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Alzheimer's disease (AD), the most common type of dementia, currently represents an extremely challenging and unmet medical need worldwide. Amyloid-β (Aβ) and Tau proteins are prototypical AD hallmarks, as well as validated drug targets. Accumulating evidence now suggests that they synergistically contribute to disease pathogenesis. This could not only help explain negative results from anti-Aβ clinical trials but also indicate that therapies solely directed at one of them may have to be reconsidered. Based on this, herein, we describe the development of a focused library of 2,4-thiazolidinedione (TZD)-based bivalent derivatives as dual Aβ and Tau aggregation inhibitors. The aggregating activity of the 24 synthesized derivatives was tested in intact Escherichia coli cells overexpressing Aβ42 and Tau proteins. We then evaluated their neuronal toxicity and ability to cross the blood-brain barrier (BBB), together with the in vitro interaction with the two isolated proteins. Finally, the most promising (most active, nontoxic, and BBB-permeable) compounds 22 and 23 were tested in vivo, in a Drosophila melanogaster model of AD. The carbazole derivative 22 (20 μM) showed extremely encouraging results, being able to improve both the lifespan and the climbing abilities of Aβ42 expressing flies and generating a better outcome than doxycycline (50 μM). Moreover, 22 proved to be able to decrease Aβ42 aggregates in the brains of the flies. We conclude that bivalent small molecules based on 22 deserve further attention as hits for dual Aβ/Tau aggregation inhibition in AD., This work was conducted by A.E.G during a scholarship at the University of Bologna, Italy, supported by the International Cooperation Program CAPES/ PDSE Process #88881.187586/ 2018-01. O.S. acknowledges the Ministry of Education, Youth and Sports of the Czech Republic (project ERDF no. CZ.02.1.01/0.0/0.0/18_069/0010054). DMT: grant PID2020-118127RB-I00 funded by MCIN/AEI/10.13039/501100011033. M.L.B. and M.B. would also like to acknowledge the University of Bologna (RFO 2019) and the Italian Ministry of Education, Universities and Research (MIUR), for financial support.

Immune checkpoint inhibitors (ICIs) have significantly improved outcomes for patients with neoplasms in advanced stages. On the other hand, ICIs have immune-related adverse events. These adverse events affect mostly other organs than the kidney, such as skin or gastrointestinal tract. The incidence of nephrotoxicity with monotherapy with any ICI is about 2%, which increases to 5% in combination therapy. Acute tubulointerstitial nephritis (AIN) is the most common pattern of kidney damage related to ICIs. Globally, without considering ICI nephrotoxicity, AIN is estimated to account for 15% to 20% of cases of acute kidney injury (AKI). This is crucial because patients who are treated with ICIs, may also be taking other drugs that potentially cause AIN, and therefore, knowing the particularities about ICI-related AIN could be helpful in clinical practice to better understand the phenotypic differences between the 2 types of AIN. In addition, several studies have now shown that being on proton pump inhibitors is a risk factor for AIN from ICI therapy.

Immunotherapy; PARP inhibitors; Pembrolizumab Immunoteràpia; Inhibidors de PARP; Pembrolizumab Inmunoterapia; Inhibidores de PARP; Pembrolizumab Metastatic triple-negative breast cancer (mTNBC) is a poor prognostic disease with limited treatments and uncertain therapeutic algorithms. We performed a systematic review and multiple Bayesian network meta-analyses according to treatment line to establish an optimal therapeutic sequencing strategy for this lethal disease. We included 125 first-line trials (37,812 patients) and 33 s/further-lines trials (11,321 patients). The primary endpoint was progression-free survival (PFS). Secondary endpoints included overall response rates (ORR), overall survival (OS) and safety, for first and further lines, separately. We also estimated separate treatment rankings for the first and subsequent lines according to each endpoint, based on (surface under the cumulative ranking curve) SUCRA values. No first-line treatment was associated with superior PFS and OS than paclitaxel ± bevacizumab. Platinum-based polychemotherapies were generally superior in terms of ORR, at the cost of higher toxicity.. PARP-inhibitors in germline-BRCA1/2-mutant patients, and immunotherapy + chemotherapy in PD-L1-positive mTNBC, performed similar to paclitaxel ± bevacizumab. In PD-L1-positive mTNBC, pembrolizumab + chemotherapy was better than atezolizumab + nab-paclitaxel in terms of OS according to SUCRA values. In second/further-lines, sacituzumab govitecan outperformed all other treatments on all endpoints, followed by PARP-inhibitors in germline-BRCA1/2-mutant tumors. Trastuzumab deruxtecan in HER2-low mTNBC performed similarly and was the best advanced-line treatment in terms of PFS and OS after sacituzumab govitecan, according to SUCRA values. Moreover, comparisons with sacituzumab govitecan, talazoparib and olaparib were not statistically significant. The most effective alternatives or candidates for subsequent lines were represented by nab-paclitaxel (in ORR), capecitabine (in PFS) and eribulin (in PFS and OS).

[cat] Les característiques immunomoduladores induïdes per la proteïna plasmàtica humana C4BP(β-) en el model in vitro de Mo-DCs inflamatòries li confereixen potencial terapèutic per al restabliment de la tolerància immunològica i la prevenció o mitigació del dany tissular en malalties autoimmunitàries i inflamatòries. Per això, els objectius d’aquesta tesi són: 1. Avaluar el potencial terapèutic de C4BP(β-) com a tractament in vivo en processos immune-inflamatoris. Per assolir aquest objectiu: ■ Avaluar l’eficàcia terapèutica de C4BP(β-) en els models experimentals de LES amb NL, NZBW F1 i MRL-lpr. ■ Avaluar l’eficàcia terapèutica de C4BP(β-) en el model experimental de CU aguda induïda per DSS. 2. Disseccionar i caracteritzar l’activitat no canònica immunomoduladora de C4BP(β-) generant una nova variant recombinant (PRP6-HO7), basada en la seva estructura però composada exclusivament pel domini immunomodulador CCP6, per avaluar la seva independència funcional i el seu potencial respecte C4BP(β-) en la inducció de tolerogènesi sobre els fagòcits mononuclears i com a tractament en processos immune-inflamatoris.

Introduction Primary hyperoxaluria (PH) is a family of 3 rare genetic disorders of hepatic glyoxylate metabolism that lead to overproduction and increased renal excretion of oxalate resulting in progressive renal damage. LDHA inhibition of glyoxylate-to-oxalate conversion by RNA interference (RNAi) has emerged as a potential therapeutic option for all types of PH. LDHA is mainly expressed in the liver and muscles. Methods Nonclinical data in mice and nonhuman primates show that LDHA inhibition by RNAi reduces urinary oxalate excretion and that its effects are liver-specific without an impact on off-target tissues, such as the muscles. To confirm the lack of unintended effects in humans, we analyzed data from the phase I randomized controlled trial of single-dose nedosiran, an RNAi therapy targeting hepatic LDHA. We conducted a review of the literature on LDHA deficiency in humans, which we used as a baseline to assess the effect of hepatic LDHA inhibition. Results Based on a literature review of human LDHA deficiency, we defined the phenotype as mainly muscle-related with no liver manifestations. Healthy volunteers treated with nedosiran experienced no drug-related musculoskeletal adverse events. There were no significant alterations in plasma lactate, pyruvate, or creatine kinase levels in the nedosiran group compared with the placebo group, signaling the uninterrupted interconversion of lactate and pyruvate and normal muscle function. Conclusion Phase I clinical data on nedosiran and published nonclinical data together provide substantial evidence that LDHA inhibition is a safe therapeutic mechanism for the treatment of all known types of PH., Graphical abstract

[eng] The herpesvirus diseases are increasing in importance as a public health problem throughout the world. Members of the human herpesvirus family are global in distribution and infect 60-95% of the world's population, both in developed and in developing countries. The terminase complex plays a key role in genome packaging and is a promising drug target for developing new antiviral compounds. The tripartite terminase 3 subunit C-terminal domain (TRM3-C) of five human herpesviruses (HSV-2, VZV, EBV, HCMV and KSHV) has been biochemically characterized, showing the need of Mn2+ cations in order to enhance the nuclease activity. The three-dimensional structure of TRM3-C HSV-2 has been determined by X-ray crystallography and showed a new position of the two Mn+2 ions in the active centre. Diverse inhibitors have been tested against the TRM3-C terminal domain of the five herpesviruses. Some of them were developed in our laboratory (BS14 and BS17) and the others are specific inhibitors of the HIV integrase. Docking models of TRM3-C have been obtained with the inhibitors that were able to inhibit the nuclease activity in a low micromolar range (BS14, BS17 and Bictegravir). Cell assays with BS14 showed that is moderately active against HCMV. Considering all the biochemical and structural data, new compounds have been designed to obtain better inhibitors against the TRM3-C target. Coronaviruses are a large family of viruses that are known to cause human illness ranging from the common cold to more severe diseases such as MERS and SARS. The emergence of SARS-CoV-2 in 2019 has triggered an ongoing global pandemic of the severe pneumonia-like disease coronavirus disease 2019 (COVID-19). The main protease (Mpro) is one of the best characterized drug targets among coronaviruses. Compound 1 inhibits the protease of the rhinovirus, which is homolog to the SARS-CoV-1 Mpro. Thus, could be a promising drug against coronaviruses. The three-dimensional structure of SARS-CoV-1 Mpro in complex with the inhibitor Compound 1 has been determined., [spa] Las enfermedades provocadas por herpesvirus están cobrando cada vez más importancia como problema de salud pública en todo el mundo. Los miembros de la familia del herpesvirus humano están distribuidos mundialmente e infectan al 60-95% de la población, tanto en países desarrollados como en vías de desarrollo. El complejo de la terminasa juega un papel clave en el empaquetamiento del genoma y es una diana farmacológica prometedora para el desarrollo de nuevos compuestos antivirales. Se ha caracterizado bioquímicamente el dominio C- terminal de la subunidad 3 de la terminasa tripartita (TRM3-C) de cinco herpesvirus humanos (HSV-2, VZV, EBV, HCMV y KSHV), mostrando la necesidad de cationes Mn2+ para potenciar la actividad nucleasa. La estructura tridimensional de TRM3-C HSV-2 ha sido determinada por cristalografía de rayos X, mostrando una nueva posición de los dos iones Mn+2 en el centro activo. Se han probado diversos inhibidores contra el dominio terminal TRM3-C de los cinco herpesvirus. Algunos de ellos fueron desarrollados en nuestro laboratorio (BS14 y BS17) y los demás son inhibidores específicos de la integrasa del VIH. Se han obtenido modelos de acoplamiento de TRM3-C con los inhibidores que fueron capaces de inhibir la actividad nucleasa en un rango micromolar bajo (BS14, BS17 y Bictegravir). Los ensayos celulares con BS14 mostraron que es moderadamente activo contra HCMV. Teniendo en cuenta todos los datos bioquímicos y estructurales, se han diseñado nuevos compuestos para obtener mejores inhibidores contra la diana TRM3-C. Los coronavirus son una gran familia de virus que causan enfermedades humanas que van desde el resfriado común hasta enfermedades más graves como MERS y SARS. La aparición del SARS-CoV-2 en 2019 ha desencadenado una pandemia mundial en curso de la enfermedad grave por coronavirus 2019 (COVID-19), una enfermedad similar a la neumonía. La proteasa principal (Mpro) es una de las dianas farmacológicas mejor caracterizadas entre los coronavirus. El compuesto 1 inhibe la proteasa del rinovirus, que es homóloga al SARS-CoV-1 Mpro. Por lo tanto, podría ser un fármaco prometedor contra los coronavirus. Se ha determinado la estructura tridimensional de SARS-CoV-1 Mpro en complejo con el inhibidor Compuesto 1.

