Assim como o número de casos de câncer vem aumentando em nível global, a busca por abordagens terapêuticas visando uma maior eficácia com um menor poder de causar efeitos prejudiciais aos pacientes também vem crescendo. As antraciclinas e antracenodionas, as quais tem como exemplos, doxorrubicina (DOX) e mitoxantrona (MTX), respectivamente, são fármacos utilizados na quimioterapia em diversas neoplasias incluindo tumores sólidos e não sólidos tais como de mama, leucemias, linfomas, sarcomas etc. Embora sejam eficazes ao que se propõem, o tratamento com estas moléculas pode acarretar em efeitos secundários, tais como arritmias e insuficiência cardíaca. Estas drogas além de interagirem com o ferro e apresentarem capacidade de gerar espécies reativas de oxigénio (ROS), apresentam como principal mecanismo a inibição da enzima topoisomerase 2 (Top2). Os inibidores de PARP-1 emergiram como uma nova alternativa para tratar determinados tipos de neoplasias em que a letalidade sintética possa ser explorada. Além disto, já foi relatado que a toxicidade cardíaca induzida por DOX seja influenciada pela atividade de PARP-1. O objetivo desta tese foi, portanto, avaliar a influência da inibição de PARP-1 na toxicidade cardíaca de DOX e MTX em células cardíacas. Células foram incubadas durante 24h com DOX ou MTX na presença ou na ausência de inibidor de PARP-1. Ensaios de viabilidade, apoptose e genotoxicidade e foram realizados. Além disso, a fosforilação de proteínas envolvidas na resposta a danos no DNA (ATM, MRE-11 e H2AX) foram avaliadas por western blot e imunofluorescência. Os resultados demonstraram que a inibição de PARP-1, apesar de diminuir a concentração de ROS, diminui a viabilidade de células H9c2 tratadas com DOX ou MTX por aumentar a geração de quebras duplas no DNA induzida por estes fármacos. / As the number of people with cancer are globally increasing, the search for therapeutic approaches that increases efficiency decreasing harmful effects to patients is also growing, giving rise to cardio-oncology. Anthracyclines, e.g., doxorubicin (DOX), and anthracenediones, e.g., mitoxantrone (MTX), are drugs used in the chemotherapy of several cancer types, including solid and non-solid malignancies such as breast cancer, leukemia, lymphomas, and sarcomas. Although they are effective in tumor therapy, treatment with these two drugs may lead to side effects such as arrhythmia and heart failure. These drugs interact with iron to generate reactive oxygen species (ROS), target topoisomerase 2 (Top2), and impair mitochondria. PARP-1 inhibitors have emerged as a new alternative for treating certain types of malignancies in which the synthetic lethality can be exploited. Furthermore, it has been reported that DOX-induced cardiac cardiotoxicity is influenced by PARP-1 activity. The main goal of this thesis was, therefore, to evaluate PARP-1 inhibition influence in cardiac toxicity of DOX and MTX in cardiac cells. Cells were incubated for 24h with MTX or DOX in presence or absence of PARP-1 inhibitor. Viability, oxidative stress and genotoxicity assays have been conducted. Furthermore, phosphorylation of proteins involved in response to DNA damage (ATM, H2AX and MRE-11) were evaluated by western blot and immunofluorescence. Results demonstrated that inhibition of PARP-1, although decreasing ROS generation, decreases H9c2 cells viability after DOX or MTX by increasing DNA double strand break generation induced by these drugs.

Many patients suffering from depressive disorders are refractory to treatment with currently available antidepressant medications, while many more exhibit only a partial response. These factors drive research to discover new pharmacological approaches to treat depression. Numerous studies demonstrate evidence of inflammation and elevated oxidative stress in major depression. Recently, major depression has been shown to be associated with elevated levels of DNA oxidation in brain cells, accompanied by increased gene expression of the nuclear base excision repair enzyme, poly(ADP-ribose) polymerase-1. Given these findings and evidence that drugs that inhibit poly(ADP-ribose) polymerase-1 activity have antiinflammatory and neuroprotective properties, the present study was undertaken to examine the potential antidepressant properties of poly(ADP-ribose) polymerase inhibitors.

