RESUMEN
Endothelial dysfunction (ED) is critical in the development and progression of cardiovascular (CV) disorders, yet effective therapeutic targets for ED remain elusive due to limited understanding of its underlying molecular mechanisms. To address this gap, we employed a systems biology approach to identify potential targets for ED. Our study combined multi omics data integration, with siRNA screening, high content imaging and network analysis to prioritise key ED genes and identify a pro- and anti-ED network. We found 26 genes that, upon silencing, exacerbated the ED phenotypes tested, and network propagation identified a pro-ED network enriched in functions associated with inflammatory responses. Conversely, 31 genes ameliorated ED phenotypes, pointing to potential ED targets, and the respective anti-ED network was enriched in hypoxia, angiogenesis and cancer-related processes. An independent screen with 17 drugs found general agreement with the trends from our siRNA screen and further highlighted DUSP1, IL6 and CCL2 as potential candidates for targeting ED. Overall, our results demonstrate the potential of integrated system biology approaches in discovering disease-specific candidate drug targets for endothelial dysfunction.
Asunto(s)
Biología de Sistemas , ARN Interferente PequeñoRESUMEN
A insuficiência cardíaca (IC) é uma síndrome de elevada morbimortalidade, correspondendo a um grave problema de saúde pública. Uma das abordagens terapêuticas para IC consiste no uso de antagonistas do receptor de angiotensina II do tipo 1 (AT1R), conhecidos como sartanas. Estudos apontam que uma nova classe de compostos, os agonistas enviesados, é capaz de induzir a sinalização da via da ß-arrestina sem ativação da via da proteína G. Essa seletividade funcional é particularmente interessante, pois a via dependente da proteína G é responsável pelo aumento da pressão arterial, morte celular e fibrose tecidual, levando a hipertrofia cardíaca e progressão da IC. No entanto, a via da ß-arrestina está associada com renovação celular e aumento do inotropismo. Além disso, estudos in vivo sugerem que agonistas enviesados poderiam corresponder a uma terapia superior à dos antagonistas convencionais, que bloqueiam ambas as vias. Apesar do potencial terapêutico, esses compostos possuem estrutura peptídica e, por isso, tem sua administração restrita à via intravenosa. A resolução da estrutura cristalográfica do AT1R permitiu estudos de modelagem molecular mais acurados. Tendo isso em mente, nesse trabalho foram propostos agonistas enviesados de natureza não peptídica para o AT1R por meio de técnicas de modelagem molecular e validação das hipóteses levantadas por ensaios in vitro. Foram realizados estudos de dinâmica molecular com o AT1R (PDB ID: 4YAY) em uma bicamada lipídica e ensaios de ancoramento molecular da angiotensina II (AngII) e do ligante enviesado TRV027. As poses de ancoramento molecular selecionadas foram utilizadas em dinâmicas de complexo, que revelaram diferenças entre os sistemas apo (sem nenhum ligante) e holo (com o ligante no sitio de ligação). Nossos resultados sugerem que o TRV027 induz um padrão exclusivo de ligações de hidrogênio e de estrutura secundária, enquanto que a AngII afeta os resíduos do bolso hidrofóbico do sitio de ligação, principalmente a conformação do Trp2536.48. Com base nas simulações, três farmacóforos foram criados e utilizados de maneira complementar em triagens virtuais na base de dados ZINC15, resultando na seleção de cinco compostos. Um desses compostos apresentou afinidade pelo receptor AT1R e, ainda que estudos complementares de ativação de vias especificas sejam necessários para que o composto possa ser classificado como agonista enviesado, já se constitui em molécula potencialmente promissora. Além disso, esses estudos permitiram a proposição de estruturas inéditas que podem vir a ser hits no processo de desenvolvimento de agonistas enviesados para AT1R. Portanto, como continuidade desse trabalho, essas moléculas serão sintetizadas e investigadas quanto à possível interação com o receptor.
