GC-MS Profiling and Pharmacological Potential of Physconia venusta (Ach.) Poelt.

Objectives: Lichens are complex symbiotic organisms that generate various bioactive compounds with significant therapeutic value. We investigated the chemical composition and bioactivity of the acetone extract of the Algerian lichen Physconia venusta (Ach.) poet. Materials and Methods: Phytochemical screening was performed using gas chromatography-mass spectrometry (GC-MS). The antibacterial activity was assessed against Escherichia coli, Pseudomonas aeruginosa, Salmonella enteritidis, Salmonella typhi, Staphylococcus aureus, Listeria monocytogenes, and Bacillus subtilis using an agar diffusion test with the determination of the minimal inhibition concentration (MIC), while the antioxidant activity was determined using different chemical methods (DPPH, ABTS, CUPRAC, reducing power, superoxide anion scavenging, ß-carotene bleaching, and metal chelate). In addition, cytotoxic activity was tested using Artemia salina (Brine shrimp) bioassay. Results: The studied extract exhibited intense antibacterial activity against E. coli and S. aureus with inhibition diameters of 28 ± 0.01 and 22 ± 0.01 mm, respectively, with a MIC value of 6.25 mg/mL and a selectivity index of 2.8. The obtained extract showed different antioxidant trends depending on the selected assay. GC-MS analysis revealed many secondary metabolites. Conclusion: P. venusta, a type of lichen, is a potential source of bioactive substances that could be used in pharmaceuticals.

Enhanced accumulation of biologically active compounds in lichens with potential functional food applications.

Lichens have great potential as food, functional food additives or flavourings. The presence of specific substances with multiple biological activities is one of the characteristics of lichens. However, research on lichens as a food source or functional food additive is limited. The present study simulated, for the first time, the potential bioaccessibility of active compounds from 6 lichen species in simulated gastric and intestinal conditions. An in vitro digestion showed that the lichen substances had different bioaccessibility and stability during digestion. It was found that the application of some metabolic modulators significantly altered the accumulation of metabolites in most species. In addition, the study demonstrated the antimicrobial activity of the tested extracts as well as of 14 isolated lichen metabolites. These multi-directional studies demonstrate the potential of lichens in terms of their use as antimicrobial functional food additives.

Usnic acid alleviates testicular ischemia/reperfusion injury in rats by modulating endoplasmic reticulum stress.

Testicular torsion (TT) is a urological condition that can result in infertility in men. The etiopathogenesis of TT includes ischemia/reperfusion injury (IRI) characterized by oxidative stress (OS), inflammation and apoptosis resulting from increased levels of free radicals. Usnic acid (UA), a dibenzofuran, is one of the most common metabolites found in lichens and is known to possess powerful antioxidant properties. The aim of this study was to investigate the potential protective activity of UA in an experimental testicular IRI model for the first time. A total of 18 rats were randomly assigned to three groups (n=6): sham control, IRI and IRI+UA. The IRI groups underwent a four-hour period of ischemia and a two-hour period of reperfusion. The OS, inflammation, endoplasmic reticulum stress (ERS) and apoptosis markers in testicular tissue were evaluated using colorimetric methods. Furthermore, tissue samples were subjected to histological examination, with staining using hematoxylin and eosin. Histopathological findings supported by increased OS, inflammation, ERS and apoptosis levels were obtained in IRI group compared with sham control group. However, UA treatment restored these pathological and biochemical changes. Although this study provides the first preliminary evidence that UA may be used as a useful molecule against testicular IRI, further extensive molecular preclinical studies should be performed before clinical use is considered.

Biomaterial Improves the Stability of Perovskite Solar Cells by Passivating Defects and Inhibiting Ion Migration.

With the rapid improvement of power conversion efficiency (PCE), perovskite solar cells (PSCs) have broad application prospects and their industrialization will be the next step. Nevertheless, the performance and long-term stability of the devices are limited by the defect-induced nonradiative recombination centers and ions' migration inside the perovskite films. Here, usnic acid (UA), an easy-to-obtain and efficient natural biomaterial with a hydroxyl functional group (-OH) and four carbonyl groups (-C═O) was added to MAPbI3 perovskite precursor to regulate the crystallization process by slowing the crystallization rate, thereby expanding the crystal size and preparing perovskite films with low defect density. In addition, UA anchors the uncoordinated Pb2+ and suppresses the migration of I-ions, which enhances the stability of the perovskite film. Consequently, an impressive PCE exceeding 20% was achieved for inverted structure MAPbI3-based PSCs. More impressively, the optimized PSCs maintained 78% of the initial PCE under air with high humidity (RH ≈ 65%, 25-30 °C) for 1000 h. UA can be extracted from the plant, usnea, making it inexpensive and easy to obtain. Our work demonstrates the application of the plant material in PSCs and their industrialization, which is significant nowadays.

