Tesis Doctorals - Departament - Farmacologia, Toxicologia i Química Terapèutica

URI permanent per a aquesta col·leccióhttps://hdl.handle.net/2445/103264

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Exploring novel target combinations for treating neurodegenerative diseases
(Universitat de Barcelona, 2025-04-25) Sampietro Pifarré, Anna; Muñoz-Torrero López-Ibarra, Diego; Galdeano Cantador, Carlos; Universitat de Barcelona. Departament de Farmacologia, Toxicologia i Química Terapèutica
[eng] Neurodegenerative diseases, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), affect millions of people worldwide and share common multifactorial characteristics, including neuroinflammation, oxidative stress, and protein aggregation. Due to the complexity of these diseases, this thesis focuses on exploring novel therapeutic strategies that have been poorly exploited to address AD and PD. Firstly, for AD, a bibliometric study on the development of multitarget drugs has revealed a significant increase in publications since 2011, with a predominant interest in linked hybrids, which mostly hit acetylcholinesterase (AChE), oxidative stress, and β-amyloid aggregation, while targets such as tau aggregation or GSK-3β remain relatively underexplored. Secondly, efforts have been made during this thesis to deepen into the understanding of protein aggregation by performing, in the frame of several collaborations, the evaluation in genetically modified Escherichia coli cells of the anti- aggregation activity of two structural series synthesized in this thesis and four different synthesized by others. It was observed that active anti-aggregating compounds display similar potencies against the aggregation of β-amyloid and tau proteins. Based on this information, new hybrid compounds have been designed to combine the inhibition of soluble epoxide hydrolase (sEH) and AChE with anti-aggregation properties. These compounds have demonstrated nanomolar potency against both targets, while effectively inhibiting the aggregation of β-amyloid, tau, and TDP-43 proteins. Moreover, they have shown to be non-neurotoxic, capable of crossing the blood-brain barrier, and have exhibited moderate aqueous solubility, making them good candidates for future in vivo evaluation. Regarding PD, this thesis has focused on the design of sEH inhibitors with specific anti-aggregation activity against α-synuclein. The new compounds have demonstrated high sEH inhibitory potency, significant α-synuclein anti-aggregation activity, and good DMPK and safety profile, thereby emerging as promising candidates for future preclinical studies in murine models. In addition to conventional multitarget strategies, targeted protein degradation (TPD) was explored as an alternative modality to tackle CNS-related proteins. One of the main needs in the field is to increase the toolbox of E3 ligases. In this context, novel ligands for KEAP1, an E3 ligase that degrades Nrf2—a key transcription factor with antioxidant effects—have been investigated. The aim is to develop a dual-action PROTACs, which could simultaneously increase Nrf2 levels to enhance antioxidant activity while degrading a protein of interest via KEAP1’s E3 ligase activity. Using MDMix calculations and virtual screening, 20 potential ligands were identified, five of which were biophysically validated. Among them three ligands showed micromolar affinity (Kd) towards the Kelch domain of KEAP1 and hints of upregulation of Nrf2-regulated genes. These results provided the foundation for the design and evaluation of new KEAP1-recruiting PROTACs incorporating the discovered ligands.
Estudio de los efectos en el neurodesarrollo de productos a base de plantas (PBPs) consumidos por la población gestante y lactante usando el modelo del pez cebra
(Universitat de Barcelona, 2025-04-08) Romero, Noelia Giselle; Teixidó Condomines, Elisabet; Barenys Espadaler, Marta; Universitat de Barcelona. Departament de Farmacologia, Toxicologia i Química Terapèutica
[spa] A nivel mundial, el uso de productos a base de plantas (PBPs) entre las mujeres durante el embarazo y la lactancia se estima en un 60% en los países occidentales y 76% en países orientales, pero no hay datos sobre este tipo de consumo en la población catalana. En algunos entornos existe la creencia que los PBPs son más efectivos, accesibles, y seguros durante el embarazo que los medicamentos convencionales, y por ello se utilizan para tratar afecciones comunes del embarazo y mejorar el bienestar materno. Sin embargo, estas prácticas plantean ciertas preocupaciones debido a la limitada información científica sobre sus posibles efectos adversos en el desarrollo. Esta tesis fue planteada en tres estudios: en el estudio 1, evaluamos por primera vez la prevalencia de consumo de PBPs entre mujeres embarazadas (a partir de la semana 22) y lactantes (hasta 9 meses después del parto) en Cataluña, a través de entrevistas personales (N=102). Los resultados revelaron una alta prevalencia de uso de PBPs, ya que el 94% (96 de 102) de las entrevistadas reportó haberlos utilizado, y entre ellas, el 75% (72/96) indicó que el consumo fue por vía oral, dando lugar a un 70% de consumo de PBPs por vía oral considerando el total de las participantes. Los PBPs más consumidos incluyeron jengibre (para aliviar náuseas y molestias gástricas), manzanilla (para dolores varios y náuseas), tomillo (resfriados), rooibos (parte de la dieta y sustituto del café), arándano rojo (infección del tracto urinario) y hojas de frambueso (preparación para el parto). La principal forma de consumo fue en infusiones, seguida de cápsulas y comprimidos. Este estudio evidenció que no existe un perfil demográfico específico entre las consumidoras de PBPs, lo que sugiere que cualquier mujer embarazada o lactante podría ser una consumidora potencial. Además, se identificó una percepción generalizada entre las entrevistadas de que las mujeres embarazadas deberían preferir los remedios herbales sobre los medicamentos convencionales, una tendencia estadísticamente significativaEn el estudio 2, se evaluaron los efectos adversos en el neurodesarrollo de los PBPs más consumidos durante el embarazo y la lactancia en Cataluña usando el modelo de embrión de pez cebra. Se utilizaron el ensayo de toxicidad del desarrollo embrionario en pez cebra (ZEDTA) y pruebas de comportamiento, como el test de Transición Luz/Oscuridad (L/O) y de Respuesta evocada al tacto (TER). Los extractos de tomillo (Thext) y de hojas de frambueso (HFext) causaron efectos en el comportamiento de las larvas de pez cebra a concentraciones realistas tras el consumo de estos extractos Por otro lado, el extracto de jengibre (JIext) presentó efectos adversos a concentraciones superiores. Los extractos de jengibre decocción (JDext) y rooibos (Rbext) también mostraron ciertos efectos adversos, aunque no pueden considerarse potencialmente tóxicos, ya que requerirían dosis muy altas y no realistas para producirse. El extracto de manzanilla (MCext) no presentó efectos adversos a ninguna de las concentraciones evaluadas. En el estudio 3, se analizaron tres productos herbales comunes de la Medicina Tradicional China (MTC): TM (tubérculo de Gastrodia elata Blume), LGT (raíz y rizoma de Tripterygium wilfordii Hook.f), y té verde (hojas de Camellia sinensis K). TM no presentó potencial tóxico en el neurodesarrollo de los embriones. Sin embargo, LGT y sus principales compuestos bioactivos, triptolide y celastrol, ocasionaron alteraciones significativas en el comportamiento de las larvas. Por su parte, la exposición al EGCG, la principal catequina del té verde, también resultó en modificaciones conductuales tras la exposición durante el desarrollo. Más estudios son necesarios para evaluar la reversibilidad de los efectos observados. En esta tesis se propone la combinación del ensayo ZEDTA con el análisis de comportamiento L/O como una herramienta efectiva para estudiar la potencial neurotoxicidad durante el desarrollo de productos herbales.
