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ResistĂȘncia aos inibidores do proteassoma: uma estratĂ©gia in silico
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Advisor(s)
Abstract(s)
O equĂlibrio entre a sĂntese e a degradação de proteĂnas Ă© fundamental para um bom funcionamento do nosso organismo.
O sistema ubiquitina-proteassoma constitui a principal via de degradação das proteĂnas intracelulares do nosso organismo. Neste sistema existem 2 processos: a ubiquitinação e a degradação das proteĂnas. Na ubiquitinação as proteĂnas a serem degradadas sĂŁo marcadas com ubiquitina para que sejam reconhecidas pelo proteassoma 26S responsĂĄvel pela sua degradação.
O proteassoma 26S Ă© um complexo multienzimĂĄtico constituĂdo por duas unidades regulatĂłrias 19S e por uma unidade catalĂtica designada de proteassoma 20S. O desequilĂbrio entre a sĂntese e a degradação de proteĂnas estĂĄ diretamente relacionado com a origem de vĂĄrias patologias, nomeadamente oncolĂłgicas, caracterizando-se cĂ©lulas cancerĂgenas por uma sobreexpressĂŁo da atividade proteassomal.
Nos Ășltimos anos a indĂșstria farmacĂȘutica tem demonstrado bastante interesse no desenvolvimento de inibidores do proteassoma 20S. No entanto, e apesar de todo o esforço e investimento apenas 4 compostos foram aprovados para uso clĂnico pela FDA e pela EMA: bortezomib, ixazomib, carfilzomib e marizomib.
Os inibidores do proteassoma 20S apresentam como limitaçÔes graves efeitos secundĂĄrios e uma enorme resistĂȘncia inata e/ou adquirida. Esta resistĂȘncia estĂĄ relacionada, por exemplo, com a sobreatividade e sobreexpressĂŁo do proteassoma, polimorfismos de nucleĂłtidos Ășnicos dos genes que codificam as subunidades ÎČ catalĂticas do proteassoma 20S, mutaçÔes do gene que codifica a subunidade ÎČ5 do proteassoma 20S e a sobreexpressĂŁo dos nĂveis dos transportadores de efluxo.
De forma a superar a resistĂȘncia, uma estratĂ©gia possĂvel Ă© o desenvolvimento de novos e mais potentes inibidores do proteassoma 20S, o desenvolvimento de terapĂȘuticas combinatĂłrias de inibidores do proteassoma 20S com outros fĂĄrmacos, como por exemplo inibidores dos transportadores de efluxo dos fĂĄrmacos (P-gp), corticosterĂłides, medicamentos citotĂłxicos, imunomoduladores e inibidores das desacetilases de histonas.
Com o objetivo de identificar novos inibidores do proteassoma 20S foi aplicada uma estratĂ©gia in silico mediante a construção de farmacĂłforos baseados na estrutura de ligandos ativos bem como na estrutura do receptor. Neste trabalho foram desenvolvidos 1483 modelos farmacofĂłricos baseados na estrutura da subunidade ÎČ5 do proteassoma 20S e 997 modelos farmacofĂłricos baseados na estrutura de 3 classes distintas de inibidores do proteassoma 20S: boronatos (bortezomib, delanzomib e ixazomib), αâ,ÎČâ-epoxicetonas (carfilzomib, dihidroeponemicina, epoxomicina e oprozomib) e um inibidor α-cetoaldeĂdo (Z-LLY-cetoaldeĂdo), que pudessem ser utilizados na procura e desenvolvimento de novos inibidores, mais potentes e inerentes a situaçÔes de resistĂȘncia inata e adquirida. Destes 2480 modelos farmacofĂłricos obtidos, 36 foram otimizados e selecionados de acordo com as suas propriedades de previsĂŁo de atividade ou inatividade inibitĂłria. Quatro modelos farmacofĂłricos foram considerados os mais adequados por serem seletivos e capazes de detetar potentes inibidores do proteassoma 20S (IC50 < 50 nM).
