Searching for New Potential Small-Molecule Modulators of Pro-Apoptotic Proteins Using the Yeast-Based Screening Assay

Verónica Sofia Leite Salazar e Costa SEARCHING FOR NEW POTENTIAL SMALL MOLECULE MODULATORS OF PRO-APOPTOTIC PROTEINS USING THE YEAST-BASED SCREENING ASSAY Dissertação do 2º Ciclo de Estudos Conducente ao Grau de Mestre em Química Farmacêutica Trabalho realizado sob a orientação da Professora Doutora Lucília Helena Ataíde Saraiva e coorientação da Professora Doutora Honorina Maria de Matos Cidade e Doutora Mariana Valencia C. Ferreira Leão July 2015 ii DE ACORDO COM A LEGISLAÇÃO EM VIGOR, NÃO É PERMITIDA A REPRODUÇÃO DE QUALQUER PARTE DESTA TESE. iii ACKNOWLEDGEMENTS “Science is a way of life. Science is a perspective. Science is the process that takes us from confusion to understanding in a manner that's precise, predictive and reliable - a transformation, for those lucky enough to experience it, that is empowering and emotional.” Brian Greene First, I would like to thank to my supervisor Professor Lucília Saraiva for opening the doors of her laboratory and allow me to join her fantastic research group. Thank you for the opportunity, accompaniment, patient and transmitted knowledge. No doubt that she is a person for whom I have great admiration for everything she has conquered so far. I would also like to thank my co-supervisors for the all the support and enormous knowledge given to me in this period. To Mariana for helping me in my first steps as a researcher, for having taught me the bases and giving me precious tips. To Professor Honorina for all the words of encouragement and the huge sense of peace that she pours. To my dear research group: Ana Sara, Sara Gomes, Joana Soares, Liliana Raimundo, Helena Ramos, Mariana Leão and Cláudia Bessa, a huge thank from my heart. Without you, this would not have been possible. Thanks for all the good advice, support, willingness and transmitted knowledge. They were always by my side and made me grow as a person. Each of you has taught me something that will be precious in my life's journey. I would particularly like to thank Cláudia, you taught me a lot during this experience, always with great care and professionalism. Much of what I’ve learned, I’ve learned with you. I cannot forget the support given by the laboratory assistant, Cristina, always ready to help me, happy and expeditious. I will not forget what you did for me. But overall, thanks to all the other assistants, Nuno and Fernanda. They are equally spectacular. I would also like to thank to Professor Madalena Pinto, as well as, Professor Honorina Cidade, Professor Emília, Professor Carla and Professor Carlos Afonso. Thank you for making this master’s degree excellent. I am sure it will open several doors in the near future. My biggest acknowledgement goes to my parents. Everything I do is in order to make you proud. Thank you for all the efforts you do for me, you are the best persons in the world. In particularly to my mother which is the best mother ever. Thank you very much for all the support, friendship, for all the laughs, for being by my side making this process easier, for all the love in general. One day I want to be like you. I hope someday to be able to return what you have done for me. iv I cannot forget to thank to my boyfriend André. Your support was crucial during these time. You always believed in my value and also made me believe too. I hope you will be part of my life forever, contending together to have success in our lives. You were always on my side in the worst and best moments making me smile. To my friends Letícia and Agostinho, thank you very much for sharing this experience with me. Your support and friendship was unconditional. But overall, thanks to all my colleagues and masters degree’s friends. I wish you plenty of success in your life. This work was developed in the Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia da Universidade do Porto and in Centro de Química Medicinal da Universidade do Porto – CEQUIMED-UP, Laboratório de Química Orgânica e Farmacêutica, Departamento de Ciências Químicas, Faculdade de Farmácia da Universidade do Porto. This research was partially supported by the Strategic Funding UID/Multi/04378/2013 and UID/Multi/04423/2013 through FCT - Foundation for Science and Technology, FEDER funds through the Operational Programme for Competitiveness Factors – COMPETE and European Regional Development Fund (ERDF), in the framework of the programme PT2020 and the Project Pest-OE/SAU/UI4040/2014 and UCIBIO-PestC/EQB/LA0006/2013. v RESUMO A pesquisa de novas terapias contra o cancro tem vindo a assentar na promoção da apoptose, bem como, em formas de ultrapassar a resistência a este processo nas células tumorais. As caspases são uma família de proteases com função crucial na apoptose. As caspases-3, -6 e -7 encontram-se na fase executora da apoptose, armazenadas como procaspases que uma vez ativadas por proteólise clivam um grande número de substratos, resultando na apoptose. Assim sendo, a pesquisa de ativadores destas procaspases merecem especial atenção na descoberta de novos potenciais fármacos anticancerígenos. Num trabalho anterior realizado pelo nosso grupo, utilizando o modelo de pesquisa da levedura Saccharomyces cerevisiae expressando as caspases-3, -6 e -7 humanas, levou à identificação de dois compostos ativadores da caspase-7: 5,6dihydroxy-7-prenyloxyflavone (1a) and 3-hydroxy-7-geranyloxyflavone (2a). Estes resultados incitaram à realização da presente tese com o objetivo de descobrir novos ativadores das caspases-3, -6 e -7. Para isso, foram sintetizados análogos dos compostos 1a e 2a, e a sua atividade foi avaliada usando o modelo de levedura desenvolvido no trabalho anterior. Desta forma, foram identificados três potenciais ativadores da caspase7: 7-(allyloxy)-5,6-dihydroxy-2-phenyl-4H-chromen-4-one (S1), 6-dihydroxy-2-phenyl-7propoxy-4H-chromen-4-one (S2) and 5,6-dihydroxy-7-(isopentyloxy)-2-phenyl-4Hchromen-4-one (S3). A proteína cinase C (PKC) é uma família de cinases de serina/treonina envolvidas na regulação da proliferação e morte celular, desempenhando assim um papel crucial no processo de carcinogénese. Desta forma, esta família representa um alvo promissor na terapia anticancerígena. Com base nisto, num trabalho prévio foi desenvolvido um ensaio de levedura para a pesquisa de moduladores isoforma-seletivos de PKCs. Utilizando este modelo foi identificado o composto C4 como potencial ativador da PKCδ. Baseado nisto, na presente tese foi analisada a atividade antitumoral in vitro deste composto em linhas celulares HCT116 do adenocarcinoma do cólon. O composto C4 exibiu uma potente inibição do crescimento nas células tumorais associada com a indução da apoptose. Concluindo, foram identificados novos reguladores farmacológicos de proteínas pro-apoptóticas no presente trabalho, representando uma alternativa de estratégia personalizada no tratamento do cancro. Presentemente, mais trabalho está a decorrer de forma a confirmar o mecanismo de ação molecular dos mesmos em células tumorais humanas. Palavras-chave: Cancro; Descoberta de novos fármacos; Modelo de pesquisa na levedura; Família de proteínas Caspases; Família de proteínas PKC. vi ABSTRACT The search for new therapies against cancer has been focused on devising ways to overcome resistance and to trigger apoptosis in tumour cells. Caspases are a family of proteases with a crucial role in apoptosis. Caspases-3, -6 and -7 are at the core of the execution phase of apoptosis, stored as procaspases that once activated cleave a large set of substrates, ultimately resulting in the hallmarks of apoptosis. Therefore, the search for activators of these procaspases has deserved particular attention in the field of anticancer drug discovery. In a previous work from our group, the use of a yeast screening assay, based on the heterologous expression of human caspases-3 or -7 in the yeast Saccharomyces cerevisiae, led to the identification of two prenylated flavonoids as potential activators of caspase-7: 5,6-dihydroxy-7-prenyloxyflavone (1a) and 3-hydroxy-7-geranyloxyflavone (2a). These results prompted us to the present work aiming to discover new activators of caspases-3, -6 and -7. For that, analogues of flavonoids 1a and 2a were synthesised, and their activities on these proteins were evaluated, using the previously developed yeastbased screening assay. Three new potential selective activators of caspase-7 were identified: 7-(allyloxy)-5,6-dihydroxy-2-phenyl-4H-chromen-4-one (S1), 6-dihydroxy-2phenyl-7-propoxy-4H-chromen-4-one (S2) and 5,6-dihydroxy-7-(isopentyloxy)-2-phenyl4H-chromen-4-one (S3). The protein kinase C (PKC) is a family of serine/threonine kinases involved in the regulation of cell proliferation and death, playing therefore a major role in the carcinogenesis process. Thus, this family represents a promising and appealing target in anticancer therapy. In this way, in a previous work, a yeast targeted screening assay was developed to search for PKC isoform-selective modulators. Using this yeast approach, the compound C4 was identified as a potential selective activator of PKCδ. Based on this, in the present dissertation, the in vitro antitumor activity of C4 was analysed in the human colon adenocarcinoma HCT116 cell lines. The tested compound exhibited a potent growthinhibitory activity in tumour cells, which was associated with apoptosis induction. In conclusion, in this dissertation, new potential pharmacological regulators of proapoptotic proteins were identified. Further work is underway to confirm their molecular mechanism of action in human tumour cells. Despite that, the data emerged from the present work demonstrated that these compounds may represent an alternative strategy for personalized cancer treatment. Keywords: Cancer; Drug discovery; Yeast-based screening assays; Caspases family proteins; PKC family proteins. vii INDEX ACKNOWLEDGMENTS ………………………………………………………………………... iii RESUMO …………………………………………………………………………………………. v ABSTRACT ………………………………………………………………………………………. vi ABBREVIATIONS ……………………………………………………………………………..... xi CHAPTER 1 – INTRODUCTION 1. PRINCIPLES ABOUT CANCER ……………………………………………………………. 