TTBK1 and CK1 inhibitors restore TDP-43 pathology and avoid disease propagation in lymphoblast from Alzheimer’s disease patients

IntroductionTDP-43 proteinopathy in Alzheimer’s disease (AD) patients is recently emerging as a relevant pathomolecular event that may have been overlooked. Recent results in immortalized lymphocytes from AD patients have shown not only an increase of post-translational modifications in TDP-43, such as hyperphosphorylation and fragmentation, but also its prionic behaviour and cell-to-cell disease transmission. With the main goal to advance therapeutic interventions, we present in this work different kinase inhibitors with potential to restore this pathological mechanism.MethodologyWe have used immortalized lymphocytes from healthy controls and AD severe patients to evaluate the correction of TDP-43 pathology after the treatment with previously synthetized TTBK1 and CK1 inhibitors. Moreover we used the conditioned mediums of these cells to perform different disease propagation experiments.ResultsTDP-43 pathology observed in lymphoblasts from severe AD patients is reduced after the treatment with TTBK1 and CK1 inhibitors (decreasing phosphorylation and increasing nuclear localisation), Furthermore, the significant increase in TDP-43 phosphorylation, cytoplasmic accumulation and aberrant F-actin protrusions (TNT-like structures) observed in control cells growing in CM from AD lymphoblasts were abolished when the CM from AD lymphoblasts treated with previously reported TTBK1 and CK1 inhibitors were used. In addition, the cytosolic transport mediated by molecular motors of the receptor cells was altered with the induced TDP-43 pathology, but it was not produced with the abovementioned pretreated CMs.ConclusionTTBK1 and CK1 inhibitors, specially VNG1.47 and IGS2.7 compounds, restore TDP-43 pathology and avoid cell-to-cell propagation in immortalized lymphocytes from AD patients, being excellent candidates for the future therapy of this prevalent and devastating disease

COVID-19: Drug targets and potential treatments

92 p.-22 fig.-1 tab.-1 graph. abst.Currently, we are immersed in a pandemic caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which severely threatens public health worldwide. Until now, no drug or vaccine has been approved to treat the severe disease caused by this coronavirus, COVID-19. We will focus on the main virus-based and host-based targets that can guide medicinal chemistry efforts to discover new drugs for this devastating disease. In principle, all CoVs enzymes and proteins involved in viral replication and the control of host cellular machineries are potentially druggable targets in the search for therapeutic options for SARS-CoV-2. This perspective provides an overview of the main targets from a structural point of view, together with reported therapeutic compounds with activity against SARS-CoV-2 and/or other CoVs. Also, the role of innate immune response to coronavirus infection and the related therapeutic options will be presented.Funding from CSIC (201980E024 and 202020E103) is acknowledged. This research was partially supported through "la Caixa" Banking Foundation (HR18-00469), Instituto de Salud Carlos III (ISCIII-COV20/01007), Spanish Ministry of Science and Innovation (RTI2018-097305-R-I00), CONICYT-PCI (REDES190074 to D. R. and A. M.) and FONDECYT (11180604 to D.R.). I. M. was funded by H2020-MSCA-ITN-2017 (grant no. 765912), V. N. holds a pre-doctoral FPU grant (FPU16/04466) and J. U. was financed by FPI-SGIT2018-04.Peer reviewe

Modulación de la neurodegeneración con nuevas aproximaciones multidiana: diseño y síntesis de compuestos innovadores