Heart Failure (HF) is a common syndrome with high morbimortality, being considered a serious public health problem. One of the therapeutic approaches for HF consists in the use of the sartan class, which are angiotensin II type 1 receptor (AT1R) antagonists. Recent studies have shown that a new class of compounds, known as biased agonists, is able to induce signaling via ß-arrestin without G-protein activation. This functional selectivity is particularly interesting since G-protein dependent signaling is responsible for cell death and cardiac tissue fibrosis, which leads to cardiac muscle hypertophy and HF progression. On the other hand, ß-arrestin signaling is associated with cellular renewal and increased inotropism. In vivo studies suggests that biased agonists could correspond to a superior therapy over conventional angiotensin II type 1 receptor antagonists, which blocks cell signaling as a whole, however their peptidic structure restricts their use to intravenous administration. Moreover, the AT1R crystal structure determination holds great promise for more accurate molecular modeling studies. With that being said, the aim of this work was to plan and develop new non-peptidic biased agonists for ATR1 employing molecular modeling techniques and in vitro tests for hypothesis validation. Molecular dynamics (MD) simulations of the refined AT1R crystal (PDB ID: 4YAY) embedded in a lipid bilayer and molecular docking studies with angiotensin II (AngII) and TRV027 (biased agonist) were conducted. Selected docking poses from both ligands underwent complex MD simulations revealing differences between apo (ligand free) and holo (ligand in the binding site) systems. Our results suggest that TRV027 induces an exclusive hydrogen bond and secondary structure pattern, while AngII affects the hydrophobic pocket conformation, mainly Trp253. Based on the simulations, three pharmacophore models were created and used in virtual screenings in the ZINC15 database, resulting in the selection of five compounds that were tested in vitro. One of the compounds displayed affinity for AT1R and is a promising molecule. Nonetheless, it needs further pathway activation characterization in order to be a classified as a biased agonist. Furthermore, these results have contributed significantly for the proposition of new structures that could be hits with biased agonist activity for AT1R. Thus, for future works, we point out the necessity for synthesis and characterization of this new compounds
Asunto(s)
Técnicas In Vitro/métodos , Angiotensina II/agonistas , Insuficiencia Cardíaca/patología , Ligandos , Organización y Administración , Receptores de Angiotensina/análisis , Receptor de Angiotensina Tipo 1/análisis , MétodosRESUMEN
Functional selectivity is a phenomenon observed in G protein-coupled receptors in which intermediate active-state conformations are stabilized by mutations or ligand binding, resulting in different sets of signaling pathways. Peptides capable of selectively activating ß-arrestin, known as biased agonists, have already been characterized in vivo and could correspond to a new therapeutic approach for treatment of cardiovascular diseases. Despite the potential of biased agonism, the mechanism involved in selective signaling remains unclear. In this work, molecular dynamics simulations were employed to compare the conformational profile of the angiotensin II type 1 receptor (AT1R) crystal bound to angiotensin II, bound to the biased ligand TRV027, and in the apo form. Our results show that both ligands induce changes near the NPxxY motif in transmembrane domain 7 that are related to receptor activation. However, the biased ligand does not cause the rotamer toggle alternative positioning and displays an exclusive hydrogen-bonding pattern. Our work sheds light on the biased agonism mechanism and will help in the future design of novel biased agonists for AT1R.
Asunto(s)
Simulación de Dinámica Molecular , Oligopéptidos/farmacología , Receptor de Angiotensina Tipo 1/agonistas , Apoproteínas/agonistas , Apoproteínas/química , Apoproteínas/metabolismo , Cristalografía por Rayos X , Ligandos , Simulación del Acoplamiento Molecular , Oligopéptidos/metabolismo , Conformación Proteica , Receptor de Angiotensina Tipo 1/química , Receptor de Angiotensina Tipo 1/metabolismoRESUMEN
Alkylphosphocholines (APC) are promising antitumor agents, which have the cellular membrane as primary target; however, red blood cell damage limits their wide therapeutic use. A variety of APC analogs has been synthesized and tested showing less hemolytic effect than the class prototype, Miltefosine (HePC). In this work, chemometric methods were applied to a set of 34 APC derivatives to identify the most relevant structural and molecular features of hemolytic activity. The APC derivatives were divided into three groups: (i) N-methylpiperidine and N-methylmorpholine derivatives with a long alkyl chain or flexible cyclopentadecyl rings, displaying a hemolytic rate of 17 %; (ii) adamantyl and cyclopentadecyl derivatives, showing an average hemolysis of 39 %; and, N,N,N-trimethylammonium, trans-N,N,N-trimethylcyclohexanamine, and trans-N,N,N-trimethylcyclopentanamine derivatives, whose average hemolysis was 41 %. The findings suggested that the presence of either bulky cationic head groups, or rings such as adamantyl and cyclohexyl, primarily increases the hemolysis of compounds with eleven atoms in the alkyl chain. Moreover, the macrocyclic cyclopentadecyl seems to be important to the hemolytic potential especially of compounds with five carbon atoms in the alkyl chain. Regarding linear carbon chain derivatives with no ring substitution, less bulky cationic head groups seem to favor hemolysis. Thus, in order to design more potent and less toxic APC antitumors, the reported structural/molecular patterns should not be included in their structure.