Addressing Alzheimer's disease through innovative mechanisms
(Universitat de Barcelona, 2025-02-07) Martínez Conde, Noemí; Muñoz-Torrero López-Ibarra, Diego; Universitat de Barcelona. Departament de Farmacologia, Toxicologia i Química Terapèutica
[eng] Alzheimer’s disease (AD) remains a major unmet medical need, with no effective disease- modifying treatments available despite decades of research and a growing socioeconomic burden associated with its increasing prevalence. This Thesis addresses AD by exploring innovative therapeutic strategies, including new combinations of biological targets as well as underexplored targets. Firstly, a new family of dual soluble epoxide hydrolase (sEH) and acetylcholinesterase inhibitors was developed. This target combination should lead to increased cognition and memory, as well as synergistic effects against the characteristic neuroinflammation of AD brains. A potent second-generation lead compound was identified, which overcame the metabolic and pharmacokinetic limitations of the first-generation lead and showed favourable physicochemical properties and in vitro pharmacokinetic and safety profile. Furthermore, it demonstrated efficacy in a mouse model of AD with enhancement of cognition and memory and reduction of several disease biomarkers, emerging as a promising drug candidate. Secondly, a novel family of small-molecule dual inhibitors of sEH and glutaminyl cyclase endowed with a high degree of structural diversity was developed, aiming to obtain disease-modifying effects by targeting neuroinflammation and beta-amyloid peptide pathologies, which are key hallmarks of AD pathological network. Its in vitro profiling led to the identification of a lead compound, which showed inhibitory activity in the nanomolar range for both targets, as well as good aqueous solubility, brain permeability, metabolic stability and an adequate safety profile. The selected compound also exhibited favourable biodistribution and safety profiles in mice, with demonstrated brain exposure and a relatively high maximum tolerated dose, as well as promising effects in a first proof of concept in a mouse model of AD (hQC x hAPP mice). The lead compound is currently undergoing additional in vivo efficacy studies in two different AD mouse models (5xFAD and SAMP8) with an adjusted dose aiming to prove its efficacy. Thirdly, the underexplored phosphatase domain of sEH, which has been related with cholesterol metabolism and might play a role in AD, has been also investigated, by the development of brain permeable compounds that selectively inhibit the phosphatase activity of the enzyme, without altering the hydrolase activity. Thus, the use of these novel compounds in additional studies will enable further exploration of the implication of this target in AD pathogenesis. Finally, the amyloidogenic Tar DNA-binding Protein 43 (TDP-43) has been addressed, due to its implication in over half of AD cases and considering the lack of drugs that directly target the protein, with two approaches: 1) a fast and unexpensive in vitro assay has been set up to facilitate the identification of TDP-43 aggregation inhibitors; 2) biophysical methods, namely surface plasmon resonance and differential scanning fluorimetry, have allowed the identification of a promising protein ligand, that will be used as the starting point for the design and optimisation of proteolysis targeting chimeras aiming to degrade TDP-43.
Development of Iron-Catalyzed Hydrogen Atom Transfer Driven Cross-Coupling and Cyclization Reactions: Use of Isocyanides, Heterocycles and Tosylhydrazones as Radical Acceptors
(Universitat de Barcelona, 2024-05-10) Puig Bosch, Jordi; Bradshaw, Ben; Bonjoch i Sesé, Josep; Universitat de Barcelona. Departament de Farmacologia, Toxicologia i Química Terapèutica
[eng] Radical reactions have been proven valuable tools in C-C bond generation because the initial radical can evolve to form additional C-C bonds until the new radical species is trapped. With the careful design of the substrates and the control of the reaction conditions, tandem radical reactions can be developed that allow the construction of complex structures starting from simple starting materials. Metal-catalyzed hydrogen atom transfer (MHAT) reactions are radical-based reactions that allow mild and non-toxic conditions, with high chemo- and site-selectivity and a wide functional group tolerance. Our group has developed several MHAT C-C bond-forming reactions of non-activated alkenes with a wide variety of acceptor groups such as aldehydes, ketones, Cbz hydrazones, and tosylhydrazones. Despite the many advantages presented by MHATbased radical reactions and their potential for developing new tandem reactions, few examples have yet been reported in the literature. In the present thesis, the aim was to expand the scope of radical acceptors that can be used in MHAT reactions as well as develop novel tandem reaction processes. First, isocyanides have been studied as radical acceptors to carry out a tandem reaction to obtain functionalized nitrogen heterocycles, highly valued compounds in medical chemistry (Scheme 2). The reaction uses MHAT conditions to generate a radical on an alkene that carries out a first radical addition step on the isocyanide, followed by subsequent intramolecular cyclization to give rise to the functionalized heterocycle. A wide range of different nitrogen-containing heterocycles can be synthesized using this methodology. In cases where the alkene substitution made it more challenging, combining HAT-Minisci conditions made the coupling possible. The reductive cyclization of these isocyanides has also been shown to be effective, in the absence of alkene, to give rise to the corresponding unfunctionalized heterocycles. Secondly, a cross-dehydrogenative coupling reaction has been developed from the initial serendipitously observed side reaction. Using conditions derived from MHAT and Minisci, it has been possible to generate radicals through C-H activation on the - position of ethers, amides, and alkanes to carry out the radical coupling on activated heterorenes. This reaction has the potential to be applied in medicinal chemistry for latestage functionalization under mild conditions. Thirdly, the use of tosylhydrazones as acceptor groups in MHAT reactions has been studied to carry out a tandem reaction that begins with intramolecular cyclization followed by in situ fragmentation of the tosylhydrazine and subsequent intermolecular trapping of the generated tertiary radical with a Michael acceptor in a Giese-type addition. This reaction allows the generation of two adjacent quaternary centers in a single reaction via the formation of two geminal C-C bonds. This methodology has been used to prepare functionalized derivatives of adamantane, a very interesting scaffold from the point of view of medicinal chemistry and other fields, using different Michael acceptors. Further transformations also allowed the preparation of adamantanesubstituted heterocycles.
Nuevas metodologías para la síntesis de espiroindolinas y indoloquinolicidinas. Aplicación a la síntesis de alcaloides indólicos
(Universitat de Barcelona, 2024-09-20) Piras, Valentina; Amat Tusón, Mercedes; Pérez Bosch, Maria; Universitat de Barcelona. Departament de Farmacologia, Toxicologia i Química Terapèutica
[spa] Los alcaloides oxindólicos son un grupo de productos naturales caracterizados por la presencia de un sistema espiro[pirrolidina-3,3'-oxindol] y poseen una amplia gama de actividades biológicas. Por esta razón, han suscitado interés en el desarrollo de diferentes estrategias sintéticas. La mitrafilina es un alcaloide oxindólico pentacíclico aislado de Mitragyna speciosa, con efecto antitumoral contra líneas celulares de cáncer de mama humano, neuroblastoma y glioma maligno. En esta Tesis Doctoral se muestra la aproximación sintética a la espiroindolina, abriendo un acceso enantioselectivo a la ent-isomitrafilina. Las etapas clave de nuestra aproximación sintética son la preparación estereoselectiva de una lactama bicíclica derivada del (S)-triptofanol mediante una reacción de ciclocondensación con un derivado de oxoéster, una espirociclización altamente estereoselectiva, la formación de una lactama -insaturada y una secuencia de Michael-Claisen para la introducción de los sustituyentes de dos carbonos en una única vez. La espirotriprostatina B es un alcaloide oxindólico con un considerable interés como un prometedor fármaco para el tratamiento del cáncer con actividad inhibidora del ciclo celular mamífero en la fase G2/M, del crecimiento de células de leucemia mieloide crónica humana K562 y células de leucemia promielocítica humana HL-60. Como una extensión de nuestro trabajo sobre el uso de lactamas bicíclicas derivadas del (S)-triptofanol para la síntesis de alcaloides indólicos, aprovechando nuestra metodología eficiente para la generación de sistemas con estructura de espiroindolina, se ha desarrollado una metodología sintética para la obtención de un intermedio avanzado a partir de una lactama -insaturada a la que se efectúan estudios para su apertura oxidativa. Además, se exploran métodos para la síntesis del alcaloide indoloquinolicidínico (+)-antirrina. Se propone una estrategia sintética basada en la generación del núcleo indolo[2,3-a]quinolicidínico a partir de lactamas derivadas del (S)-triptofanol. Los estudios de estereoselectividad se centran en la reacción de ciclación y la posterior reacción de Pictet-Spengler. La metodología permite la generación de lactamas α,β-insaturadas, que permite una posterior reacción de adición conjugada estereoselectiva para la introducción de un sustituyente en el anillo de piperidina. Se han logrado avances significativos proporcionan una base sólida para la síntesis para la síntesis de este alcaloide en forma enantiopura.