Hepatotoxicity of usnic acid and underlying mechanisms.

Since usnic acid was first isolated in 1844 as a prominent secondary lichen metabolite, it has been used for various purposes worldwide. Usnic acid has been claimed to possess numerous therapeutic properties, including antimicrobial, anti-inflammatory, antiviral, anti-proliferative, and antipyretic activities. Approximately two decades ago, crude extracts of usnic acid or pure usnic acid were marketed in the United States as dietary supplements for aiding in weight loss as a "fat-burner" and gained popularity in the bodybuilding community; however, hepatotoxicity was documented for some usnic acid containing products. The US Food and Drug Administration (FDA) received numerous reports of liver toxicity associated with the use of dietary supplements containing usnic acid, leading the FDA to issue a warning letter in 2001 on a product, LipoKinetix. The FDA also sent a recommendation letter to the manufacturer of LipoKinetix, resulting in the withdrawal of LipoKinetix from the market. These events triggered investigations into the hepatotoxicity of usnic acid and its mechanisms. In 2008, we published a review article titled "Usnic Acid and Usnea Barbata Toxicity". This review is an updated version of our previous review article and incorporates additional data published since 2008. The purpose of this review is to provide a comprehensive summary of the understanding of the liver toxicity associated with usnic acid, with a particular focus on the current understanding of the putative mechanisms of usnic acid-related hepatotoxicity.

Oxidative DNA damage contributes to usnic acid-induced toxicity in human induced pluripotent stem cell-derived hepatocytes.

Dietary supplements containing usnic acid have been increasingly marketed for weight loss over the past decades, even though incidences of severe hepatotoxicity and acute liver failure due to their overuse have been reported. To date, the toxic mechanism of usnic acid-induced liver injury at the molecular level still remains to be fully elucidated. Here, we conducted a transcriptomic study on usnic acid using a novel in vitro hepatotoxicity model employing human induced pluripotent stem cell (iPSC)-derived hepatocytes. Treatment with 20 µM usnic acid for 24 h caused 4272 differentially expressed genes (DEGs) in the cells. Ingenuity Pathway Analysis (IPA) based on the DEGs and gene set enrichment analysis (GSEA) using the whole transcriptome expression data concordantly revealed several signaling pathways and biological processes that, when taken together, suggest that usnic acid caused oxidative stress and DNA damage in the cells, which further led to cell cycle arrest and eventually resulted in cell death through apoptosis. These transcriptomic findings were subsequently corroborated by a variety of cellular assays, including reactive oxygen species (ROS) generation and glutathione (GSH) depletion, DNA damage (pH2AX detection and 8-hydroxy-2'-deoxyguanosine [8-OH-dg] assay), cell cycle analysis, and caspase 3/7 activity. Collectively, the results of the current study accord with previous in vivo and in vitro findings, provide further evidence that oxidative stress-caused DNA damage contributes to usnic acid-induced hepatotoxicity, shed new light on molecular mechanisms of usnic acid-induced hepatotoxicity, and demonstrate the usefulness of iPSC-derived hepatocytes as an in vitro model for hepatotoxicity testing and prediction.

Nanostructured Poly-l-lactide and Polyglycerol Adipate Carriers for the Encapsulation of Usnic Acid: A Promising Approach for Hepatoprotection.

The present study investigates the utilization of nanoparticles based on poly-l-lactide (PLLA) and polyglycerol adipate (PGA), alone and blended, for the encapsulation of usnic acid (UA), a potent natural compound with various therapeutic properties including antimicrobial and anticancer activities. The development of these carriers offers an innovative approach to overcome the challenges associated with usnic acid's limited aqueous solubility, bioavailability, and hepatotoxicity. The nanosystems were characterized according to their physicochemical properties (among others, size, zeta potential, thermal properties), apparent aqueous solubility, and in vitro cytotoxicity. Interestingly, the nanocarrier obtained with the PLLA-PGA 50/50 weight ratio blend showed both the lowest size and the highest UA apparent solubility as well as the ability to decrease UA cytotoxicity towards human hepatocytes (HepG2 cells). This research opens new avenues for the effective utilization of these highly degradable and biocompatible PLLA-PGA blends as nanocarriers for reducing the cytotoxicity of usnic acid.