Endothelial adenosine monophosphate-activated protein kinase (AMPK) plays a critical role in the regulation of vascular tone through stimulating nitric oxide (NO) release in endothelial cells. Since obesity leads to endothelial dysfunction and AMPK dysregulation, AMPK activation might be an important strategy to restore vascular function in cardiometabolic alterations. Here, we report the identification of a novel AMPK modulator, the indolic derivative IND6, which shows affinity for AMPKα1β1γ1, the primary AMPK isoform in human EA.Hy926 endothelial cells. IND6 shows inhibitory action of the enzymatic activity in vitro, but increases the levels of p-Thr174AMPK, p-Ser1177eNOS and p-Ser79ACC in EA.Hy926. This paradoxical finding might be explained by the ability of IND6 to act as a mixed-type inhibitor, but also to promote the enzyme activation by adopting two distinct binding modes at the ADaM site. Moreover, functional assays reveal that IND6 increased the eNOS-dependent production of NO and elicited a concentration-dependent vasodilation of endothelium-intact rat aorta due to AMPK and eNOS activation, demonstrating a functional activation of the AMPK–eNOS–NO endothelial pathway. This kinase inhibition profile, combined with the paradoxical AMPK activation in cells and arteries, suggests that these new chemical entities may constitute a valuable starting point for the development of new AMPK modulators with therapeutic potential for the treatment of vascular complications associated with obesity., his research was funded by Fundación Eugenio Rodríguez Pascual, the Spanish Ministerio de Economía y Competitividad (MDM-2017-0767), the Spanish Ministerio de Ciencia Innovación y Universidades (RTI2018- 095544-B-I00) and the Generalitat de Catalunya (2017SGR1746) for financial support. Computational resources from the Barcelona Supercomputing Center (BSC; BCV-2019-1-0009 and BCV-2019-2-0017) and the Consorci de Serveis Universitaris de Catalunya (CSUC; Molecular Recognition project) are acknowledged. E.A. thanks AGAUR (Generalitat of Catalunya; 2019FI_B2_00001) for her fellowship. M.S.G. thanks Universidad Com- plutense de Madrid for her fellowship.

Immune checkpoint inhibitors; Metastatic renal carcinoma; Second-line treatment Inhibidors del punt de control immunitari; Carcinoma renal metastàtic; Tractament de segona línia Inhibidores del punto de control inmunitario; Carcinoma renal metastásico; Tratamiento de segunda línea Background: The INMUNOSUN trial had the objective of prospectively evaluating the efficacy and safety of sunitinib as a pure second-line treatment in patients with metastatic renal cell carcinoma (mRCC) who have progressed to first-line immune checkpoint inhibitor (ICI)-based therapies. Patients and methods: A multicenter, phase II, single-arm, open-label study was carried out in patients with a histologically confirmed diagnosis of mRCC with a clear-cell component who had progressed to a first-line regimen of ICI-based therapies. All patients received sunitinib 50 mg once daily orally for 4 weeks, followed by a 2-week rest period following package insert instructions. The primary outcome was the objective response rate. Results: Twenty-one assessable patients were included in the efficacy and safety analyses. Four patients [19.0%, 95% confidence interval (CI) 2.3% to 35.8%] showed an objective response (OR), and all of them had partial responses. Additionally, 14 (67%) patients showed a stable response, leading to clinical benefit in 18 patients (85.7%, 95% CI 70.7% to 100%). Among the four assessable patients who showed an OR, the median duration of the response was 7.1 months (interquartile range 4.2-12.0 months). The median progression-free survival (PFS) was 5.6 months (95% CI 3.1-8.0 months). The median overall survival (OS) was 23.5 months (95% CI 6.3-40.7 months). Patients who had better antitumor response to first-line ICI-based treatment showed a longer PFS and OS with sunitinib. The most frequent treatment-emergent adverse events were diarrhea (n = 11, 52%), dysgeusia (n = 8, 38%), palmar-plantar erythrodysesthesia (n = 8, 38%), and hypertension (n = 8, 38%). There was 1 patient who exhibited grade 5 pancytopenia, and 11 patients experienced grade 3 adverse events. Eight (38%) patients had serious adverse events, four of which were considered to be related to sunitinib. Conclusion: Although the INMUNOSUN trial did not reach the pre-specified endpoint, it demonstrated that sunitinib is active and can be safely used as a second-line option in patients with mRCC who progress to new standard ICI-based regimens. This work was supported by Pfizer, S.L.U. (Madrid, Spain). Pfizer, S.L.U. provided an unrestricted research grant with drug funding and drug supply to conduct the study (no grant number).

Objetivo Durante los años 2019 y 2020 se realizaró una serie de reuniones en todo el territorio nacional dirigidas a explicar la metodología y los criterios de elaboración de las recomendaciones sobre la utilización de iPCSK9 publicadas por la Sociedad Española de Arteriosclerosis. Al final de cada una de las reuniones se realizó una encuesta entre los médicos participantes dirigida a describir los requerimientos de prescripción de estos fármacos en las diferentes regiones españolas. Metodología El Proyecto Efecto Mariposa fue desarrollado por un comité científico experto en lipídos. Tras la elaboración de todos los materiales necesarios para llevar a cabo el proyecto, se efectuó una reunión de formadores, impartida por los coordinadores del proyecto, a un total de 17 expertos. Posteriormente, se realizaron 16 talleres regionales con la asistencia de 169 médicos implicados en el manejo de la hipercolesterolemia, los cuales respondieron una encuesta donde se planteaban diferentes cuestiones acerca del uso de los iPCSK9 en su práctica clínica. Resultados Hubo una gran heterogeneidad entre centros en cuanto a los requerimientos y dificultades para la prescripción de iPCSK9. Un 21% de los encuestados indicaron tener dificultades escasas para prescribir iPCSK9 en su hospital, mientras que el 78% encontraba dificultades moderadas o elevadas. Las dificultades procedían en un 18% de aspectos burocrático-administrativos, en un 41% de restricciones en su indicación y en un 38% de ambas. En general, los obstáculos encontrados no dependían del nivel del hospital, de la especialidad ni de la presencia de unidades homologadas de lípidos, si bien la existencia de esta última se asociaba a un mayor número de pacientes tratados con iPCSK9. Los factores asociados con una mayor dificultad en la prescripción fueron: la presencia de una comisión de aprobación, la frecuencia de revisión del tratamiento activo por parte de la farmacia hospitalaria, la cadencia temporal de la prescripción, el perfil de pacientes atendidos y los criterios seguidos por los especialistas para la prescripción. Conclusión Los resultados demuestran importantes diferencias en el tratamiento con iPCSK9 en el contexto de la práctica clínica habitual en España. El análisis de estos resultados permitirá plantear en el futuro actuaciones orientadas a la equidad del acceso a los iPCSK9 a nivel nacional, con el objetivo principal de maximizar su beneficio potencial de acuerdo con el perfil del paciente.

Failure of second-generation tyrosine kinase inhibitors (2GTKI) is a challenging situation in patients with chronic myeloid leukemia (CML). Asciminib, recently approved by the US Federal Drug Administration, has demonstrated in clinical trials a good efficacy and safety profile after failure of 2GTKI. However, no study has specifically addressed response rates to asciminib in ponatinib pretreated patients (PPT). Here, we present data on responses to asciminib from 52 patients in clinical practice, 20 of them (38%) with prior ponatinib exposure. We analyzed retrospectively responses and toxicities under asciminib and compared results between PPT and non-PPT patients.After a median follow-up of 30 months, 34 patients (65%) switched to asciminib due to intolerance and 18 (35%) due to resistance to prior TKIs. Forty-six patients (88%) had received at least 3 prior TKIs. Regarding responses, complete cytogenetic response was achieved or maintained in 74% and 53% for non-PPT and PPT patients, respectively. Deeper responses such as major molecular response and molecular response 4.5 were achieved in 65% and 19% in non-PPT versus 32% and 11% in PPT, respectively. Two patients (4%) harbored the T315I mutation, both PPT.In terms of toxicities, non-PPT displayed 22% grade 3–4 TEAE versus 20% in PPT. Four patients (20% of PPT) suffered from cross-intolerance with asciminib as they did under ponatinib.Our data supports asciminib as a promising alternative in resistant and intolerant non-PPT patients, as well as in intolerant PPT patients; the resistant PPT subset remains as a challenging group in need of further therapeutic options.

Simple Summary Since the irruption of PARPi in the therapeutic armamentarium for ovarian cancer, concerns regarding post-progression treatment outcomes have emerged, owing to known crossed-resistance mechanisms between PARPi and platinum. In this multicentric retrospective series of ovarian cancer patients, we evaluated chemotherapy results upon progression to maintenance with PARPi in the relapsed setting. We further selected the population of platinum-sensitive patients (according to the classical definition) retreated with platinum (n = 74). In this platinum-sensitive population, overall response rate and survival outcomes of platinum rechallenge after PARPi were similar to historical series of the prePARPi era. However, within this group, analysis according to BRCA status showed that BRCA mutant patients (n = 35) presented higher rates of progression and worse survival outcomes under subsequent platinum than BRCA wild type patients (n = 39), with statistically significant differences. This is the largest real-world data series of ovarian cancer patients treated with platinum rechallenge in the post-PARPi scenario. Background: Despite impressive progression-free survival (PFS) results from PARP inhibitors (PARPi) in ovarian cancer, concerns about their effect on post-progression treatment outcomes have recently arisen, particularly when administered in the relapsed setting. Overlapping mechanisms of resistance between PARPi and platinum have been described, and optimal therapies upon progression to PARPi are unknown. We communicate real-world data (RWD) on outcomes of subsequent chemotherapy upon progression to PARPi used as maintenance in ovarian cancer relapses, particularly focusing on platinum rechallenge, according to BRCA status. Methods: Data from high-grade serous or endometrioid ovarian cancer patients who received subsequent chemotherapy after progression to maintenance PARPi in the relapsed setting, in 16 Catalan hospitals between August 2016 and April 2021, and who were followed-up until July 2021, were included. Endpoints were overall response rate (ORR), and PFS and overall survival (OS) measured from the subsequent chemotherapy starting date. Results: 111 patients were included [46 (41.4%) presented pathological BRCA1/2 mutations, 8 (7.5%) in other homologous recombination-related genes]. Sixty-four patients (57.7%) had received two prior chemotherapy lines, including the one immediately prior to PARPi. PARPi were niraparib (n = 60, 54.1%), olaparib (n = 49, 44.1%), and rucaparib (n = 2, 1.8%). A total of 81 patients remained platinum-sensitive (PS population) after progression to PARPi (when progression-free interval [PFI] was >6 months after the last cycle of prior platinum) [median PFI 12.0 months (interquartile range, IQR, 8.8-17.1)]. Of those, 74 were treated with subsequent platinum regimens, with the following results: ORR of 41.9%, median PFS (mPFS) of 6.6 months (95% CI 6-9.2), and median OS (mOS) of 20.6 months (95% CI 13.6-28.9). Analysis of these 74 patients according to BRCA status showed that PFIs for BRCA mutant and non BRCA-mutant patients were 13.6 [IQR11.2-22.2] and 10.3 [IQR 7.4-14.9] months, respectively (p = 0.010); ORR were 40.0% versus 43.6%, respectively; Rates of progression (as best response) to subsequent platinum were 45.7% versus 17.9%, respectively (p = 0.004); mPFS and mOS were 3.5 (95% CI 2.5-8.6) versus 7.5 months (95% CI 6.5-10.1, p = 0.03), and 16.4 (95% CI 9.3-27.5) versus 24.2 months (95% CI 17.2-NR, p = 0.036), respectively. Conclusion: This is the largest series of real-world data on ovarian cancer patients retreated with platinum in the post-PARPi scenario, separately analyzing BRCA mutant and non-mutant patients, to our knowledge. In our platinum-sensitive population, rechallenge with platinum after progression upon PARPi in the 3rd or later lines for ovarian cancer relapses shows relevant ORR and similar PFS outcomes to historical series of the prePARPi era. However, BRCA mutant patients presented significantly higher rates of progression under subsequent platinum and worse survival outcomes associated with subsequent platinum than non-BRCA-mutant patients.