217 p.-63 fig.-15 tab.-15 esquemas.[EN]Neurodegenerative diseases such as Alzheimer’s disease (AD) or amyotrophic lateral sclerosis (ALS), which affect millions of people worldwide, lack a therapy able to stop the characteristic neuronal death. Traditional medicinal chemistry and drug discovery strategies have failed to succeed in finding an effective treatment. In this thesis entitled: “Modulation of the neurodegeneration with new multitarget approaches: design and synthesis of innovative compounds”, two new approaches for the treatment of these pathologies are proposed. The first approach is the modulation with small molecules of the pathology of TDP-43, a protein described as the main component of the aggregates present in the motor neuron of ALS patients. This protein is at the same time, the central agent of different pathological pathways involved in other neurodegenerative diseases such AD. Taking into consideration that the hyperphosphorylation of TDP-43 caused by kinases is a crucial pathological mechanism which triggers this aggregation, several inhibitors of one of the main kinases involved in this event: the tau and tubulin kinase 1 (TTBK1), have been designed and synthetized. The binding mode of the inhibitors with the kinase domain of TTBK1 has been studied using crystallographic and computational techniques. The synthesized compounds efficiently inhibited the kinase and therefore have been tested in cellular models which have shown the ability of these inhibitors to modulate the pathology of TDP-43 causing a reduction of the cellular death. Finally, one of the compounds has been tested in a transgenic TDP-43 animal model, confirming that this TTBK1 inhibitor was able to decrease TDP-43 hyperphosphorylation and protect the motor neurons of the animals. These results demonstrate that TTBK1 inhibition is a valuable strategy for the modulation of this pathology in neurodegenerative diseases.The second innovative approach described here is the design and synthesis of multitarget ligands for the treatment of AD. The compounds were designed to modulate the two main pathological hallmarks of this dementia: neurofibrillary tangles composed of hyperphosphorylated protein tau and senile plaques, the main constituent of which is aggregated amyloid-β peptide. The designed multitarget ligands showed inhibitory activity against BACE1, the main protease involved in the processing of amyloid-β precursor protein and inhibitory activity against two different kinases responsible for the hyperphosphorylation of tau: CK1δ, LRRK2, GSK3β y TTBK1. Linkage strategy was used to connect diverse fragments originally inhibitors of the before mentioned protein kinases, designed and synthetized in this research group using a triazole ring. Besides, the in situ click chemistry methodology has been implemented using BACE1 as protein template which facilitated the discovery of new multitarget compounds efficiently. The binding mode of the compounds with BACE1 was studied using computational methods. Biological evaluation of the synthesized inhibitors in different cellular models resulted in a reduction of the amyloid-β fragment production and a neuroprotection of the cells against the toxic insult of okadaic acid, resulting in a decreased hyperphosphorylation of tau.In summary, in this thesis the design, synthesis and validation of new modulators of key proteins involved in different pathological mechanism is described, offering new therapeutic strategies for the treatment of neurodegenerative diseases which lack an effective cure.[ES]Las enfermedades neurodegenerativas como la enfermedad de Alzheimer (EA) o la esclerosis lateral amiotrófica (ELA), que sufren millones de personas en el mundo, carecen hoy en día de una terapia que frene la muerte neuronal que en ellas se produce. Las estrategias tradicionales empleadas por la química médica y el descubrimiento de fármacos no han sido capaces hasta ahora de conseguir un tratamiento efectivo y es por ello que en la presente tesis doctoral titulada: “Modulación de la neurodegeneración con nuevas aproximaciones multidiana: diseño y síntesis de compuestos innovadores”, se proponen dos nuevas aproximaciones para el tratamiento de estas patologías. La primera aproximación es la modulación a través de moléculas pequeñas de la patología de TDP-43, proteína descrita como principal componente de los agregados presentes en las motoneuronas de pacientes con ELA. Esta proteína también se ha definido como agente central de diversos mecanismos patológicos en otras enfermedades neurodegenerativas como la EA. Dado que la hiperfosforilación de TDP-43 mediada por proteínas quinasa es un evento patológico crucial para esta agregación, en esta tesis doctoral se han diseñado, sintetizado y validado inhibidores de la quinasa de tau y tubulina 1 (TTBK1), una de las principales enzimas implicadas en el proceso de hiperfosforilación de esta diana. El modo de unión de los inhibidores con la quinasa ha sido estudiado por medio de cristalografía de rayos X, así como con estudios computacionales. Los compuestos obtenidos han conseguido inhibir de manera eficiente esta quinasa y han sido ensayados en modelos celulares, en los cuales se ha demostrado su efectividad en la modulación de la patología de TDP-43, reduciendo la muerte celular. Por último, se ha demostrado que uno de los compuestos es capaz de proteger las motoneuronas de ratones transgénicos con la patología de TDP-43, disminuyendo la hiperfosforilación de esta y posicionando los inhibidores de TTBK1 como una estrategia eficaz para el tratamiento de esta proteinopatía. La segunda aproximación innovadora descrita en esta tesis doctoral es el diseño y síntesis de compuestos multidiana para el tratamiento de la EA. En concreto, se han diseñado y sintetizado compuestos dirigidos a modular los dos eventos patológicos principales que se producen en esta enfermedad: los ovillos neurofibrilares compuestos por la proteína tau hiperfosforilada y las placas seniles formadas por agregados del péptido β-amiloide. Los compuestos multidiana diseñados presentaron actividad inhibitoria frente a BACE1, la principal proteasa implicada en la digestión de la proteína precursora del β-amiloide así como actividad inhibitoria frente a dos quinasas diferentes implicadas en la hiperfosforilación de tau: CK1δ, LRRK2, GSK3β y TTBK1.De las estrategias habituales para el diseño de este tipo de ligandos, los compuestos multidiana fueron obtenidos partiendo de inhibidores de quinasa conocidos uniéndolos mediante un conector de tipo triazol. Así mismo, se ha puesto a punto una metodología de química click in situ utilizando BACE1 como molde de reacción facilitando el descubrimiento de nuevos compuesto multidiana. El modo de unión respecto a la proteasa BACE1 se estudió con métodos computacionales. La evaluación biológica de los inhibidores sintetizados en distintas líneas celulares provocó por un lado, una reducción en la producción de los fragmentos del β-amiloide así como una neuroprotección de las células frente al daño inducido por el ácido ocadaico y por tanto, por la hiperfosforilación de la proteína tau. En esta tesis doctoral se describe el diseño, síntesis y validación de nuevos moduladores de proteínas clave en distintos mecanismos patológicos, ofreciendo nuevas estrategias terapéuticas para el tratamiento de enfermedades neurodegenerativas que a día de hoy no tienen cura.Contrato FPU del Ministerio de Educación. Premios de la SEQT y de la RANF al trabajo realizado.Peer reviewe

