Publication
Evaluation of Specific Anthraquinones as New catechol-O-methyltransferase Inhibitors: Virtual Docking, Molecular Dynamics, Inhibition and Cytotoxicity In Vitro Studies
| datacite.subject.fos | CiĆŖncias Naturais::CiĆŖncias QuĆmicas | |
| datacite.subject.sdg | 03:SaĆŗde de Qualidade | |
| dc.contributor.advisor | Passarinha, LuĆs AntĆ³nio Paulino | |
| dc.contributor.advisor | Silvestre, Samuel Martins | |
| dc.contributor.author | ProenƧa, FƔbio Alexandre Esteves | |
| dc.date.accessioned | 2025-11-12T11:38:45Z | |
| dc.date.available | 2025-11-12T11:38:45Z | |
| dc.date.issued | 2025-03-07 | |
| dc.description.abstract | ParkinsonĀ“s disease (PD) is the second most prevalent age-related neurodegenerative disorder around the world, with no cure currently in sight. This condition is caused by the gradual loss of the brain's dopaminergic neurons, in the substantia nigra pars compacta of the brain. This progressive neuronal loss leads to the typical symptoms of the disease characterized by rigidity, resting tremor, bradykinesia, and postural imbalance. Actually, the conventional therapy for this pathology consists in the administration of oral levodopa (L-DOPA), a natural dopamine precursor, and two enzymatic inhibitors: one for Catechol-O-Methyltransferase (COMT) and the other for the peripheral aromatic L-amino acid decarboxylase (AADC). In terms of central nervous system, AADC can transform the administered L-DOPA in dopamine, and both substances, at central and peripheral level, can be metabolized by COMT. In humans, the COMT enzyme is present in two isoforms, a soluble isoform (S-COMT) and the membrane-bounded isoform (MB-COMT). Despite their similarities in the primary amino acid sequences, there is a accentuated difference in the kinetic behavior of both isoforms. MB-COMT tends to have a higher affinity for the substrate (lower Km) than SCOMT. On the other hand, S-COMT has a much higher catalytic reaction capacity (Vmax) than MB-COMT. Those differences in the kinetic behavior of the isoforms determined the role of the isoenzymes, with MB-COMT being physiologically more relevant due to its role in catecholamine methylation at physiological concentrations. Typically, COMT inhibitors enhance L-DOPA and dopamine bioavailability and effectiveness. However, they are often associated with toxicity and/or limited ability to cross the blood-brain barrier (BBB). This underscored the need to discover/ develop novel molecules with greater potency, reduced toxicity, and better pharmacokinetic properties than the existing inhibitors in clinical use. This dissertation investigates the potential of anthraquinones as potential COMT inhibitors, based on their molecular skeleton similarity with tolcapone. Anthraquinones are a subclass of quinones derived from anthracene with anti-tumor, anti-inflammatory, and neuroprotective properties, among others. They are characterized by having three planar rings with two ketones groups on the second ring. The inhibition of MB-COMT by nine anthraquinone family compounds was evaluated in vitro, using an analytical method previously described by the research group. From the obtained results, four of the nine compounds in study exhibited an enzymatic inhibition capacity close to 60%, at 100 ĀµM. After, a cytotoxicity evaluation was performed for these 4 compounds by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays using two different cell lines, normal human dermal fibroblasts (NHDF) and a rat dopaminergic neural