2 1.1. APOPTOSIS ..……………………………………………………………………… 4 1.2. TARGET PROTEINS OF THE APOPTOTIC PATHWAY……………………… 7 1.2.1. CASPASES…………………………………………………………….... 7 1.2.1.1. Effector caspases …………………………………………... 9 1.2.1.1.1. Small molecule activators of caspases family … 11 1.2.2. PROTEIN KINASE C FAMILY PROTEINS ………………………… 13 1.2.2.1. Modulators of PKC family proteins ...…………….............. 16 1.3. FLAVONOIDS AS PROMISING ANTICANCER AGENTS ……………………18 1.4. YEAST AS A MODEL SYSTEM ………………………………………………... 21 1.4.1. YEAST AS A MODEL TO STUDY HUMAN PROTEINS ………….. 21 1.4.2. YEAST AS A MODEL FOR TARGETED SCREENING ASSAYS .. 22 1.4.3. HUMAN PROTEINS STUDIED IN YEAST …………………………. 24 1.4.3.1. Caspase family proteins …………………………............... 24 1.4.3.1.1. Potential pharmacological modulators of caspases identified in yeast …………………………………………………….. 26 1.4.3.2. PKC family proteins…………………………………………. 27 1.4.3.2.1. Potential pharmacological modulators of PKC identified in yeast …………………………………………………….. 28 1.5. SCOPE OF THIS DISSERTATION…………………………………………… 31 CHAPTER 2 – MATERIALS AND METHODS 2.1. CASPASE FAMILY PROTEINS ………………………………………………… 33 2.1.1. CHEMISTRY …………………………………………………………… 33 2.1.1.1. General procedures for the preparation of baicalein derivatives ……………………………………………………………………... 33 2.1.1.1.1. 7-(allyloxy)-5,6-dihydroxy-2-phenyl-4H-chromen-4one (S1) ……………………………………………………………….. 34 viii 2.1.1.1.2. 5,6-dihydroxy-2-phenyl-7-propoxy-4H-chromen-4one (S2) ……………………………………………………………….. 34 2.1.1.1.3. 5,6-dihydroxy-7-(isopentyloxy)-2-phenyl-4Hchromen-4-one (S3) …………………………………………………. 34 2.1.2. BIOLOGICAL ACTIVITY ……………………………………………… 35 2.1.2.1. Yeast targeted screening assay…………………………… 35 2.1.2.2. Statistical analysis ………………………………………….. 35 2.2. PROTEIN KINASE C FAMILY PROTEINS ……………………………………..36 2.2.1. COMPOUNDS …………………………………………………………. 36 2.2.2. HUMAN TUMOUR CELL LINES AND GROWTH CONDITIONS ... 36 2.2.3. SULFORHODAMINE B (SRB) ASSAY ……………………………... 36 2.2.4. ANALYSES OF CELL CYCLE AND APOPTOSIS ………………… 36 2.2.5. WESTERN BLOT ANALYSIS ………………………………………... 37 2.2.6. FLOW CYTOMETRIC DATA ACQUISITION AND ANALYSIS …... 37 2.2.7. STATISTICAL ANALYSIS ……………………………………………. 37 CHAPTER 3 – RESULTS 3.1. CASPASE FAMILY PROTEINS ………………………………………………… 39 3.2. PKC FAMILY PROTEINS ………………………………………………………… 42 CHAPTER 4 – DISCUSSION, CONCLUSION AND FUTURE PROSPECTS 4.1. IDENTIFICATION OF FLAVONOIDS S2 AND S3 AS POTENTIAL SELECTIVE ACTIVATORS OF CASPASE-7 ……………………………………………………………….. 45 4.2. COMPOUND C4 HAS A POTENT IN VITRO TUMOUR GROWTH INHIBITORY EFFECT ASSOCIATED WITH APOPTOSIS INDUCTION ……………….......................... 47 4.3. FUTURE PROSPECTS ………………………………………………………….. 48 CHAPTER 5 – REFERENCES ……………………………………………………………….. 49 CHAPTER 6 – ATTACHMENTS ……………………………………………………………... 62 ix FIGURES INDEX Figure 1 - The hallmarks of cancer …………………………………………………………….. 3 Figure 2 - The extrinsic and intrinsic pathways of apoptosis ………………………………... 5 Figure 3 - Caspase family structure ……………………………………………………………. 7 Figure 4 - Scheme of caspase activation ……………………………………………………… 8 Figure 5 - Chemical structures of PAC-1, M1541, M1541B and WF-210 ………………… 11 Figure 6 - Mechanism of action of compound PAC-1 ………………………………………. 12 Figure 7 - PKC family structure ………………………………………………………………... 13 Figure 8 - Chemical structure of PMA ……………………………………….……………….. 17 Figure 9 - Basic skeleton and numbering pattern of some flavonoid classes ………….... 18 Figure 10 - Flavonoids antitumor activities ………………………………………………….. 19 Figure 11 - Gene overexpression technique ………………………………………………... 23 Figure 12 - Different strategies of human caspases-3, -6 or -7 activation in S. cerevisiae …………………………………………………………………………………………………….. 25 Figure 13 - The use of a yeast-based screening assay to search for modulators of procaspases-3 and -7 …………………………………………………………………………... 26 Figure 14 - Chemical structure of the flavonoids activators of caspase-7 identified using a yeast screening assay: compound 1a and compound 2a …………………………………... 27 Figure 15 - Chemical structure of the diterpene compound coleon U …........................... 28 Figure 16 - Molecular mechanism of action of coleon U in yeast cells ……………………. 29 Figure 17 - Chemical structure of compounds S1-S3 ……………………………………..... 39 Figure 18 - Effect of compounds S1-3 on the growth of yeast cells expressing procaspases3 or -7 …………………………………………………………………………………………….. 40 Figure 19 - Effect of compounds M1541, S2 and S3 on the growth of yeast cells expressing procaspase-6 ……………………………………………………………………………………. 41 Figure 20 - C4 induces a potent growth-inhibitory effect associated with apoptosis induction, in human colon adenocarcinoma HCT116 tumour cells ………………………… 43 4 Activating invasion and metastasis Tumour cells can spread to different parts of the body, in other words, they can metastasize. Metastatic cells must mimic normal cell-cell interactions, through cell-cell adhesion molecules (CAMs) and integrins. Integrins display substrate preferences, and changes in integrins are displayed by migrating cells. Matrix-degrading proteases are also necessary to facilitate invasion into stroma, across blood vessel walls, and through normal epithelial cell layers [reviewed in10]. Reprogramming energy metabolism In the presence of oxygen, normal cells process glucose into pyruvate via glycolysis in the cytosol, and thereafter to carbon dioxide in the mitochondria. In the absence of oxygen, glycolysis is favoured and relatively little pyruvate is dispatched to the oxygenconsuming mitochondria. Tumour cells are able to reprogram their energy metabolism. Even in the presence of oxygen, tumour cells can reprogram their glucose metabolism, and thus their energy production, by limiting their energy metabolism largely to glycolysis [reviewed in4]. Evading immune destruction Cells and tissues are constantly monitored by an immune surveillance that is responsible for recognizing and eliminating the wide majority of tumour cells and nascent tumours. However, it seems that the solid tumours are able to avoid detection by the immune system or have been able to limit the extent of immune killing, evading immunological destruction, in particular by T and B lymphocytes, macrophages, and natural killer cells, avoiding eradication [reviewed in4]. 1.1. APOPTOSIS Apoptosis is a type of programmed cell death that occurs when DNA damage is irreparable. This process is characterized by specific morphological and biochemical features, including blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation [reviewed in11]. The mechanisms of apoptosis are highly complex and sophisticated, involving an energy-dependent cascade of molecular events [reviewed in12]. There are two main apoptotic pathways: the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway (Figure 2) [reviewed in13]. 