Untersuchung des Wirkmechanismus von AEZS 126 auf drei triple negative Mammakarzinomzelllinien HCC1937, HCC1806 und MDA-MB468 und eine Oestrogenrezeptor positive Zelllinie MCF-7 mittels Kristallviolett assay, FACS und Western Blot. Es konnte gute Antitumorwirkung des Inhibitors in vitro gezeigt werden. / Of more than one million global cases of breastcancer diagnosed each year, a high percentage are characterized as triple-negative, lacking the oestrogen, progesterone and Her2/neu receptors. The incidence exceeds the incidence of malignancies like CML by far. Lack of effective therapies, younger age at onset and early metastatic spread have contributed to the poor prognosis and outcomes associated with these malignancies. Here, we investigate the ability of the PI3 K/AKT inhibitor AEZS 126 to selectively target the triple negative breast cancer (TNBC) cell proliferation and survival in vitro by MTT-assays and FACS-based analysis. Furthermore, the mechanism of cytotoxicity is analysed by FACS-based assays and Western blots. Results AEZS 126 showed good antitumour activity in in vitro models of TNBC as well as in MCF-7 cells. We demonstrated the highly efficient antitumour activity of AEZS 126 in in vitro models of TNBC. Due to the good anti-tumour activity and the expected favourable toxicity profile, AEZS 126 in combination with chemotherapy seems to be a promising candidate for clinical testing in TNBC especially in the basal-like subgroup of TNBC.

Carbon tetrachloride (CCl4) is a hepatotoxicant known to elevate alanine aminotransferase (ALT) and other liver enzyme levels, and cause lipid peroxidation, as well as centrilobular necrosis. A number of poly ADP-ribose polymerase (PARP) inhibitors were administered via intraperitoneal (i.p.) injections to male ICR mice as cotreatments at various time intervals relative to the CCl4. Aminophylline, a water soluble complex consisting of two molecules of theophylline bridged by ethylene diamine, was administered one-half hour, one hour and two hours after CCl4. The levels of ALT in the serum, as well as malondialdehyde and its equivalent markers of oxidative damage in the liver, were significantly reduced by aminophylline, relative to those in mice receiving only CCl4. The hepatoprotective effects of aminophylline were confirmed via the examination of histopathologic samples from the livers of mice receiving aminophylline in conjunction with CCl4 as opposed to those administered CCl4 alone. The potential benefit to society as a result of this research is that aminophylline, which has already been approved by the Food and Drug Administration (FDA), could potentially be administered in the event of an overexposure to CCl4 or similar halocarbons to minimize the free radical-mediated hepatotoxicity resulting from overexposure.