Treballs Finals de Grau de Química, Facultat de Química, Universitat de Barcelona, Any: 2022, Tutor: Jaime Rubio Martínez, The aim of the following work is to find the computational calculation method capable of quantitatively ranking a list of inhibitors from natural products1 of the main protease of SARS-CoV-2, the protein in charge of reproducing the virus related to the recent COVID-19 pandemic (WHO), according to its binding energy with the protein (receptor). Given the complexity of both the system and the reaction conditions, this simulation is complicated and it is necessary to evaluate it in different ways in order to find the method that most closely matches, thus understanding its behaviour and being able to develop different drugs or remedies against this disease and against other upcoming diseases with similar characteristics. Such inhibitors are present in nature and have been found by different molecular dynamics assays, starting from a database of approximately 2000 compounds providing a total of 11 inhibitors, of which 5 have inhibitory activity in vivo. Finding the calculation method that ranks them can help to expand this number and thus increase the chances of finding the substance with the highest inhibitory capacity. To rank the inhibitors, we will assess how well different methods calculate their binding energy to the receptor (as we will call the protein) relative to the inhibitory activity they have demonstrated in in vivo assays. This binding energy is the difference in energies of the individual receptor and inhibitor and the ligand-receptor complex. The ideal stage of this work would be to obtain energies close to 0 for ligands with no inhibitory activity and a low energy for those with inhibitory activity. Results obtained do not give this exact order but give the first steps to achieve the final method. Previous order1 give 2 true positive compounds and the final method in this work can give 3, a part of obtaining interesting conclusions

The research leading to these results has received funding from AIRC under IG 2019 - ID. 22805 project - P.I., Katia Scotlandi, and Ministry of University and Research under FISR2019_00374 MeDyCa project - P.I. Antonello Mai. The authors would also thank Cristina Ghinelli for graphic support. Altres ajuts: This work was supported by the Italian Ministry of Health (RF-2016-02361373). DNA methylation is an important component of the epigenetic machinery that regulates the malignancy of Ewing sarcoma (EWS), the second most common primary bone tumor in children and adolescents. Coordination of DNA methylation and DNA replication is critical for maintaining epigenetic programming and the DNMT1 enzyme has been demonstrated to have an important role in both maintaining the epigenome and controlling cell cycle. Here, we showed that the novel nonnucleoside DNMT inhibitor (DNMTi) MC3343 induces a specific depletion of DNMT1 and affects EWS tumor proliferation through a mechanism that is independent on DNA methylation. Depletion of DNMT1 causes perturbation of the cell cycle, with an accumulation of cells in the G1 phase, and DNA damage, as revealed by the induction of γH2AX foci. These effects elicited activation of p53-dependent signaling and apoptosis in p53wt cells, while in p53 mutated cells, persistent micronuclei and increased DNA instability was observed. Treatment with MC3343 potentiates the efficacy of DNA damaging agents such as doxorubicin and PARP-inhibitors (PARPi). This effect correlates with increased DNA damage and synergistic tumor cytotoxicity, supporting the use of the DNMTi MC3343 as an adjuvant agent in treating EWS.

Different EGFR tyrosine kinase inhibitors (TKIs) have been developed for the treatment of non-small cell lung cancer (NSCLC) patients harboring sensitizing mutations in the EGFR gene. Apart from acquired secondary mutations, multiple resistance mechanisms have been reported, such as the overexpression of fatty acid synthase (FASN), a multi-functional enzyme essential for the de novo lipogenesis, or the increase of cancer stem cells, a small subpopulation within the tumor responsible for relapse, metastasis, and resistance to therapies. Hence, the purpose of this work is to evaluate the novel FASN inhibitor AZ12756122, both alone and in combination with gefitinib and osimertinib, in EGFR-mutated (EGFRm) lung adenocarcinoma cell models sensitive and resistant to EGFR-TKIs. The molecular effect of AZ12756122 (alone and in combination with EGFR-TKI) on FASN, EGFR/ STAT3, Akt/mTOR, and MAPK signaling pathways was analyzed using RT-qPCR and Western blot. FASN expression was also evaluated in samples from patients with EGFRm NSCLC through immunohistochemistry. Our findings revealed that AZ12756122 caused cytotoxic effects inducing apoptosis, downregulated FASN expression and activity, decreased the activation of EGFR and Akt/mTOR pathway, and reduced cancer stem-like cells. Furthermore, the combination of AZ12756122 and osimertinib sensitized cells to EGFR-TKI, showing a syner- gistic effect that resulted in a reduction in the activation of EGFR, Akt/mTOR, and MAPK signaling pathways. Our study also showed that FASN+ EGFRm NSCLC patients exhibited a longer mPFS in patients who responded to EGFR-TKI treatment. In conclusion, FASN inhibition should be further studied for the treatment, alone or in combination with EGFR-TKIs, for EGFRm NSCLC patients

BackgroundIncreasing evidence indicates that redox stress participates in MFS aortopathy, though its mechanistic contribution is little known. We reported elevated reactive oxygen species (ROS) formation and NADPH oxidase NOX4 upregulation in MFS patients and mouse aortae. Here we address the contribution of xanthine oxidoreductase (XOR), which catabolizes purines into uric acid and ROS in MFS aortopathy.Methods and ResultsIn aortic samples from MFS patients, XOR protein expression, revealed by immunohistochemistry, increased in both the tunicae intima and media of the dilated zone. In MFS mice (Fbn1C1041G/+), aortic XOR mRNA transcripts and enzymatic activity of the oxidase form (XO) were augmented in the aorta of 3-month-old mice but not in older animals. The administration of the XOR inhibitor allopurinol (ALO) halted the progression of aortic root aneurysm in MFS mice. ALO administrated before the onset of the aneurysm prevented its subsequent development. ALO also inhibited MFS-associated endothelial dysfunction as well as elastic fiber fragmentation, fibrotic collagen remodeling, nuclear translocation of pNRF2 and increased 3’-nitrotyrosine levels all occurring in the tunica media. ALO reduced the MFS-associated large aortic production of H2O2, and NOX4 and MMP2 transcriptional overexpression.ConclusionsAllopurinol interferes in aortic aneurysm progression acting as a potent antioxidant. This study strengthens the concept that redox stress is an important determinant of aortic aneurysm formation and progression in MFS and warrants the evaluation of ALO therapy in MFS patients.HIGHLIGHTSXanthine oxidoreductase (XOR) is upregulated in the aortic aneurysm of Marfan syndrome (MFS) both in patients and young mice.Allopurinol halts the formation and progression of aortic aneurysm in MFS miceAllopurinol reduces a variety of oxidative stress-associated molecular reactions.Allopurinol prevents MFS endothelial-dependent vasodilator dysfunction.The antioxidant action of allopurinol suggests its repositioning for pharmacological use in MFS aortopathy.GRAPHICAL ABSTRACT

The complement system has long been recognized as a potential druggable target for a variety of inflammatory conditions. Very few complement inhibitors have been approved for clinical use, but a great number are in clinical development, nearly all of which systemically inhibit complement. There are benefits of targeting complement inhibition to sites of activation/disease in terms of efficacy and safety, and here we describe P-selectin targeted complement inhibitors, with and without a dual function of directly blocking P-selectin-mediated cell-adhesion. The constructs are characterizedin vitroand in murine models of hindlimb ischemia/reperfusion injury and hindlimb transplantation. Both constructs specifically targeted to reperfused hindlimb and provided protection in the hindlimb ischemia/reperfusion injury model. The P-selectin blocking construct was the more efficacious, which correlated with less myeloid cell infiltration, but with similarly reduced levels of complement deposition. The blocking construct also improved tissue perfusion and, unlike the nonblocking construct, inhibited coagulation, raising the possibility of differential application of each construct, such as in thromboticvs. hemorrhagic conditions. Similar outcomes were obtained with the blocking construct following vascularized composite graft transplantation, and treatment also significantly increased graft survival. This is outcome may be particularly pertinent in the context of vascularized composite allograft transplantation, since reduced ischemia reperfusion injury is linked to a less rigorous alloimmune response that may translate to the requirement of a less aggressive immunosuppressive regime for this normally nonlife-threatening procedure. In summary, we describe a new generation of targeted complement inhibitor with multi-functionality that includes targeting to vascular injury, P-selectin blockade, complement inhibition and anti-thrombotic activity. The constructs described also bound to both mouse and human P-selectin which may facilitate potential translation.

[eng] Nucleoside transporters are essential for nucleotide homeostasis as well as for nucleoside-derived drugs bioavailability. Non-canonical natural nucleosides or “epigenetic nucleosides” are endogenously produced and possess pharmacological properties. Their pharmacological action depends on cytidine deaminase activity, which is a typical mechanism of chemoresistance induction for nucleoside-derived drugs. Thus, epigenetic nucleosides show a potential singular status in terms of cancer therapy approach. However, the interplay between these nucleosides and nucleoside transporters which might facilitate their bioavalability remains unknown. In order to unveil that question both interaction and translocation aspects of epigenetic nucleosides towards nucleoside transporters are addressed within this work. In addition to epigenetic nucleosides, the possible interaction of ruxolitinib with nucleoside transporters is also studied in this work. Ruxolitinib is a Janus kinase inhibitor used for the treatment of negative Philadelphia- chromosome chronic myeloproliferative neoplasms. Since it is well known and perfectly established that several kinase inhibitors are recognized by nucleoside transporters, the same question has been raised for ruxolitinib. Additionally, the molecular structure of ruxolitinib contains a heterocycle which is highly similar to hypoxanthine, a key structural determinant for purine nucleoside recognition by nucleoside transporters. That particularity together with the fact that the main cell target of ruxolitinib are hematopoietic precursors which lack de novo nucleotide synthesis and completely depend on nucleoside salvage pathway make ruxolitinib a good candidate in terms of its interaction with nucleoside transporters. As a result of this work, it is shown that epigenetic nucleosides are recognized by nucleoside transporters being even translocated by them in some cases. To that extent, two associations stand out over the rest: 5hmdC with hCNT1 and 5fdC with hENT1. The critical factor which determines molecular recognition turns out to be the chemical environment provided by the functional group at position 5 of the pyrimidine ring of the nucleoside. Unexpectedly, 5fdC turns out to be recognized and even transported by the purine-preferring transporter hCNT2. As to ruxolitinib, its interaction with hENT1 is elucidated. Due to this interaction, ruxolitinib has the capacity to modulate the pharmacological activity of nucleoside-derived drugs such as gemcitabine by inhibiting hENT1 activity.

