PMEG [9-(2-phosphonomethoxyethyl)guanine] and PMEDAP [9-phosphonomethoxy- ethyl)-2,6-diaminopurine] are acyclic nucleoside phosphonates possessing cytotoxic properties. Antiproliferative effect of PMEG was demonstrated in various tumor cell lines in vitro. PMEG also represents an active component of some experimental prodrugs with enhanced selectivity and efficacy (such as GS-9219). PMEDAP seems to have weaker effect in vitro compared to PMEG, however it exhibited pronounced antitumor effect in SD-rats with spontaneous lymphoma. Therefore it was included in the present study as well. The aim of this study was to describe the interactions of PMEG and PMEDAP with p38 MAP kinase signaling and its relationship to the apoptosis. We investigated the influence of these compounds on the expression of four genes encoding p38 MAPK isoforms and whether this change is translated into the protein. It was found that PMEG up-regulates p38β and γ mRNA in CCRF-CEM cells and p38 β and δ in HL-60 cells. The effect of PMEDAP was less pronounced than that of PMEG. However, total p38 protein level remained unaffected by PMEG and PMEDAP. Activation of p38 MAPK cascade was also measured in the cells exposed to these agents using phospho-specific antibodies. We found that neither PMEG nor PMEDAP activated p38 kinase...