The Potential of Usnic-Acid-Based Thiazolo-Thiophenes as Inhibitors of the Main Protease of SARS-CoV-2 Viruses.

Although the COVID-19 pandemic caused by SARS-CoV-2 viruses is officially over, the search for new effective agents with activity against a wide range of coronaviruses is still an important task for medical chemists and virologists. We synthesized a series of thiazolo-thiophenes based on (+)- and (-)-usnic acid and studied their ability to inhibit the main protease of SARS-CoV-2. Substances containing unsubstituted thiophene groups or methyl- or bromo-substituted thiophene moieties showed moderate activity. Derivatives containing nitro substituents in the thiophene heterocycle-just as pure (+)- and (-)-usnic acids-showed no anti-3CLpro activity. Kinetic parameters of the most active compound, (+)-3e, were investigated, and molecular modeling of the possible interaction of the new thiazolo-thiophenes with the active site of the main protease was carried out. We evaluated the binding energies of the ligand and protein in a ligand-protein complex. Active compound (+)-3e was found to bind with minimum free energy; the binding of inactive compound (+)-3g is characterized by higher values of minimum free energy; the positioning of pure (+)-usnic acid proved to be unstable and is accompanied by the formation of intermolecular contacts with many amino acids of the catalytic binding site. Thus, the molecular dynamics results were consistent with the experimental data. In an in vitro antiviral assay against six strains (Wuhan, Delta, and four Omicron sublineages) of SARS-CoV-2, (+)-3e demonstrated pronounced antiviral activity against all the strains.

Enhancement of the Antitumor and Antimetastatic Effect of Topotecan and Normalization of Blood Counts in Mice with Lewis Carcinoma by Tdp1 Inhibitors-New Usnic Acid Derivatives.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is an important DNA repair enzyme and one of the causes of tumor resistance to topoisomerase 1 inhibitors such as topotecan. Inhibitors of this Tdp1 in combination with topotecan may improve the effectiveness of therapy. In this work, we synthesized usnic acid derivatives, which are hybrids of its known derivatives: tumor sensitizers to topotecan. New compounds inhibit Tdp1 in the micromolar and submicromolar concentration range; some of them enhance the effect of topotecan on the metabolic activity of cells of various lines according to the MTT test. One of the new compounds (compound 7) not only sensitizes Krebs-2 and Lewis carcinomas of mice to the action of topotecan, but also normalizes the state of the peripheral blood of mice, which is disturbed in the presence of a tumor. Thus, the synthesized substances may be the prototype of a new class of additional therapy for cancer.

Bioactivities and Structure-Activity Relationships of Usnic Acid Derivatives: A Review.

Usnic acid has a variety of biological activities, and has been widely studied in the fields of antibacterial, immune stimulation, antiviral, antifungal, anti-inflammatory and antiparasitic. Based on this, usnic acid is used as the lead compound for structural modification. In order to enhance the biological activity and solubility of usnic acid, scholars have carried out a large number of structural modifications, and found some usnic acid derivatives to be of more potential research value. In this paper, the structural modification, biological activity and structure-activity relationship of usnic acid were reviewed to provide reference for the development of usnic acid derivatives.

Non-photochemical quenching may contribute to the dominance of the pale mat-forming lichen Cladonia stellaris over the sympatric melanic Cetraria islandica.