In this study, we aim to explore the effects on lipids of integrase strand transfer inhibitors (INSTIs) in naïve and switch randomised controlled trials, and compare them with protease inhibitors (PIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). We reviewed phase 3/4 randomised clinical trials in the Cochrane and PubMed databases that compare an INSTI with a boosted PI, an NNRTI, or another INSTI plus one or two nucleoside/nucleotide reverse transcriptase inhibitors (NtRTIs) in naïve patients and switching strategies in HIV-infected patients. We reported the baseline plasma concentration of total cholesterol (TC), low and high-density lipoprotein cholesterol (LDL-c, HDL-c), triglycerides (TG), and the TC/HDL-c ratio, as well as the change at weeks 48 and 96, when available. In naïve HIV-infected patients, raltegravir (RAL) and dolutegravir (DTG) have a more favourable lipid profile compared with NNRTI and boosted PI. Elvitegravir (EVG/c) has a superior lipid profile compared with efavirenz and is similar to that observed with ritonavir-boosted atazanavir except in TG, which increases less with EVG/c. In naïve patients, RAL, DTG, and bictegravir (BIC) produce a similar, slight increase in lipids. In switching trials, the regimen change based on a boosted PI or efavirenz to RAL, DTG, or BIC is associated with clinically significant decreases in lipids that are minor when the change is executed on EVG/c. No changes were observed in lipids by switching trials between INSTIs. In summary, RAL, DTG, and BIC have superior lipid profiles compared with boosted-PI, efavirenz, and EVG/c, in studies conducted in naïve participants, and they are associated with a clinically significant decrease in lipoproteins by switching studies.

Triple-negative breast cancer (TNBC) is characterized by the absence of hormone receptor and HER2 expression, and therefore a lack of therapeutic targets. Anthracyclines and taxane-based neoadjuvant chemotherapy have historically been the cornerstone of treatment of early TNBC. However, genomic and transcriptomic analyses have suggested that TNBCs include various subtypes, characterized by peculiar genomic drivers and potential therapeutic targets. Therefore, several efforts have been made to expand the therapeutic landscape of early TNBC, leading to the introduction of platinum and immunomodulatory agents into the neoadjuvant setting. This review provides a comprehensive overview of the currently available evidence regarding platinum agents and immune-checkpoint-inhibitors for the neoadjuvant treatment of TNBC, as well as the novel target therapies that are currently being evaluated in this setting. Taking into account the economic issues and the side effects of the expanding therapeutic options, we focus on the potential efficacy biomarkers of the emerging therapies, in order to select the best therapeutic strategy for each specific patient.

Succinate dehydrogenase (SDH) inhibition with malonate during reperfusion reduced myocardial infarction in animals, whereas its endogenous substrate, succinate, is detected in plasma from STEMI patients. We investigated whether protection by SDH inhibition is additive to that of remote ischemic perconditioning (RIC) in pigs submitted to transient coronary artery occlusion, and whether protective maneuvers influence plasma levels of citric acid cycle metabolites. Forty pigs were submitted to 40 min coronary occlusion and reperfusion, and allocated to four groups (controls, sodium malonate 10 mmol/L, RIC, and malonate + RIC). Plasma was obtained from femoral and great cardiac veins and analyzed by LC-MS/MS. Malonate, RIC, and malonate + RIC reduced infarct size (24.67 ± 5.98, 25.29 ± 3.92 and 29.83 ± 4.62% vs. 46.47 ± 4.49% in controls, p &lt, 0.05), but no additive effects were detected. Enhanced concentrations of succinate, fumarate, malate and citrate were observed in controls during initial reperfusion in the great cardiac vein, and most were reduced by cardioprotective maneuvers. Concentrations of succinate, fumarate, and malate significantly correlated with infarct size. In conclusion, despite the combination of SDH inhibition during reperfusion and RIC did not result in additive protection, plasma concentrations of selected citric acid cycle metabolites are attenuated by protective maneuvers, correlate with irreversible injury, and might become a prognosis tool in STEMI patients.

Salinity is a major constraint in improving agricultural productivity due to its adverse impact on various physiological and biochemical attributes of plants, and its effect on reducing nitrogen (N) use efficiency due to ion toxicity. To understand the relationship between sodium chloride (NaCl) and increased N application rates, a pot study was performed in which the ammonical (NH4+) form of N was applied as urea to maize crops at different rates (control, 160, 186, 240, 267, 293, and 320 kg N ha−1) using two salinity levels (control and 10 dS m−1 NaCl). The results indicate that all biochemical and physiological attributes of the maize plant improved with increased concentration of N up to 293 kg ha−1, compared to those in the control treatment. Similarly, the optimal N concentration regulated the activities of antioxidant enzymes, i.e., catalase activity (CAT), peroxidase activity (POD), and superoxide dismutases (SOD), and also increased the N use efficiencies of the maize crop up to 293 kg N ha−1. Overall, our results show that the optimum level of N (293 kg ha−1) improved the salinity tolerance in the maize plant by activating stress coping physiological and biochemical mechanisms. This may have been due to the major role of N in the metabolic activity of plants and N assimilation enzymes activity such as nitrate reductase (NR) and nitrite reductase (NiR).

Summary Many metabolic pathways, including lipid metabolism, are rewired in tumors to support energy and biomass production and to allow adaptation to stressful environments. Neuroblastoma is the second deadliest solid tumor in children. Genetic aberrations, as the amplification of the MYCN-oncogene, correlate strongly with disease progression. Yet, there are only a few molecular targets successfully exploited in the clinic. Here we show that inhibition of fatty acid synthesis led to increased neural differentiation and reduced tumor burden in neuroblastoma xenograft experiments independently of MYCN-status. This was accompanied by reduced levels of the MYCN or c-MYC oncoproteins and activation of ERK signaling. Importantly, the expression levels of genes involved in de novo fatty acid synthesis showed prognostic value for neuroblastoma patients. Our findings demonstrate that inhibition of de novo fatty acid synthesis is a promising pharmacological intervention strategy for the treatment of neuroblastoma independently of MYCN-status., Graphical Abstract, Highlights • Fatty acid synthesis inhibition reduces neuroblastoma growth in vitro and in vivo • Decreased availability or reduced lipid synthesis downregulates MYC levels • Impaired fatty acid synthesis induces neural differentiation through ERK activation • High expression of fatty-acid-synthesis-related genes correlates with bad prognosis, Biological sciences; molecular biology; cell biology; cancer

Aromatase inhibitors (AI) are one of the main therapies to treat estrogen-receptor positive breast cancer. AI use is associated with several side effects that affects patient’s quality of life and reduces treatment adherence. Hence, it is necessary to make further efforts in elucidating and diminishing the AI-related side effects. In this line, this thesis provided new and additional evidence for this purpose. Starting by the importance of assessing vitamin D levels during AI treatment, especially to those who underwent to chemotherapy. We also studied the bone health evolution at the end and one-year after AI cessation, and the impact of oral bisphosphonates (BP). Moreover, we analyzed the arthralgia (VAS score) and health-related quality of life in osteoporosis (ECOS-16 score) progression during the AI treatment until one- year post-treatment. Then, fracture incidence and risk during AI therapy compared to tamoxifen (TAM) was analyzed, as well as the protective effect of BP. Finally, we studied the cardiovascular and thromboembolic risk, and overall survival benefit of AI compared to TAM. Our research leads us to state that bone health and circulant vitamin D levels monitoring, plus calcium and vitamin D supplementation is key for the clinical management of AI patients. BP treatment is proved to diminish bone loss and fracture risk, but cannot reverse risk levels towards patients at low fracture risk. Furthermore, prior TAM treatment enhances the odds to withdraw during the first year, increases bone loss during AI treatment, and restricts the recovery in lumbar spine location at one-year post-treatment. On the other hand, since there are no differences in cardiovascular and thromboembolic risk between AI and TAM users, but AI users have lower all-cause mortality, AI should be the preferable choice. In summary, it is mandatory to clinical monitoring AI patients, especially those who were previously treated with TAM, including fracture risk and related risk factors assessments. These would reduce early cessation of treatment and improve patients’ quality of life.

Neuroactive substances are the largest group of chemicals detected in European surface waters. Mixtures of neuroactive substances occurring at low concentrations can induce adverse neurological effects in humans and organisms in the environment. Therefore, there is a need to develop new screening tools to detect these chemicals. Measurement of behavior or motor effects in rodents and fish are usually performed to assess potential neurotoxicity for risk assessment. However, due to pain and stress inflicted on these animals, the scientific community is advocating for new alternative methods based on the 3R principle (reduce, replace and refine). As a result, the behavior measurement of early stages of zebrafish embryos such as locomotor response, photomotor response and spontaneous tail coiling are considered as a valid alternative to adult animal testing. In this study, we developed a workflow to investigate the spontaneous tail coiling (STC) of zebrafish embryos and to accurately measure the STC effect in the KNIME software. We validated the STC protocol with 3 substances (abamectin, chlorpyrifos-oxon and pyracostrobin) which have different mechanisms of action. The KNIME workflow combined with easy and cost-effective method of video acquisition makes this STC protocol a valuable method for neurotoxicity testing.•Video acquisition duration of 60 s at 25 ± 1 hpf was used•20 embryos exposed per dish and acclimatized for 30 min before video acquisition•Capability to inspect and correct errors for high accuracy, Graphical abstract Image, graphical abstract

Neuroactive chemicals are frequently detected in the environment. At sufficiently high concentrations or within mixtures, they could provoke neurotoxic effects and neurological diseases to organisms and humans. Fast identification of such neuroactive compounds in the environment could help in hazard assessment and risk mitigation. Behavior change is considered as an important endpoint and might be directly or indirectly connected to a neuroactive mode of action. For a fast evaluation of environmental samples and pure substances, we optimized the measurement of a behavioral endpoint in zebrafish embryos - the spontaneous tail coiling (STC). Evaluation of results is automated via the use of a workflow established with the KNIME (R) software. Analysis duration and developmental stage were optimized to 1 min and 25 +/- 1 hpf respectively during measurement. Exposing the embryos in a group of 10 or 20 and acclimatizing for 30 min at room temperature proved to be reliable. The optimized method was used to investigate neurotoxic effects of 18 substances with different modes of action (MoA). The STC WA accurately detected the effect of 8 out of 11 neuroactive substances (chlorpyrifos, chlorpyrifos-oxon, diazinon, paraoxon-methyl, abamectin, carbamazepine, propafenone and diazepam). Aldicarb and nicotine showed subtle effects which were considered to be conditional and imidacloprid showed no effect. For substances with unknown neuroactive MoA, 3 substances did not provoke any effect on the STC (pyraclostrobin, diuron and daunorubicin-hydrochloride) while 4 other substances provoked an increased STC (hexaconazole, aniline, dimethyl-sulfoxide and 3,4-dichloroaniline). Such unexpected effects indicate possible neuroactive side effects or unknown mechanisms of action that impact on the STC. In conclusion, the optimized STC parameters and the automated analysis in KNIME (R) indicate opportunities for the harmonization of the STC WA and further development for prospective and diagnostic testing.