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L’équilibre rédox entre les niveaux des espèces réactives de l’oxygène et de l’azote (ROS, RNS) et les espèces antioxydantes cellulaires est déterminant pour le fonctionnement normal de la cellule et sa viabilité. Le déséquilibre redox ou « stress oxydant » peut altérer les voies de signalisation cellulaires et générer des dommages sur les protéines, les lipides et l’ADN des cellules. Il est ainsi associé à de nombreuses pathologies, notamment les cancers. Les cellules cancéreuses présentent une dérégulation redox importante et un stress oxydant basal intrinsèque plus élevé par rapport aux cellules normales. Elles sont donc très dépendantes des systèmes antioxydants pour leur viabilité. Ainsi, l’administration de drogues qui i) génèrent des ROS / RNS additionnelles ou ii) inhibent les systèmes antioxydant cellulaires, permet une cytotoxicité sélective contre les cellules cancéreuses. C’est la base biologique de la « thérapie anticancéreuse basée sur la modulation de l’équilibre redox». Dans ce contexte, nos travaux ont pour but de décrypter les mécanismes redox derrière l’activité anticancéreuse de la vitamine C (VitC) et de l’auranofin (AUF), seuls ou en combinaison, dans le modèle du cancer du sein. La VitC à des concentrations pharmacologiques élevées présente des propriétés pro-oxydantes. Dans cette étude, l’activité anticancéreuse de la VitC contre les lignées du cancer du sein est associée à une génération extracellulaire et intracellulaire de peroxyde d'hydrogène (H₂O₂) accompagnée d'une oxydation intracellulaire du glutathion (GSH). L’approche protéomique «redoxome» a révélé que la VitC induit une altération de l'état rédox d’enzymes antioxydantes clés et d'un certain nombre de protéines contenant des cystéines, impliquées dans les métabolismes de l’ARN et l’ADN et dans les processus énergétiques. La VitC est également responsable d’un retard dans la progression du cycle cellulaire et d’une inhibition de la traduction. Finalement, des analyses bioinformatiques ont montré que les niveaux d'expression de la peroxiredoxine 1 (PRDX1) sont corrélés à la cytotoxicité différentielle de la VitC dans les cellules cancéreuses du sein. L'AUF, un antirhumatismal, est un inhibiteur des thiorédoxines réductases qui a reçu une attention croissante pour son activité anticancéreuse. Nos travaux montrent que l’AUF inhibe également le système antioxydant du GSH et que cette inhibition est primordiale pour son activité anticancéreuse. L’AUF modifie l'état redox de nombreuses protéines impliquées dans la prolifération et le cycle cellulaire, et provoque une déplétion des dNTPs et un arrêt du cycle cellulaire. De façon remarquable, nous avons démontré que la combinaison de l’AUF et de la VitC présente une cytotoxicité accrue, synergique, médiée par H₂O₂ dans les cellules MDA-MB-231 et d'autres lignées cellulaires du cancer du sein sans trop affecter les cellules normales. In vivo, l’efficacité de la combinaison AUF/VitC a été validée sur des xénogreffes de MDA-MB-231 chez les souris sans présenter une toxicité notable, tandis que l'administration de l’AUF ou de la VitC en monothérapie n’inhibe pas la croissance tumorale. Enfin, les analyses protéomiques, bioinformatiques et fonctionnelles ont identifié la prostaglandine réductase 1 (PTGR1) comme biomarqueur prédictif de la réponse des cellules cancéreuses du sein à la combinaison AUF/VitC. En résumé, ces résultats contribuent à une meilleure compréhension des mécanismes anticancéreux de la VitC et de l'AUF, seuls et en combinaison. En particulier, la combinaison de ces deux médicaments disponibles et non toxiques pourrait être efficace contre le cancer du sein triple négatif et potentiellement d'autres cancers présentant des propriétés redox similaires. Ainsi, une évaluation préclinique et clinique de ces traitements ouvrira la voie à des nouvelles thérapies anticancéreuses basées sur la modulation de l’équilibre redox cellulaire. / Reactive oxygen and nitrogen species (ROS, RNS) homeostasis and intracellular reductive/oxidative (redox) dynamics play a key role in regulating cell fate and are critical for normal cellular functions. Oxidative stress via the disruption of redox homeostasis can lead to aberrant cell signaling and toxic oxidative damage of DNA, lipids and proteins, and is therefore associated with human pathologies such as cancers. Cancer cells experience extensive redox deregulation and generally exhibit higher intrinsic basal oxidative stress than normal cells, as a consequence, they are more dependent on their antioxidant systems for survival. Thus, the administration of a drug generating additional ROS / RNS or inhibiting cellular antioxidant systems will exert a selective cytotoxicity towards cancer cells while sparing their normal counterparts. This is the biological basis for « redox-based anticancer therapy ». The work described here aims to investigate the redox-based anticancer activity of vitamin C (VitC) and auranofin (AUF), as single drugs or in combination, in breast cancer model. VitC at high pharmacological concentrations shows pro-oxidant properties. In this study, we showed that VitC anticancer activity against breast cancer cell lines was associated to extracellular and intracellular generation of hydrogen peroxide (H₂O₂), accompanied by the oxidation of intracellular glutathione (GSH). A “redoxome” proteomics approach revealed that VitC induces alterations of the redox state of key antioxidant enzymes and a number of cysteines-containing proteins including many proteins involved in RNA and DNA metabolisms and energetic processes. Cell cycle arrest and translation inhibition are associated with VitC-induced cytotoxicity. Finally, bioinformatics analysis and biological experiments identified that peroxiredoxin 1 (PRDX1) expression levels correlate with VitC differential cytotoxicity in breast cancer cells. AUF, an antirheumatic drug and known inhibitor of thioredoxin reductases, has been repurposed recently as a potent anticancer drug. We showed that AUF acts on both the thioredoxin and GSH systems and its impact on GSH system is essential for its anticancer activity. AUF alters the redox state of a number of nucleic acid-binding proteins involved in cell proliferation, cell division and cell cycle, triggering dNTP depletion and cell cycle arrest. Importantly, we observed that the combination of AUF and VitC reveals a synergetic and H₂O₂-mediated cytotoxicity towards MDA-MB-231 cells and other breast cancer cell lines without much impact on normal cells, thus decreasing the cytotoxic concentrations of AUF or VitC single drug. The anticancer potential of AUF/VitC combinations was validated in vivo on MDA-MB-231 xenografts in mice without notable side effects, while administration of AUF or VitC as a single agent failed to suppress tumor growth. Finally, SILAC proteomics, bioinformatics analysis, and functional experiments linked prostaglandin reductase 1 (PTGR1) expression levels with breast cancer cell response to AUF/VitC combination, thus identifying a potential predictive biomarker. Overall, these results provide new insights into the anticancer mechanisms of VitC and AUF, as single drugs and in combination. In particular, this combination of two non-toxic and commonly available drugs could be efficient against triple-negative breast cancer and potentially other cancers with similar redox properties. Further assessment in preclinical and clinical studies of these drugs and combinations could open new avenues for redox-based anticancer therapy.

