Screening of Synergistic Interactions of Epigallocatechin-3-gallate with Antiangiogenic and Antitumor Compounds

This is the accepted manuscript of the article that finally was published in Synergy with DOI: 10.1016/j.synres.2016.05.001Purpose: To screen for possible synergistic interactions of epigallocatechin-3-gallate (EGCG) with a selection of 10 anti-angiogenic or anti-tumor compounds on the survival of endothelial and tumor cells. Methods: Human HMEC endothelial and MDA-MB231 breast cancer cells were treated with different concentrations of EGCG and the 10 tested compounds either as single agents or in paired combinations with EGCG for 3 days and final survival of cells was determined by the MTT assay. IC50 values, sensitization factors and combination indexes were calculated. Results; IC50 values of 140±2 and 45±6 μM were determined for EGCG-treated endothelial and tumor cells, respectively. IC50 values for all tested compounds were within the micromolar and the submillimolar range. The values of the sensitization factor increased and those of the combination index decreased for paired combinations of EGCG with 4-methylumbelliferone. The opposite was true for the combination of EGCG with vitamin D3. Other tested combinations did not exhibit a clear monotonic effect but rather a biphasic behaviour. Conclusion: Combinations of EGCG and 4-methylumbelliferone synergistically decrease endothelial and tumor cell survival. In contrast, the presence of EGCG antagonizes with the antiproliferative effect exerted by vitamin D3 on endothelial and tumor cells.Supported by grants BIO2014-56092-R (MINECO and FEDER), P12-CTS-1507 (Andalusian Government and FEDER) and funds from group BIO-267 (Andalusian Government). The "CIBER de Enfermedades Raras" is an initiative from the ISCIII (Spain). JAGV has the financial support of Vicerrectorado de Investigación y Transferencia (University of Málaga, Spain). The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript

Histamine inhibits adrenocortical cell proliferation but does not affect steroidogenesis.

Histamine (HA) is a neurotransmitter synthesized in most mammalian tissues exclusively by histidine decarboxylase enzyme. Among the plethora of actions mediated by HA, the modulatory effects on steroidogenesis and proliferation in Leydig cells (LCs) have been described recently. To determine whether the effects on LCs reported could be extrapolated to all steroidogenic systems, in this study, we assessed the effect of this amine on adrenal proliferation and steroidogenesis, using two adrenocortical cell lines as experimental models, murine Y1 cells and human NCI-H295R cells. Even when steroidogenesis was not modified by HA in adrenocortical cells, the biogenic amine inhibited the proliferation of H295R cells. This action was mediated by the activation of HRH1 subtype and an increase in the production of inositol phosphates as second messengers, causing cell-cycle arrest in the G2/M phase. These results indicate a new role for HA in the proliferation of human adrenocortical cells that could contribute to a better understanding of tumor pathology as well as to the development of new therapeutic agents.Fil: Pagotto, Romina María del Luján. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Pereyra, Elba Nora. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Monzón, Casandra Margarita. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Mondillo, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Pignataro, Omar Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin

HUMAN AROMATIC L-AMINO ACID DECARBOXYLASE: WHEN STRUCTURE AND MOBILITY DRIVE EFFICIENT CATALYSIS. IMPLICATIONS FOR AADC DEFICIENCY