5 Figure 2 – The extrinsic and intrinsic pathways of apoptosis. Abbreviations: AIF - apoptosis inducing factor; Apaf-1 - apoptotic protease-activating factor 1; BID - BH3-interacting domain death agonist; tBID - truncated BID; Cyt. c - cytochrome c; DISC - death-inducing signalling complex; Endo G - endonuclease G; FADD - Fasassociated protein with death domains; IAP - inhibitors of apoptosis family; MOMP - mitochondrial outer membrane permeabilization; Smac/DIABLO - second mitochondria-derived activator of caspases. The extrinsic pathway requires the activation of cell death receptors, namely tumour necrosis factor (TNF) superfamily, such as TNF, CD95 (Fas), and TNF-related apoptosis inducing ligand (TRAIL) receptors, found on the surface of the cell membranes, and their respective ligands transmitting the death signal from the surface to the intracellular signalling pathways. The activation of some receptors, as CD95 and TNF, leads to receptor clustering and intracellular recruitment of proteins such as the protein adaptor FADD (Fasassociated protein with death domains). In turn, FADD recruits the initiator procaspase-8, forming the death-inducing signalling complex (DISC). The formation of this complex leads to the activation of procaspase-8, an initiator caspase. This activation induces the execution phase of apoptosis via the activation of the executioner procaspases-3, -6 and -7 or induces the cleavage of the BH3-interacting domain death agonist (BID), leading to the generation of a mitochondrion-permeabilizing fragment, the truncated BID (tBID) [reviewed in13-14]. On the other hand, the mitochondrial pathway is activated by stress signals such as DNA damage, oxidative stress, hypoxia or chemotherapeutic drugs. These external stimuli lead to modifications in the inner mitochondrial membrane permeability. These alterations induce changes at the mitochondrial outer membrane permeabilization (MOMP), loss of 6 mitochondrial transmembrane potential (Δѱm) and to the release of pro-apoptotic proteins such as cytochrome c (cyt c), the second mitochondria-derived activator of caspases (Smac/DIABLO), the apoptosis inducing factor (AIF) and the endonuclease G (Endo G) [reviewed in13]. After release into the cytoplasm, cyt c binds to the apoptotic protease-activating factor 1 (Apaf-1) and to procaspase-9, stimulating the apoptosome formation. In turns, the apoptosome activates procaspase-9, which in turn activates the executioner procaspases3, -6 and -7, resulting in an apoptotic cell death [reviewed in15]. Smac/DIABLO binds and inhibits several members of the inhibitor of apoptosis (IAP) family, preventing the IAPs from arresting the apoptotic process, thereby activating the executioner procaspases-3, -6, and -7, allowing apoptotic progression [reviewed in13]. In some models, the release of AIF and Endo G is a late event in apoptosis, which occurs once the cells are committed to die. Following the release of AIF and Endo G, these proteins are able to translocate to the nucleus, promoting DNA fragmentation through a caspase-independent manner [reviewed in14].The intrinsic pathway is mainly controlled by Bcl-2 family members, once they play a central role in the regulation of cyt c release from mitochondria. This family consists of anti-apoptotic members, such as Bcl-2 and Bcl-xL, and pro-apoptotic members, such as Bax, Bak and Bid. Bcl-2 and Bcl-xL are able to inhibit cyt c release by the protection of the membrane integrity through the interaction of these antiapoptotic proteins with mitochondrial proteins such as the adenine nucleotide translocase (ANT) or the voltage dependent anion channel (VDAC), thus preventing them from forming mitochondrial pores. On the other hand, Bax can homodimerize or heterodimerize with Bak or tBib, leading to the disruption of the mitochondrial membrane integrity through the formation of mitochondrial pores, with the subsequent cyt c release [reviewed in13]. The apoptotic process is essential for the maintenance of tissue homeostasis in multicellular organisms. It is well-known that the deregulation of this process has been implicated in numerous pathological conditions such as in cancer [reviewed in16]. In this way, the search for new anticancer therapies has been focused on devising ways to overcome increasing tumour resistance and to trigger apoptosis in tumour cells. It is clear that a number of anticancer drugs induce apoptosis in tumour cells. However, the problem is that some of these drugs usually induce cell death in normal cells as well. Therefore, the goal is to selectively modulate genes/proteins involved in apoptosis in tumour cells. In this way, understanding how those genes/proteins work reveals to be a crucial task for the development of more potent and effective compounds [reviewed in17]. 7 1.2. TARGET PROTEINS OF THE APOPTOTIC PATHWAY 1.2.1. CASPASES It became evident that the cellular and biochemical features of apoptosis are a consequence of the cleavage of a subset of proteins by proteases of the caspase family [reviewed in18]. The central role in the regulation and in the execution of apoptotic cell death belongs to caspases. In fact, an inappropriate caspase activity can cause deficient cell death, potentially leading to carcinogenesis and other apoptotic-associated pathologies [reviewed in19]. Caspases are a conserved family of cysteine-dependent aspartate-specific proteases that are expressed in an inactive proenzyme form (zymogens). Structurally caspases consist of an N-terminal prodomain, followed by a large subunit with 20 kDa (p20) and a small subunit with 10 kDa (p10). The N-terminal prodomain contains structural motifs that belong to the death domain superfamily: the death effector domain (DED) and the caspase recruitment domain (CARD) [reviewed in20] (Figure 3). Depending on the structure and cellular functions, caspases are divided in inflammatory (Group I: caspases-1, -4, -5, -11, - 12 e -13) and apoptotic (caspases-2, -3, -6, -7, -8, -9 e -10). In addition, apoptotic caspases are divided in initiator caspases (Group II: -2, -8, -9 e -10) and effector caspases (Group III: -3, -6 e -7) (Figure 3) [reviewed in21]. Figure 3 – Caspase family structure. Three major groups of caspases are presented: group I: inflammatory caspases; group II: initiator caspases; group III: effector caspases. Caspases consist of a single chain constituted by three domains: N-terminal prodomain (DED and CARD), large (p20) and small (p10) catalytic subunits. Adapted from20. 8 In most cells, caspases are activated through proteolytic cleavage in specific asparagines residues (Asp-X). This results in the generation of mature caspases, allowing the initiation of a protease cascade that is implicated in the inflammatory process and in apoptosis [reviewed in22] (Figure 4). This strategy provides a control over the activity of caspases, in order to reduce the possibility of, inadvertently, they enter apoptosis [reviewed in23]. Initiator caspases are presented in cells as inert monomers, which require dimerization and inter-domain cleavage for their activation. In contrast, effector caspases present themselves as inactive dimers, requiring cleavage of its catalytic domain, leading to intermolecular rearrangements with consequent formation of enzymatically active dimer [reviewed in24]. Figure 4 – Scheme of caspase activation. Cleavage of the procaspase leads to the formation of mature caspase, which comprises the heterotetramer p202–p102 without the prodomain. Adapted from20. There are two pathways through which the caspase family proteases can be activated: one is the signal-induced death receptor-mediated pathway and the other is the stressinduced, mitochondria-mediated pathway [reviewed in24]. This proteolytic cascade, in which one caspase can activate other caspases, amplifies the apoptotic signalling pathway, leading to a rapid cell death. Basically, caspases may be activated in response to different pro-death stimuli through the formation of activating complexes in the presence of adaptor proteins [reviewed in25]. The outcome of death receptor--mediated activation of caspase-8 depends of the cell type [reviewed in21]. In type I cells, procaspase-8 activation can directly activate downstream effector caspases (caspases-3, -6, -7) to promote DNA damage and apoptosis without requirement of the mitochondria dependent pathway. On type II cells, the apoptotic process is mostly controlled by Bcl-2 family members. Both caspase-8 and -2 can cleave and activate the pro-apoptotic Bcl-2 family protein. Indeed, Bax can homodimerize or heterodimerize with Bak or tBib, leading to the disruption of the mitochondrial membrane integrity, release of cyt c, and to the subsequent induction of the intrinsic apoptosis pathway. Once released, cyt c binds to Apaf-1, which recruits procaspase-9 to form the apoptosome. Subsequently, caspase-9 is activated and cleaves the effector caspases-3,-6 and -7, leading to the induction of apoptosis in a caspase-dependent manner [reviewed in26]. 9 Caspases activity can be suppressed by caspase inhibitor proteins, such as cowpox virus cytokine response modifier A (crmA), baculoviral p35/p49 and IAPS, a family of cellular proteins including, neuronal apoptosis inhibitory protein (NAIP), cellular IAP1 (cIAP1) and 2 (cIAP2), X-linked IAP (XIAP), survivin, bruce, melanoma IAP (ML-IAP) and IAP-like protein 2 (ILP2) [reviewed in27]. Mutation in CASP genes is not frequent in human tumour cells. In fact, it is believed that the inactivation of individual caspases is not usually sufficient to prevent the progression of caspase cascade. However, the reduced expression of pro-apoptotic caspases has been reported in manifold cancers, as well as specific inactivating mutations have been reported in many tumour types and stages of malignant transformation [reviewed in21]. Despite of the enormous relevance of initiator caspases, in this dissertation a particular attention will be given to effector caspases. 