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Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Nikola Jaščevská Supervisor: PharmDr. Anna Jirkovská, Ph.D. Title of diploma thesis: The effects of topoisomerase II beta on the sensitivity of the cancer cells to the antineoplastics Topoisomerase II (TOP II) is a cellular enzyme responsible for solving topological problems of double-stranded DNA. Alpha and beta isoforms of TOP II are different gene products having similar catalytic activities. The expression of TOP IIα is cell-cycle dependent, peaking in G2/M phase, while TOP II isoform is expressed constitutively throughout the cell cycle. It is therefore present also in non-proliferating differentiated cells. Anthracycline antibiotics are an old class of anticancer drugs, belonging to TOP II poisons. Although their clinical usefulness is high, the incidence of side effects (especially myelotoxicity and cardiotoxicity) may limit the therapy. The key role of TOP II inhibition, which is present also in cardiomyocytes, has been increasingly discussed. Dexrazoxane, the only clinically used cardioprotective, leads to depletion of TOP II in cardiomyocytes, which may explain its cardioprotection. Although TOP II was previously shown to be dispensable for cellular proliferation, its possible...

Natural products have been a significant source of new drugs, especially in treating cancer, infectious diseases, hypertension, and neurological disorders. Although many natural metabolites have been screened and yielded pharmaceutically important drugs, many potential sources of natural product drug therapies still need to be investigated, including lichens. Lichens are symbiotic systems consisting of a filamentous fungus and a photosynthetic partner (an eukaryotic alga and/or cyanobacterium). Lichens produce an impressive variety of unique secondary compounds and have been used as ingredients in folk medicines for centuries. Demonstrated biological roles based on lichen chemistry include: antibiotics, anti-proliferative, antioxidants, anti-HIV, anti-cancer, immunomodulation, and anti-protozoans. Although North America is home to an impressive variety of lichen species, there is limited research to examine the biological potentials of these lichens. The core goal of this dissertation research has been to investigate some of the biological roles including, antibiotic, antioxidant, and anticancer potentials using lichen crude extracts and their metabolites collected from various locations in the United States. Antibiotic screening of crude extracts of 36 lichen species demonstrated inhibitory effects against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Methicillin-resistant S. aureus (MRSA). Generally, acetone extractions were found to be more effective than methanol extractions. It has also been shown that L. vulpina extract was bacteriocidal against MRSA with a relatively slow kill rate that disrupts cell membrane integrity and cell division as possible modes of action. Antioxidant screening of extracts from 11 lichen species, using the Oxygen Radical Absorbance Capacity (ORAC) assay, showed that lichen extracts inhibited the oxidative degradation of the fluorescent molecule (fluorescein-sodium salt) by the oxygen free radical initiator AAPH (2,2'-azobis(2-aminidopropane) dihydrochloride Acetone extracts as well as pure compounds from lichen species showed cytotoxic effects against Burkitt's lymphoma (Raji) cells and a colon cancer cell line (HT29 and SW620). They decreased proliferation, arrested cell cycle at various stages and force the cell to undergo apoptosis. The tested extracts or pure compounds were not toxic to normal cells. In colon cancer apoptosis took place independent of casapase-3. The results of this dissertation showed that lichen compounds merits for further investigation.

