently observed in cancer patients treated with B-Raf inhibitors.
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| 4. |
- Larsson, Ida, et al.
(författare)
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Reconstructing the regulatory programs underlying the phenotypic plasticity of neural cancers
- 2024
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Nervous system cancers contain a large spectrum of transcriptional cell states, reflecting processes active during normal development, injury response and growth. However, we lack a good understanding of these states' regulation and pharmacological importance. Here, we describe the integrated reconstruction of such cellular regulatory programs and their therapeutic targets from extensive collections of single-cell RNA sequencing data (scRNA-seq) from both tumors and developing tissues. Our method, termed single-cell Regulatory-driven Clustering (scRegClust), predicts essential kinases and transcription factors in little computational time thanks to a new efficient optimization strategy. Using this method, we analyze scRNA-seq data from both adult and childhood brain cancers to identify transcription factors and kinases that regulate distinct tumor cell states. In adult glioblastoma, our model predicts that blocking the activity of PDGFRA, DDR1, ERBB3 or SOX6, or increasing YBX1-activity, would potentiate temozolomide treatment. We further perform an integrative study of scRNA-seq data from both cancer and the developing brain to uncover the regulation of emerging meta-modules. We find a meta-module regulated by the transcription factors SPI1 and IRF8 and link it to an immune-mediated mesenchymal-like state. Our algorithm is available as an easy-to-use R package and companion visualization tool that help uncover the regulatory programs underlying cell plasticity in cancer and other diseases.
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| 5. |
- Larsson, Ida, et al.
(författare)
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Using drug-induced cell states to build therapeutic combinations against nervous system cancers
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Evidence is amounting that nervous system cancers are heterogeneous at the single cell level, yet data are currently scarce on how therapeutic agents affect this heterogeneity. Here, we describe a new, data-driven strategy to identify drugs that modulate the intratumoral heterogeneity of nervous system cancers. First, we demonstrate that drugs elicit structured changes in pathway activation in patient-derived cells from glioblastomas, neuroblastomas and medulloblastomas. Second, we present a mathematical model to estimate how drugs induce changes in tumor heterogeneity, as defined by single cell RNA sequencing atlases of each disease. Finally, as an evaluation of our method we use it to identify candidate synergistic drug pairs based on the drugs' effects on intratumoral heterogeneity.
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| 6. |
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| 7. |
- Popova, Gergana
(författare)
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Dihydroorotate dehydrogenase : new insights into an old target
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Purine and pyrimidine nucleotides are important for both biosynthetic and regulatory pathways in cells. As constituents of the DNA and RNA molecules, the requirements for nucleotides in actively dividing cells are increased. Therefore, the enzymes from the purine and pyrimidine nucleotide synthesis pathways are considered attractive targets for treatment of diseases associated with deregulated cell proliferation, such as cancer. Dihydroorotate dehydrogenase (DHODH), an enzyme from the de novo pyrimidine ribonucleotide synthesis pathway located on the inner mitochondrial membrane, has been intensively studied in the past few years as a target for anti-cancer therapy. As a result, several inhibitors of DHODH are currently in clinical trials for treatment of myeloid malignancies. In a screen for p53 activators, we discovered a novel class of DHODH inhibitors with a tetrahydroindazole core structure (Paper I). The hit compound from the screen, the chiral compound named HZ00, increased p53 translation without increasing p53 mRNA levels or the stability of the protein. Furthermore, treatment with HZ00 accumulated cancer cells in the S-phase of the cell cycle while it led to cell cycle arrest in normal fibroblasts. After screening of commercially available HZ analogues, we discovered the more potent compound HZ05. The active enantiomer of the chiral HZ compound was the (R)-enantiomer, confirmed by the co-crystallization of DHODH with HZ05 where only (R)-HZ05 was bound to the quinone tunnel of DHODH (membrane binding domain). Additionally, both compounds acted synergistically with the MDM2 inhibitor nutlin-3 (which protects p53 from degradation) in different tumor cell lines and in a melanoma xenograft model. Following the discovery of the HZ compounds as inhibitors of DHODH, we investigated the structure and function of DHODH using mass spectrometry and molecular dynamics simulations (Paper II) and afterwards optimized the HZ compounds in a structure activity relationship study (SAR, Paper III). Using non-denaturing mass spectrometry, we established that the interactions between DHODH and its co-factor FMN, the lipids present in the mitochondrial membrane, as well as ligands such as the DHODH inhibitor brequinar, have stabilizing effect on the protein. Furthermore, molecular dynamic stimulations demonstrated the flexibility of the membrane binding domain and the transmembrane helix. We also illustrated that the lipid interactions that stabilize the membrane binding domain may affect the binding of longer ligands, which is important for the design of new inhibitors. The optimization of the HZ-compounds was based on a metabolic soft spot study and the crystal structure of DHODH with (R)-HZ05 obtained in Paper I. The activity of many of the new HZ inhibitors was improved in the in vitro enzymatic assay as well as in cellular assays using a melanoma cell line. Based on in vitro metabolic stability analysis we identified a lead compound in the series, which demonstrated high specificity towards DHODH. The new active HZ-analogues were also able to trigger p53 transcription factor function and affected the cell growth/viability of a melanoma cell line with only a small effect on a normal fibroblast, suggesting a reasonably wide therapeutic window. Supplementation with an excess of uridine ameliorated both, the activation of p53 by the HZ compounds and their effect on cell growth/viability. Under our experimental conditions, the new HZ analogues did not activate DNA damage markers. Additionally, we demonstrated that the ability of DHODH inhibitors to induce apoptosis related g-H2AX was prevented by co-treatment with the pan-caspase inhibitor Z-VAD-FMK. Due to the re-emerging interest in DHODH as a drug target, we investigated the early events triggered after inhibition of this enzyme (Paper IV). For that purpose, we performed RNA sequencing analysis of cells treated with two DHODH inhibitors for up to 4 h. One of the earliest significant changes was the fast downregulation of PNUTS mRNA. This event was observed at the protein level as well. The PNUTS decrease was accompanied by an increase of p53 protein, although the correlation between the two changes requires further investigation. Nevertheless, due to the early decrease in PNUTS we propose that the downregulation of PNUTS mRNA may be a marker for DHODH inhibition.
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| 8. |
- Popova, Gergana, et al.
(författare)
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Optimization of Tetrahydroindazoles as Inhibitors of Human Dihydroorotate Dehydrogenase and Evaluation of Their Activity and In Vitro Metabolic Stability
- 2020
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 63:8, s. 3915-3934
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Tidskriftsartikel (refereegranskat)abstract
- Human dihydroorotate dehydrogenase (DHODH), an enzyme in the de novo pyrimidine synthesis pathway, is a target for the treatment of rheumatoid arthritis and multiple sclerosis and is re-emerging as an attractive target for cancer therapy. Here we describe the optimization of recently identified tetrahydroindazoles (HZ) as DHODH inhibitors. Several of the HZ analogues synthesized in this study are highly potent inhibitors of DHODH in an enzymatic assay, while also inhibiting cancer cell growth and viability and activating p53-dependent transcription factor activity in a reporter cell assay. Furthermore, we demonstrate the specificity of the compounds toward the de novo pyrimidine synthesis pathway through supplementation with an excess of uridine. We also show that induction of the DNA damage marker gamma-H2AX after DHODH inhibition is preventable by cotreatment with the pancaspase inhibitor Z-VAD-FMK. Additional solubility and in vitro m etabolic stability profiling revealed compound 51 as a favorable candidate for preclinical efficacy studies.
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