The mat-forming fruticose lichens Cladonia stellaris and Cetraria islandica frequently co-occur on soils in sun-exposed boreal, subarctic, and alpine ecosystems. While the dominant reindeer lichen Cladonia lacks a cortex but produces the light-reflecting pale pigment usnic acid on its surface, the common but patchier Cetraria has a firm cortex sealed by the light-absorbing pigment melanin. By measuring reflectance spectra, high-light tolerance, photosynthetic responses, and chlorophyll fluorescence in sympatric populations of these lichens differing in fungal pigments, we aimed to study how they cope with high light while hydrated. Specimens of the two species tolerated high light equally well but with different protective mechanisms. The mycobiont of the melanic species efficiently absorbed excess light, consistent with a lower need for its photobiont to protect itself by non-photochemical quenching (NPQ). By contrast, usnic acid screened light at 450-700 nm by reflectance and absorbed shorter wavelengths. The ecorticate usnic species with less efficient fungal light screening exhibited a consistently lower light compensation point and higher CO2 uptake rates than the melanic lichen. In both species, steady state NPQ rapidly increased at increasing light with no signs of light saturation. To compensate for less internal shading causing light fluctuations with a larger amplitude, the usnic lichen photobiont adjusted to changing light by faster induction and faster relaxation of NPQ rapidly transforming excess excitation energy to less damaging heat. The high and flexible NPQ tracking fluctuations in solar radiation probably contributes to the strong dominance of the usnic mat-forming Cladonia in open lichen-dominated heaths.

Usnic acid attenuates 7,12-dimethylbenz[a] anthracene (DMBA) induced oral carcinogenesis through inhibiting oxidative stress, inflammation, and cell proliferation in male golden Syrian hamster model.

In this study, we investigated the chemopreventive efficacy of usnic acid (UA), an effective secondary metabolite component of lichens, against 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral squamous cell carcinoma (OSCC) in the hamster model. Initially, the buccal pouch carcinogenesis was induced by administering 0.5% DMBA to the HBP (hamster buccal pouch) region about three times a week until the 10th week. Then, UA was orally treated with different concentrations (25, 50, 100 mg/kg b.wt) on alternative days of DMBA exposure, and the experimental process ended in the 16th week. After animal experimentation, we observed 100% tumor incidence with well-differentiated OSCC, dysplasia, and hyperplasia lesions in the DMBA-induced HBP region. Furthermore, the UA treatment of DMBA-induced hamster effectively inhibited tumor growth. In addition, UA upregulated antioxidant levels, interfered with the elevated lipid peroxidation by-product of thiobarbituric acid reactive substances, and changed the activities of the liver detoxification enzyme (Phase I and II) in DMBA-induced hamsters. Furthermore, immunohistochemical staining of inflammatory markers (iNOS and COX-2) and proliferative cell markers (cyclin-D1 and PCNA) were upregulated in the buccal pouch part of hamster animals induced with DMBA. Notably, the oral administration of UA significantly suppressed these markers during DMBA-induced hamsters. Collectively, our findings revealed that UA exhibits antioxidant, anti-inflammatory, antitumor, and apoptosis-inducing characteristics, demonstrating UA's protective properties against DMBA-induced HBP carcinogenesis.

Enhancing Wound Healing and Bactericidal Efficacy: A Hydrogel Membrane of Bacterial Cellulose and Sanxan Gel for Accelerating the Healing of Infected Wounds.

Bacterial cellulose is an extracellular polysaccharide produced by microorganisms, offering advantages such as high water-holding capacity, flexibility, and biocompatibility. However, its lack of bactericidal activity hampers its wide application. Usnic acid, a secondary metabolite derived from lichens of the Usnea genus, is recognized for its antibacterial and anti-biofilm efficiency, coupled with anti-inflammatory properties. Its water insolubility presents challenges for wide utilization and stable release. Sanxan gel, a novel polysaccharide, exhibits exceptional freeze-thaw stability, suspension properties, and high elasticity, rendering it effective as a suspending agent to improve the bioavailability of water-insoluble drugs. In this study, a hydrogel membrane is designed by combining bacterial cellulose and usnic acid suspended in sanxan gel through a simple in situ microorganism fermentation. The obtained membranes demonstrate excellent ability for sustained drug release, strong eradication capability against tested bacteria in both in vitro and in vivo experiments, effective inhibition of biofilm formation, and excellent hemocompatibility and cytocompatibility. Additionally, the composite membranes promote wound healing with reduced inflammation and bacterial infection in a full-thickness wound infection model in mice. This study provides innovative insights and strategies for the development of functional dressings for infected wounds in future clinical applications.

Usnea aurantiaco-atra (Jacq) Bory: Metabolites and Biological Activities.

