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    Targeting metabolic dependencies in cancer cells in vitro
    (2024) Sahlén, Julia; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Molin, Mikael; Sayin, Volkan; Alvarez, Samantha
    Lung adenocarcinoma (LUAD) is a prevalent and aggressive form of lung cancer. Targeting metabolic pathways, particularly the serine and glycine biosynthesis pathways, represents a promising therapeutic strategy. Phosphoglycerate dehydrogenase (PHGDH), a key enzyme in serine biosynthesis, is frequently overexpressed in LUAD. NCT-503, a PHGDH inhibitor, has emerged as a potential therapeutic agent against LUAD. This study aimed to evaluate the efficacy of NCT-503 in inhibiting cancer cell viability and altering metabolic pathways in LUAD cell lines. Cell Titer Glow (CTG) assays were employed to assess cell viability across varying drug concentrations, while uptake assays measured the absorption and secretion of serine and glycine in treated and untreated cells. Experiments were conducted on two LUAD cell lines, A1 and Y2, representing elder and younger populations, respectively. CTG assays revealed a dose-dependent response to NCT-503, with significant cell viability reduction at higher drug concentrations (200 µM). Interestingly, the A1 cell line showed higher sensitivity to lower concentrations (<100 µM) compared to Y2. Uptake assays presented complex and varied responses; Y2 cells exhibited decreased serine secretion and glycine absorption post-treatment, while A1 cells showed increased secretion of these metabolites. Notably, the patterns of nutrient uptake were inconsistent across different trials, indicating potential variability in cellular responses to NCT-503. The study confirmed that PHGDH inhibition by NCT-503 effectively hinders cancer cell growth, particularly in the A1 cell line. However, the discrepancies between CTG and uptake assays highlight the complexity of cellular metabolic responses. Variability in results suggests the need for further investigation to ensure reliability and reproducibility. Factors such as cell age, genetics, and experimental conditions may influence the drug’s efficacy. The study highlights the necessity for ongoing research, including repeated uptake assays, intracellular metabolite analysis, and in vivo trials, to validate NCT-503's therapeutic potential and ensure minimal side effects. NCT-503 shows promise as a therapeutic agent against LUAD, particularly for elder patients, by effectively inhibiting cancer cell viability through PHGDH inhibition. However, the complexity of cellular responses and potential laboratory errors necessitate further research to confirm these findings and optimize treatment protocols.
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    Investigating the effect of KEAP1 knockout and lactate treatment on histone lysine lactylation in KRAS-driven lung adenocarcinoma
    (2024) Karlsson, Elin; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Wittung-Stafshede, Pernilla; Raj , Dorota; Wiel, Clotilde
    Lung cancer is a major global health concern, responsible for the highest number of deaths among all cancer types. Whereof, the subtype lung adenocarcinoma is the most common form, accounting for 40% of all lung cancer cases. Loss-of-function mutations in the gene KEAP1, found in 20% of lung adenocarcinoma patients, are associated with treatment resistance and lower overall survival, highlighting the importance of investigating this mutation in research. Additionally, KEAP1 loss of function are linked to metabolic reprogramming, a known hallmark of cancer. The Warburg effect, a metabolic reprogramming in cancer cells characterized by high rates of aerobic glycolysis , leads to elevated lactate concentrations in cancerous tissue compared to healthy tissue. Lactate, earlier considered to be a waste product, is now recognized for its role as a signaling molecule and its function in coordinating metabolism. Recent studies has identified histone lysine lactylation, a post-translational modification induced by lactate, which affects gene expression and chromatin structure. The aim of this thesis project was to investigate the effect of KEAP1 knockout and lactate treatment on histone lysine lactylation levels in lung adenocarcinoma. This study utilized mouse lung adenocarcinoma cell lines derived from genetically engineered mouse models with an activating mutation in the oncogene Kras. Using the protein detection method Western blot, the results demonstrated that histone lysine lactylation levels on H3 and H4 were lower in Keap1 knockout cells compared to Keap1 wild-type cells in one of the cell lines, while no definitive conclusion could be drawn for the other cell line. Inhibition of the KEAP1 protein without affecting the Keap1 gene directly, showed the same results. Additionally, lactate treatment induced histone lysine lactylation levels on H3 and H4 in Keap1 wild-type cells, but no increase was observed in Keap1 knockout cells. By measuring intracellular lactate concentration it was observed that the lactate treatment induced intracellular lactate levels for both Keap1 wild-type cells and Keap1 knockout cells. Indicating that there is no direct correlation between the intracellular lactate levels and histone lysine lactylation in cells with a Keap1 knockout.