cell line (N27). From that cytotoxic evaluation it was observed that the compounds in study displayed a cytotoxicity profile similar to the described inhibitor, tolcapone. In silico trials were performed using Autodock Vina complementing the in vitro findings by analyzing interactions and the positioning of the compounds in relation to the proteinĀ“s active center. The results were ranked based on the binding energy and key interactions with critical residues for MB-COMTĀ“s catalytic capacity. Promising molecules were further analyzed using molecular dynamics simulations, using the commercial inhibitor tolcapone as a positive control. The principal Adsorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) properties were also predicted intending to complement the pharmacokinetic analysis and toxicity of the molecules. The in vitro and in silico obtained results suggest that purpurin, alizarin, 3-nitroalizarin and lucidin are promising candidates as COMT inhibitors, however, half maximal inhibitory concentration (IC50) assays for the enzymatic inhibition and for cytotoxicity, need to be performed to properly evaluate the potential of this molecules. | eng |
| dc.description.abstract | A doenƧa de Parkinson (DP) Ć© a segunda doenƧa neurodegenerativa, relacionada com o envelhecimento, mais prevalente em todo o mundo e, atĆ© Ć data, nĆ£o existe perspetiva de cura. Esta patologia Ć© causada pela perda gradual dos neurĆ³nios dopaminĆ©rgicos do cĆ©rebro, na substĆ¢ncia nigra pars compacta do cĆ©rebro. Esta diminuiĆ§Ć£o progressiva dos neurĆ³nios dopaminĆ©rgicos conduz ao aparecimento dos sintomas tĆpicos da doenƧa, caracterizados pela rigidez, o tremor de repouso, a bradicinĆ©sia e o desequilĆbrio postural. Atualmente, a terapia convencional para esta patologia consiste na administraĆ§Ć£o oral de levodopa (L-DOPA), um precursor natural de dopamina, e de dois inibidores enzimĆ”ticos: um da catecol-O-metiltransferase (COMT) e outro da LaminoĆ”cido descarboxilase aromĆ”tica (AADC) perifĆ©rica. Ao nĆvel do sistema nervoso central, a AADC pode transformar a L-DOPA administrada em dopamina, e ambas as substĆ¢ncias, ao nĆvel central e perifĆ©rico, podem ser metabolizadas pela COMT. Nos seres humanos, a enzima COMT estĆ” presente em duas isoformas, uma isoforma solĆŗvel (SCOMT) e a isoforma ligada Ć membrana (MB-COMT). Apesar das suas semelhanƧas nas sequĆŖncias primĆ”rias de aminoĆ”cidos, existem diferenƧas acentuadas no comportamento cinĆ©tico de ambas as isoformas. A MB-COMT tende a ter maior afinidade pelo substrato (menor Km) em comparaĆ§Ć£o a S-COMT. Por outro lado, a S-COMT apresenta uma capacidade catalĆtica de reaĆ§Ć£o muito maior (Vmax) do que a MB-COMT. Essas diferenƧas no comportamento cinĆ©tico das isoformas determinam o papel das isoenzimas, tornando a proteĆna membranar fisiologicamente a mais relevante, pela metilaĆ§Ć£o tĆpica de catecolaminas em concentraƧƵes fisiolĆ³gicas. Tipicamente, os inibidores da COMT podem aumentar a biodisponibilidade e a eficĆ”cia da L-DOPA e da dopamina. No entanto, estĆ£o frequentemente associados a toxicidade e/ou incapacidade de atravessarem a barreira hematoencefĆ”lica (BBB). Assim, Ć© crucial descobrir/desenvolver novas molĆ©culas que apresentem maior potĆŖncia, toxicidade reduzida e melhores propriedades farmacocinĆ©ticas do que os inibidores atualmente existentes em uso clĆnico. Nesta dissertaĆ§Ć£o, analisamos o potencial de antraquinonas como potenciais inibidores da COMT, com base na semelhanƧa com o esqueleto