10th German Pharm-Tox Summit / 91. Jahrestagung der Deutschen Gesellschaft für Experimentelle und Klinische Pharmakologie und Toxikologie (DGPT)

Prof. Dr.

Chair

PD Dr. rer. nat. habil.

Dr. rer. nat.

Prof., Dr. rer. nat.

Tackling Antimicrobial Resistance Addressing Energy-coupling Factor Transporters by Drug and Target Repurposing

Ion channels and membrane transporters

BR05

Problem. Antimicrobial resistance (AMR) of virulent pathogens against available antibiotics represents a global socio-economic threat. Over 5,000,000 deaths per year are attributed to AMR worldwide. The identification and validation of novel bacterial targets as well as the development of antibacterial drugs is imperative.Challenge and Opportunity. Energy-coupling factor transporters (ECFTs) are bacteria-specific ATP-binding cassette transporters (ABCs) of B-type vitamins essential for bacterial survival, growth, and prosperity, and were suggested as potential bacterial drug targets. However, only 54 inhibitors of 3 structural classes with mostly very low inhibitory potency have been found. ECFTs share structural motifs with human ABCs, allowing for the application of "target repurposing" – the translation of knowledge gained from well-studied proteins of a family toward under-studied members. Of particular interest are polypharmacological drugs as they may target conserved structural motifs within bacterial and human ABCs.Strategy and Results. Several public databases were searched for polypharmacological ligands of ABCs. 280 ligands were identified, of which 46 had extended polypharmacological profiles (≥4 ABCs addressed). 31 ligands that collectively addressed the entire ABC proteome were acquired and assessed against ECFTs, of which 9 showed inhibitory activity (hit rate: 29.0%), and 4 demonstrated moderate (benzbromarone: 19.5 µM; MK571: 50.0 µM) and strong (ivermectin: 4.27 µM; pranlukast: 9.82 µM) inhibition. All compounds showed efficacy against ECFTs-expressing S. pneumoniae, with the avermectin derivative ivermectin as the strongest inhibitor (MIC50: 5.58 µM, MIC95: 22.8 µM). Investigation of other avermectin derivatives revealed abamectin (11.4 µM), doramectin (4.97 µM), emamectin (30.2 µM), eprinomectin (16.2 µM), and moxidectin (2.23 µM) as in part very potent ECFT inhibitors. Particularly moxidectin had a very strong antibacterial activity against S. pneumoniae (MIC50: 2.10 µM, MIC95: 2.91 µM), revealing it as one of the most potent and efficacious ECFTs-targeting agents.Conclusion. Target repurposing is a valid strategy to gain structurally novel bioactive compounds of high originality and potency for (almost) undruggable targets of, e.g., bacterial origin. Many hit molecules are approved drugs that can easily be repurposed for adjuvant use against resistant bacteria tackling the challenges emerging from AMR.

Basic Research

Short talk

Entry and impacts of cystic fibrosis transmembrane conductance regulator modulator Elexacaftor/Tezacaftor/Ivacaftor on fetal cortex in a rat model

Drug transport/delivery and metabolism

BR06

Background: The novel cystic fibrosis modulators therapy Elexacaftor /Tezacaftor/Ivacaftor (ETI) is currently the most recommended drug targeting cystic fibrosis. However, its safety and efficiency in pregnancy and breastfeeding remain hugely unknown. This study aims to assess the impacts of in utero ETI exposure on fetal brain.Methods: Pregnant Sprague Dawley rats were fed with ETI (human equivalent dose: 6.7 mg/kg/d Elexacaftor + 3.5 mg/kg/d Tezacaftor + 25 mg/kg/d Ivacaftor) orally twice daily from embryonic day (E) 12 to E19. Fetal cortex were collected at E19 and analyzed with 3 techniques: (1) liquid chromatography mass spectrometry; (2) RNA sequencing; (3) histopathology with hematoxylin & eosin (H&E) staining.Results: For all three ETI components, the fetal cortex/plasma ratio is much higher than 100%, and the entry to cortex is as comparably high as to other fetal tissues. After chronic ETI exposure in late pregnancy, 44 downregulated genes were identified, some of which were associated with neurogenesis and neuronal differentiation. However, no visible histological changes were identified in terms of cortical layer thickness and cell density.Conclusions: The highly lipophilic ETI crosses the fetal blood brain barrier, accumulate in fetal brain and potentially affect CNS development.