Study of new strategies for steatotic liver disease: SIRT1-mediated modulation of VLDLR levels and evaluation of a soluble epoxyde hydrolase-targeted PROTAC
(Universitat de Barcelona, 2024-05-31) Peyman, Mona; Vázquez Carrera, Manuel; Barroso Fernández, Emma; Universitat de Barcelona. Departament de Farmacologia, Toxicologia i Química Terapèutica
[eng] Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common cause of chronic liver disease (CLD). The first stage in the development of MASLD is metabolic dysfunction-associated fatty liver (MASL), which is defined as a condition where excessive levels of triglycerides (TG) accumulate in the liver. Little is known about the role played by the uptake of lipoproteins, such as very low-density lipoproteins (VLDL) that predominantly transport TG in plasma, in the development of hepatic steatosis. Interestingly, endoplasmic reticulum (ER) stress-mediated increase in the levels of the VLDL receptor (VLDLR) results in remarkable hepatic steatosis via enhanced triglyceride-rich lipoprotein delivery to the liver. A better understanding of how hepatic VLDLR is regulated might help to develop new effective therapies against MASLD. On the other hand, soluble epoxide hydrolase (sEH), an enzyme highly expressed in the liver, converts epoxyeicosatrienoic acids (EETs) and other epoxy fatty acids (EpFAs) to their corresponding diols. These diols are much less bioactive than their parents’ epoxides. As a result, compounds that inhibit sEH increase the levels of EETs and other EpFAs, which are opposing counterparts to the largely pro-inflammatory prostanoids and leukotrienes, thereby providing therapeutic efficacy for the treatment of several diseases, including hepatic steatosis. An additional new strategy for targeting sEH might be the use of proteolysis-targeting chimeras (PROTACs), due to its capacity to induce its degradation. In the present thesis, we show that induction of hepatic steatosis by fructose-drinking water in rats caused a reduction in the levels of hepatic sirtuin 1 (SIRT1), a NAD (+)-dependent deacetylase, and upregulation of VLDLR, suggesting a potential relationship between these two proteins. Consistent with this, Sirt1-/- mice displayed increased hepatic VLDLR levels and enhanced expression of HIF-1α-target genes. Likewise, the increase in VLDLR protein levels induced by pharmacological inhibition or gene knockdown of SIRT1 in a human hepatic cell line was abolished by a HIF-1α inhibitor. Moreover, SIRT1 activation in mice prevented the increase in hepatic VLDLR protein levels caused by ER stress. Additionally, we report that the sEH-targeting PROTAC ALT-PG2 degrades this protein in the human hepatoma-derived Huh-7 cell line and in primary mouse hepatocytes as well as in the liver of mice. PROTAC-mediated degradation of the sEH protein in cells resulted in adenosine monophosphate-activated protein kinase (AMPK) activation, while phosphorylated extracellular-signal-regulated kinase 1/2 (ERK1/2) was reduced. Consistent with the role of these two kinases in ER stress, ALT-PG2 reduced both ER stress and inflammatory markers. Altogether, the findings of the present doctoral thesis shed light on a new mechanism of VLDLR regulation and its contribution to hepatic steatosis and demonstrate that targeting sEH with a PROTAC molecule is an effective strategy to activate AMPK and to prevent ER stress and inflammation in hepatic cells.
GDF15, AMPK, PPAR β/δ: Estudio de su relación en el control de la glucemia
(Universitat de Barcelona, 2024-09-27) Jurado Aguilar, Javier; Vázquez Carrera, Manuel; Universitat de Barcelona. Departament de Farmacologia, Toxicologia i Química Terapèutica
[spa] La diabetes mellitus tipo 2, una enfermedad multifactorial cuyo principal factor de riesgo es la obesidad, donde se produce la acumulación anormal o excesiva que puede ser dañina para la salud. Esta enfermedad se ha convertido en una epidemia con un importante impacto social, económico y sanitario en el mundo entero. Esta patología suele iniciarse con el desarrollo de resistencia a la insulina , debido a un estado proinflamatorio crónico , condición en la que el organismo es incapaz de responder adecuadamente a esta hormona, y que precede y predice la aparición de la diabetes mellitus de tipo 2 A pesar del aumento de la incidencia de la diabetes mellitus tipo 2, los tratamientos actualmente disponibles para esta patología son limitados, y presentan efectos secundarios y escasa eficacia a la hora de controlar la patología. El fármaco más prescrito para el tratamiento de la diabetes mellitus tipo 2 es la metformina, cuyo mecanismo de acción no está completamente descrito. Este fármaco actúa principalmente reduciendo la gluconeogénesis hepática, uno de los procesos que más contribuye a aumentar los n iveles de glucosa sérica de estos pacientes en ayunas. En las últimas décadas han surgido moléculas que podrían presentar un prometedor papel terapéutico en las alteraciones metabólicas, dos de ellas son GDF15 ( growth differentiation factor 15 ) y PPAR β/δ (peroxisome proliferator activated receptor). El estudio de los mecanismos por los que metformina o GDF15 y PPAR β/δ mejoran la hiperglucemia pueden ayudar a diseñar nuevas estrategias terapéuticas para el tratamiento de la diabetes mellitus tipo 2. Los resultados de la presente tesis doctoral demuestran que los ratones Gdf15 presentan intolerancia a la glucosa, niveles reducidos de AMPK ( AMP activated protein kinase ) a sí como un aumento de los niveles de TGF β (transforming growth factor β) y de la fosforilación del mediador de fibrosis SMAD3 ( mothers against decapentaplegic homolog 3 ), todo ello acompañado de un aumento de genes implicados en la gluconeogénesis y fibrosis. Todos estos cambios fueron revertidos mediante la inhibición farmacológica de SMAD3, lo que sugería que GDF15 puede regular la gluconeogénesis hepática, la fibrosis y la esteatosis hepática a través de la vía TGF β1 /SMAD3. Asimismo, los resultados de esta tesis también indican que la metformina incrementa los niveles de GDF15 en hígado, músculo y riñón al aumentar los niveles proteicos de PCSK6 y β/arrestina 1, proteínas implicadas en su maduración y secreción a través de PPAR beta/delta AMPK ( AMP activated protein kinase ). No se observó el incremento de PCSK3 y PCSK5 sugiriendo que el efecto de la metformina es específico para PCSK6. Ambos resultados refuerzan la teoría de que GDF15 es una prometedora diana terapéutica para el tratamiento de enfermedades metabólicas al es tar implicado en numerosas vías alteradas durante la obesidad y la diabetes.
