Karin Thevissen

Objective: Drug‐resistant epilepsy (DRE) affects >25 million people worldwide and is often associated with neuroinflammation. Increasing evidence links deficiency or malfunctioning of the enzyme phosphoglycerate dehydrogenase (PHGDH), which converts 3‐phosphoglycerate to generate serine and the neurotransmitter glycine, with (drug‐resistant) epilepsy. Moreover, PHGDH, which is primarily expressed in astrocytes within the brain, has been identified as a critical enzyme in driving macrophage polarization toward an anti‐inflammatory state. Hence, PHGDH activators may be beneficial for treating DRE by exhibiting both antiseizure and anti‐inflammatory activity. The objective of this study was to identify such PHGDH activators. Methods: We screened a drug repurposing library for PHGDH activators and assessed their antiseizure and anti‐inflammatory properties using various zebrafish and mouse epilepsy models and explored the mechanistic consequences of activating PHGDH in a cell line, in astrocytes, and in zebrafish heads. Finally, we assessed the efficacy of clioquinol as add‐on treatment in three severe DRE patients in a clinical open pilot proof‐of‐concept study. Results: We identified haloquinolines from a drug repurposing library as potent activators of PHGDH. The most promising haloquinoline clioquinol can increase the catalytic activity of PHGDH up to 2.5‐fold, thereby increasing de novo glycine biosynthesis and resulting in reduced glutamate levels. Moreover, we show that clioquinol has PHGDH‐dependent antiseizure activity as well as anti‐inflammatory properties in vivo using various zebrafish and mouse epilepsy models. Finally, we demonstrate the efficacy of clioquinol as add‐on treatment in severe DRE patients; two patients showed a 37%–47% reduction in seizure frequency, and all three patients noted a positive impact on quality of life and seizure severity. Significance: Increasing activity of PHGDH is a promising new approach to treat DRE.

Fungal infections represent a significant global health concern, with a growing prevalence of antifungal drug resistance. Targeting glucosylceramides (GlcCer), which are functionally important glycosphingolipids (GSL) present in fungal membranes, represents a promising strategy for the development of antifungal drugs. GlcCer are associated with the antifungal activity of certain plant and insect defensins. The 44-residue ETD151 peptide, optimized from butterfly defensins, is active against several fungal pathogens. ETD151 has been shown to induce a multifaceted mechanism of action (MOA) in Botrytis cinerea , a multiresistant phytopathogenic fungus. However, the target has yet to be identified. Our findings demonstrate that the presence of GlcCer in membranes determines the susceptibility of Pichia pastoris and Candida albicans toward ETD151. To ascertain whether this is due to direct molecular recognition, we demonstrate that ETD151 selectively recognizes liposomes containing GlcCer from B. cinerea , which reveals a methylated-sphingoid base structure. The dissociation constant was estimated by microscale thermophoresis to be in the µM range. Finally, fluorescence microscopy revealed that ETD151 localizes preferentially at the surface of B. cinerea . Furthermore, the majority of prokaryotic cells do not contain GSL, which explains their resistance to ETD151. We investigated the susceptibility of Novosphingobium capsulatum , one of the rare GSL-containing bacteria, to ETD151. ETD151 demonstrated transient morphological changes and inhibitory growth activity (IC ₅₀ ~75 µM) with an affinity for the cell surface, emphasizing the critical importance of GSL as target. Understanding the MOA of ETD151 could pave the way for new perspectives in human health and crop protection.