Respiratory viruses cause ferroptosis features in lung epithelial cells

Disease models, drug development

BR07

Introduction: Influenza A virus (IAV) and human adenovirus type C5 (HAdV-C5) are two common causes of respiratory infections that can lead to severe illness in high-risk groups. Both viruses have been linked to exacerbations in patients with underlying chronic respiratory diseases. IAV is a enveloped, single-stranded RNA virus that cause seasonal influenza. Contrastingly, HAdV-C5 is non-enveloped, containing double-stranded DNA genome and causes acute respiratory distress. Despite profound differences in infection mechanisms, both viruses can infect epithelial cells of the upper and lower respiratory tract subsequently leading to cell death, and thus loss of lung tissue. A recent study reported that swine influenza A virus drives alveolar (A549) epithelial cells to a ferroptosis-like cell death1. Here we studied if IAV and HAdV-C5 infections cause ferroptosis-like features in bronchial (BEAS-2B) and A549 epithelial cells.Methods: BEAS-2B and A549 epithelial cells were infected with IAV A/PR8/(H1N1) or HAdV-C5 at a multiplicity of infection (MOI) of 1. The ferroptosis inducer, RSL3, served as control. The ferroptosis inhibitor ferrostatin (Fer-1, 5 µM) was added 1 h before infection. Virus infection was monitored with the IAV-specific nuclear protein (NP) and the HAdV-C5-specific hexon protein after the indicated hours post-infection (h.p.i.). Metabolic activity was measured using by the MTT assay, malondialdehyde (MDA), served as a marker for lipid peroxidation and was quantified following the thiobarbituric acid reactive substances assay (TBARS)2, intracellular iron was measured using the FerroZine method3, ferritin expression was quantified by western blot, and mitochondrial morphology cells was assessed using Mitotracker Deep Red.Results and discussion: IAV and HAdV-C5 infections altered the metabolic activity in BEAS-2B and A549 epithelial cells. Both viruses significantly increased lipid peroxidation in both BEAS-2B and A549 epithelial cells, a process sensitive to the ferroptosis inhibitor Fer-1. IAV and HAdV-C5 infections altered in both BEAS-2B and A549 epithelial cells the relative expression of ferritin and the intracellular iron level. Likewise, IAV and HAdv-C5 infections alter the mitochondrial morphology.Conclusion: IAV and HAdV-C5 infections alter metabolic activity, lipid peroxidation, ferritin expression, intracellular iron and mitochondrial morphology in BEAS-2B and A549 epithelial cells. Lipid peroxidation is a marker of ferroptotic cell death and ferritin is the cellular iron stock protein. Our current result suggest that respiratory viral infections cause ferroptosis subsequently leading to a loss of lung tissue.1. Cheng, J., Tao, J., Li, B.et al. Swine influenza virus triggers ferroptosis in A549 cells to enhance virus replication. Virol J 19, 104 (2022). https://doi.org/10.1186/s12985-022-01825-y2. Eva Feldman. Thiobarbituric acid reactive substances (TBARS) Assay. AMDCC Protocols (2019).3. Missirlis, F., Holmberg, S., Georgieva, T. et al. Characterization of mitochondrial ferritin in Drosophila. Proc Natl Acad Sci USA 103, 15(2006). https://doi.org/ 10.1073/pnas.0601471103

Treatment of stress and sleep disorders with Valerian root extract: Microbiota-derived enterolactone and enterodiol as active constituents?