Reaction space charting of multicomponent processes: a novel approach in organic and biomedical research
(Universitat de Barcelona, 2024-10-04) Nadal Rodríguez, Pau; Lavilla Grífols, Rodolfo; Ghashghaei, Ouldouz; Universitat de Barcelona. Departament de Farmacologia, Toxicologia i Química Terapèutica
[eng] To discover new scaffolds and to streamline the production of novel chemical entities with biological activity is essential for accelerating drug discovery. In this regard, multicomponent reactions (MCRs) offer efficient access to complex and unconventional connectivities. This thesis applies the reaction space charting strategy to systematically explore and describe MCRs, potentially leading to new scaffolds with biomedical applications. Through this approach, the investigation of the MCR involving carbonyls, amines, and isocyanoacetates revealed new multicomponent processes, yielding unsaturated imidazolones, and novel reactivity of the generated core. These scaffolds show promise in various biomedical applications, such as fluorescent probes. were tested as fluorescent probes and antimicrobial activity. Additionally, the reactivity of indole-2-carboxaldehyde (indole-2-CHO) in MCRs was studied, leading to the discovery of 6-substituted indolo[3,2-b]carbazoles (6-ICZs), potent ligands for the Aryl hydrocarbon Receptor (AhR). These synthesized 6-ICZs, obtained in one step, show promising anti-inflammatory activity and offer a tunable platform for AhR drug discovery, addressing key challenges in the field.
Developmental toxicity screening in the zebrafish embryo – a metabolomics approach
(Universitat de Barcelona, 2024-07-19) Wilhelmi, Pia Rosa Maria; Barenys Espadaler, Marta; Zickgraf, Franziska; Flick, Burkhard; Universitat de Barcelona. Departament de Farmacologia, Toxicologia i Química Terapèutica
[eng] Birth defects occur in about 3-6% of human births worldwide (CDC, 2023). To mitigate the risk to the unborn child, the assessment of embryo-fetal developmental toxicity is an integral part of chemical registration and regulation. This currently requires testing on mammals, which raises ethical concerns, is costly and time-consuming. The zebrafish embryotoxicity test (ZET) has been proposed as an alternative approach to developmental toxicity assessment. However, the ZET is not widely accepted by regulatory authorities as the relevance of morphological findings from zebrafish embryo (ZFE) to humans is questioned. Thus, mechanistic endpoints are included as these are more conserved between vertebrate species and could improve interspecies transferability. Metabolome analysis has proven to be a valuable tool to reveal molecular changes indicative of various toxicity mechanisms. The main objective of this thesis was to evaluate the use of metabolome analysis in ZFE for the assessment of developmental toxicity. Two mechanisms of developmental toxicity, thyroid hormone disruption and antiangiogenesis, were examined through exposure of ZFEs to corresponding model substances. A targeted metabolomics approach measuring pooled ZFEs exposed to 6-propyl-2-thiouracil (PTU) was adopted for the initial study on thyroid hormone disruption. In the second study two specific inhibitors of angiogenesis, SU4312 and sorafenib, and two developmental toxicants, methotrexate, and rotenone, with a presumed antiangiogenic mode of action (MoA) were investigated. An untargeted metabolomics approach was employed to overcome the limitations of the previous study. This enabled the analysis of metabolome changes in the individual embryo and the optimization of metabolite coverage. The initial study demonstrated two main aspects: (i) reproducibility of the metabolome response in ZFE, and (ii) correlations between metabolite changes, the MoA of PTU and developmental toxicity. In the second study, 247 concordant metabolite changes were identified between SU4312, sorafenib, and methotrexate. Among the common changes were references to disturbed metabolic pathways of antiangiogenesis and developmental toxicity. Furthermore, 183 joint effects of methotrexate and rotenone suggested a common MoA related to disturbances in energy metabolism. Both studies support the use of metabolome analysis in ZFE for the assessment of developmental toxicity.
Study of the effects of PPAR-β/δ activators in the treatment of MASH
(Universitat de Barcelona, 2024-07-19) Zhang, Meijian; Vázquez Carrera, Manuel; Barroso Fernández, Emma; Universitat de Barcelona. Departament de Farmacologia, Toxicologia i Química Terapèutica
[eng] Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as nonalcoholic fatty liver disease (NAFLD), is the most common chronic liver disease around the world, affecting more than 30% of the global population. MASLD ranges from isolated lipid accumulation or steatosis to its active inflammatory form, metabolic dysfunction-associated steatohepatitis (MASH). MASH is a serious progressive liver disease in which liver inflammation may lead to liver fibrosis and liver dysfunction over time. MASH is often associated with other health problems [e.g. hypertension and type 2 diabetes mellitus (T2DM)] and is a leading cause of liver- related mortality. The increasing burden of MASH on global health systems has created an urgent need to develop effective and safe treatments. In this regard, peroxisome proliferator-activated receptor (PPAR)-β/δ agonists have been proven to be effective in attenuating the progression of MASLD by ameliorating insulin resistance (IR), reducing lipogenesis, and alleviating inflammation and endoplasmic reticulum (ER) stress. However, the role of PPAR-β/δ in hepatic fibrosis remains controversial. In the present thesis, we show that two PPAR-β/δ agonists, elafibranor and GW501516, prevented glucose intolerance and IR and reduced collagen accumulation in the liver of MASH mice. Surprisingly, elafibranor, a dual PPAR-α/-β/δ agonist, increased the levels of the EMT-promoting protein S100A4 via PPAR-β/δ activation, as confirmed in liver cells. Additionally, it decreased the levels of ASB2, a protein promoting S100A4 degradation. Conversely, GW501516, a specific PPAR-β/δ ligand, inhibited TGF-β1-induced HSC activation by reducing the suppressor of mothers against decapentaplegic (SMAD)3, as well as the levels of the SMAD3 co-activator p300 via AMP-activated protein kinase (AMPK) activation and the subsequent inhibition of extracellular signal-regulated kinase-1/2 (ERK1/2). Overall, these findings reveal novel mechanisms underlying the therapeutic effects of PPAR-β/δ agonists in liver diseases.
Chiral tricyclic lactams as efficient enantiomeric scaffolds for the synthesis of cis-decahydroquinolines substituted on the carbocyclic ring. Total synthesis of Myrioneuron alkaloids
(Universitat de Barcelona, 2024-02-16) Calbó Zabala, Arnau; Amat Tusón, Mercedes; Griera Farres, Rosa; Universitat de Barcelona. Departament de Farmacologia, Toxicologia i Química Terapèutica
[eng] The Myrioneuron alkaloids constitute a family of natural products found in East and South-East Asia. They feature a cis- or trans-decahydroquinoline (DHQ) as a common unit, attached to an oxazine, a diazine, or a cyclohexane ring, forming complex polycyclic ring systems. Despite exhibiting promising biological properties, their study has been hindered by the limited quantities available from natural sources. This thesis presents our advances on the exploration of new cyclocondensation reactions leading to tricyclic lactams bearing substituents at positions of the carbocyclic ring that were previously inaccessible by the group's established methodology. These studies have culminated in the synthesis of various Myrioneuron alkaloids. In the first chapter, the synthesis of cis-decahydroquinolines substituted on the carbocyclic ring is explored. Starting from racemic diastereomeric mixtures of dimethyl-2-oxocyclohexanepropionic acids the synthesis of both enantiomers of 7,8-, 6,8-, and 5,8-dimethyl-substituted cis-decahydroquinolines is reported. The procedure involves a dynamic kinetic asymmetric transformation in the cyclocondensation of the ketoacids with (R)-phenylglycinol to give in each case two major oxazoloquinolone lactams, which differ in the absolute configuration of all the stereogenic centers except that of the chiral inductor. A subsequent two-step stereoselective removal of the phenylethanol residue with simultaneous reduction of the lactam carbonyl afforded the enantiopure cis-decahydroquinolines in both enantiomeric series. The second chapter is centered on the total synthesis of (–)-schoberine B using the previously developed methodology. Firstly, the synthesis of a δ-ketoacid, bearing the appropriate functional groups for the synthesis of the alkaloid, is reported. Subsequently, the application of the aforementioned methodology with this compound allowed the obtention of two major lactams, that differ in the configuration of the four stereocenters on the decahydroquinoline moiety. From the above lactams, the removal of the chiral inductor, the introduction of a 2-piperidone ring, and the closure of the diazine ring completes the first enantioselective total synthesis of the Myrioneuron alkaloid (−)-schoberine B and its enantiomer (+)-schoberine B. In the third chapter, we assess the scope and limitations in the introduction of substituents at the C-7a and/or C-11 positions of simple (R)-phenylglycinol-derived oxazolo-DHQs. The reaction of this compound with various electrophiles allowed the regio- and stereoselective formation of various C-7a-subsituted oxazolo-DHQs bearing an all-carbon quaternary stereocenter. In a selected example, the regioselectivity of the process has been successfully modulated leading to the obtention of an 11-hydroxymethyl-substituted oxazolo-DHQ. A subsequent two-step stereoselective removal of the phenylethanol moiety afforded various C-4a and C-8 substituted cis-DHQs. Using this methodology, the most efficient enantioselective synthesis of (+)-myrioxazine A has been achieved.