A microwave reactor was used to synthesize a series of novel 3,6-disubstituted or 3-substituted pyrazolo[1,5-a]pyrimidines in a total of 1 hour reaction time over 3 steps. The products were obtained in good to excellent yields (34-92%, ave = 52%) using a straightforward synthesis starting with the reaction of dimethylformamide-dimethylacetal with commercially available aryl acetonitriles (120C, 20 min). This was followed by treatment with H2NNH2 • HBr (120C, 20 min), and then reacted with either 1,1,3,3-tetramethoxypropane or a 2-aryl-substituted malondialdehydes (120C, 20 min). The resulting product was either collected on a sintered glass funnel or purified via column chromatography. The compounds were screened for anti-cancer activity against A2780 Ovarian and/or MCF7 breast cancer cell lines in vitro. The most active compounds were the 3-(4-(trifluoromethyl)phenyl)-6-[4-(2-(piperidin-1-yl)ethoxy]phenyl analogue and the 3-(2-fluorophenyl)-6-[4-(2-(4-methylpiperzin-1-yl)ethoxy]phenyl analogue, exhibiting EC50 values of 0.84 and 0.52 M respectively, which is 2-3 times more potent than Dorsomorphin. Several of the derivatives also showed promising activities against several viruses of emerging concern, including HBV, MERS Coronavirus, Zika, and Ebola. Use of a microwave reactor to synthesize N’-aryl/(alkyl) substituted N-[(4-hydroxy-6-phenyl)pyrimidin-2-yl]guanidines or N-[(4-hydroxy-6-trifluoromethyl)pyrimidin-2-yl]guanidines from the corresponding cyanamides with alkyl/aryl amines was achieved in good to excellent yields (39-96%, ave = 62%) in 10 minutes at 120C using only 1 equivalent of amine. Work-up was exceptionally simple, and involved collecting precipitated solids on a sintered glass funnel and washing with cold 2-propanol. Products were obtained in analytically pure form and required approximately 1 hour to prepare, start to finish. Compounds in this series showed early promise as potential inhibitors of A2780 Ovarian cancer, in vitro.

A library of 25 novel 3,6-disubstituted and 3-substituted pyrazolo[1,5-a]pyrimidines were synthesized using a microwave chemical reactor in 3 steps with a total reaction time of 1 hour. The products were obtained in good to excellent yields (20-93%, ave. = 62%). The synthesis began with the reaction of aryl acetonitriles with dimethylformamide-dimethylacetal (120C, 20 min), followed by treatment of the resulting 2-arylacrylonitrile with H2NNH2 • HBr (120C, 20 min). The intermediate 4-arylpyrazol-5-amine obtained was finally reacted with either 2-aryl-substituted malondialdehydes or 1,1,3,3-tetramethoxypropane (120C, 20 min) to give the final products. The products were either collected directly on a Buchner funnel or purified via flash chromatography. The compounds were screened for anti-cancer activity against the A2780 Ovarian cancer cell line in vitro at 10 µM. The most active compound was the 2-(pyrazolo[1,5-a]pyrimidin-3-yl)benzothiazole, herein referred to as RD-I-53, which had an EC50 value of 0.9 µM nearly mirroring the experimental control, Dorsomorphin, which had an EC50 of 1.1 µM. RD-I-53 was screened against a panel of 453 kinases by DiscoverX in a KinomeScan™, a competitive binding inhibition assay wherein RD-I-53 selectively inhibited VPS34 kinase and JAK1-JH2 pseudokinase (Kd VPS34 = 0.4µM, Kd JAK-1 JH2 = 0.5µM). NCI-60 data revealed selective anticancer activity of RD-I-53 against the MCF-7 and MDA-MB-468 breast cancer cell lines. Virtual docking studies of RD-I-53 against the VPS34 active site and its derivatives resulted in the creation of a virtual library of new compounds with potentially improved anticancer activity. A highly convergent route was developed to facilitate the ease of access to derivatives of RD-I-53. In the process, new methodologies for the synthesis of 2-aminobenzothiazoles and the thiocyanation of non-C4-substituted anilines and heterocycles were investigated and reported. A library of derivatives of RD-I-53 has been synthesized to be screened for potentially improved kinase inhibitory and anti-cancer activity.

Editorial on the Research Topic. Antimicrobial and Anticancer Peptides.

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