1.2.1.1. EFFECTOR CASPASES Caspases-3, -6 and -7 are at the core of the execution phase of apoptosis [reviewed in28]. Structurally these proteases are composed by large and small catalytic subunits, existing in the cells as inactive homodimers. The effector caspases are activated through proteolytic cleavage mediated by an initiator caspase [reviewed in19]. Once activated, besides to lead to cell death through cleavage of specific target proteins, the executioner caspases will be able to activate other effector caspases, leading to the generation of a feedback loop of caspase activation [reviewed in21]. Caspase-3 Caspase-3 is encoded by the CASP3 gene located in chromosome 4q35.1 and is the most well-characterized effector caspase, since exerts a key role in both extrinsic and intrinsic apoptotic pathways. Caspase-3 has both distinct and overlapping roles with caspase-6 and -729. The divergent amino-terminal sequences between caspase-3 and -7 appear to contribute to different modes of regulation30. In fact, Slee et al. (2001) performed a study where caspases-3, -6 and -7 were silenced in order to analyse the impact of the absence of these proteases on the execution phase of apoptosis. Indeed, the authors demonstrated that caspase-3 is a key component of the apoptotic process by participating in several events among this process, while caspase-6 and -7 exert more specialized roles31. 10 Many studies demonstrated that alterations in the CASP3 gene might promote human tumorigenesis, suggesting that caspase-3 may play a crucial role in cancer prevention. In fact, CASP3 polymorphism and their haplotypes help to define the individual’s genetic susceptibility to cancer development [reviewed in21]. This may happen since the CASP3 gene can give rise to an alternative splicing variant known as caspase-3s, which exert an anti-apoptotic function [reviewed in32]. Additionally, distinct works have demonstrated that procaspase-3 is overexpressed in a variety of human tumours, namely, colon cancer33, lung cancer34, melanoma35, hepatoma36, breast cancer37, among others. Caspase-6 Caspase-6 is encoded by the CASP6 gene located in chromosome 4q25. This executioner caspase are much more weakly apoptotic than caspase-3 and -738. Nevertheless, caspase-6 also cleaves a different set of cellular substrates39. The substrates of this effector caspase include PARP, lamin and procaspase-3. In fact, caspase-6 can activate procaspase-3 by a positive feedback pathway [reviewed in22]. The way how caspase-6 is activated is still controversial. In 2009, it has been reported that caspase-6 is activated only following activation of caspases-3 and -7 and thus is not activated directly by an initiator caspase29. However, in contrast, previous works showed that this caspase can be activated in the absence of caspase-3 and -740. More recently, it has been reported that, in vivo, caspase-6 can activate itself41. Expression of caspase-6 in gastric cancer is decreased, suggesting that loss of caspase-6 expression might be involved in the mechanism of gastric cancer development42. In fact, mutations in the CASP6 gene have been found in 2% of human cancers of colonic and gastric origin43. Caspase-7 Caspase-7 is encoded by the CASP7 gene located in chromosome 10q25.3. This caspase shares many similarities with caspase-3. Both proteases are effector caspases and are substrates of initiator caspases in the extrinsic or intrinsic apoptotic pathways. Despite that, caspases-3 and -7 exhibit distinct activities in vivo [reviewed in32]. This may be due to the presence in caspase-7 of an inhibitor of apoptosis binding motif (IBM), which is absent in caspase-3, that appears to be more efficiently ubiquitinated by IAPs, leading to a more readily proteasomal degradation of caspase-7 compared to caspase-344. In fact, several substrates are specifically cleaved by caspase-7, such as cochaperone p23 [reviewed in30a, 45]. 11 Mutations in the CASP7 gene affect the pathogenesis of some human solid cancers, since the function of caspase-7 is compromised [reviewed in21]. In fact, in the analysis of multiple types of cancer, developed in 2003, somatic mutations in CASP7 were detected in 2% of colon and esophageal carcinomas and 3% in head/neck carcinomas46. 1.2.1.1.1. SMALL MOLECULE ACTIVATORS OF CASPASES FAMILY The activation of effector caspases with small molecules could be important in the activation of these proteases and could have a direct impact on important physiological processes [reviewed in28]. Indeed, caspase activation can be particularly applied as an anticancer strategy by selective induction of apoptosis in cancer cells [reviewed in47]. Thus, the discovery of small molecule caspase activators has deserved particular relevance in the cancer field [reviewed in32]. However, to date, only few small molecule caspase activators have been reported [reviewed in28, 47]. Putt et al. (2006) discovered the Procaspase-Activating Compound 1 (PAC-1) (Figure 5A) as the first small molecule caspase activator that induces apoptosis in cancer cells through the activation of procaspases-3 and -733. Figure 5 - Chemical structures of (A) PAC-1 which activates caspases-3 and -7, (B) M1541 which activates caspases-3 and -6 (C) M1541B, (D) WF-210 which activate caspase-3. 12 In fact, PAC-1 does not act by direct activation of executioner caspases, but through the zinc ion chelation, the more physiologically relevant metal in inhibiting procaspases activity (Figure 6)48. Despite that, some studies stated that PAC-1 is able to kill cerebellar granule neurons in vitro and causes neurotoxicity in vivo49. Therefore, these evidences led to intensive efforts in the development of novel procaspase activators with minimal side effects. Figure 6 – Mechanism of action of compound PAC-1. Adapted from33. In 2009, Wolan and colleagues verified that compound M1541 (Figure 5B) was able to induce apoptosis in the p53-deficient breast cancer cell line BT549. In this study, it was reported that M1541 can bypass initiator caspase activation and directly induce apoptosis in a caspase-3/-6-dependent manner without requiring apoptotic stimuli or upstream factor50. This can be a promising strategy in cancer therapy where apoptosis of cancer cells can be directly triggered, resulting in cell death [reviewed in28]. In the same study, it was reported a potent activator of procaspase-3, compound M1545B (Figure 5C), an analogue of M154150. Compound M1541B appears to activate procaspase-3 via a binding-induced shift in the on-off state equilibrium50, or trough formation of nanofibriles51. More recently, a novel procaspase-3 activator, WF-210, (Figure 5D) was discovered. WF-210 induces apoptosis in human leukemia cell lines HL-60 and U-937 by activating procaspase-3. This compound showed higher in vivo efficacy and safety characteristics compared with PAC-152. 13 1.2.2. PROTEIN KINASE C FAMILY PROTEINS The protein kinase C (PKC) is a family of serine/threonine kinases that are involved in relevant processes, including neoplastic transformation, carcinogenesis and tumour cell invasion. Hence, in these processes, the activity and expression of some of these kinases are altered. Therefore, they are potentially suitable targets for anticancer therapy [reviewed in53]. The PKC family consists of at least 10 isozymes with distinct and in some cases opposing roles in cellular processes, such as cell growth, differentiation, survival and apoptosis. This family is divided into three subfamilies according to their primary structure and cofactors required for activation: conventional or classic PKCs (cPKCs: PKCα, PKCβI, PCKβII and PKCγ), novel PKCs (nPKC: PKCδ, PKCε, PKCη and PKCθ) and atypical PKCs (aPCKS: PKCζ and PKCλ) (Figure 7) [reviewed in53b, 54]. The principal co-factors required for PKC activity are calcium, phosphatidylserine (PS) and diacylglycerol (DAG), a lipid second messenger transiently generated upon stimulation of membrane receptors such as tyrosine-kinase and G-protein coupled receptors [reviewed in53b, 55]. The differences between the distinct subfamilies reside in the regulatory domain. The C1 domain that comprises two cysteine-rich motifs forming the DAG binding site has only one cysteine-rich in aPKCs, and is not able to bind DAG. The C2 domain, that binds calcium and PS in cPKCs, is altered in nPKCs and aPKCs, losing the ability to bind calcium. In turn, C3 domain acts as an ATP binding site and C4 domain as a kinase catalytic centre [reviewed in56]. Figure 7 – PKC family structure. The three subfamilies are represented, as well as, their distinct bindings. The table on the right summarizes the second messengers that bind to each subfamily of PKC, with (+) representing binding, (++) representing binding with∼100-fold higher affinity, (+++) representing binding with >100-fold higher affinity, and (−) representing lack of binding. Abbreviations: aC1 – atypical C1 domain; DAG – diacylglycerol; nC2 – novel C2 domain; PB1 - Phox and Bem 1 domain; PS – phosphatidylserine. Adapted from57. 