Introduction: For alleviating mild mental stress and sleep disorders, Valeriana officinalis L. extracts are a commonly used treatment option. However, like in many herbal extracts, there remains a lack of scientific consensus regarding its active ingredients (Shinjyo et al., 2020).Material and Methods: With the aim to investigate its potential gut microbiome-mediated biotransformation, we subjected valerian root extract to fermentation with human fecal microbiota from three healthy donors (24h, anoxic, 37 °C) (Pérez-Burillo et al., 2021). Annotation of the constituents present in the native preparation and of the metabolites formed during anaerobic fermentation was accomplished by UHPLC-HRMS.Results: In the extract, lignans and caffeic acid derivatives were detectable as major constituents and valerenic acids, valepotriates and baldrinals as minor constituents. During incubation with human microbiota preparations, the phenylpropanoid derivatives were found to be readily catabolized by gut microbiota. The mammalian lignans enterolactone and enterodiol were found as the major metabolites in all donor samples.Discussion and conclusion: It is noteworthy that enterolactone was recently associated with lower prevalence of depressive symptoms (Cui et al., 2022) and sleep disorders (Sun et al., 2020). This indicates that the metabolic activation of V. officinalis constituents by the gut microbiota, leading to newly produced mammalian lignans, could contribute to the observed therapeutic effects of Valerian root preparations.References: Cui, K., Luo, J., Zhang, L., Zhang, T., Feng, Z., Zhang, D., 2022. The association between urinary phytoestrogens and depressive symptoms. Int. J. Food Sci. Nutr. 73, 657-668.Pérez-Burillo, S., Molino, S., Navajas-Porras, B., et al., 2021. An in vitro batch fermentation protocol for studying the contribution of food to gut microbiota composition and functionality. Nat. Protoc. 16, 3186–3209.Shinjyo, N., Waddell, G., Green, J., 2020. Valerian root in treating sleep problems and associated disorders-a systematic review and meta-analysis. J. Evid. Based Integr. Med. 25, 1-31.Sun, J., Jiang, H., Wang, W., Dong, X., Zhang, D., 2020. Associations of urinary phytoestrogen concentrations with sleep disorders and sleep duration among adults. Nutrients. 12, 2103.

Dr. Dr.

Fine-tuning preclinical animal models for providing proof-of-concept for targeted click-to-release activated antibacterials

High doses of colistin as a last-resort antibacterial, in case of antimicrobial resistance needed to confer an effect, often result in potential kidney toxicity as adverse side effect.[1,2] We aimed to explore local chemically-triggered activation of colistin derivatives by using targeted click-to-release reactions bearing in mind that local activation of colistin might reduce systemic toxicity. For drug development, classic PK-PD models such as the neutropenic thigh infection model are used. However, these cannot demonstrate an effect for these novel chemical entities. Thus, novel methods are needed to advance systemically administered compounds that are specifically activated at the infection site in preclinical development.For the neutropenic thigh infection model, it is known that bacteria can disseminate from the primary infection site, i.e. thigh, toward e.g. lung tissue.[3] To demonstrate the potential of the concept of targeted click-to-release, we decided to make use of the secondary infection site and employ this classical model with small adjustments. Therefore, we administered a tetrazine derivative, D-Ubi-Tz [4,5], intended for triggering release by click chemistry, locally via nebulization into the lung. The colistin-conjugate, Col-(TCO-Asp)1 was administered intravenously. Colistin itself served as a control. Bacterial burden was determined in thigh and lung tissue.Our results show that colistin reduced bacterial burden back to baseline in thigh and lung tissue. Col-(TCO-Asp)1 and D-Ubi-Tz alone did neither impact bacterial burden in thigh nor lung tissue demonstrating no residual activity. Combination of nebulized D-Ubi-Tz with systemically administered Col-(TCO-Asp)1 reduced bacterial burden in the lung, in a similar range as colistin, whereas no effect was seen in thigh tissue. This demonstrated that Col-(TCO-Asp)1 was locally activated with D-Ubi-Tz.[5]In summary, our study provides the first in vivo proof-of-concept of locally activated targeted click-to-release antibacterials. Future studies need to investigate in detail the dimension of reduced toxicity of local activation. Finally, our study shows that small adjustments of a classic PK-PD model serve as a good surrogate model for non-classical, novel antibacterials.[1] Poirel et al., Clin Microbiol Rev. 2017[2] Chien et al., J Antimicrob Agents 2020[3] Rox, Sci Rep 2022[4] Tegge et al., Angew Chem Int Ed 2021[5] Charoenpattarapreeda et al., Angew Chem Int Ed 2024

Short talk 8
Disease models and drug development

Termin

Chair

Programm

Short talk 8Disease models and drug development

Termin

Chair

Programm

Short talk 8
Disease models and drug development