G9a, a histone lysine methylatransferase, as a potential target for Alzheimer’s disease
(Universitat de Barcelona, 2024-04-22) Bellver Sanchis, Aina; Griñán Ferré, Christian; Universitat de Barcelona. Departament de Farmacologia, Toxicologia i Química Terapèutica
[eng] Alzheimer’s disease (AD) is the most common cause of dementia that currently lacks effective pharmacological treatment. Identifying new targets to halt disease progression is a pressing need. In most cases, AD has a complex etiology likely to involve multiple susceptibility genes and environmental factors. Epigenetics plays a crucial role in linking the genome to the environment and regulating the transcription of genes related to learning and memory. In particular, G9a is a histone lysine methyltransferase identified as having a critical role in various neurodegenerative diseases, including AD. On the one hand, I first conducted a study to confirm the involvement of G9a in the neurodegenerative processes that underlie the Senescence Accelerated Mouse-Prone 8 (SAMP8) model at both a behavioural and molecular level. Secondly, we demonstrated the efficacy of G9a inhibition as a therapy for AD using the SAMP8 mouse. We identified its main altered pathways and a novel mechanism of action based on Glia maturation factor beta (GMFB) modulation. The third study validated additional epigenetic mechanisms, specifically microRNAs, that participate in AD and are present in the SAMP8 model. These mechanisms were reversed after the inhibition of G9a. Notably, the downregulation of miRNA-128 after G9ai partially explains the modulations of pathways related to Peroxisome proliferator-activated receptor gamma (PPARγ). On the other hand, given the problems associated with G9a inhibitors (G9ai), such as toxicity, poor blood-brain barrier (BBB) permeability, and selectivity, it was decided to focus efforts on the search for chemical structures that would overcome these challenges. The fourth study involved structure- based virtual screening and in vitro and in vivo screening of the chosen compounds. This screening shows that a candidate with promising results in in vitro and in vivo studies, FLAV-27, is under patent. Optimization and characterization studies for FLAV-27 are being developed in our Knowledge Industry projects. In addition, the Multi Target Directed Ligands (MTDL)-based approach shows attractive advantages for multifactorial diseases such as AD. Therefore, we have also conducted in silico studies to obtain dual G9a/ Glycogen synthase kinase-3 beta (GSK-3β) compounds. In vivo studies were shown with Caenorhabitis elegans (C. elegans) and SAMP8, demonstrating their potential for AD. This doctoral thesis presents results for candidate T2, who is currently in the patent process. Finally, the drug discovery process is characteristically expensive and lengthy. Therefore, the fifth article focuses on the repurposing technique, which is often less risky, more cost-effective, and can be completed in less time. An in silico study proposed a cancer drug, Raltitrexed, as a possible G9ai. This work evaluated its efficacy in inhibiting G9a through in vitro and in vivo studies using the C. elegans model. The results suggest that Raltitrexed could be a candidate for treating AD, although further experiments are required.
El déficit cognitivo y la enfermedad de Alzheimer en presencia de alteraciones metabólicas: Estudio in vivo de una nueva diana terapéutica y de la estrategia multidiana para el tratamiento de estas condiciones
(Universitat de Barcelona, 2023-09-26) Espinosa Jiménez, Triana; Camins Espuny, Antoni; Ettcheto Arriola, Miren; Universitat de Barcelona. Departament de Farmacologia, Toxicologia i Química Terapèutica
[spa] La enfermedad de Alzheimer (EA) es el tipo de demencia más común. En los últimos años su prevalencia se ha incrementado preocupantemente, considerándose la epidemia del siglo XXI. Hasta ahora solo se han conseguido desarrollar fármacos capaces de retrasar el avance de la enfermedad, pero ninguno de ellos ha sido capaz de curarla. Esto podría deberse a que la patofisiología de la EA comienza décadas antes de que aparezcan los síntomas clínicos, por lo que la intervención terapéutica en la fase de demencia es demasiado tardía. Por otro lado, diferentes estudios han relacionado el consumo de una dieta rica en grasa, la cual puede llevar a obesidad y a diabetes mellitus tipo 2 (DMT2), con un mayor riesgo de desarrollar EA. Sin embargo, las vías moleculares que relacionan ambas patologías todavía no han sido descritas completamente, necesitándose nuevas investigaciones sobre posibles mecanismos que relacionen las alteraciones metabólicas y las cognitivas. Por ello, el primer estudio de esta tesis estuvo centrado en conocer el papel del receptor PPARβ/δ en el desarrollo del déficit cognitivo y las consecuencias de su asociación con una dieta rica en grasa, pudiendo representar una interesante diana para el control de la EA antes de su aparición. Este estudio se llevó a cabo en ratones PPARβ/δ-/- de seis meses de edad alimentados con una dieta rica en grasa. En él, se demostró que estos ratones presentaban una disminución del número de espinas dendríticas y marcadores sinápticos, hechos que fueron acompañados por la alteración de la memoria. Además, también se observó un aumento de la activación astrocítica y microglial, junto con un aumento de biomarcadores neuroinflamatorios. Asimismo, este estudio mostró una alteración de la vía del receptor de la insulina a nivel hipocampal. Interesantemente, mientras que algunas alteraciones causadas por la falta del receptor PPARβ/δ no se vieron afectadas por la alimentación con la dieta rica en grasa, otras sufrieron una exacerbación o necesitaron la combinación de ambos factores. Por otro lado, la EA se caracteriza por su carácter multifactorial en la que numerosas vías moleculares se ven afectadas. Por este motivo, el desarrollo de moléculas multidiana está tomando más protagonismo, y en la segunda parte de la presente tesis se estudió el fármaco multidiana rheina-huprina (RHE-HUP), inhibidor de la acetilcolinesterasa (AchE) y de la agregación de Tau, como posible tratamiento de la EA exacerbada por una dieta rica en grasa. En este trabajo, la inyección intraperitoneal de esta molécula a ratones hembra APP/PS1 de seis meses de edad alimentados con una dieta rica en grasa redujo los principales eventos característicos de la EA, incluyendo la hiperfosforilación de Tau, los niveles de Aβ42 y la formación de placas. Todo ello fue acompañado de un aumento de diferentes proteínas sinápticas y factores neurotróficos y de un incremento del número de espinas dendríticas, lo que resultó en una mejora de la memoria. Es de destacar que la mejora observada en este modelo puede ser atribuida directamente a una regulación a nivel central, ya que no se observaron modificaciones de las alteraciones periféricas originadas por la dieta rica en grasa. En resumen, esta tesis doctoral demuestra que la falta del receptor PPARβ/δ a nivel cerebral constituye no solo un nuevo factor de riesgo para la pérdida cognitiva en desórdenes neurológicos, sino que también se trata de una proteína clave dirigida a las vías fundamentales que conducen al deterioro de la memoria y a la EA, y que el fármaco RHE-HUP es un posible candidato para el tratamiento de la EA, incluso en presencia de alteraciones metabólicas, gracias al efecto observado en la mejora de algunos de los eventos neuropatológicos más importantes de esta enfermedad.