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    Exploring the metabolism and mixture effects of the isothiazolinone biocides BIT and OIT in PLHC-1 cells
    (2024) Carvalho Nejstgaard, Aline; Blomqvist, Oskar; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Scheers, Nathalie; Celander, Malin
    The powerful antimicrobial properties of isothiazolinones make them a common preservative in both industrial and consumer products. However, methylated deriva tives of isothiazolinones have been associated with concerning allergenic effects leading to stricter regulations. As a consequence, industries have turned to more lipophilic isothiazolinones, such as benzisothiazolinone (BIT) and octylisothiazoli none (OIT), which are less regulated. Similar effects have however been reported for exposures of BIT and OIT, highlighting the importance of expanding the knowledge about these compounds, to enforce stricter regulation and potentially controlling the isothiazolinones as a group. This study aimed to investigate the toxicodynamic and toxicokinetic responses in a fish liver cell model system, Poeciliopsis lucida hep atocellular carcinoma (PLHC-1) following exposure to BIT and OIT, alone and in mixture. Results from the ethoxyresorufin-O-deethylase (EROD) assay revealed no apparent induction of the common detoxification enzyme CYP1A after exposure to BIT and OIT. However, more data is needed for a more robust evaluation of whether the enzyme is involved in their metabolism. Analysis of cultured exposure media in high performance liquid chromatography (HPLC) demonstrated cellular biotransformation of BIT into a more polar metabolite. A rapid cellular uptake of OIT was observed, however no potential OIT metabolite could be detected, nei ther extracellularly nor intracellularly. A cytotoxic assessment was also conducted on exposed PLHC-1 cells investigating the mitochondrial activity and membrane in tegrity, common markers for cellular viability. Significant toxic effects were observed on both markers after exposure to 10 µM OIT, while no toxic effect was observed for the same concentration of BIT. However, co-exposure to these concentrations of OIT and BIT, displayed an even greater toxicity, indicating a synergistic mixture effect between the two. While this study provides valuable insights into the toxi codynamic and toxicokinetic profiles of BIT and OIT, highlighting the importance of considering mixture effects in assessing their potential health and environmental impacts, additional research is needed to fully understand their specific metabolic pathways and interaction mechanisms, to assess their long-term exposure effects.
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    Machine Learning for Structural Predictions of PROTACs
    (2024) Källberg, Anders; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Wittung-Stafshede, Pernilla; Mercado, Rocío; Nittinger, Eva; Tyrchan, Christian
    PROteolysis TArgeting Chimeras (PROTACs) are molecules that induce the degradation of targeted proteins by hijacking the ubiquitin–proteasome system in the cell. A PROTAC binds simultaneously to an E3 ligase and a protein of interest (POI), forming a ternary complex. The ubiquitin–proteasome system tags the POI with ubiquitin, marking it for degradation by the proteasome. The formation of a good ternary complex is essential for the ubiquitination and subsequent degradation of the POI. Being able to accurately model ternary complexes thus provides critical advantages in the development of PROTACs; however, data on PROTACs and their crystallized ternary complexes are limited. Accurate predictions of these structures are desirable, but current computational methods struggle to simulate the interactions between the PROTAC and both proteins simultaneously. AlphaFold, a machine learning tool, has been shown to accurately predict protein complexes. Yet, research on applying AlphaFold to predict ternary complexes is scarce. In the first part of this thesis, the ternary complex was modeled using AlphaFold by utilizing the sequences of both natural and artificially linked POIs and E3 ligase. Nevertheless, it was determined that AlphaFold was unable to accurately predict these complexes, reasonably because it was not able to take the PROTAC into account in the predictions. The second part of this thesis focused on generating data on PROTAC substructures, essential for the development of these molecules. Despite the availability of such data, obtaining high-quality data on substructures of specific PROTACs can be challenging and time-consuming. To address this, the PROTAC Splitter, a novel machine learning tool based on graph neural networks, was developed to predict these substructures. The PROTAC Splitter predicts 99.7% of PROTACs, with known substructures, to a maximal error of 6 atoms wrong between the boundaries of the ligands and linker. It generalizes to PROTACs with three unknown substructures, where 23.1% of these predictions satisfy the same criteria. The code for the PROTAC splitter is available at https://github.com/AndersKallberg/PROTAC_splitter. Although accurate predictions of ternary complexes remain challenging, the PROTAC Splitter makes the substructures easily accessible to anyone in this field of research. In summary, the work presented in this thesis answers scientific questions in two complementary areas of PROTAC development: (1) ternary (protein) structure prediction, and (2) PROTAC component prediction. This information is limited and valuable, and accurate predictions of these could accelerate the discovery of effective PROTACs and help in the fight against disease.
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    Non-equilibrium stabilisation of proteins by chaperones - The Hsp70 molecular chaperone
    (2024) López Bautista, María Li; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Wittung-Stafshede, Pernilla; De Los Rios, Paolo
    Within living organisms, proteins are essential components. They are responsible for carrying out almost every function in the cell. Proteins must fold into a specific three-dimensional shape to perform their diverse roles effectively. Indeed, improper folding is the root cause of many diseases. Not surprisingly, there is a specific group of proteins whose function is to assist and safeguard the folding process of other proteins. These are known as molecular chaperones. Here, we focus on the 70 kiloDalton heat shock protein, Hsp70, a molecular chaperone that has been under the spotlight of scientists for decades due to its ubiquitous presence across all living systems and its assistance in a wide range of cellular processes. It is well accepted that the mechanism of action of Hsp70 chaperones consists of a biochemical energy-consuming cycle, which allows them to drive the system out-of-equilibrium and escape the inherent limitations of equilib rium thermodynamics to perform their functions efficiently. Considering both the molecular details of chaperones and their client protein, along with a correct inclusion of the energy consumed in each step of the cycle and all relevant conformational transitions, we present a kinetic rate model for the description of the functional cycle of Hsp70 chaperones in protein folding that aims to elucidate the fundamental principles that govern their complex behaviour.