[eng] Alzheimer’s disease (AD) is the most common form of dementia and one of the most important health-care problems in the world, due to its high prevalence and unaffordable personal and economic impact. Moreover, current commercialised treatments are only symptomatic, but are not capable of preventing, curing or even delaying the disease progression. Because AD arises from a complex network of pathological events, such as dysfunction in neurotransmitter systems (mainly cholinergic and glutamatergic), β-amyloid and tau proteins disorders, oxidative stress or neuroinflammation, amongst others, the traditional medicinal chemistry paradigm of “one molecule-one target” is increasingly regarded as clearly ineffective. On the contrary, it becomes evident that a more comprehensive, complex pharmacological approach is needed to tackle AD. As a consequence, the use of multitarget directed ligands, where one single molecule is able to interact simultaneously with multiple targets of the pathological network, is emerging as a promising and more realistic way to confront this disease. In this context, the purpose of the present Thesis was the design, synthesis and biological evaluation of three novel families of compounds, endowed with multitarget biological profile, in order to find novel treatments for AD: 1) firstly, a new series of compounds designed by substitution of the rhein subunit of a rhein–huprine hybrid lead, previously developed in our group, by more simplified scaffolds, with the aim of finding optimized hybrids with reduced lipophilicity and better drug-like properties, while maintaining favourable activities against cholinesterases, BACE1, β- amyloid and tau aggregation, and antioxidant properties; 2) secondly, a novel family of huprine- derived hybrids, designed to perform a dual binding site interaction within BACE1 through the linkage of a huprine moiety to new scaffolds, selected by their predicted binding affinities towards a secondary transient pocket in BACE1, which were expected to combine cholinesterases and BACE1 inhibitory activities, as well as activity against β-amyloid and tau aggregation, and antioxidant properties; 3) finally, a family of huprine–TPPU and tacrine–TPPU hybrids, which were designed to be dual inhibitors of acetylcholinesterase (AChE) and soluble epoxide hydrolase (sEH). The blood–brain barrier permeability was also assessed for all these compounds, as it is a crucial factor for drugs acting in the central nervous system, while other important physicochemical and pharmacokinetic parameters, such as solubility and microsomal stability were determined for the latter series of compounds. Also, the toxicity of some compounds was evaluated. Finally, using the same assay that was employed for the determination of the β-amyloid and tau antiaggregating activity of the first two families and other compounds synthesised by our group, we demonstrated that a single compound can be able of inhibiting the aggregation of different types of amyloid-prone proteins, with these results supporting the notion that common mechanisms exist for the aggregation of different amyloidogenic proteins and that a generic treatment of conformational diseases is possible.

The DKK1 gene encodes an extracellular inhibitor of the Wnt pathway with an important role in bone tissue development, bone homeostasis, and different critical aspects of bone biology. Several BMD genome-wide association studies (GWASs) have consistently found association with SNPs in the DKK1 genomic region. For these reasons, it is important to assess the functionality of coding and regulatory variants in the gene. Here, we have studied the functionality of putative regulatory variants, previously found associated with BMD in different studies by others and ourselves, and also six missense variants present in the general population. Using a Wnt-pathway-specific luciferase reporter assay, we have determined that the variants p.Ala41Thr, p.Tyr74Phe, p.Arg120Leu, and p.Ser157Ile display a reduced DKK1 inhibitory capacity as compared with WT. This result agrees with the high-bone-mass (HBM) phenotype of two women from our cohort who carried mutations p.Tyr74Phe or p.Arg120Leu. On the other hand, by means of a circularized chromosome conformation capture- (4C-) sequencing experiment, we have detected that the region containing 24 BMD-GWA variants, located 350-kb downstream of DKK1, interacts both with DKK1 and the LNCAROD (LncRNA-activating regulator of DKK1, AKA LINC0148) in osteoblastic cells. In conclusion, we have shown that some rare coding variants are partial loss-of-function mutations that may lead to a HBM phenotype, whereas the common SNPs associated with BMD in GWASs belong to a putative long-range regulatory region, through a yet unknown mechanism involving LNCAROD. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research. Funds for the study included grants SAF2014‐56562‐R and SAF2016‐75948‐R (Spanish MINECO), and CIBERER (U720). NMG is a recipient of a FI predoctoral fellowship from AGAUR (Generalitat de Catalunya); NRA is a recipient of a FPU predoctoral fellowship from the Spanish Ministerio de Educación Cultura y Deporte. EB is a recipient of a postdoctoral fellowship (12A3814N) from the Research Foundation‐Flanders. We thank O. Monclús and M. Cozar for relevant technical assistance. Authorsʼ roles: NMG, WVH, DG, SB were involved in the study conception and design. Material preparation and data collection were performed by NMG, NRA, NA, NGG, EB, DO, LM, XN, and ADP. The analyses were performed by NMG. The statistical analyses were performed by MPM. The first draft of the manuscript was written by NMG, DG, SB, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

miR-155-5p is a well-known oncogenic microRNA, showing frequent overexpression in human malignancies, including breast cancer. Here, we show that high miR-155-5p levels are associated with unfavorable prognostic factors in two independent breast cancer cohorts (CSS cohort, n = 283; and TCGA-BRCA dataset, n = 1,095). Consistently, miR-155-5p results as differentially expressed in the breast cancer subgroups identified by the surrogate molecular classification in the CSS cohort and the PAM50 classifier in TCGA-BRCA dataset, with the TNBC and HER2-amplified tumors carrying the highest levels. Since the analysis of TCGA-BC dataset also demonstrated a significant association between miR-155-5p levels and the presence of mutations in homologous recombination (HR) genes, we hypothesized that miR-155-5p might affect cell response to the PARP-1 inhibitor Olaparib. As expected, miR-155-5p ectopic overexpression followed by Olaparib administration resulted in a greater reduction of cell viability as compared to Olaparib administration alone, suggesting that miR-155-5p might induce a synthetic lethal effect in cancer cells when coupled with PARP-1-inhibition. Overall, our data point to a role of miR-155-5p in homologous recombination deficiency and suggest miR-155-5p might be useful in predicting response to PARP1 inhibitors in the clinical setting.

Background: There is a lack of head-to-head studies comparing the efficacy of fingolimod (FIN) and natalizumab (NTZ) as second-line therapy for relapsing-remitting multiple sclerosis (RRMS). Methods: Multicenter, observational study, in which, information of 388 patients randomly selected and treated with FIN or NTZ in routine clinical practice was retrospectively collected with the main objective of comparing the annualized relapse rate (ARR) over the first year, after FIN or NTZ treatment initiation. Results: Mean ARR during the first year of treatment was 0.28 in FIN group and 0.12 in NTZ group (p = 0.0064); nevertheless, the difference between groups lost statistical significance when the propensity score analysis was performed. Time to disability ­progression was similar in both treatment groups (12.3 ± 6.7 months in FIN, and 12.8 ± 0.1 months in NTZ; p = 0.4654). Treatment persistence after the first year of treatment was higher in patients treated with FIN (95%) than in those treated with NTZ (84%; p = 0.0014). Conclusions: After 12 months of treatment, both FIN and NTZ reduced the ARR, but ARR percent reduction was significantly higher with NTZ. Treatment persistence was higher in patients receiving FIN.

[eng] Donepezil (DPZ) is one of the most widely prescribed drugs to treat the neuropsychiatric symptoms of Alzheimer's disease (AD) which are commercially available in oral tablet form. However, these can lead to inconveniences, especially among geriatric patients or those who are in advanced stages of the disease. In addition to this, there are marked disadvantages of the oral route, including difficulties in crossing the blood-brain barrier. The intranasal route presents an alternative due to its direct connection to the brain, which could increase the bioavailability of the drug. On the other hand, pioglitazone (PGZ) is a peroxisome proliferator-activated receptor γ (PPAR-γ) agonist used to treat type 2 diabetes mellitus. There is mounting scientific evidence of the anti-inflammatory effect of PGZ which suggests it as a promising candidate for the treatment of inflammatory disorders. The incorporation of these drugs in micro- and nanoemulsions could be used as a strategy to facilitate their administration. Based on these findings, the aim of this thesis was to design, develop and characterize intranasal DPZ micro- and nanoemulsions for the treatment of AD, as well as topical PGZ nanoemulsions for the treatment of inflammatory skin diseases. Two PGZ nanoemulsions (PGZ-NE and PGZ-NE2) along with one microemulsion and two nanoemulsions of DPZ (DPZ-ME, DPZ-NE, and DPZ-PNE) were formulated by constructing pseudo-ternary phase diagrams. All formulations were physically stable with spherical nanodroplets distributed uniformly in the system. The rheological analysis confirmed the Newtonian behavior in four formulations, while DPZ-PNE presented a pseudoplastic behavior. The five formulations were able to release the drug following a hyperbolic kinetic model. DPZ exhibited greater permeation through the porcine nasal mucosa from microemulsion compared to nanoemulsions, possibly due to its higher content of surfactants and cosurfactants. PGZ-NE and PGZ-NE2 favored the passage of the drug through the stratum corneum and promoted its retention in the skin thereby guaranteeing a local effect, especially for PGZ-NE2. The tolerance results demonstrated the biocompatibility and suitability of the formulations for intranasal or dermal administration. Finally, PGZ-NE treatment inhibited approximately 44% of inflammation, significantly decreased the expression of proinflammatory cytokines IL-6, IL-1β, and TNF-α, and counteracted histopathological alterations in acute inflammation assays, while PGZ-NE2 demonstrated efficacy in the treatment of atopic dermatitis, exhibiting reduction of skin lesions, restoration of elasticity and biomechanical properties of the skin, as well as reduction in the expression of proinflammatory cytokines associated with the pathophysiology of the disease. In conclusion, the obtained results encourage further clinical research into new therapeutic indications for PGZ as well as the use of intranasal-administered DPZ micro- and nanoemulsions to increase the bioavailability of the drug in the brain and facilitate its administration.

[cat] Aquesta tesi doctoral està enfocada a la investigació de nous agents amb potencial activitat antitumoral per inhibició enzimàtica. S’ha descrit la implicació de la G6PDH en el desenvolupament i progressió del càncer degut a la seva participació en la síntesi d’àcids nucleics. Es busca la preparació de compostos que actuïn inhibint de forma més selectiva les cèl·lules canceroses, actuant sobre els enzims implicats en el seu metabolisme, com és la G6PDH, per tal de millorar l’eficàcia i reduir els efectes adversos dels tractaments dels que disposem actualment. S’ha dut a terme la preparació sintètica de tres series de compostos diferents. La primera pertany a les arilmetilenindolinona i derivats, la segona consta de sistemes tiofen-condensats, dissenyats a partir de modelització i amb canvis estructurals a partir de farmacomodulacions modulatives i finalment la tercera sèrie, la corresponent a les urees. Els compostos s’han preparat mitjançant reaccions clàssiques de química orgànica i s’han purificat posteriorment mitjançant tècniques de separació (cromatografia). L’elucidació estructural corresponent s’ha dut a terme mitjançant tècniques de Ressonància Magnètica Nuclear (RMN), Espectroscòpia de masses (EM) i d’Infraroig (IR). Finalment, s’ha dut a terme part de l’avaluació biològica d’alguns dels compostos preparats, utilitzant assajos in vitro de viabilitat cel·lular en diferents línies cel·lulars de leucèmia (TPH-1, MOLT3, NB4, HL-60, K562, KU812 i OCIAML-3) i de càncer de pròstata (PC-3) mitjançant la tècnica MTT. Per tal d’estudiar l’activitat inhibitòria enzimàtica, s’han realitzat assajos d’inhibició directa de la G6PDH, mesurada a partir de la producció d’NADPH., [eng] This PhD has been focused on the discovery of new compounds with antitumoral potential activity for inhibition of enzymes, like G6PDH. The implications of G6PDH for the development and progression of cancer due to its participation in nucleic acid synthesis have been described. Our objective was to prepare compounds that can be able to inhibit enzymes such as G6PDH that involved in metabolism of cancer cells with a good level of selectivity which have a perfect efficacy without significant side effects. Synthetic preparation of three different compounds has been carried out. The first one has arilmetilenindolinone and its derivatives. The second one contains thiophen systems and designed by computational modulator and changed structure from pharmacomodulations and finally, the third series of compounds is based on the urea-derivatives. Compounds have been prepared using classical organic chemistry reactions and further purification with separation techniques (chromatography). Corresponding structural elucidation by NMR, Mass and IR. Finally, part of the biological evaluation of some of the compounds have been prepared and carried out using in vitro assays based on cellular viability on different prostate (PC-3) and leukemic (TPH-1, MOLT3, NB4, HL-60, K562, KU812 and OCIAML-3) cell lines using the MTT assay. The direct inhibition of G6PDH measured by the production quantity of NADPH was used to study enzymatic inhibition activity.