L’enzima Decarbossilasi degli L-amino acidi aromatici (AADC) è responsabile della sintesi di due neurotrasmettitori essenziali: la dopamina e la serotonina. AADC deve la sua attività catalitica alla chimica del suo cofattore, il piridossale 5’-fosfato (PLP). La struttura cristallografica dell’enzima da mammifero (precisamente da maiale che ha il 90% di identità con l’enzima umano) nella sua forma olo venne risolta venti anni fa e tale risoluzione aprì la strada ad importanti studi strutturali. Dieci anni dopo venne pubblicata la struttura umana di AADC nella sua forma apo evidenziando quali cambiamenti conformazionali avvengono quando il PLP viene legato dall’enzima. Le strutture apo e olo AADC hanno avuto notevole importanza per la comprensione della patogenicità di varianti enzimatiche associate alla malattia chiamata ‘Deficit da AADC’ (AADCd, OMIM#608643). Questa malattia autosomica recessiva molto rara è dovuta prevalentemente a mutazioni missenso sul gene AADC. I pazienti affetti da AADCd mostrano un’amAromatic L-Amino Acid Decarboxylase (AADC) is the enzyme responsible for the synthesis of two essential neurotransmitter dopamine and serotonin from L-Dopa and L-hydroxytryptophan. AADC owes its specific catalytic activity to the chemistry of its cofactor, pyrydoxal-5’-phosphate (PLP). Almost 20 years ago, the crystal structure of a mammalian holoAADC (porcine, sharing 90% of sequence identity) was solved and the availability of its 3D structure paved the way to structural studies. Moreover, 10 years later, human apoAADC structure was published, shedding light on the conformational rearrangement occurring on the apo enzyme upon addition of PLP. Importantly, apo and holoAADC structures provided crucial insights for the comprehension of the pathogenicity of a number of AADC deficiency associated variants. AADC deficiency (OMIM#608643) is a rare autosomal recessive inborn disease due to missense mutations in the AADC gene. Patients bearing these mutations show mild to severe phenotypes, whose destiny is often fatal. Due to the rarity of the disease and to the heterogeneous response to the treatments, medications are not often satisfactory. In the past years, some efforts on human recombinant AADC pathogenic variants have tried to provide support to the research on AADC deficiency by means of biochemical and biophysical approaches determining the impact of the amino acid substitutions on the enzyme features. Here, a further contribution to the comprehension of the AADC deficiency is provided. The crystal structure of human holoAADC has been solved under different conditions, both in its native and ligand bound form. The combination of crystallographic studies, molecular dynamics simulations (MD) and site directed mutagenesis uncovered novel aspects of the AADC structure-function relationship. Moreover, the characterization of 21 novel identified pathogenic variants (spread on each AADC domain, N-terminal, Large and C-terminal Domains) led to the widening of the range of enzymatic phenotypes associated to AADC deficiency. The proposed combination of biochemical and kinetic studies permitted to determine correlations between structural and functional signals. Enzymatic phenotypes span from variants characterized by a mild phenotypes to variants (mainly located at the NTD-CTD interface) whose dramatic structural defects lead to a catalytic incompetence. In addition, MD simulations and in solutions data point out a critical role for the loop3 element that contains the essential catalytic residue Tyr332. A group of variants affecting loop3 has been identified as catalytically incompetent and their structural features have been dissected thanks also to the solving of the crystal structure of pathogenic variant L353P, which constitutes the first solved structure of an AADC variant. Altogether, this study on human AADC provides new elements for the comprehension of the structure-function relationship of AADC with a particular focus on protein dynamics and mobility. Lastly, structural details might represent the basis for both the designing of novel specific inhibitors and for a better comprehension of the molecular aspects of the variants associated with the AADC deficiency

Aromatic amino acids decarboxylase and histidine decarboxylase: deep functional investigations give insights into pathophysiological mechanisms with possible therapeutic implications