20 Table 2 – Flavonoids with antitumor activity in distinct human tumour cells. Flavonoid Antitumor activity Human tumour cells References Luteolin Inhibits proliferation; Induces G2/M-phase cell cycle arrest; Induces Apoptosis. Human esophageal squamous adenocarcinoma KYSE-510 cells; lung carcinoma CH27 cells and human colon cancer HT-29 cells. 82 Genistein Induces G2/M-phase cell cycle arrest; Induces apoptosis. ---- 83 Quercetin Induces G2/M-phase cell cycle arrest; Induces apoptosis trough a p53dependent mechanism and trough the release of cyt c. Human leukaemia HL-60 cells. 84 Baicalein Induces apoptosis Human colorectal adenocarcinoma HT-29 cells and human epithelial colorectal adenocarcinoma Caco-2 cells. 84b, 85 Apigenin Induces apoptosis Human colorectal adenocarcinoma HT-29 cells; human epithelial colorectal adenocarcinoma Caco-2 cells and human leukaemia HL-60 cells. 84b, 86 In conclusion, flavonoids interact with a wide range of molecular targets involved in apoptosis and cell proliferation pathways by affecting their expression or activity. Accordingly, it is reasonable to consider this scaffold as a valuable basis for the development of new and more potent pro-apoptotic agents that could be relevant for cancer treatment [reviewed in87]. 21 1.4. YEAST AS A MODEL SYSTEM 1.4.1. YEAST AS A MODEL TO STUDY HUMAN PROTEINS In the last few years, the budding yeast Saccharomyces cerevisiae has provided a major contribution on the understanding of the pathobiology of some human diseases. In fact, the high degree of conservation of cellular and molecular processes between the yeast S. cerevisiae and higher eukaryotes have made this microorganism a valuable model system for numerous studies on devastating human disorders such as cancer [reviewed in60, 76, 88]. Easy manipulation, short generation time, a well-defined genetic system and a high amenability to genetic modifications represent advantages of yeast cells over other cell models. However, as a unicellular organism, the limitation of this cell system for the study of human diseases concerns in the analysis of disease aspects that rely on multicellularity and cell-cell interactions. In fact, some disease-associated genes may not be present in yeast [reviewed in60, 89]. In spite of this limitation, yeast has provided a valuable contribution in the discovery of mechanistic processes involved in the cancer disease. For instance, with the finding that yeast can undergo an apoptotic cell death exhibiting phenotypic features and basic molecular machinery similar to those found in higher eukaryotes, the mechanisms of apoptosis could also be intensively addressed in yeast and the knowledge obtained transposed to human cells, providing clues towards an understanding of apoptosis-related diseases, such as cancer [reviewed in90]. In fact, the yeast model system has been considered a useful tool to study cancer-related proteins. If a gene encoding a protein is conserved in yeast, it is possible to directly study its function in this organism. On the other hand, if the gene has no orthologues in yeast, its heterologous expression in this cell model can still be highly informative because yeast may conserve protein interactions that may give crucial clues to the gene function. In fact, the absence of orthologues of a protein or an entire pathway can sometimes be an advantage because the protein can be independently studied in a simpler eukaryotic system, without the interference of other proteins that can share similar or overlapping functions [reviewed in60]. Based on the exposed above, in the last few years, the yeast cell model has been used not only to study the biology of important molecular processes, but also to study the pharmacology of proteins involved in cancer. 22 1.4.2. YEAST AS A MODEL FOR TARGETED SCREENING ASSAYS The simplicity of yeast genetics has made this cell model a promising system for identifying and testing target-specific drug therapies. Indeed, due to several advantages over cell-free and human cell-based assays (Table 3), yeast has emerged as a valuable tool for genetic and chemical large-scale screenings [reviewed in89a, 91]. Table 3 – Advantages and drawbacks of yeast targeted screening assays over other widely used assays. Adapted from76. Yeast target directed screening assays Advantages Drawbacks Remarks Over cell-free assays Over human cell based assays Are not the most physiologically relevant model system; Tests in mammalian are crucial to validate the pharmacological relevance of the targets identified. Cell wall does not limit compound permeability; Yeast pleiotropic drug resistance exporter proteins are structurally similar to the mammalian multiple drug resistance efflux pumps. Purification of the target is not required; Target proteins are examined in a cellular environment; Selection against compounds that are generally cytotoxic, instable in a cell culture or that cannot permeate plasma membrane. A clean read-out in a null background environment for the expression of the human protein; Less expensive to culture; Faster growth in defined media; Easier to propagate in HTS automated systems; Multitude of genetic tools and cellular selection systems readily converted to HTS formats. In the last years, humanized yeast cells have emerged as a useful model in the first-line drug screening, particularly in targeted screenings where the activity of a defined human protein is measured. Indeed, yeast can contribute to drug discovery through the identification of compounds that cause a desired physiological change through modulation of a specific protein [reviewed in76]. 23 Most of the human proteins involved in cancer (e.g. caspases) are toxic when overexpressed in yeast, since are able to interfere with the growth-regulatory pathways of this microorganism. Based on this, the restoration of the yeast cell growth is the basis of several chemical and genetic screenings for inhibitors of human target proteins. In the same way, the increase of the growth arrest induced by the foreign protein is the basis of several screenings for activators of human target proteins92. Indeed, when used a yeast model in the first step of the drug discovery process, coupled with other complementary approaches in mammalian systems, it can have an important impact in the discovery of new therapeutic opportunities [reviewed in93]. The yeast targeted screening assays are developed based on the use of the gene overexpression technique. In the last years, genome-wide yeast overexpression libraries have been created, taking advantage of the high amenability of this microorganism to genetic alterations. In fact, due to this organism be easily transformed by extrachromosomal plasmids, the selection of transformed cells is quite straightforward, and there are several regulatable promoters available that allow a considerable control over gene expression [reviewed in76]. The gene overexpression technique consists on the use of a high-copy library, in which a cDNA from a library of human cDNAs, is expressed under a regulatable promoter, inserted in yeast expression vectors (Figure 11) [reviewed in76]. These overexpression libraries have been applied to studies concerning human diseaseassociated proteins that induce a screenable phenotype, like growth arrest, such as caspases [reviewed in76]. Figure 11 – Gene overexpression technique. The insert of a gene encoding a protein of interest in yeast results in an engineered yeast cell, which can be used to study the biology and the pharmacology of proteins that play a central role in human disorders. 24 1.4.3. HUMAN PROTEINS STUDIED IN YEAST Based on the exposed above, during the last years, our research group has been focused on the development of yeast targeted screening assays to search for modulators of the activity of distinct cancer-related human proteins, such as caspases, PKC family proteins, among others [reviewed in56, 60, 76, 94]. 1.4.3.1. CASPASE FAMILY PROTEINS Based on the high complexity of caspases-signalling pathways, the yeast cell mode has been used as a simpler cell system for an independent analysis of human caspase family members in order to identify specific regulators and substrates [reviewed in76]. Indeed, several yeast assays have recently been developed for the HTS of modulators of human caspases, using either chemical or cDNA libraries. The exploitation of these assays to screen for activators and inhibitors of individual caspase family members may help in the discovery of new therapeutic agents against cancer [reviewed in60]. Yeast encodes a metacaspase (Yca1p)95. Metacaspases are found only in eukaryotes that are devoid of caspases, such as yeast. In fact, Yca1p shares structural homology and mechanical characteristics with mammalian caspases. However, metacaspases have a different cleavage specificity than mammalian caspases96. Although the controversy, recent studies have demonstrated that the activity of human caspases in yeast is independent of Yca1p97. The caspase activity in yeast has been frequently associated with the induction of a cell death phenotype. However, it was found that the phenotypic consequences of the expressed human caspase in yeast were highly dependent on the yeast strain used, probably a result of differences in the expression levels of endogenous caspase substrates or of other proteases with similar functions among the yeast strains [reviewed in76]. In fact, several aspects of the regulation of mammalian caspases could be recapitulated in yeast. For instance, it was reported in 2000 that IAPs in mammals, namely XIAP, suppressed the growth defect of S. cerevisiae expressing active human caspase-398. 