BACKGROUND: Lichens are complex symbiotic associations between a fungus and an alga or cyanobacterium. Due to their great adaptability to the environment, they have managed to colonize many terrestrial habitats, presenting a worldwide distribution from the poles to the tropical regions and from the plains to the highest mountains. In the flora of the Antarctic region, lichens stand out due to their variety and development and are a potential source of new bioactive compounds. METHODS: A phytochemical study of the Antarctic lichen Usnea aurantiaco-atra (Jacq) Bory was conducted with the intention of determining the most important metabolites. In addition, the cytotoxic and antioxidant activities of its extracts were determined. RESULTS: Cytotoxicity studies revealed that the hexane extract contains usnic acid as a majority metabolite, in addition to linoleic acid, ergosterols and terpenes, and demonstrates cytotoxic activity against an A375 melanoma cell line. On the other hand, the presence of total phenols in the extracts did not influence their antioxidant activity. CONCLUSIONS: U. aurantiaco-atra contains mainly usnic acid, although there are terpenes and ergosta compounds that could be responsible for its cytotoxic activity. The presence of phenols did not confer antioxidant properties.

Strategic and Innovative Roles of lncRNAs Regulated by Naturally-Derived Small Molecules in Cancer Therapy.

In the field of precision and personalized medicine, the next generation sequencing method has begun to take an active place as genome-wide screening applications in the diagnosis and treatment of diseases. Studies based on the determination of the therapeutic efficacy of personalized drug use in cancer treatment in the size of the transcriptome and its extension, lncRNA, have been increasing rapidly in recent years. Targeting and/or regulating noncoding RNAs (ncRNAs) consisting of long noncoding RNAs (lncRNAs) are promising strategies for cancer treatment. Within the scope of rapidly increasing studies in recent years, it has been shown that many natural agents obtained from biological organisms can potentially alter the expression of many lncRNAs associated with oncogenic functions. Natural agents include effective small molecules that provide anti-cancer effects and have been used as chemotherapy drugs or in combination with standard anti-cancer drugs used in routine treatment. In this review, it was aimed to provide detailed information about the potential of natural agents to regulate and/or target non-coding RNAs and their mechanisms of action to provide an approach for cancer therapy. The discovery of novel anti-cancer targets and subsequent development of effective drugs or combination strategies that are still needed for most cancers will be promising for cancer treatment.

Virtual screening of pyrazole derivatives of usnic acid as new class of anti-hyperglycemic agents against PPARγ agonists.

The finest sources of therapeutic agents are natural products, and usnic acid is a secondary metabolite derived from lichen that has a wide range of biological actions, including anti-viral, anti-cancer, anti-bacterial, and anti-diabetic (hyperglycemia). Based on the hyperglycemia activity of UA, this work seeks to identify new anti-hyperglycemia medicines by virtual screening of pyrazole derivatives of UA. Seven hit compounds (Compounds 1, 5, 6, 7, 17, 18 and 33), which finally go through docking-based screening to produce the lead molecule, were identified by the physicochemical attributes, drug-likeliness, and ADMET prediction. The docking score for the chosen compounds containing PPARγ agonists ranged from -7.6 to -9.2 kcal/mol, whereas the docking goal for compounds 5, 6, and 7 was -9.2 kcal/mol. Based on the binding energy and bound amino acid residues as well as compared to the reference compound, compound-6 considered as lead compound. Furthermore, the MD simulation of 3CS8-Compound-6 and 3CS8-Rosiglitazone complexes were performed to verify the stability of these complexes and the binding posture acquired in docking experiments. The compound-6 had strong pharmacological characteristics, bound to the PPARγ agonist active site, and was expected to reduce the activity of the receptor, according to the virtual screening results. It must be justified to conduct both in-vitro and in-vivo experiments to examine the efficacy of this compound. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-023-00176-y.

Discovery and Development of 4-Hydroxyphenylpyruvate Dioxygenase as a Novel Crop Fungicide Target.

Plant pathogenic fungi pose a significant threat to crop yields and quality, and the emergence of fungicide resistance has further exacerbated the problem in agriculture. Therefore, there is an urgent need for efficient and environmentally friendly fungicides. In this study, we investigated the antifungal activity of (+)-Usnic acid and its inhibitory effect on crop pathogenic fungal 4-hydroxyphenylpyruvate dioxygenases (HPPDs) and determined the structure of Zymoseptoria tritici HPPD (ZtHPPD)-(+)-Usnic acid complex. Thus, the antifungal target of (+)-Usnic acid and its inhibitory basis toward HPPD were uncovered. Additionally, we discovered a potential lead fungicide possessing a novel scaffold that displayed remarkable antifungal activities. Furthermore, our molecular docking analysis revealed the unique binding mode of this compound with ZtHPPD, explaining its high inhibitory effect. We concluded that HPPD represents a promising target for the control of phytopathogenic fungi, and the new compound serves as a novel starting point for the development of fungicides and dual-purpose pesticides.