Mitochondrial metabolism and the generation of reactive oxygen species (ROS) contribute to the acquisition of DNA mutations and genomic instability in cancer. How genomic instability influences the metabolic capacity of cancer cells is nevertheless poorly understood. Here, we show that homologous recombination‐defective (HRD) cancers rely on oxidative metabolism to supply NAD + and ATP for poly(ADP‐ribose) polymerase (PARP)‐dependent DNA repair mechanisms. Studies in breast and ovarian cancer HRD models depict a metabolic shift that includes enhanced expression of the oxidative phosphorylation (OXPHOS) pathway and its key components and a decline in the glycolytic Warburg phenotype. Hence, HRD cells are more sensitive to metformin and NAD + concentration changes. On the other hand, shifting from an OXPHOS to a highly glycolytic metabolism interferes with the sensitivity to PARP inhibitors (PARPi) in these HRD cells. This feature is associated with a weak response to PARP inhibition in patient‐derived xenografts, emerging as a new mechanism to determine PARPi sensitivity. This study shows a mechanistic link between two major cancer hallmarks, which in turn suggests novel possibilities for specifically treating HRD cancers with OXPHOS inhibitors., Homologous recombination‐defective (HRD) cancers need high levels of NAD + and ATP for alternative PARP‐dependent DNA repair. HRD cancer cells undergo a characteristic metabolic shift that include enhanced OXPHOS, opening new opportunities for treatment with OXPHOS inhibitors like metformin.

Epidermal growth factor receptor (EGFR) tyrosine kinases inhibitors (TKIs) are effective therapies for non-small cell lung cancer (NSCLC) patients whose tumors harbor an EGFR activating mutation. However, this treatment is not curative due to primary and secondary resistance such as T790M mutation in exon 20. Recently, activation of transducer and activator of transcription 3 (STAT3) in NSCLC appeared as an alternative resistance mechanism allowing cancer cells to elude the EGFR signaling. Overexpression of fatty acid synthase (FASN), a multifunctional enzyme essential for endogenous lipogenesis, has been related to resistance and the regulation of the EGFR/Jak2/STAT signaling pathways. Using EGFR mutated (EGFRm) NSCLC sensitive and EGFR TKIs&rsquo, resistant models (Gefitinib Resistant, GR) we studied the role of the natural polyphenolic anti-FASN compound (&minus, )-epigallocatechin-3-gallate (EGCG), and its derivative G28 to overcome EGFR TKIs&rsquo, resistance. We show that G28&rsquo, s cytotoxicity is independent of TKIs&rsquo, resistance mechanisms displaying synergistic effects in combination with gefitinib and osimertinib in the resistant T790M negative (T790M&minus, ) model and showing a reduction of activated EGFR and STAT3 in T790M positive (T790M+) models. Our results provide the bases for further investigation of G28 in combination with TKIs to overcome the EGFR TKI resistance in NSCLC.

Hydrophilic matrix tablets are a type of sustained release dosage form characterized by distributing a drug in a matrix that is usually polymeric. Tolcapone is a drug that inhibits the enzyme catechol-O-methyl transferase. In recent years, it has been shown that tolcapone is a potent inhibitor of the amyloid aggregation process of the transthyretin protein, and acts by stabilizing the structure of the protein, reducing the progression of familial amyloid polyneuropathy. The main objective of this study was to obtain a sustained release tablet of tolcapone for oral administration with a preferred dosage regimen of 1 administration every 12 or 24 h and manufactured, preferably, by direct compression. The SeDeM Diagram method has been used for the formulation development of hydrophilic matrix tablets. Given the characteristics of tolcapone, the excipient selected for the formation of the polymeric matrix was a high viscosity hydroxypropylmethylcellulose (Methocel&reg, K100M CR). A decrease in the particle size of tolcapone resulted in a slower dissolution release of the formulation when the concentration of the polymer Methocel&reg, K100M CR was below 29%. These surprising and novel results have given rise to patent number WO/2018/019997.

Thanks to recent guidelines, the design of safe and effective covalent drugs has gained significant interest. Other than targeting non‐conserved nucleophilic residues, optimizing the noncovalent binding framework is important to improve potency and selectivity of covalent binders toward the desired target. Significant efforts have been made in extending the computational toolkits to include a covalent mechanism of protein targeting, like in the development of covalent docking methods for binding mode prediction. To highlight the value of the noncovalent complex in the covalent binding process, here we describe a new protocol using tethered and constrained docking in combination with Dynamic Undocking (DUck) as a tool to privilege strong protein binders for the identification of novel covalent inhibitors. At the end of the protocol, dedicated covalent docking methods were used to rank and select the virtual hits based on the predicted binding mode. By validating the method on JAK3 and KRas, we demonstrate how this fast iterative protocol can be applied to explore a wide chemical space and identify potent targeted covalent inhibitors.

Acquired resistance to trastuzumab is a major clinical problem in the treatment of HER2-positive (HER2+) breast cancer patients. The selection of trastuzumab-resistant patients is a great challenge of precision oncology. The aim of this study was to identify novel epigenetic biomarkers associated to trastuzumab resistance in HER2+ BC patients. Methods: We performed a genome-wide DNA methylation (450K array) and a transcriptomic analysis (RNA-Seq) comparing trastuzumab-sensitive (SK) and trastuzumab-resistant (SKTR) HER2+ human breast cancer cell models. The methylation and expression levels of candidate genes were validated by bisulfite pyrosequencing and qRT-PCR, respectively. Functional assays were conducted in the SK and SKTR models by gene silencing and overexpression. Methylation analysis in 24 HER2+ human BC samples with complete response or non-response to trastuzumab-based treatment was conducted by bisulfite pyrosequencing. Results: Epigenomic and transcriptomic analysis revealed the consistent hypermethylation and downregulation of TGFBI, CXCL2, and SLC38A1 genes in association with trastuzumab resistance. The DNA methylation and expression levels of these genes were validated in both sensitive and resistant models analyzed. Of the genes, TGFBI presented the highest hypermethylation-associated silencing both at the transcriptional and protein level. Ectopic expression of TGFBI in the SKTR model suggest an increased sensitivity to trastuzumab treatment. In primary tumors, TGFBI hypermethylation was significantly associated with trastuzumab resistance in HER2+ breast cancer patients. Conclusions: Our results suggest for the first time an association between the epigenetic silencing of TGFBI by DNA methylation and trastuzumab resistance in HER2+ cell models. These results provide the basis for further clinical studies to validate the hypermethylation of TGFBI promoter as a biomarker of trastuzumab resistance in HER2+ breast cancer patients This work was supported in part by the Spanish Instituto de Salud Carlos III (ISCIII; FIS PI11/00692 and PI14/00329; to’ T. Puig), Fundacion Ramon Areces (to T. Puig), the support of Catalonian Government (2017SGR00385), and La Marató de TV3 (20131530, TPuig), financial support was from the University of Girona (MPCUdG2016/036), and the University of Girona and La Caixa Foundation awarded S. Palomeras with a predoctoral grant. A.D.-L. is funded by a “Juan Rodés” contract (JR17/00016) from ISCIII. Ana B Crujeiras is funded by a research contract “Miguel Servet” (CP17/00088) from the ISCIII

Background The effect of neuraminidase inhibitor (NAI) treatment on length of stay (LoS) in patients hospitalized with influenza is unclear. Methods We conducted a one-stage individual participant data (IPD) meta-analysis exploring the association between NAI treatment and LoS in patients hospitalized with 2009 influenza A(H1N1) virus (A[H1N1]pdm09) infection. Using mixed-effects negative binomial regression and adjusting for the propensity to receive NAI, antibiotic, and corticosteroid treatment, we calculated incidence rate ratios (IRRs) and 95% confidence intervals (CIs). Patients with a LoS of, We found that neuraminidase inhibitor (NAI) treatment initiated on hospital admission to patients with clinically diagnosed or laboratory-confirmed A(H1N1)pdm09 virus infection was associated with a reduction in hospital length of stay when compared to later or no NAI treatment.

Multiple sclerosis; SERPINA3 Esclerosi múltiple; SERPINA3 Esclerosis múltiple; SERPINA3 Objective To identify biomarkers associated with progressive phases of MS and with neuroprotective potential. Methods Combined analysis of the transcriptional and proteomic profiles obtained in CNS tissue during chronic progressive phases of experimental autoimmune encephalomyelitis (EAE) with the transcriptional profile obtained during the differentiation of murine neural stem cells into neurons. Candidate biomarkers were measured by ELISA in the CSF of 65 patients with MS (29 with relapsing-remitting MS [RRMS], 20 with secondary progressive MS, and 16 with primary progressive MS [PPMS]) and 30 noninflammatory neurologic controls (NINCs). Results Integrative analysis of gene and protein expression data identified 2 biomarkers, the serine protease inhibitor Serpina3n and the calcium-binding protein S100A4, which were upregulated in chronic progressive EAE and whose expression was induced during neuronal differentiation. Immunofluorescence studies revealed a primarily neuronal expression of S100A4 and Serpina3n during EAE. CSF levels of SERPINA3, the human ortholog of murine Serpina3n, and S100A4 were increased in patients with MS compared with NINCs (SERPINA3: 1,320 vs 838.6 ng/mL, p = 0.0001; S100A4: 1.6 vs 0.8 ng/mL, p = 0.02). Within the MS group, CSF SERPINA3 levels were significantly elevated in patients with progressive forms, mainly patients with PPMS compared with patients with RRMS (1,617 vs 1,129 ng/mL, p = 0.02) and NINCs (1,617 vs 838.6 ng/mL, p = 0.0001). Of interest, CSF SERPINA3 levels significantly correlated with CSF neurofilament light chain levels only in the PPMS group (r = 0.62, p = 0.01). Conclusion These results point to a role of SERPINA3 as a biomarker associated with the progressive forms of MS, particularly PPMS. This work was supported by grants from the Fondo de Investigación Sanitaria (FIS; grant number PI17/00596), Ministry of Science and Innovation, Spain; Generalitat de Catalunya Suport Grups de Recerca (2017 SGR 0527); and the Red Española de Esclerosis Múltiple (RD16/0015/0004) funded by the FIS.

Cancer origin and development is associated not only with genetic alterations, but also with the disturbance of epigenetic profiles.1 In this regard, the tumoral epigenome is characterized by both specific and general shifts in the DNA methylation and histone-modification landscapes.1 However, in contrast to genetic disruption, the effect of epigenetic modifications or marks may potentially be reversed by the use of drugs that target enzymes involved in adding, removing or signaling DNA methylation and histone modifications.1 This basic knowledge has been adopted into clinical practice, and inhibitors of histone deacetylases and DNA demethylating agents have been approved for use in the therapy of hematologic malignancies, such as cutaneous T-cell lymphoma and myelodysplastic syndrome, respectively.2 Other promising epigenetic drugs include inhibitors of histone methyltransferases,2 histone demethylases,2 histone kinases,3 and bromodomain proteins that interfere with the 'reading' of acetylated histone residues.