A balance between the synthesis and degradation of proteins is essential for a well-functioning organism. The ubiquitine-proteasome system is the main channel of degradation of intracellular proteins of our organism. In this system 2 processes take place: ubiquitination and the degradation of proteins. During ubiquitination proteins being degradaded are marked with ubiquitine so that they can be recognized by the 20S proteasome responsible for their degradation. The 26S proteasome is a multienzymatic complex consisting of two 19S regulatory units and a catalytic unit called 20S proteasome. The imbalance between protein synthesis and protein degradation is related to the origin of several pathologies, namely oncological, and cancer cells are characterized by an overexpression of proteasomal activity. Over the last years, the pharmaceutical industry has shown great interest in the development of 20S proteasome inhibitors. However, despite all efforts and investments, only 4 were approved for clinical practice by the FDA and EMA. These are: bortezomib, ixazomib, carfilzomib and marizomib. Limitations of these inhibitors include dangerous secondary effects and inactive and/or acquired resistance. Such resistance is associated, for example, with proteasomal overactivation and overexpression, single nucleotide polymorphisms of the genes encoding the catalytic subunits ÎČ of the 20S proteasome, ÎČ5 subunit mutations and upregulation of the expression levels of the transporters of efflux. To overcome resistance, one possible strategy is the development of novel and more potent inhibitors, the development of combination therapies of 20S proteasome inhibitors with other agents, such as inhibitors of efflux transporters (P-gp), corticosteroids, cytotoxic agents, immunomodulatory agents and inhibitors of histone deacetylases. In order to identify new 20S proteasome inhibitors, an in silico strategy consisting of the construction of pharmacophores based on the structure of active ligands as well as the receptorâs structure was applied. In this work, 1483 pharmacophoric models were developed based on the structure of ÎČ5 subunit of 20S proteasome and 997 based on the structure from 3 different classes of 20S proteasome inhibitors: boronates (bortezomib, delanzomib and ixazomib), αâ,ÎČâ-epoxyketones (carfilzomib, dihydroeponemycin, epoxomicin and oprozomib) and an α-ketoaldehyde inhibitor (Z-LLY-ketoaldehyde), which could be used to search and develop new inhibitors, more potent and inherent in situations of innate and acquired resistance. Of these 2480 pharmacophoric models, 36 were optimized and selected in accordance with their properties of prevision of inhibitory activity or inactivity. Four pharmacophoric models were considered more favorable due to their selectivity and their capacity to detect potent inhibitors of 20S proteasome inhibitors (IC50 < 50 nM).
A balance between the synthesis and degradation of proteins is essential for a well-functioning organism. The ubiquitine-proteasome system is the main channel of degradation of intracellular proteins of our organism. In this system 2 processes take place: ubiquitination and the degradation of proteins. During ubiquitination proteins being degradaded are marked with ubiquitine so that they can be recognized by the 20S proteasome responsible for their degradation. The 26S proteasome is a multienzymatic complex consisting of two 19S regulatory units and a catalytic unit called 20S proteasome. The imbalance between protein synthesis and protein degradation is related to the origin of several pathologies, namely oncological, and cancer cells are characterized by an overexpression of proteasomal activity. Over the last years, the pharmaceutical industry has shown great interest in the development of 20S proteasome inhibitors. However, despite all efforts and investments, only 4 were approved for clinical practice by the FDA and EMA. These are: bortezomib, ixazomib, carfilzomib and marizomib. Limitations of these inhibitors include dangerous secondary effects and inactive and/or acquired resistance. Such resistance is associated, for example, with proteasomal overactivation and overexpression, single nucleotide polymorphisms of the genes encoding the catalytic subunits ÎČ of the 20S proteasome, ÎČ5 subunit mutations and upregulation of the expression levels of the transporters of efflux. To overcome resistance, one possible strategy is the development of novel and more potent inhibitors, the development of combination therapies of 20S proteasome inhibitors with other agents, such as inhibitors of efflux transporters (P-gp), corticosteroids, cytotoxic agents, immunomodulatory agents and inhibitors of histone deacetylases. In order to identify new 20S proteasome inhibitors, an in silico strategy consisting of the construction of pharmacophores based on the structure of active ligands as well as the receptorâs structure was applied. In this work, 1483 pharmacophoric models were developed based on the structure of ÎČ5 subunit of 20S proteasome and 997 based on the structure from 3 different classes of 20S proteasome inhibitors: boronates (bortezomib, delanzomib and ixazomib), αâ,ÎČâ-epoxyketones (carfilzomib, dihydroeponemycin, epoxomicin and oprozomib) and an α-ketoaldehyde inhibitor (Z-LLY-ketoaldehyde), which could be used to search and develop new inhibitors, more potent and inherent in situations of innate and acquired resistance. Of these 2480 pharmacophoric models, 36 were optimized and selected in accordance with their properties of prevision of inhibitory activity or inactivity. Four pharmacophoric models were considered more favorable due to their selectivity and their capacity to detect potent inhibitors of 20S proteasome inhibitors (IC50 < 50 nM).
Description
Tese de mestrado, QuĂmica FarmacĂȘutica e TerapĂȘutica, Universidade de Lisboa, Faculdade de FarmĂĄcia, 2018
Keywords
Proteassoma 20S Modelo farmacofĂłrico Inibidores do proteassoma 20S ResistĂȘncia proteassomal Teses de mestrado - 2018