Preparació i Avaluació Biològica de Nous Sistemes Heterocíclics Nitrogenats
(Universitat de Barcelona, 2023-04-14) Viñas Simón, Miquel; Pujol Dilmé, M. Dolors; Universitat de Barcelona. Departament de Farmacologia, Toxicologia i Química Terapèutica
[cat] Aquest treball està orientat en l’estudi de nous sistemes heterocíclics nitrogenats amb potencial activitat biològica, concretament, en la recerca de nous antitumorals per inhibició enzimàtica. S’ha descrit la implicació de múltiples dianes en el desenvolupament i progressió del càncer i, és per això, que en aquest treball s’ha centrat en la cerca de compostos capaços d’inhibir aquestes dianes, entre les quals, es troben les CDKs, la proteïna KRAS, l’EZH2 i l’enzim G6PDH, sent aquesta darrera un dels objectius fonamentals en que versa aquest treball. En aquesta tesi es cerca la preparació de nous compostos que actuïn inhibint de forma selectiva i amb el menor nombre d’efectes adversos possible, les cèl·lules canceroses actuant sobre les diferents dianes esmentades que estan implicades en les diferents etapes de la malaltia cancerosa. S’ha dut a terme la preparació sintètica de tres sèries de compostos diferents, amb nuclis heterocíclics nitrogenats i sofrats. Els compostos obtinguts s’han preparat mitjançant reaccions clàssiques i innovadores de química orgànica i s’han purificat, posteriorment, mitjançant tècniques cromatogràfiques o bé per cristal·lització dels mateixos. La corresponent elucidació estructural s’ha dut a terme mitjançant Ressonància Magnètica Nuclear (RMN), Espectrometria de Masses (EM) i Espectroscòpia d’Infraroig (IR).
Generació de "building blocks" enantiopurs amb un estereocentre quaternari. Aplicació a la síntesi de productes naturals i compostos biològicament actius
(Universitat de Barcelona, 2022-11-11) Ordeix i Utiel, Sergi; Amat Tusón, Mercedes; Llor Brunés, Núria; Universitat de Barcelona. Departament de Farmacologia, Toxicologia i Química Terapèutica
[cat] Els alcaloides indòlics monoterpènics del grup leuconolam-leuconoxina-mersicarpina [(–)- mersicarpina, (–)-leuconoxina, (+)-melodinina E] i la (–)-kopsiyunnanina K presenten una interessant activitat biològica juntament amb estructures moleculars complexes, contenint tots ells un estereocentre quaternari, fet que els converteix en objectius interessants pels químics orgànics de síntesi. A la present Tesi Doctoral es descriu una metodologia per a la síntesi enantioselectiva de compostos lineals enantiopurs amb funcionalització d’1,5-aminoalcohol que contenen un estereocentre quaternari de 4 carbonis a partir de lactames derivades del fenilglicinol (Figura 2). Com etapes claus s’estudia la dialquilació estereoselectiva de les lactames (Figura 1) i la doble obertura reductiva dels anells d’oxazolidina i lactama. La posterior eliminació reductiva (en condicions compatibles amb la presència de insaturacions) o oxidativa de l’inductor quiral permet obtenir building blocks acíclics amb un estereocentre quaternari a la seva estructura. A la primera part del manuscrit es presenta la metodologia desenvolupada per a la generació de centres quaternaris de forma estereocontrolada utilitzant oxazolopiperidones que contenen fenilglicinol com a inductor quiral. La formació del centre quaternari es realitza a través de la reacció d’alquilació de l’enolat de la lactama, incorporant dos substituents a la posició C-6 de l’oxazolopiperidona. Addicionalment, les lactames poden presentar un segon estereocentre a la posició C-8. L’obtenció d’un dels dos diastereòmers de forma majoritària és degut a la rigidesa conformacional que presenten aquest tipus de compostos. Així doncs, l’agent alquilant entra preferencialment per la cara més accessible del sistema bicíclic. Cal destacar que ambdós enantiòmers són accessibles segons s’utilitzi (R) o (S)-fenilglicinol com a inductor quiral. Un cop s’ha format el centre quaternari de les lactames d’oxazolopiperidona, per trencament de l’enllaç C-O i posterior eliminació de l’inductor quiral per hidrogenació catalítica es descriu la síntesi enantioselectiva de piperidines amb un estereocentre quaternari diferentment substituïda. D’altra banda, el tractament de les oxazolopiperidines amb la combinació animo-borà/base permet l’obtenció estereoselectiva de compostos lineals enantiopurs amb funcionalització d’1,5- aminoalcohol (Figura 2). L’eliminació de la resta de feniletanol sota condicions reductores o oxidatives permet l’obtenció d’N-Boc aminopentanols, 5-hidroxipentàcids o 5-hidroxipentanitrils forma enantiopura i amb un estereocentre quaternari. Per tal de demostrar el potencial sintètic d’aquesta metodologia s’ha abordat la síntesis formal de la (–)-kopsiyunnanina K i la síntesi enantioselectiva de l’intermedi de Kerr. La síntesi d’aquest intermedi representa la síntesis formal dels alcaloides del grup leuconolam-leuconoxina- mersicarpina. La síntesi de l’intermedi de Kerr es va realitzar a partir de la lactama derivada de l’(S)-fenilglicinol que es mostra a la Figura 3. Les etapes clau d’aquesta síntesi varen ser una doble obertura reductiva, seguit de l’eliminació reductiva de l’inductor quiral per obtenir l’1,5-aminoalcohol representat (building block que conté l’estereocentre quaternari amb la configuració absoluta adequada). Una homologació de Bestmann-Ohira per tal d’introduir un grup alquí a la molècula va permetre obtenir un precursor avançar del compost diana. La incorporació d’una unitat d’anilina seguit d’una ciclació indòlica intramolecular va proporcionar la part indòlica del compost. Seguidament, una oxidació i tancament de l’anell lactàmic va rendir l’intermedi de Kerr, descrivint així la síntesi formal dels alcaloides indòlics monoterpènics (-)-mersicarpina, (-)-leuconoxina i (+)-melodinina E. La síntesi formal de la (–)-Kopsiyunnanina K s’inicia amb la mateixa lactama derivada de l’(S)- fenilglicinol utilitzada per a la síntesi de l’intermedi de Kerr. La doble obertura reductiva i l’eliminació reductiva de l’inductor quiral va permetre obtenir l’1,5-aminoalcohol necessari per a la síntesi del producte natural. Seguidament, es va introduir una unitat de triptamina al compost i es va realitzar un trencament oxidatiu del doble enllaç amb una reacció de Lemieux-Johnson per obtenir-ne l’aldehid. Aquest compost implica l’assoliment de la síntesi formal de la (–)-Kopsiyunnanina K.
Safety and efficacy investigations for new prenatal neuroprotective therapies. Applications in a model of intrauterine growth restriction (IUGR).