Aromatic amino acids decarboxylase and histidine decarboxylase (AADC and HDC) are two homologous enzymes responsible for the synthesis of dopamine/serotonin and histamine, respectively, and other minor signalling aromatic amines. All these molecules are main protagonists or regulators of several physiological pathways, which are fundamental both in central nervous system and in peripheral tissues. Alterations of their homeostasis, indeed, as well as of AADC and HDC functioning or expression, cause and/or participate in the development and progression of several often severe and disabling pathological conditions, such as AADC Deficiency and cholangiocarcinoma. Consequently, AADC and HDC characterization might be useful in the pathophysiological understanding of several diseases and in improving/developing new therapeutic strategies. However, the knowledge of the biochemical features of these two crucial enzymes is still rather limited. Thus, the aim of this thesis is to biochemically characterise human HDC, mostly unknown, and to individuate some possible regulative mechanisms for both HDC and AADC. In addition, a neuronal AADC Deficiency cell model, derived from patient induced pluripotent stem cells (iPSCs), was used to evaluate endogenous AADC features, as well as to research further alterations in dopaminergic pathway. Investigations on human recombinant HDC allowed to discover that, surprisingly, its conformation and catalytic efficiency are influenced by redox state: increasing oxidizing conditions, indeed, favour a more stable and active form of the dimeric enzyme, due to the presence of an intermolecular reversible disulphide bridge involving residue Cys180 of both subunits. Then, in solution analyses of a possible phosphorylation of AADC identified Ser193 as protein kinase A target site, and allowed the detection of an effect on enzyme kinetic parameters, in particular an increased affinity for its substrates. Finally, endogenous AADC levels analyses in dopaminergic neurons derived from AADC Deficiency patients suggested a possible positive feedback mechanism that could tend to increase AADC expression, and the same cell model showed alterations in other cell types besides neurons, in particular glia cells, suggesting that variations in neurons-glia cells Abstract 5 interplay could participate in the pathophysiology mechanisms of AADC Deficiency. Altogether, data and information obtained from the performed experiments have increased AADC and HDC knowledge, as well as paved the way for new hypothesis regarding possible efforts in the development of new disease treatments

Monoaminergic Neuropathology in Alzheimer's disease

Acknowledgments This work was supported by The Croatian Science Foundation grant. no. IP-2014-09-9730 (“Tau protein hyperphosphorylation, aggregation, and trans-synaptic transfer in Alzheimer’s disease: cerebrospinal fluid analysis and assessment of potential neuroprotective compounds”) and European Cooperation in Science and Technology (COST) Action CM1103 (“Stucture-based drug design for diagnosis and treatment of neurological diseases: dissecting and modulating complex function in the monoaminergic systems of the brain”). PRH is supported in part by NIH grant P50 AG005138.Peer reviewedPostprin

Desarrollo y utilización de métodos computacionales en la mejora del proceso de obtención de nuevos fármacos