Rationale: The influence of the lung bacterial microbiome, including potential pathogens, in patients with influenza-associated pulmonary aspergillosis (IAPA) or coronavirus disease (COVID-19)–associated pulmonary aspergillosis (CAPA) has yet to be explored. Objectives: To explore the composition of the lung bacterial microbiome and its association with viral and fungal infection, immunity, and outcome in severe influenza versus COVID-19 with or without aspergillosis. Methods: We performed a retrospective study in mechanically ventilated patients with influenza and COVID-19 with or without invasive aspergillosis in whom BAL for bacterial culture (with or without PCR) was obtained within 2 weeks after ICU admission. In addition, 16S rRNA gene sequencing data and viral and bacterial load of BAL samples from a subset of these patients, and of patients requiring noninvasive ventilation, were analyzed. We integrated 16S rRNA gene sequencing data with existing immune parameter datasets. Measurements and Main Results: Potential bacterial pathogens were detected in 20% (28/142) of patients with influenza and 37% (104/281) of patients with COVID-19, whereas aspergillosis was detected in 38% (54/142) of patients with influenza and 31% (86/281) of patients with COVID-19. A significant association between bacterial pathogens in BAL fluid and 90-day mortality was found only in patients with influenza, particularly patients with IAPA. Patients with COVID-19, but not patients with influenza, showed increased proinflammatory pulmonary cytokine responses to bacterial pathogens. Conclusions: Aspergillosis is more frequently detected in the lungs of patients with severe influenza than bacterial pathogens. Detection of bacterial pathogens associates with worse outcome in patients with influenza, particularly in those with IAPA, but not in patients with COVID-19. The immunological dynamics of tripartite viral–fungal–bacterial interactions deserve further investigation.

Background/Objectives: The rise and spread of antimicrobial resistance complicates the treatment of bacterial wound pathogens, further increasing the need for newer, effective therapies. Azoles such as miconazole have shown promise as antibacterial compounds; however, they are currently only used as antifungals. Previous research has shown that combining azoles with quaternary ammonium compounds yields synergistic activity against fungal pathogens, but the effect on bacterial pathogens has not been studied yet. Methods: In this study, the focus was on finding active synergistic combinations of imidazoles and quaternary ammonium compounds against (multidrug-resistant) bacterial pathogens through checkerboard assays. Experimental evolution in liquid culture was used to evaluate the possible emergence of resistance against the most active synergistic combination. Results: Several promising synergistic combinations were identified against an array of Gram-positive pathogens: miconazole/domiphen bromide, ketoconazole/domiphen bromide, clotrimazole/domiphen bromide, fluconazole/domiphen bromide and miconazole/benzalkonium chloride. Especially, miconazole with domiphen bromide exhibits potential, as it has activity at a low concentration against a broad range of pathogens and shows an absence of strong resistance development over 11 cycles of evolution. Conclusions: This study provides valuable insight into the possible combinations of imidazoles and quaternary ammonium compounds that could be repurposed for (topical) wound treatment. Miconazole with domiphen bromide shows the highest application potential as a possible future wound therapy. However, further research is needed into the mode of action of these compounds and their efficacy and toxicity in vivo.

Drug-resistant epilepsy (DRE) affects over 25 million people worldwide and is associated with neuroinflammation. We identified haloquinolines from a drug repurposing library as potent activators of phosphoglycerate dehydrogenase (PHGDH) enzyme, which converts 3-phosphoglycerate to generate serine and the neurotransmitter glycine, and steers anti-inflammatory responses. The most promising haloquinoline clioquinol can increase the catalytic activity of PHGDH up to 2.5-fold, thereby increasing de novo glycine biosynthesis and resulting in reduced glutamate levels. Moreover, we show that clioquinol has PHGDH-dependent antiseizure activity as well as anti-inflammatory properties in vivo using various zebrafish and mouse epilepsy models. Finally, we demonstrate the efficacy of clioquinol as add-on treatment in severe DRE patients. Therefore, increasing activity of PHGDH is a promising new approach to treat DRE.