(Universitat de Barcelona, 2022-10-21) Kühne, Britta Anna; Barenys Espadaler, Marta; Illa Armengol, Míriam; Universitat de Barcelona. Departament de Farmacologia, Toxicologia i Química Terapèutica
[eng] Intrauterine growth restriction (IUGR) is defined as a significant reduction on fetal growth rate, resulting in a birth weight below the 10th percentile for the corresponding gestational age (Sharma et al., 2016). The prevalence accounts for 5-10% of all pregnancies, and amounts to approximately 600.000 cases in Europe, being a serious health problem worldwide (Kady and Gardosi, 2004). Placental insufficiency, the main cause of IUGR, chronically decreases the blood flow and nutrient supply to the developing fetus resulting in an adverse intrauterine environment with chronic hypoxia conditions and undernutrition. This situation results to a wide range of changes in brain development including grey (GM) and white matter (WM) injury (Esteban et al., 2010; Pla et al., 2020), which are associated with short- and long-term neurodevelopmental damage and cognitive dysfunctions (Mwaniki et al., 2012; Batalle et al., 2014; Eixarch et al., 2016). The most prevalent causes of brain damage of prenatal origin manifest as subtle neurological abnormalities. Indeed, IUGR has been proposed as the cause of one-quarter of special educational needs postnatally (Mackay et al., 2013). But currently, there is no efficient treatment which avoids deleterious consequences related to IUGR, especially in the neurodevelopmental field. To better understand, which basic cellular processes of brain development are altered due to IUGR, we established a novel in vitro model based on primary rabbit neuronal progenitor cells (NPCs) (Barenys et al., 2021). IUGR was surgically induced in one uterus horn in pregnant rabbits on gestational day (GD) 25. Neural progenitor cells (NPCs) growing as three-dimensional (3D) cell aggregates known as neurospheres were obtained from rabbit pups’ brains immediately after caesarean delivery at GD30. Neurospheres are able to mimic basic processes of brain development such as NPC proliferation, migration, differentiation into the brain effector cells neurons, oligodendrocytes and astrocytes, synaptogenesis, and network formation (Barenys et al., 2017; Breier et al., 2010; Gassmann et al., 2010a; Moors et al., 2009, 2007a; Schreiber et al., 2010). We revealed a significantly lower ability to form oligodendrocytes due to a slower differentiation rate in IUGR neurospheres. This result correlates very well with the clinical outcome of IUGR-generated white matter alterations. In addition, IUGR neurospheres presented an increased neurite length, which is consistent with previous in vivo studies (Pla et al., 2020). We have discovered for the first time that IUGR neurospheres respond differently than control to the exposure of the compound EGCG, which triggers migration alterations, and we have revealed the mechanism behind this difference: an overexpression of the adhesion molecule Integrin-β1 in IUGR. Because Integrin-β1 is implied in NPC migration but also in axonal growth and neuronal branching, this discovery gives new insights into the characterization of IUGR-induced neurodevelopmental alterations. The thesis addresses the medical necessity by assessing the safety and efficacy of the potential neuroprotective therapies docosahexaenoic acid (DHA), melatonin (MEL), 3,3',5-triiodothyronine (T3), zinc, lactoferrin (LF) and its main metabolite sialic acid (SA), epigallocatechin gallate (EGCG) and derivatives in two different approaches: (1) exposure in vitro and (2) prenatal administration in vivo, both followed by the evaluation in vitro. DHA, and MEL, were identified as the best therapeutical agents for preventing/reverting impaired oligodendrogenesis caused by IUGR. LF and its main metabolite SA were revealed to reduce IUGR-induced neurite length extension. Finally, we integrated the discovered results about IUGR-induced changes in neurodevelopment into an “adverse outcome pathway” (AOP) approach and developed the putative AOP “Disrupted laminin-β1-integrin interaction leading to developmental neurotoxicity”. Overall, the novel neurosphere model is well suited for characterizing so far unknown neurodevelopmental effects on the cellular level induced by chemicals or IUGR. This new method opens the door to testing possible neuroprotective therapies for IUGR easily and cost- efficiently.
Nous antagonistes del receptor NMDA pel tractament de la malaltia d’Alzheimer. Caracterització farmacològica in vivo
(Universitat de Barcelona, 2022-07-15) Companys Alemany, Júlia; Pallàs i Llibería, Mercè, 1964-; Griñán Ferré, Christian; Universitat de Barcelona. Departament de Farmacologia, Toxicologia i Química Terapèutica
[cat] Actualment, el tractament de la malaltia d’Alzheimer (MA) és una necessitat mèdica no coberta, ja que només existeixen tres grups de fàrmacs aprovats que no són suficientment eficaços pel control de la malaltia. La naturalesa complexa de la fisiopatologia de la MA i la manca de biomarcadors són els principals obstacles per al desenvolupament de nous fàrmacs pel tractament de la MA. Tenint en compte l’alta taxa de fracàs, l’optimització dels fàrmacs ja en el mercat representa una eina interessant ja que es reduirien els costos en comparació amb l’assaig de nous compostos dirigits a noves dianes. La memantina, un antagonista dels receptors N-metil-D-aspartat (NMDA), ha demostrat beneficis clínics en relació tant en aspectes cognitius i no cognitius, com en vies moleculars implicades en el procés neurodegeneratiu. Malgrat tot, la seva efectivitat és limitada. Per aquest motiu, aquesta tesi doctoral s’ha centrat en caracteritzar farmacològicament en models in vivo dos nous compostos anàlegs a la memantina (l’RL-208 i l’UB-ALT-EV) que s’han desenvolupat amb l’objectiu de millorar l’eficàcia terapèutica de la memantina. Durant la tesi doctoral, es van dur a terme estudis cognitius i de comportament en el nematode C.elegans, en un model de ratolí amb senescència accelerada (SAMP8) i en un model de ratolí transgènic de la MA, el 5XFAD, després del tractament amb els nous antagonistes. A més, es va avaluar molecularment vies associades a la MA per poder avaluar l’efecte modulador dels antagonistes dels receptors NMDA sobre elles. Els resultats obtinguts en el models animals de ratolí demostren com el tractament crònic per via oral a una dosi baixa amb els nous compostos és capaç de millorar les alteracions de la memòria, així com regular les alteracions de la conducta social i el comportament de tipus ansiós. Per altra banda, el compost UB-ALT-EV va demostrar efectes positius sobre els dèficits locomotors i del comportament els cucs C.elegans causats per l’acumulació del pèptid β-amiloide. Així mateix, els antagonistes del receptor NMDA avaluats van reduir els marcadors neuropatològics típics de la MA com la formació i l’acumulació del pèptid Aβ i la fosforilació de la proteïna tau. De la mateixa manera es va demostrar l’efectivitat dels nous compostos en la modulació de vies de senyalització molecular implicades en la neurodegeneració, com la desregulació dels nivells de calci intracel·lular, l’estrès oxidatiu, el flux autofàgic i la neuroinflamació. De manera global, els estudis duts a terme en aquesta tesi permeten demostrar l’efecte neuroprotector dels dos antagonistes del receptor NMDA a més de suggerir que l’optimització de fàrmacs aprovats suposa una estratègia de desenvolupament de nous fàrmacs que permetria l’abordatge de la MA de manera més eficaç. Particularment, donada la capacitat dels nous compostos de regular no només aspectes cognitius i no cognitius sinó també vies moleculars implicades en el procés neurodegeneratiu, aquestes noves teràpies podrien acabar sent considerades com teràpies modificadores de la malaltia i no només tractaments simptomatològics.
Synthesis of nitrogenated compounds with potential interest for the treatment of Unmet Medical Needs
(Universitat de Barcelona, 2022-09-23) Bagán Polonio, Andrea; Vázquez Cruz, Santiago; Escolano Mirón, Carmen; Universitat de Barcelona. Departament de Farmacologia, Toxicologia i Química Terapèutica
[eng] The synthesis of heterocyclic organic compounds allows access to a wide arsenal of compounds with potential pharmacological activity. This Thesis is part of the field of pharmaceutical chemistry, developing new therapeutic strategies through the design, synthesis and pharmacological evaluation of various families of compounds aimed at treating diseases that currently do not have a therapeutic solution. In the first chapter, the steps followed for the development, in its preclinical phase, of a representative compound that was the object of intellectual protection by the Fundació Bosch i Gimpera (UB) are explained. In the second chapter, the synthetic studies resulting from the exploration of the reactivity of different positions of the family of bicyclic α-iminophosphonates are exposed, which have allowed access to new structures that have been subjected to pharmacological evaluation. Specifically, it has been possible to access a family of compounds that have shown a very relevant biological activity and with potential application to Alzheimer's disease, whose diffusion will be preserved as it is being evaluated for intellectual protection by of the Bosch i Gimpera Foundation. In the third chapter, the synthetic studies resulting from the in-depth study of a multicomponent reaction that has given rise to a family of compounds with antiproliferative activity in a glioblastoma cell line are presented. This activity is similar to that of the drug used in the first line of treatment and, therefore, preliminary studies have been carried out that have made it possible to characterize the representative compound, promoting its development at the preclinical level. In the fourth chapter, a family of compounds that inhibit an enzyme directly related to inflammatory processes is provided. This new family has improved metabolic characteristics compared to previous families developed in the group and that were the subject of intellectual protection. The fifth chapter includes results derived from joint work with collaborating research groups, aimed at finding new applications for both compounds already previously published by us, with which we continue to work, and new compounds that we are developing.