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 19-02-201

Development of new Catechol-Omethyltransferase inhibitors

The Catechol-O-methyltransferase (COMT, EC 2.2.1.6) is an enzyme responsible for the Omethylation of catechol substrates, such as catecholamines and catechol estrogens. Considering its physiological functions and the existence of polymorphisms, several studies associate COMT with the pathogenesis of several neurological disorders, especially with Parkinson’s Disease (PD) as well as with cardiovascular and hormone-dependent cancers, like breast cancers. Given the important role that COMT has in the catecholamines and catechol estrogens metabolism, COMT has become a relevant therapeutic target. Currently, the most effective and clinically approved by the Federal Drug Administration and the European Medicines Agency for the PD therapy consists of the use of Levodopa, combined with COMT inhibitors. Since the commercially available inhibitors for this enzyme still display a lot of disadvantages, like hepatoxicity, difficulty to reach the brain, among others, the main goal of this work was to develop new COMT inhibitors with potential clinical interest for the PD therapy. For this, triazolopyrimidinics were prepared through the Biginelli reaction, that can be considered catechol bioisosteres, therefore have a higher potential to interact with COMT. This hypothesis was confirmed through molecular docking, being predicted similar interactions as the ones that the catecholic substrates forms with the COMT active site. Their inhibitory properties were evaluated in human recombinant COMT lysates, after the compounds’ incubation at 10 and 100 µM. Contrary to what was expected, the compounds increased the enzyme specific activity, being considered COMT stabilizers. The compounds cytotoxicity was also evaluated in neural dopaminergic rat cells (N27), in the same concentrations. The vast majority of compounds at 10 µM did not exhibited cytotoxicity, being observed similar values to those of the commercial COMT inhibitors, Entacapone and Tolcapone, in the studied cell line. As expected, with the increase in compounds’ concentration (100 µM) a decrease in the relative cell proliferation was observed, reaching values considered to be cytotoxic. Altogether, the synthesized compounds at the concentration of 10 µM stabilized COMT and did not induce cytotoxicity in the N27 cells. In sum, these compounds may be useful for thermal stability assays, crystallography, structure-activity relationship studies and display potential to be studied in specific breast cancers cell lines.A Catecol-O-metiltransferase (COMT, EC 2.2.1.6)) é a enzima responsável pela O-metilação de substratos catecólicos, como as catecolaminas e os estrogénios com estrutura catecólica. Considerando as suas funções fisiológicas e a existência de polimorfismos, vários estudos associam a COMT com a patogénese de várias desordens neurológicas, especialmente com a Doença de Parkinson (DP) e também com doenças cardiovasculares e cancros hormonodependentes, como cancros da mama. Devido à importância que a COMT tem no metabolismo das catecolaminas e dos estrogénios catecólicos, a COMT tornou-se nas últimas décadas num importante alvo terapêutico. Atualmente, a terapia mais eficaz e clinicamente aprovadas pela Federal Drug Administration e European Medicines Agency para a doença de Parkinson consiste no uso de Levodopa, combinada com inibidores da COMT. Uma vez que os inibidores desta enzima comercialmente disponíveis ainda apresentam diversas desvantagens, como hepatotoxicidade, dificuldade em alcançar o cérebro, entre outras, o objetivo principal deste trabalho foi desenvolver novos inibidores da COMT com potencial clínico para a terapia da DP. Para isto, foram preparados triazolopirimidínicos através da reação de Biginelli, os quais podem ser considerados bioisósteros de catecóis, podendo por isso ter potencial para interagir com a COMT. Esta hipótese foi confirmada através de docking molecular, prevendo-se interações moleculares semelhantes às dos substratos catecóis com o centro ativo da COMT. As suas propriedades inibitórias foram avaliadas em lisados recombinantes da enzima, após incubação dos compostos nas concentrações de 10 e 100 µM. Contrariamente ao expectável, os compostos aumentaram a atividade específica da enzima, podendo ser considerados estabilizadores da COMT. Foi ainda avaliada a citoxicidade dos mesmos em células dopaminérgicas neuronais de rato (N27) nas mesmas concentrações. A grande maioria dos compostos a 10 µM não mostrou citotoxicidade, observando-se valores semelhantes aos dos inibidores comerciais da COMT, Entacapone e Tolcapone, na linha celular N27. Como esperado com o aumento da concentração (100 µM) ocorreu um decréscimo na proliferação celular, atingindo valores já considerados citotóxicos. No geral, os compostos sintetizados, na concentração de 10 µM estabilizaram a COMT e não induziram citoxicidade nas células N27. Em suma, as moléculas sintetizadas podem ser úteis para estudos de estabilidade térmica, de cristalografia, de relação estrutura-atividade e apresentam potencialidade para ser estudados em linhas celulares especificas do cancro da mama

Nanoparticle-guided brain drug delivery: Expanding the therapeutic approach to neurodegenerative diseases

Neurodegenerative diseases (NDs) represent a heterogeneous group of aging-related disorders featured by progressive impairment of motor and/or cognitive functions, often accompanied by psychiatric disorders. NDs are denoted as ‘protein misfolding’ diseases or proteinopathies, and are classified according to their known genetic mechanisms and/or the main protein involved in disease onset and progression. Alzheimer’s disease (AD), Parkinson’s disease (PD) and Huntington’s disease (HD) are included under this nosographic umbrella, sharing histopathologically salient features, including deposition of insoluble proteins, activation of glial cells, loss of neuronal cells and synaptic connectivity. To date, there are no effective cures or disease-modifying therapies for these NDs. Several compounds have not shown efficacy in clinical trials, since they generally fail to cross the blood-brain barrier (BBB), a tightly packed layer of endothelial cells that greatly limits the brain internalization of endogenous substances. By engineering materials of a size usually within 1–100 nm, nanotechnology offers an alternative approach for promising and innovative therapeutic solutions in NDs. Nanoparticles can cross the BBB and release active molecules at target sites in the brain, minimizing side effects. This review focuses on the state-of-the-art of nanoengineered delivery systems for brain targeting in the treatment of AD, PD and HD. © 2021 by the authors. Licensee MDPI, Basel, Switzerland