Restoring Colistin Sensitivity and Combating Biofilm Formation: Synergistic Effects of Colistin and Usnic Acid against Colistin-Resistant Enterobacteriaceae.

Colistin (COL), the last line of defense in clinical medicine, is an important therapeutic option against multidrug-resistant Gram-negative bacteria. In this context, the emergence of colistin-resistant (COL-R) bacteria mediated by broad-spectrum efflux pumps, mobile genetic elements, and biofilm formation poses a significant public health concern. In response to this challenge, a novel approach of combining COL with usnic acid (UA) has been proposed in this study. UA is a secondary metabolite derived from lichens and is well-known for its anti-inflammatory properties. This study aimed to investigate the synergistic effects of UA and COL against COL-R Enterobacteriaceae both in vitro and in vivo. The exceptional synergistic antibacterial activity exhibited by the combination of COL and UA was demonstrated by performing a comprehensive set of assays, including the checkerboard assay, time-dependent killing assay, and Live/Dead bacterial cell viability assay. Furthermore, crystal violet staining and scanning electron microscopy assays revealed the inhibitory effect of this combination on the biofilm formation. Mechanistically, the combination of UA and COL exacerbated cell membrane rupture, induced DNA damage, and generated a significant amount of reactive oxygen species, which ultimately resulted in bacterial cell death. In addition, erythrocyte hemolysis and cell viability tests confirmed the biocompatibility of the combination. The evaluation of the COL/UA combination in vivo using Galleria mellonella larvae and a mouse infection model showed a significant improvement in the survival rate of the infected larvae as well as a reduction in the bacterial load in the mouse thigh muscle. These findings, for the first time, provide strong evidence for the potential application of COL/UA as an effective alternative therapeutic option to combat infections caused by COL-R Enterobacteriaceae strains.

Synthesis of aminomethyl linked (+)-usnic acid derivatives via the Mannich reaction and evaluation of their biological activities.

(+)-Usnic acid (UA), a natural dibenzofuran derivative, abundantly produced by lichens and possess wide number of biomedical applications including antibacterial, anti-inflammatory, anti-oxidant and anticancer activities. In the present study, as series of usnic acid derivatives (3a-3i) were synthesised using Mannich reaction assessed for their antioxidant, α-glucosidase, and anticancer activities. The in vitro antioxidant activity showed that compound 3d displayed potent antioxidant activity by scavenging the activities of DPPH and ABTS+. The compounds 3d and 3e showed potent cytotoxic activity against HepG2 cancer cells by arresting the cell cycle at S phase and regulating the Bax/BcL2 expression and subsequently induce the apoptosis. Overall, the results clearly indicated that (+)-usnic acid derivatives bearing secondary amines are useful scaffolds for the development of drug candidates for treatment of oxidative stress mediated cancer and metabolic disorders.

Usnic Acid Derivatives Inhibit DNA Repair Enzymes Tyrosyl-DNA Phosphodiesterases 1 and 2 and Act as Potential Anticancer Agents.

Tyrosyl-DNA phosphodiesterase 1 and 2 (Tdp1 and Tdp2) are DNA repair enzymes that repair DNA damage caused by various agents, including anticancer drugs. Thus, these enzymes resist anticancer therapy and could be the reason for resistance to such widely used drugs such as topotecan and etoposide. In the present work, we found compounds capable of inhibiting both enzymes among derivatives of (-)-usnic acid. Both (+)- and (-)-enantiomers of compounds act equally effectively against Tdp1 with IC50 values in the range of 0.02-0.2 µM; only (-)-enantiomers inhibited Tdp2 with IC50 values in the range of 6-9 µM. Surprisingly, the compounds protect HEK293FT wild type cells from the cytotoxic effect of etoposide (CC50 3.0-3.9 µM in the presence of compounds and 2.4 µM the presence of DMSO) but potentiate it against Tdp2 knockout cells (CC50 1.2-1.6 µM in the presence of compounds against 2.3 µM in the presence of DMSO). We assume that the sensitizing effect of the compounds in the absence of Tdp2 is associated with the effective inhibition of Tdp1, which could take over the functions of Tdp2.