Diphtheria Toxin-like ADP-Ribosyltransferases The Human ADP-ribosyl transferases (ARTDs) are a group of poorly studied enzymes which are believed to be involved in e.g. DNA repair, protein degradation, transcription regulation and cell death. Medicinal chemistry programmes aimed at developing selective inhibitors of these ARTDs were initiated. A suitable starting compound for one of these enzymes, ARTD3, was found by screening a library of NAD-mimics using a thermal shift assay. A virtual screening protocol was instead developed in order to find novel inhibitors of ARTD7, 8, and 10. The hit compounds were then further developed into selective inhibitors of the corresponding ARTDs by systematically varying different structural features using a combination of synthetic organic chemistry, computational chemistry and structural biology. Compounds were initially characterized using differential scanning fluorimetry which was later replaced with an enzymatic assay to obtain IC50 values. Biotinylated analogs were also synthesized in an attempt to develop an AlphaScreen assay. A selective ARTD3 inhibitor was ultimately identified and found to delay DNA repair in cells after γ-irradiation. These compounds are potentially valuable tools for elucidating the biological role of the poorly characterized ARTD-family of proteins. Total Synthesis of Resveratrol Based Natural Products The polyphenolic natural product (-)-hopeaphenol was found to inhibit the type III secretion system present in certain gram-negative bacteria. (-)-Hopeaphenol is a tetramer of resveratrol and in order to investigate whether the entire structure was essential for inhibition two resveratrol dimers, ε-viniferin and ampelopsin B, were synthesized using a flexible and divergent synthetic route. Highlights of the synthetic strategy include the use of cyclopropylmethyl protecting groups, allowing an acid mediated three-step-one-pot deprotection-epimerization-cyclization of an advanced intermediate to form ampelopsin B. All previously reported syntheses of these two natural products include a dimerization of resveratrol which severly limits the possibilities to synthesize structural analogs. This new strategy enables the synthesis of a wide variety of analogs to ε-viniferin and ampelopsin B. / Populärvetenskaplig sammanfattning Små molekyler för att identifiera proteiners funktion Vår arvsmassa innehåller cirka 24000 gener som i sin tur innehåller information för hur de tusentals proteiner vi är uppbyggda av ska framställas. Många läkemedel fungerar genom att en molekyl interagerar med ett av dessa proteiner och hämmar dess funktion för att på så sätt framkalla en önskad effekt. Vi vet dock inte vilken funktion många av våra proteiner fyller vilket ofta gör utvecklingen av nya läkemedel svår eller omöjlig. Den första delen av denna avhandling beskriver en grupp proteiner kallade ARTDs och hur små molekyler kan framställas och systematiskt förbättras för att till slut helt kunna slå ut vissa av dessa ARTDs. Genom att sedan studera vilka effekter detta medför kan man ta reda på vilken funktion proteinet fyller. På längre sikt skulle denna kunskap sedan kunna användas för att utveckla nya läkemedel genom att till exempel slå ut de proteiner som orsakar en sjukdom. Totalsyntes av naturprodukter Naturprodukter defineras inom kemin som naturligt förekommande molekyler som produceras av levande organismer. De kan hittas i allt från mikroorganismer och växter till djur och kan vara en del av deras ämnesomsättning, en restprodukt eller ha någon annan funktion, känd eller okänd. Människor, och i vissa fall även andra djur, har sedan urminnes tider ovetandes använt dessa molekyler för en mängd olika syften, som gifter, färgämnen eller läkemedel. Penicillin är en av de mest kända, men mer än hälften av de nya läkemedel som godkänts de senaste trettio åren bygger på naturprodukter eller har inspirerats av dessa. De fortsätter således att vara viktiga för utvecklingen av nya läkemedel trots att vi idag har möjligheten att utveckla sådana från grunden. Att framställa naturprodukter på konstgjord väg kallas totalsyntes och är ofta en mycket svår och tidskrävande process. Vanligtvis rör det sig om mycket stora och komplexa molekyler och det finns sällan ett uppenbart sätt att genomföra totalsyntesen. För att bättre klara av detta måste nya metoder utvecklas. Den andra delen av denna avhandling beskriver nya metoder för att framställa komplexa molekyler kallade polyfenoler. Målet var att dessa metoder skulle vara så pass flexibla att de även kan användas för att framställa nya polyfenoler som aldrig tidigare existerat men som har förbättrade egenskaper.