Phenotyping of sigma-1 receptor knock-out rodents
(Universitat de Barcelona, 2022-06-02) Codony Soler, Xavier; Laguna Egea, Juan Carlos; Merlos Roca, Manuel; Universitat de Barcelona. Departament de Farmacologia, Toxicologia i Química Terapèutica
[eng] The sigma-1 receptor is a chaperone that is primarily expressed in the mitochondria-associated endoplasmic reticulum (MAM). It was cloned years ago from different tissues of various species and its structure has been recently elucidated.In 2003, our laboratory generated the first knock-out (KO) mouse for the sigma-1 receptor. These mice served to demonstrate the involvement of the sigma-1 receptor in acute and chronic pain. In addition to pain, the sigma-1 receptor has been implicated in other physiological processes and pathological conditions including depression and addiction. The sigma-1 receptor has also been shown to be associated with the regulation of other proteins, including dopamine (DAT) transporter. In this work, the generation of a sigma-1 receptor KO rat model is reported. The behavioural response of sigma-1 KO mice in two models of depression has been characterized, and the physiological and behavioural phenotyping of sigma-1 KO rats has been carried out, with special attention to the possible effects of the absence of the receptor in models of depression and addiction. By means of CRISPR / Cas9 technology, two KO strains for the sigma-1 receptor with deletions of 218bp and 7bp were obtained. Wild-type (WT) and the two KO strains showed no significant differences in the Irwin test, spontaneous locomotor activity, open field test, startle response, or pre-pulse inhibition. In contrast, the results obtained in response to mechanical or thermal stimulation led us to select the strain with a deletion of 218bp. Using this strain, WT (+/+), KO heterozygous (+/-) and KO homozygous (-/-) rats were phenotyped. No significant differences were found in growth or survival curves, nor in most of the assessed physiological or behavioural parameters. Regarding depression, no significant difference was found in the acute study after administration of fluoxetine (an antidepressant with sigma-1 affinity) or venlafaxine (without sigma-1 affinity) on the day of the test. This was an expected outcome, based on the literature and previous findings regarding the ineffectiveness of antidepressants in an acute treatment. In the sub-acute study, the tendency for lower immobility during training observed in mice was confirmed. On test days 1 and 7, neither fluvoxamine nor venlafaxine showed any efficacy in reducing immobility. In contrast, at day 14, the two antidepressants significantly reduced immobility only in KO rats. Given that there is no sigma-1 receptor in these rats and that both antidepressants showed activity, regardless of whether they had sigma-1 affinity, it seems that the efficacy may be due to some change in their action on SERT.Regarding locomotor activity, as a surrogate measure of addictive potential, the set of results seems to indicate a greater role of the sigma-1 receptor in the rearing activity, especially as stereotyped behaviour, rather than its merely exploratory activity, being the difference between the two genotypes mainly at high doses. There have been fewer differences in locomotor activity and, surprisingly, no differences after cocaine administration, that has a sigma-1 affinity. Differences between mobility and rearing can be assigned to the dopaminergic pathways involved, meso-limbic for mobility, and nigro-striatal for stereotypes.In conclusion, in rats, deletion of the gene encoding the sigma-1 receptor generates a viable phenotype very similar to the WT strain under normal conditions. Behavioural response under conditions of environmental stimulation and / or pharmacological treatment, reveals some differences between WT and KO, in both mice and rats. The absence of the receptor seems to lead to the adaptation of other proteins. The results further support the concept that the sigma-1 receptor may be involved in the development of depression and addiction and drugs acting on sigma-1 receptors could be useful in such pathologies.
Validación de dianas farmacológicas para la prevención y el tratamiento de la hipertrigliceridemia y el hígado graso inducidos por el consumo de fructosa líquida y dieta rica en grasa saturada
(Universitat de Barcelona, 2021-11-09) Velázquez Chávez, Ana Magdalena; Laguna Egea, Juan Carlos; Alegret i Jordà, Marta; Universitat de Barcelona. Departament de Farmacologia, Toxicologia i Química Terapèutica
[spa] La enfermedad del hígado graso no alcohólico es una patología sin tratamiento farmacológico específico que posee una alta prevalencia a nivel mundial, cuya etapa inicial se caracteriza por la esteatosis hepática simple. En esta tesis estudiamos dos fármacos candidatos para su tratamiento. El primero, la cafeína (CAF), sugerido por su efecto en la reducción de la infiltración de lípidos en el hígado a través de mecanismos antiinflamatorios, antioxidantes y relacionados con la autofagia y, el segundo, el ácido bempedoico (BemA), aprobado recientemente para el tratamiento de la hipercolesterolemia, que posee efectos en el hígado sobre la síntesis de ácidos grasos. Esta tesis fue dividida en tres estudios: en el estudio 1, caracterizamos un modelo animal de esteatosis hepática inducida por el consumo de una dieta alta en grasas (HFD) suplementada con fructosa líquida al 10% (HFHFr) durante tres meses en ratas hembras Sprague Dawley. A pesar de la mayor ingesta calórica, el peso corporal no varió en ratas HFD o HFHFr, debido a la inducción de termogénesis en el tejido adiposo marrón. La adición de fructosa al HFD incrementó la esteatosis hepática debido a la inhibición del catabolismo de ácidos grasos y la inducción de la DNL a través de ChREBP y SREBP, sin signos de inflamación, estrés del retículo endoplásmico o resistencia a la insulina hepática. Además, en el hígado de las ratas HFHFr, la activación de ChREBP aumentó la expresión de PNPLA3, desencadenando hipertrigliceridemia y aumentando el flujo de ácidos grasos hacia el músculo. Este efecto cambió el principal sustrato energético en el músculo, mostrado por un incremento de la β-oxidación de ácidos grasos en este tejido. En el estudio 2, analizamos el efecto de una dosis moderada de CAF (5 mg/kg), o como parte del extracto de café verde (GCE), en ratas HFHFr. Ni la CAF ni el GCE mejoraron la esteatosis hepática, sin embargo, las ratas tratadas con GCE, mostraron niveles más bajos de triglicéridos hepáticos en comparación con el grupo CAF. Los efectos diferenciales de GCE podrían estar relacionados con reducciones en la (a) fosforilación de mTOR hepático, lo que conduce a niveles más altos de lipina-1 nuclear y limita la expresión de genes lipogénicos; (b) niveles de diacilglicerol y ratio de hexosilceramida/ceramida; y (c) expresión del receptor de lipoproteínas de muy baja densidad. En el estudio 3, tratamos ratas HFHFr con BemA (30 mg/kg/día). El tratamiento mejoró la acumulación de lípidos en el hígado a través de nuevos mecanismos, además de la ya descrita inhibición de ACLY hepática: (a) disminución de DNL a través de la reducción de la expresión de la KHK hepática; b) aumento de la lipólisis por inducción de la expresión de PNPLA3, también responsable de la falta de efecto del fármaco sobre la hipertrigliceridemia; y c) aumento de la β-oxidación de ácidos grasos debido a un efecto agonista de PPARα, también observado sobre PPARγ.

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