25 Meanwhile, while the majority of mammalian caspases are auto-activated in yeast when expressed at high levels, downstream caspases, as caspases-3, -6 and -7, and the initiator caspase-9 cannot undergo auto-activation. In fact, these caspases require for their activation mammalian upstream proteins, such as initiator caspases or the Apaf-1, which are absent in yeast [reviewed in18, 23]. Indeed, this has been bypassed using engineered autoactivated variants that allow auto-activation of these caspases even in the absence of their upstream activators (Figure 12). Figure 12 - Different strategies of human caspases-3, -6 or -7 activation in S. cerevisiae. (A) Natural caspase activators; (B) Generation of reverse caspase, in which the small subunit precedes its prodomain and large subunit; (C) Removal of the N-terminal prodomain from the caspase coding sequence; (D) Separately coexpressing the large and small subunits of an active caspase; (E) Joining in-frame the caspase cDNA to the coding regions for Escherichia coli β-galactosidase (lacZ). Activated caspases lead to yeast growth arrest. Catalytic cysteine represented in purple. Abbreviations: LS, large subunit; SS, small subunit. Adapted from60. The most commonly used strategy for studying human caspases in yeast has relied on the fact that high expression levels of a caspase lead to a pronounced yeast cell growth arrest. Based on this, yeast can be used as a system to search for pharmacological modulators of this family of proteins (Figure 13) [reviewed in60]. 26 Figure 13 - The use of a yeast-based screening assay to search for modulators of procaspases-3 and -7, followed by an in vitro processing assay and assays in human tumour cell lines, led to the identification of activators of these proteins99. 1.4.3.1.1. POTENTIAL PHARMACOLOGICAL MODULATORS OF CASPASES IDENTIFIED IN YEAST In 2011, Glória and colleagues developed a yeast targeted screening assay to search for caspase-3 pharmacological activators. This assay is based on the measurement of yeast cell growth, particularly on the correlation established between the reversion of caspase-3induced yeast growth inhibition by a caspase-3 inhibitor and the inhibition of caspase-3 activity. The use of this assay to screen for modulators of individual caspase family members may help in the discovery of new therapeutic agents, namely, against cancer. In fact, this assay led us to the discovery of new inhibitors of caspase-3 among a chemical library of vinyl sulfones100. More recently, the use of a yeast approach developed to search for caspase-7 modulators also led us to the identification of potential small molecule activators of caspase7 with a flavonoid scaffold, being this activity also supported by in vitro processing assays and by assays in human tumour cell lines99. In this study, flavonoids 1a (5,6-dihydroxy-7prenyloxyflavone) and 2a (3-hydroxy-7-geranyloxyflavone) (Figure 14) were identified as activators of caspase-7, without interfering with the activity of caspase-3. Indeed, the yeast model provided important data concerning structural requirements for the activity of flavonoids towards caspase-799. 27 Figure 14 – Chemical structure of the flavonoids activators of caspase-7 identified using a yeast screening assay: compound 1a and compound 2a. 1.4.3.2. PKC FAMILY PROTEINS In 2003 Riedel and collaborators showed, for the first time, that yeast was an efficient model system for the evaluation of the activity of specific mammalian PKC isoforms, due to the yeast Pkc1p be a structural but not a functional homologue of mammalian PKC isoforms101. Indeed, yeast arose as a promising cell system for an individual analysis of mammalian PKC isoforms expressed in a simpler eukaryotic cellular environment without the interference of other isoforms or endogenous regulators [reviewed in76]. The catalytic function of mammalian PKC isoforms expressed in yeast was confirmed by monitoring the phosphorylation of specific PKC substrates. Particularly, the ability of mammalian PKC isoforms (namely PKCα, δ and ζ) to modulate the phosphorylation state of human proteins, such as Bcl-xL102 and p53103 in yeast was also demonstrated. The activation of a mammalian PKC isoform in yeast induced a specific phenotype and an increase in the cell doubling time proportional to the level of its enzymatic activity. These features led to the establishment of a yeast PKC phenotypic assay [reviewed in56]. The use of this yeast assay as a tool for the screening of several chemical libraries has led to the identification of new potential PKC pharmacological modulators [reviewed in56]. In fact, in this assay the PMA-induced growth inhibition in yeast cells expressing a mammalian PKC isoform is proportional to the degree of PKC activation, whilst known PKC inhibitors, such as chelerythrine or NPC 15437, reverted the effects of PKC activators. Indeed, the use of the yeast-based PKC assay in the screening of several chemical libraries led to the identification of new potent PKC activators and inhibitors, some of them selective to a particular isoform, such as PCKδ, PKCη and PKCζ [reviewed in56, 76]. 28 1.4.3.2.1. POTENTIAL PHARMACOLOGICAL MODULATORS OF PKC IDENTIFIED IN YEAST In 2004, with yeast cells expressing PKCα, PKCβI, PKCδ, PKCη or PKCζ, the potency and selectivity of the known PKC activator, PMA and its analogues MPMA (phorbol-12myristate-13-acetate-4-O-methyl-ether) and 4αPMA have been characterised104. Interestingly, MPMA and 4αPMA were identified as PKC inhibitors in contrast to their starting material104. In a previous work from the same group, the activity of daphnetoxin and its derivative mezerein has been compared, in yeast expressing PKCα, PKCβI, PKCδ or PKCζ, in order to investigate differences in their selectivity as PKC activators. In fact, daphnetoxin showed much lower potency to activate the PKCδ isoform and higher potency to activate PKCα, comparatively to mezerein72. Once again, using yeast, the molecular mechanism of action of the PKC activator euxanthone was ascertained, showing remarkable selectivity for PKCζ105. More recently, the activity of coleon U (6,11,12,14-tetrahydroxy-abieta-5,8,11,13tetraene-7-one) (Figure 15), a diterpene compound with an antiproliferative effect on human tumour cells (e.g. MCF-7 and UACC62), was demonstrated on individual isoforms of the distinct PKC subfamilies, using a yeast PKC assay (Figure 16). Figure 15 – Chemical structure of coleon U. 29 This study showed that coleon U induces the translocation of PKCδ and ε, in yeast expressing these proteins, to the nucleus, an effect associated with the induction of a Yca1pand mitochondrial-dependent apoptosis. Additionally, it was also demonstrated that the PKC activator PMA was able to activate all PKCs tested except aPCKζ, while coleon U had no effect on aPKC and cPKCs. Besides, the effect of coleon U on nPKCs was higher than that of PMA. Therefore, using the yeast screening assay it was possible to determine that coleon U was a potent and selective activator of nPKCs, inducing an apoptotic yeast cell death only in yeast cells expressing PKCδ or ε isoforms (Figure 16)74. Figure 16 - Molecular mechanism of action of coleon U in yeast cells. Coleon U had no effect on yeast cells expressing aPKC and cPKCs. On the other hand, in yeast expressing PKCδ or ε, coleon U induced the translocation of these proteins to the nucleus and a Yca1pand mitochondrial-dependent apoptosis, which was abolished by the co-expression in yeast of the human anti-apoptotic protein Bcl-xL. Taken together, these results showed that the yeast PKC model can efficiently contribute not only to the discovery of new PKC isoform-selective modulator with promising therapeutic applications, but also to unravel the molecular mechanism of action underlying their pharmacological activities, as observed with coleon U [reviewed in56]. Additionally, important insights concerning the mechanisms of apoptotic function of PKC isoforms, and physiologically relevant substrates have been provided by the yeast PKC assays (Table 4) [reviewed in60]. 