The cyclin D/cyclin-dependent kinases 4 and 6 (CDK4/6)–retinoblastoma protein (RB) pathway plays a key role in the proliferation of both normal breast epithelium and breast cancer cells. A strong rationale for inhibiting CDK4/6 in breast cancers has been present for many years. However, potent and selective CDK4/6 inhibitors have only recently become available. These agents prevent phosphorylation of the RB tumor suppressor, thereby invoking cancer cell cycle arrest in G1. CDK4/6 inhibitors have transited rapidly from preclinical studies to the clinical arena, and three have already been approved for the treatment of advanced, estrogen receptor (ER)-positive breast cancer patients on account of striking clinical trial results demonstrating substantial improvements in progression-free survival. ER-positive breast cancers harbor several molecular features that would predict their sensitivity to CDK4/6 inhibitors. As physicians gain experience with using these agents in the clinic, new questions arise: are CDK4/6 inhibitors likely to be useful for patients with other subtypes of breast cancer? Are there other agents that could be effectively combined with CDK4/6 inhibitors, beyond endocrine therapy? Is there a rationale for combining CDK4/6 inhibitors with novel immune-based therapies? In this review, we describe not only the clinical data available to date, but also the biology of the CDK4/6 pathway and discuss answers to these questions. In particular, we highlight that CDK4 and CDK6 govern much more than the cancer cell cycle, and that their optimal use in the clinic depends on a deeper understanding of the less well characterized effects of these enzymes.

Alpha-synuclein (α-SYN) is the main component of anomalous protein aggregates (Lewy bodies) that play a crucial role in several neurodegenerative diseases (synucleinopathies) like Parkinson’s disease and multiple system atrophy. However, the mechanisms involved in its transcriptional regulation are poorly understood. We investigated here the role of the cyclin-dependent kinase (Cdk) inhibitor and transcriptional regulator p27Kip1 (p27) in the regulation of α-SYN expression. We observed that selective deletion of p27 by CRISPR/Cas9 technology in neural cells resulted in increased levels of α-SYN. Knock-down of the member of the same family p21Cip1 (p21) also led to increased α-SYN levels, indicating that p27 and p21 collaborate in the repression of α-SYN transcription. We demonstrated that this repression is mediated by the transcription factor E2F4 and the member of the retinoblastoma protein family p130 and that it is dependent of Cdk activity. Chromatin immunoprecipitation analysis revealed specific binding sites for p27, p21 and E2F4 in the proximal α-SYN gene promoter. Finally, luciferase assays revealed a direct action of p27, p21 and E2F4 in α-SYN gene expression. Our findings reveal for the first time a negative regulatory mechanism of α-SYN expression, suggesting a putative role for cell cycle regulators in the etiology of synucleinopathies., This work was supported by grants from Fundación Tatiana Pérez de Guzman el Bueno, from Ministerio de Economia y competitividad (MINECO) SAF2015- 64244-R, La Caixa2016-052.407 and from the Instituto de Salud Carlos III CB16/12/00244. AB is supported by grants from the Fondation ARC pour la Reserche sur le Cancer, Ligue National contre le Cancer and Institut National du Cancer. Work in the laboratory of IF is supported by Fundación Botín-Banco Santander.

[cat] ANTECEDENTS: els inhibidors d’histona deacetilasa (HDACi) han emergit els últims anys com potencials tractaments dirigits, amb la finalitat de revertir els canvis epigenètics associats a diferents tipus de càncer, incloent les neoplàsies hematològiques. La falta d’especificitat dels HDACi, però, dificulta la predicció dels seus efectes biològics i provoca una toxicitat no menyspreable, el que ha limitat el seu ús en la pràctica clínica. L’expressió de les HDACs no ha estat estudiada en les leucèmies agudes pediàtriques més que de manera parcial i en sèries amb un limitat número de pacients; per tant, són necessaris estudis que ajudin a aclarir el paper de cadascuna de les HDACs com a possibles biomarcadors i com a dianes terapèutiques, de cara a dirigir el tractament específic i personalitzat amb HDACi. HIPÒTESI: en aquest context, l’estudi global del perfil transcripcional de les HDACs en una sèrie àmplia i representativa de pacients pediàtrics amb diferents subtipus de leucèmia aguda permetria definir millor el seu impacte pronòstic i definir el seu rol com a dianes terapèutiques, ajudant a dirigir el tractament específic amb HDACi de forma més racional i individualitzada. OBJECTIUS: estudiar de forma global el perfil d’expressió de les HDAC1-11, SIRT1, SIRT7 i d’altres gens co-reguladors, MEF2C i MEF2D, en una sèrie de pacients pediàtrics amb leucèmia aguda diagnosticats i tractats en un sol centre. METODOLOGIA: anàlisi de l’expressió de l’mRNA de les HDAC1-11, SIRT1, SIRT7, MEF2C i MEF2D mitjançant PCR quantitativa en 211 pacients pediàtrics (0-18 anys), diagnosticats de leucèmia de novo des de l’any 2003 al 2017 en el nostre centre. Els pacients es van tractar uniformement d’acord als protocols consecutius de la Sociedad Española de Hematología y Oncología Pediátrica (SEHOP). Es va emprar un pool de pacients no-neoplàsics com a calibrador i també es va analitzar l’expressió dels diferents gens en cèl·lules CD34+ normals de moll d’os i a cèl·lules B CD19+ madures i limfòcits T madurs de sang perifèrica. RESULTATS: l’expressió de les HDACs va diferir en els diferents subtipus de cèl·lules hematopoètiques normals d’acord amb el grau de maduració i el llinatge. En els pacients amb leucèmia es va observar, en general, una sobreexpressió de les HDACs, amb perfils específics que correlacionaven amb característiques clíniques i biològiques i, fins i tot en algunes ocasions, amb la supervivència dels pacients. Així, alguns perfils d’expressió d’HDAC i el perfil de MEF2C semblaven reflectir, probablement, el llinatge i la maduresa dels blasts, mentre que d’altres identificaven la via oncogènica activa en les cèl·lules leucèmiques. Concretament, es va identificar un perfil d’expressió distintiu pels pacients amb reordenament del gen KMT2A (MLL), amb nivells elevats d’HDAC9 i MEF2D, independentment de l’edat, el partner del gen de fusió i el llinatge. A més, es va observar en els pacients amb LLA-B un pronòstic advers de la sobreexpressió de l’HDAC9, independentment de l’estat del gen KMT2A. CONCLUSIONS: en aquest treball s’han observat diferents perfils d’expressió segons els diferents subtipus de leucèmia aguda pediàtrica. Concretament, s’ha identificat un perfil distintiu dels pacients amb reordenament del gen KMT2A, amb la sobreexpressió de l’ HDAC9 i MEF2D, independentment del llinatge, de l’edat i del partner del gen de fusió. Aquests resultats aporten una imatge global de l’expressió de les HDACs en els pacients pediàtrics amb leucèmia aguda i recolzen el tractament dirigit amb HDACi més específics en seleccionats grups de pacients d’alt risc, com els pacients amb reordenament del gen KMT2A., [eng] Histone deacetylase inhibitors (HDACi) emerged as promising drugs in leukaemia, but their toxicity due to lack of specificity limited their use. Therefore, there is a need to elucidate the role of HDACs in specific settings. The study of HDAC expression in childhood leukaemia could help to choose more specific HDACi for selected candidates in a personalized approach. We analysed HDAC1-11, SIRT1, SIRT7, MEF2C and MEF2D mRNA expression in 211 paediatric patients diagnosed with acute leukaemia. We found a global overexpression of HDACs, while specific HDACs correlated with clinical and biological features, and some even predicted outcome. Thus, some HDAC and MEF2C profiles probably reflected the lineage and the maturation of the blasts and some profiles pointed out specific oncogenic pathways active in the leukaemic cells. Specifically, we identified a distinctive signature for patients with MLL rearrangement, with high HDAC9 and MEF2D expression, regardless of age, MLL-partner and lineage. Moreover, we observed an adverse prognostic value of overexpression of HDAC9, regardless of MLL rearrangement. Our results provide useful knowledge on the complex picture of HDACs expression in childhood leukaemia and support the directed use of specific HDACi to selected paediatric patients with acute leukaemia.

Treballs Finals de Grau de Química, Facultat de Química, Universitat de Barcelona, Any: 2018, Tutor: Xavier Ariza Piquer, Obesity has become a priority health issue for many governments and organisations such as the WHO, it has gained the status of epidemic and according to the past years trends, the forecasts are not hopeful. The research for anti-obesity drugs has grown in the past years as the percentage of obese people grows globally. A relatively new anti-cancer drug (C75) was tested as anti-cancer drug to rodents, and it was observed that the specimens were showing an anorexigenic behaviour. The molecule was interacting in the hypothalamus reducing the food intake. Since this side effect was discovered many have been the attempts to find derivatives and analogues of C75 focusing on its food intake modulator basal. In this project, we have focused on the synthesis of C75 analogue called (±)-UB-207 that was first reported in 2012. The novelty of the present work consists in modifying the initial synthetic route in order to simplify the obtainment of the pure derivative

Sorafenib, systemic treatment for advanced hepatocellular carcinoma (HCC), and regorafenib, novel second line treatment after sorafenib failure, have efficacy limited by evasive mechanisms of acquired-drug resistance. BCL-2 proteins participate in the response to tyrosine kinase inhibitors; however, their role in HCC therapy with sorafenib/regorafenib remains uncertain. BH3-mimetic ABT-263 (navitoclax) enhanced sorafenib activity, inducing cell death via a mitochondrial caspase-dependent mechanism, after BCL-xL/BCL-2 inhibition. Sorafenib-resistant hepatoma cells (HepG2R and Hep3BR) exhibited altered mRNA expression of BCL-2 and other anti-apoptotic family members, such as MCL-1, priming drug-resistant cancer cells to death by BH3-mimetics. ABT-263 restored sorafenib efficacy in sorafenib-resistant cell lines and HCC mouse models. Moreover, in mice xenografts from patient-derived BCLC9 cells, better tumor response to sorafenib was associated to higher changes in the BCL-2 mRNA pattern. HCC non-treated patients displayed altered BCL-2, MCL-1 and BCL-xL mRNA levels respect to adjacent non-tumoral biopsies and an increased BCL-2/MCL-1 ratio, predictive of navitoclax efficacy. Moreover, regorafenib administration also modified the BCL-2/MCL-1 ratio and navitoclax sensitized hepatoma cells to regorafenib by a mitochondrial caspase-dependent mechanism. In conclusion, sorafenib/regorafenib response is determined by BCL-2 proteins, while increased BCL-2/MCL-1 ratio in HCC sensitizes drug resistant-tumors against ABT-263 co-administration. Thus, changes in the BCL-2 profile, altered in HCC patients, could help to follow-up sorafenib efficacy, allowing patient selection for combined therapy with BH3-mimetics or early switch them to second line therapy., Study funded by grants from Instituto de Salud Carlos III (FIS PI15/00145 to M.R., FIS PI14/00962 to J.B., PI13/00374 and PI16/00930 to M.M., CIBEREHD and CIBERNED), Ministerio de Economía y Competitividad (SAF2015-69944-R to J.F.C., SAF2015- 66515-R to A.M. and P.G.F., and SAF2013-47246-R to A.C.) and co-funded by FEDER (Fondo Europeo de Desarrollo Regional, Unión Europea); center grant P50- AA-11999 from Research Center for Liver and Pancreatic Diseases, US NIAAA to J.F.C.). Fundació Marató de TV3 (to A.C), AGAUR (2014_SGR_605 to J.B. and 2014_ SGR_785 to J.F.C.) and CERCA Programme / Generalitat de Catalunya.