Many pathological conditions, including renal disease, are associated with oxidative stress. 2,3,5-tris(Glutathion-S-yl)hydroquinone (TGHQ), a potent nephrotoxic and nephrocarcinogenic metabolite of benzene and hydroquinone, generates reactive oxygen species (ROS), can cause DNA strand breaks, and the subsequent activation of DNA repair proteins, including poly(ADP-ribose) polymerase (PARP)-1. Under robust oxidative damage, PARP-1 is hyper-activated, which causes elevations in intracellular calcium concentrations (iCa²⁺), NAD⁺ and ATP depletion, and ultimately necrotic cell death. The role of Ca²⁺ in PARP-dependent necrotic cell death remains unclear. We therefore sought to determine the relationship between Ca²⁺ and PARP-1 during TGHQ-induced necrotic cell death in human renal proximal tubule epithelial cells (HK-2). Extracellular Ca²⁺ is responsible for coupling PARP-1 activation to increases in iCa²⁺ during TGHQ-induced cell death. Moreover, organelles such as the endoplasmic reticulum and the mitochondria, which contain intracellular Ca²⁺ stores play no role in increases of iCa²⁺. PARP-1 inhibition attenuates increases in iCa²⁺ induced by TGHQ, and treatment with 2-aminoethoxydiphenyl borate (2-APB), a store-operated Ca²⁺ channel (SOC) inhibitor, restored cell viability, levels of NAD⁺, and attenuated PAR protein-ribosylation (PARylation). Concurrent with SOC activation having a direct effect on PARP-1 activity, and PARP-1 inhibition attenuating increases in iCa²⁺, the results suggest that PARP-1 and SOCs are coupled during TGHQ-induced cell death. We also explored the relationship between SOC activation and PARP-1 downstream of PARP-1 activity. Poly(ADP-ribose)glycohydrolase (PARG), which catalyzes the degradation of PARs to yield free ADP-ribose (ADPR), is known to activate SOCs. Interestingly, siRNA knockdown of PARG modestly increased PAR ribosylation, but did not restore cell viability in the presence of TGHQ, indicating that free ADPR is not responsible for SOC activation in HK-2 cells. Overall, our results suggest that PARP-1 and Ca²⁺ are coupled through SOC entry, and that this relationship may involve alternative PAR-mediated signaling that leads to necrotic cell death. To further elucidate the role of PAR polymers in response to TGHQ, we determined the cellular co-localization of PAR by immunofluorescent staining. PAR polymers originally co-localized in the nucleus, and in the cytosol at later time points. Immunoprecipitation with a pADPr antibody and further analysis via mass spectrometry revealed PARylation of many stress-related proteins and Ca²⁺-related proteins upon TGHQ treatment. We therefore speculate that cytosolic PAR may cause downstream signaling, PARylating proteins that activate store-operated Ca²⁺ entry either directly through Ca²⁺-related proteins or PARylation of stress-related proteins. Thus, PARylation of proteins may contribute to increases in iCa²⁺ concentrations, leading to PARP-1-dependent necrotic cell death. Our studies provide new insight into PARP-mediated necrotic cell death. Ca²⁺ is coupled to PARP-1 hyperactivation through SOCs, where iCa²⁺ increases are independent of PARG activity, demonstrating a novel signaling pathway for PARP-dependent necrotic cell death.