Tau Tubulin Kinase 1 (TTBK1), a new player in the fight against neurodegenerative diseases

22 p.-6 fig.Tau-tubuline kinases (TTBK) are a family of serine/threonine and tyrosine kinases recently discovered and implicated in the phosphorylation of important substrates such as tau, tubuline or TDP-43. Its two homologs, TTBK1 and TTBK2, show different expression patterns and different involvements in physiological mechanisms of great importance such as mitosis, ciliogenesis and neurotransmission. Their phosphorylation activity has also linked them to the development of neurodegenerative diseases like Alzheimer’s disease, amyotrophic lateral sclerosis or spinocerebellar ataxia type 11. There are currently only three inhibitors of these kinases described in the literature. This review intends to give an overview of the structure, expression, physiological and pathological mechanisms of both kinases as well as an extended analysis on the molecules that can inhibit them. The final analysis of all this information led us to propose TTBK1 as a new target for the treatment of neurodegenerative diseases and its selective inhibitors as potential effective drugs for the treatment of these severe unmet disorders.This work has been funding by grants from Comunidad de Madrid (B2017/BMD3813 ELA-Madrid) and MINECO (SAF2016-76693-R). V.N. holds a pre-doctoral FPU grant (FPU16/04466).Peer reviewe

Running title: Kinase inhibitors for ALS therapy

46 p.-3 fig.-2 tab.Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that causes the progressive loss of motoneurons and, unfortunately, there is no effective treatment for this disease. Interconnecting multiple pathological mechanisms are involved in the neuropathology of this disease, including abnormal aggregation of proteins, neuroinflammation and dysregulation of the ubiquitin proteasome system. Such complex mechanisms, together with the lack of reliable animal models of the disease have hampered the development of drugs for this disease. Protein kinases, a key pharmacological target in several diseases, have been linked to ALS as they play a central role in the pathology of many diseases. Therefore several inhibitors are being currently trailed for clinical proof of concept in ALS patients. In this review, we examine the recent literature on protein kinase inhibitors currently in pharmaceutical development for this diseaseas future therapy for AS together with their involvement in the pathobiology of ALS.Comunidad de Madrid, Grant/Award Number:B2017/BMD3813; La Caixa Banking Foundation, Grant/Award Number:LCF/BQ/PR18/11640007; MECD, Grant/Award Numbers: FPU14-00204,FPU16/04466; ISCiii, Grant/Award Number:CIBERNED, CB18/05/00040; FEDERPeer reviewe

Optimización de la estrategia de unión en el diseño de compuestos multidiana prometedores para el tratamiento de la enfermedad de Alzheimer

Este trabajo ha sido financiado por el MINECO (SAF2016-76693-R). V.N. poseee un contrato pre-doctoral FPU (FPU16/04466). V.P. quiere agradecer a la Caixa por su beca Junior Leade

Protein kinase inhibitors for amyotrophic lateral sclerosis therapy

46 p.-3 fig.-2 tab.Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that causes the progressive loss of motoneurons and, unfortunately, there is no effective treatment for this disease. Interconnecting multiple pathological mechanisms are involved in the neuropathology of this disease, including abnormal aggregation of proteins, neuroinflammation and dysregulation of the ubiquitin proteasome system. Such complex mechanisms, together with the lack of reliable animal models of the disease have hampered the development of drugs for this disease. Protein kinases, a key pharmacological target in several diseases, have been linked to ALS as they play a central role in the pathology of many diseases. Therefore several inhibitors are being currently trailed for clinical proof of concept in ALS patients. In this review, we examine the recent literature on protein kinase inhibitors currently in pharmaceutical development for this diseaseas future therapy for AS together with their involvement in the pathobiology of ALS.Comunidad de Madrid, Grant/Award Number:B2017/BMD3813; La Caixa Banking Foundation, Grant/Award Number:LCF/BQ/PR18/11640007; MECD, Grant/Award Numbers: FPU14-00204,FPU16/04466; ISCiii, Grant/Award Number:CIBERNED, CB18/05/00040; FEDERPeer reviewe