molecular do tolcapone. As antraquinonas sĆ£o uma subclasse de quinonas derivadas do antraceno com propriedades anti-tumorais, anti-inflamatĆ³rias e neuroprotectoras, entre outras. Estas caracterizamse por terem trĆŖs anĆ©is planares com dois grupos cetona no anel central. A inibiĆ§Ć£o da MB-COMT por parte de 9 compostos da famĆlia das antraquinonas foi avaliada atravĆ©s de estudos in vitro, com base num mĆ©todo analĆtico jĆ” descrito pelo grupo de investigaĆ§Ć£o. De acordo com os resultados obtidos, 4 dos 9 compostos em estudo apresentaram uma capacidade de inibiĆ§Ć£o enzimĆ”tica prĆ³xima de 60%, a uma concentraĆ§Ć£o de 100 ĀµM. Posteriormente, foi efetuada uma avaliaĆ§Ć£o da citotoxicidade para estes 4 compostos, atravĆ©s de ensaios de brometo de 3-(4,5-dimetiltiazol-2-il)-2,5- difeniltetrazĆ³lio (MTT), utilizando duas linhas celulares diferentes, fibroblastos dĆ©rmicos humanos normais (NHDF) e uma linha celular neural dopaminĆ©rgica de rato (N27). Desta avaliaĆ§Ć£o citotĆ³xica verificou-se que os compostos em estudo, apresentam um perfil citotĆ³xico similar ao do inibidor descrito, o tolcapone. Ensaios in silico foram realizados utilizando o Autodock Vina de forma a complementar os resultados in vitro, atravĆ©s do estudo das interaƧƵes e do posicionamento dos compostos em relaĆ§Ć£o ao centro ativo da proteĆna. Os resultados alcanƧados foram escalonados com base na energia de ligaĆ§Ć£o e nas interaƧƵes principais dos resĆduos alvo fundamentais para a capacidade catalĆtica da enzima MB-COMT. Posteriormente, as molĆ©culas que se destacaram foram avaliadas por ensaios de dinĆ¢mica molecular, utilizando o inibidor comercial tolcapone como controlo positivo. As principais propriedades de AbsorĆ§Ć£o, DistribuiĆ§Ć£o, Metabolismo, ExcreĆ§Ć£o e Toxicidade (ADMET) foram tambĆ©m previstas com o intuito de complementar a anĆ”lise farmacocinĆ©tica e de toxicidade das molĆ©culas. Os resultados obtidos, quer por estudos in vitro, quer in silico, sugerem que os compostos purpurina, alizarina, 3-nitroalizarina e lucidina, sĆ£o promissores como potenciais novos inibidores da COMT; no entanto, Ć© necessĆ”rio efetuar estudos de concentraĆ§Ć£o inibitĆ³ria mĆ©dia (IC50) para a inibiĆ§Ć£o enzimĆ”tica e para a citotoxicidade, para avaliar adequadamente o potencial destas molĆ©culas. | por |
| dc.identifier.tid | 204027810 | |
| dc.identifier.uri | http://hdl.handle.net/10400.6/19212 | |
| dc.language.iso | eng | |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-sa/4.0/ | |
| dc.subject | DoenƧa de Parkinson | |
| dc.subject | Catecol-O-metiltransferase | |
| dc.subject | Inibidores | |
| dc.subject | Antraquinonas | |
| dc.subject | ParkinsonĀ“s disease | |
| dc.subject | Catechol-O-methyltransferase | |
| dc.subject | Inhibitors | |
| dc.subject | Anthraquinones | |
| dc.title | Evaluation of Specific Anthraquinones as New catechol-O-methyltransferase Inhibitors: Virtual Docking, Molecular Dynamics, Inhibition and Cytotoxicity In Vitro Studies | por |
| dc.type | master thesis | |
| dspace.entity.type | Publication | |
| person.familyName | ProenƧa | |
| person.givenName | FƔbio Alexandre Esteves | |
| person.identifier.ciencia-id | B71B-AA9C-CA5B | |
| person.identifier.orcid | 0009-0007-1451-1222 | |
| relation.isAuthorOfPublication | 2b381a7b-d79d-469d-8098-92faaa5895ef | |
| relation.isAuthorOfPublication.latestForDiscovery | 2b381a7b-d79d-469d-8098-92faaa5895ef | |
| thesis.degree.name | 2Āŗ Ciclo em BioquĆmica |
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