Mechanisms of cell death extend beyond the simple apoptosis/necrosis relationship to include regulated modes of cell death that do not readily fit either of the classic descriptors. One such mechanism of cell death involves poly(ADP-ribose)polymerase-1 (PARP-1)-mediated cell death. 2,3,5-Tris(Glutathion-S-yl)-hydroquinone (TGHQ), a reactive oxygen species (ROS) generating nephrotoxic and nephrocarcinogenic metabolite of hydroquinone, causes necrotic renal cell death, the basis for which is unclear. We therefore investigated TGHQ-mediated cell death in human renal proximal tubule epithelial HK-2 cells. TGHQ induced ROS generation, DNA strand breaks, hyperactivation of PARP-1, rapid depletion of nicotinamide adenine dinucleotide (NAD), elevations in intracellular Ca2+ concentrations, loss of mitochondrial membrane potential, and subsequent necrotic cell death. Interestingly, PARP-1 hyperactivation was not accompanied by the translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus, a process usually associated with PARP-dependent cell death. Inhibition of PARP-1 with PJ34 blocked TGHQ-mediated accumulation of poly(ADP-ribose) polymers, NAD consumption, and the consequent necrotic cell death. However, HK-2 cell death was only delayed by PJ34, and cell death remained necrotic in nature. In contrast, chelation of intracellular Ca2+ with BAPTA-AM completely abrogated TGHQ-induced necrotic cell death. Ca2+ chelation not only prevented the collapse in the mitochondrial potential but also attenuated PARP-1 hyperactivation. Conversely, inhibition of PARP-1 modulated TGHQ-mediated changes in Ca2+ homeostasis. Moreover, TGHQ caused a sequential oxidation of peroxiredoxin III (PrxIII), a protein considered the primary antioxidant defense within mitochondria. Thus, TGHQ induced two acidic shifts in PrxIII, with both pI shifted spots representing oxidized forms of PrxIII. Transient expression of a dominant negative version of PrxIII resulted in a significant increase in TGHQ-induced cytotoxicity, whereas overexpression of wild-type PrxIII significantly attenuated cytotoxicity. Our studies provide new insights into PARP-1-mediated necrotic cell death. Changes in intracellular Ca2+ concentrations appear to couple PARP-1-hyperactivation to subsequent cell death, but in the absence of AIF release from mitochondria. NAD depletion, mitochondrial membrane depolarization, Ca2+-mediated calpain activation, and PrxIII oxidation, all contribute to TGHQ-driven ROS-mediated necrotic cell death.

Glutamate is the primary excitatory neurotransmitter in the central nervous system. Extracellular glutamate concentrations are tightly regulated to avoid over-stimulation of glutamate receptors, which leads to a cascade of deleterious processes collectively known as excitotoxicity. Excitotoxicity is common to several neurodegenerative disorders and CNS injuries, including stroke and Alzheimer’s disease (AD). The projects described in this thesis were designed to uncover novel protective pathways in excitotoxic neurodegeneration. Excessive activation of the DNA repair enzyme, poly(ADP-ribose) polymerase-1 (PARP-1), is a convergence point for neuron death signaling in excitotoxic pathways. In AD, the peptide amyloid-β1-42 (Aβ1-42) is aberrantly produced, leading to excitotoxic neuron death in vitro. To investigate links between Aβ1-42 and PARP, we treated cultured cortical neurons with Aβ1-42 and determined whether PARP-1 contributes to neuron death. Increased neuron death was observed after Aβ1-42 exposure. A non-selective PARP-1/2 inhibitor significantly reduced Aβ1-42-induced death while elimination of PARP-1 alone was not neuroprotective. This suggests that PARP-2 or combined effects of PARP-1 and PARP-2 are required for Aβ1-42-induced neuron death. A hallmark of PARP over-activation is depletion of intracellular NAD+ and ATP levels, yet nearly all studies examining adenine nucleotide levels use separate biochemical samples to measure nucleotides individually. We developed two HPLC methods for simultaneous separation of NAD+, ATP, ADP and AMP. We determined that PARP-1 activation in astrocytes leads to near complete NAD+ depletion, followed by partial loss of ATP pools and total adenine nucleotide pools. Finally, we hypothesized that conjugated linoleic acid (CLA), a naturally occurring polyunsaturated fatty acid, is capable of enhancing neuron survival after an excitotoxic insult. Cultured cortical neurons were exposed to glutamate in the presence and absence of CLA. CLA levels likely achievable in human plasma and brain tissue during dietary supplementation regimens, protected neurons against glutamate excitotoxicity when given during or up to five hours after glutamate exposure. Several markers of mitochondrial damage and intrinsic apoptosis were examined. CLA stabilized mitochondrial membrane potential and permeability, shedding light on the mechanism of CLA neuroprotection. Overall, our research suggests a role for PARP in Aβ1-42 toxicity and identifies a novel role for CLA in neuroprotection following excitotoxicity.