36 2.2. PROTEIN KINASE C FAMILY PROTEINS 2.2.1. COMPOUNDS Phorbol 12-myristate 13-acetate (PMA) was obtained from Enzo Life Sciences (Group Taper, Sintra, Portugal) and 7α-acetoxy-6β-hydroxyroyleanone (C4) was synthesized as described in106. All tested compounds were dissolved in DMSO (Sigma-Aldrich, Sintra Portugal). 2.2.2. HUMAN TUMOUR CELL LINES AND GROWTH CONDITIONS Human colon adenocarcinoma HCT116 cell line was kindly provided by Dr. A. Inga (CIBIO, University of Trento, Italy). HCT116 cell line was routinely cultured in RPMI-1640 with ultraglutamine medium from Lonza (VWR, Carnaxide, Portugal) supplemented with 10% fetal bovine serum from Gibco (Alfagene, Carcavelos, Portugal) and maintained in a humidified incubator at 37°C with 5% CO2. 2.2.3. SULFORHODAMINE B (SRB) ASSAY For the analysis of the effect of C4 on the in vitro growth of human HCT116 cell line, cells were incubated in 96-well plates, at a final density of 5.0 × 103 cells/well, for 24 h. Cells were thereafter treated with serial dilutions of PMA (from 1.25 to 20.0 µM) and C4 (from 0.23 to 3.6 μM). The effect of compounds on cellular integrity was analysed following 48 h incubation, using sulforhodamine B (SRB) assay. Briefly, following fixation with 10% trichloroacetic acid, plates were stained with 0.4% SRB both from Sigma-Aldrich (Sintra, Portugal), and washed with 1% acetic acid. The bound dye was then solubilized with 10 mM Tris Base and the absorbance was measured at 510 nm in a microplate reader (Biotek Instruments Inc., Synergy MX, USA). The solvent of the compound (DMSO) corresponding to the maximum concentration used in this assay (0.1%) was included as control. The concentration of PMA and C4 that causes a 50% reduction in the net protein increase in cells during treatment (GI50, growth inhibition of 50%) was thereafter determined. 2.2.4. ANALYSES OF CELL CYCLE AND APOPTOSIS HCT116 cells were plated in 6-well plates at a final density of 1.5 × 105 cells/well. After 24 h incubation, cells were treated with the GI50 concentration of PMA, C4 or DMSO only for 24 h. For cell cycle analysis, cells were thereafter fixed in ice-cold 70% ethanol and incubated at 37ºC with RNase A from Sigma-Aldrich (Sintra, Portugal) at a final 37 concentration of 20 µg/mL for 15 min, and further incubated with 50 µg/mL propidium iodide (PI) from Fluka (Sigma-Aldrich, Sintra, Portugal) for 30 min, followed by flow cytometric analysis. For apoptosis analysis, cells were analysed by flow cytometry using the Annexin V-FITC Apoptosis Detection Kit I from BD Biosciences (Enzifarma, Porto, Portugal), according to the manufacturer’s instructions. 2.2.5. WESTERN BLOT ANALYSIS Whole protein extracts from HCT116 human tumour cell lines were obtained as described 107. Protein extracts were quantified using the Coomassie staining Bradford from Sigma-Aldrich (Sintra, Portugal). Proteins (40 µg) were electrophoresed using a 10% SDS– PAGE and transferred to a Whatman nitrocellulose membrane from Protan (VWR, Carnaxide, Portugal). Membranes were blocked with 5% milk and probed with a mouse monoclonal anti-PARP (C2-10) followed by an anti-mouse horseradish-peroxidase (HRP)- conjugated secondary antibody. For loading control, membranes were stripped and reprobed with a mouse monoclonal anti-GAPDH (6C5). All antibodies were purchased from Santa Cruz Biotechnology (Frilabo, Porto, Portugal). The signal was detected with the ECL Amersham kit from GE Healthcare (VWR, Carnaxide, Portugal) and with the Kodak GBX developer and fixer from Sigma-Aldrich (Sintra, Portugal). 2.2.6. FLOW CYTOMETRIC DATA ACQUISITION AND ANALYSIS For the flow cytometric analysis an Accuri™ C6 flow cytometer and the included acquisition software from BD Biosciences (Enzifarma, Porto, Portugal) were used. Cell cycle phases were identified and quantified using the FlowJo software (TreeStar Inc., Oregon, USA). 2.2.7. STATISTICAL ANALYSIS Data were analysed statistically using the GraphPad Prism 6.0. Differences between means were tested for significance using the Student’s t-test. Data are mean ± SEM of tree independent experiment; *p<0.05, **p<0.01. 38 CHAPTER 3 Results 39 3.1. CASPASE FAMILY PROTEINS IDENTIFICATION OF FLAVONOIDS S1, S2 AND S3 AS POTENTIAL ACTIVATORS OF CASPASE-7 USING A YEASTBASED SCREENING ASSAY In a previous work from our group, a yeast targeted screening assay was developed to search for small molecule modulators of human procaspases-3 and 7. Using this yeast approach, combined with in vitro assays and assays in human tumour cells, the prenylated flavonoids 1a (5,6-dihydroxy-7-prenyloxyflavone) and 2a (3-hydroxy-7-geranyloxyflavone) (Figure 14) were identified as potential activators of caspase-799. In the present dissertation, this yeast screening approach was used to analyse the effect on the activity of human procaspases-3 and -7, of several baicalein derivatives [S1 (7-(allyloxy)-5,6-dihydroxy-2-phenyl-4H-chromen-4-one), S2 (6-dihydroxy-2-phenyl-7propoxy-4H-chromen-4-one) and S3 (5,6-dihydroxy-7-(isopentyloxy)-2-phenyl-4Hchromen-4-one)] (Figure 17), which are analogues of compounds 1a and 2a. Figure 17 – Chemical structure of compounds S1, S2 and S3. The results obtained demonstrated that compounds S1-3 induced growth inhibition in yeast cells expressing procaspase-7 at 1-15µM (Figure 18A), without interfering with the growth of control yeast (data not shown). Interestingly, for the same concentration range, the effect of these compounds was less pronounced in yeast cells expressing procaspase3 (Figure 18B). 40 Figure 18 – Effect of compounds S1-S3 on the growth of yeast cells expressing procaspases3 or -7. Concentration-response curves for the effects of flavonoids S1-S3 on the growth of yeast cells expressing human (A) procaspase-7 or (B) procaspase-3, for 24 h treatment. The percentage of drug-induced growth inhibition was estimated considered 100% growth the number of CFU obtained with DMSO only. Data are mean ± SEM of six independent experiments; values significantly different from DMSO are indicated (*p=0.01, **p=0.001; unpaired). In addition, the GI25 values obtained from the concentration-response curves for the effects of 0.1-15 μM compounds S1-S3 and 0.1-50 μM PAC-1 (a known activator of procaspases-3 and -733) (data not shown), in yeast cells expressing procaspases-3 or -7, revealed that compounds S1 and S3 were significantly more potent than the positive control PAC-1, in yeast expressing procaspase-7 (Table 5). Despite the low GI25 value of compound S2 compared to PAC-1, in yeast expressing procaspase-7, the difference between both compounds is not statistically significant. More experiments must be performed in a future work to better clarify the potency of compound S2. Table 5 - GI25 values obtained for PAC-1 and flavonoids S1, S2 and S3. GI25 values correspond to the concentration that caused 25% growth inhibition of yeast cells expressing procaspases-3 or -7 after treatment with 0.1-15 μM flavonoids S1-S3, and 0.1-50 μM PAC-1 (positive control). Data are mean ± SEM of six independent experiments; values significantly different from PAC-1 are indicated (*p=0.01, **p=0.001; unpaired). ND – Not determined. 41 Interestingly, based on the GI25 values, in opposition to PAC-1, that showed to have activity in yeast cells expressing procaspases-3 or -7, compounds S1-S3 appear to be selective for procaspase-7 (Table 5). A similar yeast phenotypic assay was developed, by our group, for procaspase-6. The suitability of this yeast approach for the screening of activators of human procaspase6 was validated, in the present dissertation, by testing the effect of a known activator of procaspase-6 in yeast cells, M154150. As expected, for 1 and 5 µM, M1541 induced growth inhibition in yeast cells expressing procaspase-6 (Figure 19A), without interfere with the growth of control yeast (data not shown). These results demonstrated that as in human cells, M1541 was also able to activate procaspase-6 in yeast. This yeast screening approach was thereafter used to analyse the effect of compounds S2 and S3 on procaspase-6 activity. The results obtained showed that, for the concentration range tested (0.1-15 µM), both compounds did not interfere with the growth of yeast cells expressing procaspase-6 (Figure 19B). These results suggest that flavonoids S2 and S3 were not activators of procaspase-6. Figure 19 - Effect of compounds M1541, S2 and S3 on the growth of yeast cells expressing procaspase-6. Concentration-response curves for the effects of the (A) positive control M1541 and (B) flavonoids S2 and S3 on the growth of yeast cells expressing human procaspase-6 for 24 h treatment. The percentage of drug-induced growth inhibition was estimated considered 100% growth the number of CFU obtained with DMSO only. Data are mean ± SEM of six independent experiments; values significantly different from DMSO are indicated (*p=0.01, **p=0.001; unpaired). 