Metabolic rewiring is a hallmark of cancer that supports tumor growth, survival, and chemotherapy resistance. Although normal cells often rely on extracellular serine and glycine supply, a significant subset of cancers becomes addicted to intracellular serine/glycine synthesis, offering an attractive drug target. Previously developed inhibitors of serine/glycine synthesis enzymes did not reach clinical trials due to unfavorable pharmacokinetic profiles, implying that further efforts to identify clinically applicable drugs targeting this pathway are required. In this study, we aimed to develop therapies that can rapidly enter the clinical practice by focusing on drug repurposing, as their safety and cost-effectiveness have been optimized before. Using a yeast model system, we repurposed two compounds, sertraline and thimerosal, for their selective toxicity against serine/glycine synthesis–addicted breast cancer and T-cell acute lymphoblastic leukemia cell lines. Isotope tracer metabolomics, computational docking, enzymatic assays, and drug–target interaction studies revealed that sertraline and thimerosal inhibit serine/glycine synthesis enzymes serine hydroxymethyltransferase and phosphoglycerate dehydrogenase, respectively. In addition, we demonstrated that sertraline's antiproliferative activity was further aggravated by mitochondrial inhibitors, such as the antimalarial artemether, by causing G1–S cell-cycle arrest. Most notably, this combination also resulted in serine-selective antitumor activity in breast cancer mouse xenografts. Collectively, this study provides molecular insights into the repurposed mode-of-action of the antidepressant sertraline and allows to delineate a hitherto unidentified group of cancers being particularly sensitive to treatment with sertraline. Furthermore, we highlight the simultaneous inhibition of serine/glycine synthesis and mitochondrial metabolism as a novel treatment strategy for serine/glycine synthesis–addicted cancers.

Metabolic rewiring is a hallmark of cancer that supports tumor growth, survival, and chemotherapy resistance. Although normal cells often rely on extracellular serine and glycine supply, a significant subset of cancers becomes addicted to intracellular serine/glycine synthesis, offering an attractive drug target. Previously developed inhibitors of serine/glycine synthesis enzymes did not reach clinical trials due to unfavorable pharmacokinetic profiles, implying that further efforts to identify clinically applicable drugs targeting this pathway are required. In this study, we aimed to develop therapies that can rapidly enter the clinical practice by focusing on drug repurposing, as their safety and cost-effectiveness have been optimized before. Using a yeast model system, we repurposed two compounds, sertraline and thimerosal, for their selective toxicity against serine/glycine synthesis–addicted breast cancer and T-cell acute lymphoblastic leukemia cell lines. Isotope tracer metabolomics, computational docking, enzymatic assays, and drug–target interaction studies revealed that sertraline and thimerosal inhibit serine/glycine synthesis enzymes serine hydroxymethyltransferase and phosphoglycerate dehydrogenase, respectively. In addition, we demonstrated that sertraline's antiproliferative activity was further aggravated by mitochondrial inhibitors, such as the antimalarial artemether, by causing G1–S cell-cycle arrest. Most notably, this combination also resulted in serine-selective antitumor activity in breast cancer mouse xenografts. Collectively, this study provides molecular insights into the repurposed mode-of-action of the antidepressant sertraline and allows to delineate a hitherto unidentified group of cancers being particularly sensitive to treatment with sertraline. Furthermore, we highlight the simultaneous inhibition of serine/glycine synthesis and mitochondrial metabolism as a novel treatment strategy for serine/glycine synthesis–addicted cancers.

This file describes all yeast experiments and includes supplementary tables S1-S3 and supplementary figures S1-S10.

This file describes all yeast experiments and includes supplementary tables S1-S3 and supplementary figures S1-S10.

Advancements in lab‐on‐a‐chip technologies have revolutionized the single‐cell analysis field. However, an accessible platform for in‐depth screening and specific retrieval of single cells, which moreover enables studying diverse cell types and performing various downstream analyses, is still lacking. As a solution, FLUIDOT is introduced, a versatile microfluidic platform incorporating customizable microwells, optical tweezers and an interchangeable cell‐retrieval system. Thanks to its smart microfluidic design, FLUIDOT is straightforward to fabricate and operate, rendering the technology widely accessible. The performance of FLUIDOT is validated and its versatility is subsequently demonstrated in two applications. First, drug tolerance in yeast cells is studied, resulting in the discovery of two treatment‐tolerant populations. Second, B cells from convalescent COVID‐19 patients are screened, leading to the discovery of highly affine, in vitro neutralizing monoclonal antibodies against SARS‐CoV‐2. Owing to its performance, flexibility, and accessibility, it is foreseen that FLUIDOT will enable phenotypic and genotypic analysis of diverse cell samples and thus elucidate unexplored biological questions.