Glutamate is the primary excitatory neurotransmitter in the central nervous system. Extracellular glutamate concentrations are tightly regulated to avoid over-stimulation of glutamate receptors, which leads to a cascade of deleterious processes collectively known as excitotoxicity. Excitotoxicity is common to several neurodegenerative disorders and CNS injuries, including stroke and Alzheimer’s disease (AD). The projects described in this thesis were designed to uncover novel protective pathways in excitotoxic neurodegeneration. Excessive activation of the DNA repair enzyme, poly(ADP-ribose) polymerase-1 (PARP-1), is a convergence point for neuron death signaling in excitotoxic pathways. In AD, the peptide amyloid-β1-42 (Aβ1-42) is aberrantly produced, leading to excitotoxic neuron death in vitro. To investigate links between Aβ1-42 and PARP, we treated cultured cortical neurons with Aβ1-42 and determined whether PARP-1 contributes to neuron death. Increased neuron death was observed after Aβ1-42 exposure. A non-selective PARP-1/2 inhibitor significantly reduced Aβ1-42-induced death while elimination of PARP-1 alone was not neuroprotective. This suggests that PARP-2 or combined effects of PARP-1 and PARP-2 are required for Aβ1-42-induced neuron death. A hallmark of PARP over-activation is depletion of intracellular NAD+ and ATP levels, yet nearly all studies examining adenine nucleotide levels use separate biochemical samples to measure nucleotides individually. We developed two HPLC methods for simultaneous separation of NAD+, ATP, ADP and AMP. We determined that PARP-1 activation in astrocytes leads to near complete NAD+ depletion, followed by partial loss of ATP pools and total adenine nucleotide pools. Finally, we hypothesized that conjugated linoleic acid (CLA), a naturally occurring polyunsaturated fatty acid, is capable of enhancing neuron survival after an excitotoxic insult. Cultured cortical neurons were exposed to glutamate in the presence and absence of CLA. CLA levels likely achievable in human plasma and brain tissue during dietary supplementation regimens, protected neurons against glutamate excitotoxicity when given during or up to five hours after glutamate exposure. Several markers of mitochondrial damage and intrinsic apoptosis were examined. CLA stabilized mitochondrial membrane potential and permeability, shedding light on the mechanism of CLA neuroprotection. Overall, our research suggests a role for PARP in Aβ1-42 toxicity and identifies a novel role for CLA in neuroprotection following excitotoxicity.

Less than half of patients with epithelial ovarian cancer (EOC) survive five years following diagnosis, underscoring the imperative need for improved treatment. Many patients, including those with advanced disease, initially respond to platinum agents, which constitute the backbone of therapy. However, tumors ultimately become resistant, rendering further treatment ineffective. Additionally, the poor tolerability of these agents warrants the exploration of more targeted treatments – one such strategy is exploiting synthetic lethal genetic relationships. Recent genomic sequencing efforts have revealed that as many of half of EOCs have homologous recombination (HR) alterations. HR is a critical pathway for the repair of platinum-induced ICLs, thus compromised HR is hypothesized to explain the initial response to chemotherapy in many patients. Accordingly, women whose tumors harbor mutations in the critical HR genes, BRCA1 or BRCA2 (BRCA1/2), demonstrate improved prognosis. BRCA1/2 mutations also confer exquisite sensitivity to inhibitors of the enzyme, poly(ADP-ribose) polymerase 1 (PARPis), hence loss of BRCA1/2 and PARP1 is synthetic lethal. A number of models have been proposed to explain this synthetic lethality, yet a consensus model that accounts for the diverse cellular roles of BRCA1/2 and PARP1 has yet to be established. Delineating the precise molecular underpinnings of PARPi action in BRCA1/2-deficient cells will aid clinicians in identifying the appropriate population of women with EOC likely to benefit from PARPi treatment and provide insight into resistance mechanisms that arise in these patients. Combining this approach with retrospective analysis of PARPi clinical trials will best define the proper indication for PARPi in EOC and other human cancers.