Improved linkage design for the discovery of multitarget ligands as powerful drugs for Alzheimer’s disease

14 p.-5 fig.-2 tab.[ENG] Multitarget drugs are molecular entities that are designed to present more than one biological activity. They are arising as powerful tools to tackle complex diseases including bacterial resistances, cancer or neurodegenerative diseases. Typically, the rational strategies to design multitarget drugs are linkage, fusion and incorporation or merge. Here we present the creation of a multitarget drug combining active fragments in a way that could inhibit an additional third target with the objective to create powerful modulating agents for neurodegenerative diseases. Multitarget compounds are ideally suited for the treatment of these pathologies due to their unknown etiology,multifactorial pathology and lack of efficient treatments.To achieve this aim we have combined fragments that inhibit kinases involved in the main pathomolecular pathways of Alzheimer’s disease such as tau aggregation, neuroinflammation and decreased neurogenesis, looking for a third action in BACE1,responsible of β-amyloid production. Finally, and after the successful results obtained using this methodology,we have started to implement the in situ click chemistry technique to better select the multitarget compounds using BACE1 as a template.[ESP] Los fármacos multidiana son entidades moleculares diseñadas para presentar más de una actividad biológica. Debido a esta propiedad, estos compuestos son considerados herramientas privilegiadas para el tratamiento de enfermedades complejas como las infecciones bacterianas, el cáncer o las enfermedades neurodegenerativas. Las estrategias de diseño para crear fármacos multidiana han sido típicamente unión, fusión e incorporación. En este trabajo presentamos la creación de compuestos multidiana combinando fragmentos activos de tal manera que puedan inhibir una tercera diana adicional una vez unidos, con el objetivo de crear fármacos prometedores para el tratamiento de enfermedades neurodegenerativas. Este tipo de fármacos multidiana resultan muy apropiados para el tratamiento de estas patologías multifactoriales, de las que a día de hoy se desconoce su etiología y que carecen de tratamientos efectivos. Para conseguir este objetivo hemos combinado fragmentos de moléculas que inhiben quinasas involucradas en los mecanismos patomoleculares principales de la enfermedad de Alzheimer como la agregación de tau, la neuroinflamación y la disminución de la neurogénesis.Además se ha buscado una tercera actividad en la enzima BACE1, responsable patología del β-amiloide en la enfermedad de Alzheimer. Finalmente, y tras los resultados prometedores obtenidos con los fármacos multidiana, hemos comenzado a implementar la técnica de química click in situ para optimizar la selección de inhibidores utilizando la enzima BACE1 como molde de reacción.Este trabajo ha sido financiado por el MINECO (SAF2016-76693-R). V.N. poseee un contrato pre-doctoral FPU (FPU16/04466).Peer reviewe

Designing multitarget ligands for neurodegenerative diseases with improved permeability trough PLGA nanoencapsulation

Multitarget ligands (MTLs) have emerged as an interesting alternative for addressing complex multifactorial pathologies such as neurodegenerative diseases. However, a common challenge associated with these compounds is often their high molecular weight and low solubility, which becomes a hurdle when trying to permeate over the blood-brain barrier (BBB). In this study, we have designed two new MTLs that modulate three pharmacological targets simultaneously (tau, beta-amyloid and TAR DNA-binding protein 43). To enhance their brain penetration, we have formulated organic polymeric nanoparticles using poly(lactic-co-glycolic acid). The characterization of the formulations, evaluation of their permeability through an in vitro BBB model, and assessment of their activity on disease-representative cellular models, such as Alzheimer's disease and amyotrophic lateral sclerosis, have been conducted. The results demonstrate the potential of the new MTLs and their nanoparticle encapsulation for the treatment of neurodegenerative diseases.This work was supported by AIE (grant PID2019–105600RB-I00), La Caixa and Luzón foundations (grant HR21-00937) and MCIN/AEI/10.13039/501100011033 (grant RYC2019-027489-I).S

O2 Improved controlled release and brain penetration of small molecules with therapeutic potential using PLGA nanoparticles

1 p.-1 fig.Peer reviewe