[eng] Matrix metalloproteinases (MMPs) are a family of zinc-containing endopeptidases involved in the degradation of the extracellular matrix. They make a major contribution to the progression of a vast number of diseases, particularly cancer and some central nervous system (CNS) disorders, such as epilepsy and Alzheimer’s disease. Although a number of MMP inhibitors (MMPi) have been developed for the treatment of cancer, they have all failed in clinical trials due to lack of efficacy and/or, most importantly, severe side effects. The latter can be explained by their lack of selectivity. Elevated levels of gelatinases (MMP-2 and MMP-9) have been described in various animal models and in clinical studies in humans with epilepsy after seizures, being MMP-9 the most overexpressed in all these cases. Thus, a selective inhibitor for MMP- 9, and to some extent MMP-2, might be beneficial for the treatment of this disease. In this thesis, computer-aided drug design was used to design novel MMP-9 inhibitors which were synthesised and evaluated using in vitro enzyme assays. Following this approach, we obtained several molecules that show selectivity for gelatinases over other MMP family members (MMP-1, MMP-3 and MMP-7). These compounds may be of particular interest for the treatment of epileptogenesis, the process by which a normal brain is functionally altered and leads to the development of epilepsy, because of the prominent role of MMP-9 in this process. The permeability across the Blood-brain barrier (BBB) is the main obstacle and a prerequisite for drugs targeting the central nervous system. In this thesis, I worked in the refinement of the structure of the molecules to improve the BBB permeability of the compounds under development. One of the developed compounds, was selected for further studies because its enzymatic and selectivity for Gelatinases and high proteolytic stability. Once the lead candidate was selected, this compound was studied in vivo. First, the pharmacokinetics of the compound were studied in mice and rats, indicating that the molecule was absorbed in the body and distributed through the BBB. Later, I evaluated the effects of the lead compound on the progression of epilepsy in three animal models (the pentylenetetrazole (PTZ) acute injection mouse model, the intrahippocampal kainic acid (KA) mouse model and the rapid kindling rat model) of epilepsy. Results from these experiments indicate that the selected compound has the capacity to inhibit MMP-9 in vivo, has anticonvulsive effects and is able to attenuate the progression of epilepsy in the tested epilepsy models, without any side effects. Moreover, the effects of increased MMP-9 activity in memory and learning were also reduced by using the tested compound. The last part of this thesis was focused on the lead compound optimization addressing metabolic deficiencies through structural modification. Based on this approach, two optimized lead candidates were obtained showing higher potency and selectivity for gelatinases and improved drug-like properties (in vitro BBB permeability, cytotoxicity and stability in serum, intestinal fluids and liver microsomes). As a conclusion, this thesis followed the general structure of the drug discovery phase in the drug research process. MMP-9 was identified and selected as a potential therapeutic target for the treatment of epileptogenesis. The hit identification and hit- to-lead process were achieved using in silico calculations in combination with in vitro tools to design MMPi that selectively target gelatinases and have the ability to cross the BBB. The pharmacokinetic study, the efficacy and toxicity of the lead compounds were demonstrated in different models of epilepsy. Finally, the lead compound was optimized for oral administration., [cat] Les metal·loproteinases de matriu (MMPs) constitueixen una família de proteases dependents del zinc i involucrades en la degradació de la matriu extracel·lular. Participen en la progressió d’un gran nombre de malalties, principalment en càncer i en malalties del sistema nerviós central, com ara l’Alzheimer i l’epilèpsia. En les últimes dècades, s’han desenvolupat una serie d’inhibidors de MMPs (MMPi) pel tractament del càncer, però aquests han fallat en estudis clínics degut a la seva ineficàcia o efectes secundaris adversos, principalment deguts a la falta de selectivitat del inhibidors. En la literatura s’ha descrit un increment en els nivells d’expressió i activitat de gelatinases (MMP-2 i MMP-9) en diferents models animals d’epilèpsia, així com també en humans amb aquesta condició, sent la MMP-9 les més sobreexpressada. Per això, un inhibidor selectiu de la MMP-9, i en part també de la MMP-2, podria ser beneficial pel tractament d’aquesta malaltia. En aquesta tesis, el disseny de fàrmacs assistit per ordinador s’ha utilitzat per a dissenyar inhibidors de la MMP-9 que han estat sintetitzats i avaluats in vitro mitjançant assajos enzimàtics. Els compostos més potents i selectius obtinguts per la inhibició de gelatinases s’han seleccionat i optimitzat per creuar la barrera hematoencefàlica. Per la molècula més prometedora, s’han dut a terme en ratolins i rates. A més, la seva eficàcia i potencial toxicitat s’ha estudiat en 3 models animals d’epilèpsia, demostrant que aquest candidat a fàrmac inhibeix la MMP-9 in vivo i pot atenuar els efectes convulsius i epileptogènics presents en els models animals, a més a més no s’han observat símptomes clínics adversos durant els experiments amb animals. Finalment, aquest compost s’ha optimitzat amb l’objectiu de millorar la seva estabilitat metabòlica i poder ser administrat per via oral. Després d’aquest procés d’optimització s’han obtingut dos compostos potents i selectius amb bones propietats ADME .

Treballs Finals de Grau de Química, Facultat de Química, Universitat de Barcelona, Any: 2018, Tutor: Xavier Ariza Piquer Obesity has become a priority health issue for many governments and organisations such as the WHO, it has gained the status of epidemic and according to the past years trends, the forecasts are not hopeful. The research for anti-obesity drugs has grown in the past years as the percentage of obese people grows globally. A relatively new anti-cancer drug (C75) was tested as anti-cancer drug to rodents, and it was observed that the specimens were showing an anorexigenic behaviour. The molecule was interacting in the hypothalamus reducing the food intake. Since this side effect was discovered many have been the attempts to find derivatives and analogues of C75 focusing on its food intake modulator basal. In this project, we have focused on the synthesis of C75 analogue called (±)-UB-207 that was first reported in 2012. The novelty of the present work consists in modifying the initial synthetic route in order to simplify the obtainment of the pure derivative

A stereoselective synthesis of 2-acetamido-1,2-dideoxyaltronojirimycin (8) and its manno epimer 9 is described. The synthetic approach is based on key bicyclic carbamate 7, which is easily accessible with high enantiomeric purity on a multigram scale by Sharpless asym. epoxidn. of 1,4-pentadien-3-ol or 2,4-pentadien-1-ol. This procedure completes an efficient stereodivergent approach to five isomers of 2-acetamido-1,2-dideoxyiminosugars in high overall yields starting from the same key intermediate 7. The approach described in this paper is based on control of the stereoselectivity of the sulfite ring-opening reaction to give retention of configuration through anchimeric assistance from the endocyclic amine.

Transcriptional repressor complexes containing p130 and E2F4 regulate the expression of genes involved in DNA replication. During the G1 phase of the cell cycle, sequential phosphorylation of p130 by cyclin-dependent kinases (Cdks) disrupts these complexes allowing gene expression. The Cdk inhibitor and tumor suppressor p27(Kip1) associates with p130 and E2F4 by its carboxyl domain on the promoters of target genes but its role in the regulation of transcription remains unclear. We report here that p27(Kip1) recruits cyclin D2/D3-Cdk4 complexes on the promoters by its amino terminal domain in early and mid G1. In cells lacking p27(Kip1), cyclin D2/D3-Cdk4 did not associate to the promoters and phosphorylation of p130 and transcription of target genes was increased. In late G1, these complexes were substituted by p21(Cip1)-cyclin D1-Cdk2. In p21(Cip1) null cells cyclin D1-Cdk2 were not found on the promoters and transcription was elevated. In p21/p27 double null cells transcription was higher than in control cells and single knock out cells. Thus, our results clarify the role of p27(Kip1) and p21(Cip1) in transcriptional regulation of genes repressed by p130/E2F4 complexes in which p27(Kip1) and p21(Cip1) play a sequential role by recruiting and regulating the activity of specific cyclin-Cdk complexes on the promoters.

The protein p27Kip1 (p27), a member of the Cip-Kip family of cyclin-dependent kinase inhibitors, is involved in tumorigenesis and a correlation between reduced levels of this protein in human tumours and a worse prognosis has been established. Recent reports revealed that p27 also behaves as a transcriptional regulator. Thus, it has been postulated that the development of tumours with low amounts of p27 could be propitiated by deregulation of transcriptional programs under the control of p27. However, these programs still remain mostly unknown. The aim of this study has been to define the transcriptional programs regulated by p27 by first identifying the p27-binding sites (p27-BSs) on the whole chromatin of quiescent mouse embryonic fibroblasts. The chromatin regions associated to p27 have been annotated to the most proximal genes and it has been considered that the expression of these genes could by regulated by p27. The identification of the chromatin p27-BSs has been performed by Chromatin Immunoprecipitation Sequencing (ChIP-seq). Results revealed that p27 associated with 1839 sites that were annotated to 1417 different genes being 852 of them protein coding genes. Interestingly, most of the p27-BSs were in distal intergenic regions and introns whereas, in contrast, its association with promoter regions was very low. Gene ontology analysis of the protein coding genes revealed a number of relevant transcriptional programs regulated by p27 as cell adhesion, intracellular signalling and neuron differentiation among others. We validated the interaction of p27 with different chromatin regions by ChIP followed by qPCR and demonstrated that the expressions of several genes belonging to these programs are actually regulated by p27. Finally, cell adhesion assays revealed that the adhesion of p27-/- cells to the plates was much higher that controls, revealing a role of p27 in the regulation of a transcriptional program involved in cell adhesion.

Ignacio Blanco,1 Patricia Bueno,2 Isidro Diego,3 Sergio Pérez-Holanda,4 Francisco Casas-Maldonado,5 Cristina Esquinas,6 Marc Miravitlles6,7 1Alpha1-Antitrypsin Deficiency Spanish Registry (REDAAT), Fundación Respira, Spanish Society of Pneumology and Thoracic Surgery (SEPAR), Barcelona, 2Internal Medicine Department, County Hospital of Jarrio, 3Materials and Energy Department, School of Mining Engineering, Oviedo University, 4Surgical Department, University Central Hospital of Asturias (HUCA), Oviedo, Principality of Asturias, 5Pneumology Department, Complejo Hospitalario Universitario de Granada, Granada, 6Pneumology Department, Hospital Universitari Vall d’Hebron, 7CIBER de Enfermedades Respiratorias (CIBERES), Barcelona, Spain Abstract: In alpha-1 antitrypsin deficiency (AATD), the Z allele is present in 98% of cases with severe disease, and knowledge of the frequency of this allele is essential from a public health perspective. However, there is a remarkable lack of epidemiological data on AATD worldwide, and many of the data currently used are outdated. Therefore, the objective of this study was to update the knowledge of the frequency of the Z allele to achieve accurate estimates of the prevalence and number of Pi*ZZ genotypes worldwide based on studies performed according to the following criteria: 1) samples representative of the general population, 2) AAT phenotyping characterized by adequate methods, and 3) measurements performed using a coefficient of variation calculated from the sample size and 95% confidence intervals. Studies fulfilling these criteria were used to develop maps with an inverse distance weighted (IDW)-interpolation method, providing numerical and graphical information of Pi*Z distribution worldwide. A total of 224 cohorts from 65 countries were included in the study. With the data provided by these cohorts, a total of 253,404 Pi*ZZ were estimated worldwide: 119,594 in Europe, 91,490 in America and Caribbean, 3,824 in Africa, 32,154 in Asia, 4,126 in Australia, and 2,216 in New Zealand. In addition, the IDW-interpolation maps predicted Pi*Z frequencies throughout the world even in some areas that lack real data. In conclusion, the inclusion of new well-designed studies and the exclusion of the low-quality ones have significantly improved the reliability of results, which may be useful to plan strategies for future research and diagnosis and to rationalize the therapeutic resources available. Keywords: SERPINA1, alpha-1 antitrypsin deficiency, protease inhibitor, genetic epidemiology, inverse distance weighted interpolation, geographic information system