42 3.2. PKC FAMILY PROTEINS In a previous work from our group a yeast targeted screening assay was developed to search for modulators of PKC family proteins74. Using this approach a diterpene compound (coleon U) was identified as a potent and selective activator of the novel PKC δ and ε isoforms74. In this way, this yeast screening approach was used to analyse the effect of a coleon U derivative (compound C4) on the activity of several PKC isoforms. The results obtained in the yeast model indicated that C4 was a potent and selective activator of PKC δ (unpublished results). Based on this finding, in the present dissertation, several assays were performing in human tumour cell lines in order to analyse the in vitro antitumor potential of C4. C4 INDUCES A CYTOTOXIC EFFECT ASSOCIATED WITH APOPTOSIS IN HUMAN TUMOUR CELLS The tumour cell growth-inhibitory potential of C4 was ascertained using the human colon adenocarcinoma HCT116 cell lines (Figure 20). The GI50 value obtained for C4 of 0.63 µM, in HCT116 cells, after 48 h treatment, showed that C4 has a potent growth inhibitory effect in human tumour cells. Additionally, the results obtained in HCT116 cells showed that C4 was significantly more potent than the known activator of classical and novel PKCs (used as positive control77), PMA with a GI50 value of 6.5 µM (*p<0.05) (Figure 20A). Moreover, it was verified that the growth-inhibitory effect of compound C4, at the GI50 concentration, was not associated with cell cycle arrest (Figure 20B). Whereas, the growth-inhibitory effect of PMA, at the GI50 concentration, demonstrated to be associated with the induction of S-phase cell cycle arrest (Figure 20B). It was also observed that the growth-inhibitory effects of both PMA and C4, at the GI50 concentrations, were associated with the induction of late apoptosis (Figure 20C). By western blot analysis, it was verified that the growth-inhibitory effect induced by C4 led to the induction o PARP cleavage, a product of caspase-3 activation, in HCT116 tumour cells (Figure 20D). Altogether, these results showed that C4 has a potent in vitro tumour growthinhibitory activity associated with the induction of apoptosis. 43 Figure 20 – C4 induces a potent growth-inhibitory effect associated with apoptosis induction, in human colon adenocarcinoma HCT116 tumour cells. (A) The GI50 concentration of PMA (positive control) and C4 was determined in HCT116 cells, after 48 h treatment, using the SRB assay. Data are mean ± S.E.M. of three independent experiments; values significantly different from PMA (*p<0.05) are indicated. (B) 0.63 µM C4 does not induce cell cycle arrest in HCT116 cells, after 24 h treatment. Cell cycle phases were analyzed by flow cytometry using PI staining; data are mean ± S.E.M. of three independent experiments. (C) 0.63 µM C4 induces late apoptosis in HCT116 cells, after 24 h treatment. Apoptosis was analyzed by flow cytometry using FITC-Annexin V and PI; data are mean ± S.E.M. of three independent experiments. (D) 0.63 µM C4 leads to PARP cleavage in HCT116 cells, after 24 h treatments. Immunoblots represent one of three independent experiments; GAPDH was used as loading control. In B and C, values significantly different from DMSO only: *p<0.05, **p<0.01. 44 CHAPTER 4 Discussion Conclusion and Future Prospects 45 Cancer is a major public health problem worldwide and the number of new cases is expected to rise significantly in the next decades. An additional concern is the increase of the resistance to the chemotherapy associated with many cancers. Apoptosis is a crucial cellular process for the maintenance of tissue homeostasis in multicellular organisms. In fact, the mechanisms of apoptosis are highly complex and sophisticated, involving an energy-dependent cascade of molecular events. The deregulation of this process has been implicated in carcinogenesis [reviewed in4]. In this way, in the last years, the search for new anticancer therapies that selectively modulate proteins involved in the apoptotic process has been the focus of an intense research in order to overcome the resistance and to trigger tumour cells into apoptosis. The yeast cell model has emerged as a powerful tool to study the biology and pharmacology of cancer-related proteins. In fact, yeast-based screening assays have already proven to be widely effective in the first-line screening of genetic and chemical libraries, leading to the successful identification of promising pharmacological modulators of human proteins involved in apoptosis, such as caspases and PKCs [reviewed in56, 60, 76]. Hence, with the present dissertation it was intended to use the yeast cell system to discover new small molecule modulators of pro-apoptotic proteins, with improved pharmacological properties. In fact, in the present work, promising modulators of caspases activity were identified. Additionally, the in vitro antitumor potential of a selective activator of PKCδidentified in yeast was confirmed. 4.1. IDENTIFICATION OF FLAVONOIDS S2 AND S3 AS POTENTIAL SELECTIVE ACTIVATORS OF CASPASE-7 At the core of the execution phase of apoptosis are the executioner caspases-3, -6 and -7. Like most proteases, these caspases are stored as procaspases that once activated, cleave a large set of substrates, ultimately resulting in the characteristic morphological and biochemical hallmarks of apoptosis. The reduced expression of proapoptotic caspases has been reported in manifold cancers and specific inactivating mutations in many tumour types and stages of malignant transformation [reviewed in21]. Indeed, the search for activators of these proteins can be considered as a novel strategy in cancer therapy by selective induction of apoptosis in cancer cells [reviewed in21, 108]. 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Baicalein induces apoptosis in SCC-4 human tongue cancer cells via a Ca2+-dependent mitochondrial pathway. in vivo 2007, 21 (6), 1053-1058. 68 69 Oral Communication in II SIMPOSIO DE JOVENES INVESTIGADORES DE LA SEQT Madrid, Espanha, June 12 2015 70 NEW POTENTIAL SELECTIVE ACTIVATORS OF CASPASE-7 IDENTIFIED USING THE YEAST SCREENING ASSAY Sofia Salazar1, Mariana Leão1, Madalena Pinto2, Honorina Cidade2, Lucília Saraiva1 1 UCIBIO/REQUIMTE, Laboratory of Microbiology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Portugal 2 CIIMAR and Laboratory of Organic and Pharmaceutical Chemistry, Department of Chemistry, Faculty of Pharmacy, University of Porto, Portugal The search for new anticancer therapies has been focused on devising ways to overcome resistance and to trigger apoptosis in tumour cells [1]. Caspases are a family of proteases with a crucial role in apoptosis [2]. At the core of the execution phase of apoptosis are the executioner caspases-3 and -7. These caspases are stored as procaspases that once activated by proteolysis cleave a large set of substrates, ultimately resulting in the morphological and biochemical hallmarks of apoptosis. Thus, the search for procaspase activators has deserved particular attention in anticancer drug discovery [2]. Yet, to date, few small-molecule caspase activators have been reported [3,4]. Our previous study reported the identification of two potential activators of caspase-7: 5,6-dihydroxy-7prenyloxyflavone (1a) and 3-hydroxy-7-geranyloxyflavone (2a) using a yeast screening assay, based on the heterologous expression of these human proteins in Saccharomyces cerevisiae [5]. These results prompted us to discover new activators of caspases-3 and -7. For that, several analogues of compounds 1a and 2a were synthesized, and their activities on these caspases were evaluated, using the developed yeast assay [5]. Potential selective activators of caspase-7 were identified with improved pharmacological properties compared to their starting analogues. Further work is underway to confirm their molecular mechanisms in human tumor cells. Acknowledgments: This work is funded through national funds from FCT under the projects REQUIMTE-PestC/EQB/LA0006/2013 and CEQUIMED-PEst-OE/SAU/UI4040/2014 and partially supported by the European Regional Development Fund (ERDF) through the COMPETE - Operational Competitiveness Programme and national funds through FCT under the project PEst-C/MAR/LA0015/2013. [1] Igney, F. H. and P. H. Krammer (2002), Death and anti-death: tumour resistance to apoptosis, Nature Reviews Cancer, 2 (4), 277-288. [2] McIlwain, D. R., Berger, T., Mak, T. W. (2013), Caspase Functions in Cell Death and Disease, Cold Spring Harbor Perspectives in Biology, 5 (4), a008656. [3] Häcker HG1, Sisay MT, Gütschow M. (2011), Allosteric modulation of caspases, Pharmacol Ther., 132(2):180-95. [4] Ottmann, C.; Hauske, P.; Kaiser, M. (2010), Activation instead of inhibition: targeting proenzymes for smallmolecule intervention, ChemBioChem, 11, 637-639. [5] Pereira, C., Lopes-Rodrigues, V., Coutinho, I., Neves, M. P., Lima, R. T., Pinto, M., Cidade, H., Vasconcelos, M. H., Saraiva L. (2014), Potential Small-molecule Activators of Caspase-7 Identified Using Yeast-based Caspase-3 and -7 Screening Assays, European Journal of Pharmaceutical Sciences, 54, 8-16. 71