ATPasas tipo P de Mycobacterium tuberculosis como dianas para el diseño racional de compuestos antituberculosos
Tuberculosis (TB) is an infectious disease caused by the acid-fast bacillus Mycobacterium tuberculosis (Mtb), which is one of the most important public health problems worldwide. Furthermore, the emergence of resistant Mtb strains to current anti-TB drugs has increased the search for alternative the...
- Autores:
-
Santos Ruiz, Paola Andrea
- Tipo de recurso:
- Work document
- Fecha de publicación:
- 2020
- Institución:
- Universidad Nacional de Colombia
- Repositorio:
- Universidad Nacional de Colombia
- Idioma:
- spa
- OAI Identifier:
- oai:repositorio.unal.edu.co:unal/77933
- Acceso en línea:
- https://repositorio.unal.edu.co/handle/unal/77933
- Palabra clave:
- 572 - Bioquímica
614 - Medicina Forense; incidencia de lesiones, heridas, enfermedades; medicina preventiva pública
P-type ATPases
tuberculosis
antituberculous compounds
cyclopiazonic acid
molecular docking
ATPasas tipo P
tuberculosis
compuestos antituberculosos
ácido ciclopiazónico
acoplamiento molecular
- Rights
- openAccess
- License
- Atribución-NoComercial-SinDerivadas 4.0 Internacional
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| dc.title.spa.fl_str_mv |
ATPasas tipo P de Mycobacterium tuberculosis como dianas para el diseño racional de compuestos antituberculosos |
| title |
ATPasas tipo P de Mycobacterium tuberculosis como dianas para el diseño racional de compuestos antituberculosos |
| spellingShingle |
ATPasas tipo P de Mycobacterium tuberculosis como dianas para el diseño racional de compuestos antituberculosos 572 - Bioquímica 614 - Medicina Forense; incidencia de lesiones, heridas, enfermedades; medicina preventiva pública P-type ATPases tuberculosis antituberculous compounds cyclopiazonic acid molecular docking ATPasas tipo P tuberculosis compuestos antituberculosos ácido ciclopiazónico acoplamiento molecular |
| title_short |
ATPasas tipo P de Mycobacterium tuberculosis como dianas para el diseño racional de compuestos antituberculosos |
| title_full |
ATPasas tipo P de Mycobacterium tuberculosis como dianas para el diseño racional de compuestos antituberculosos |
| title_fullStr |
ATPasas tipo P de Mycobacterium tuberculosis como dianas para el diseño racional de compuestos antituberculosos |
| title_full_unstemmed |
ATPasas tipo P de Mycobacterium tuberculosis como dianas para el diseño racional de compuestos antituberculosos |
| title_sort |
ATPasas tipo P de Mycobacterium tuberculosis como dianas para el diseño racional de compuestos antituberculosos |
| dc.creator.fl_str_mv |
Santos Ruiz, Paola Andrea |
| dc.contributor.advisor.spa.fl_str_mv |
Soto Ospina, Carlos Yesid López Vallejo, Fabián Harvey |
| dc.contributor.author.spa.fl_str_mv |
Santos Ruiz, Paola Andrea |
| dc.contributor.researchgroup.spa.fl_str_mv |
Bioquímica y Biología Molecular de las Micobacterias |
| dc.subject.ddc.spa.fl_str_mv |
572 - Bioquímica 614 - Medicina Forense; incidencia de lesiones, heridas, enfermedades; medicina preventiva pública |
| topic |
572 - Bioquímica 614 - Medicina Forense; incidencia de lesiones, heridas, enfermedades; medicina preventiva pública P-type ATPases tuberculosis antituberculous compounds cyclopiazonic acid molecular docking ATPasas tipo P tuberculosis compuestos antituberculosos ácido ciclopiazónico acoplamiento molecular |
| dc.subject.proposal.eng.fl_str_mv |
P-type ATPases tuberculosis antituberculous compounds cyclopiazonic acid molecular docking |
| dc.subject.proposal.spa.fl_str_mv |
ATPasas tipo P tuberculosis compuestos antituberculosos ácido ciclopiazónico acoplamiento molecular |
| description |
Tuberculosis (TB) is an infectious disease caused by the acid-fast bacillus Mycobacterium tuberculosis (Mtb), which is one of the most important public health problems worldwide. Furthermore, the emergence of resistant Mtb strains to current anti-TB drugs has increased the search for alternative therapeutic targets and methods for the rational design of new effective drugs. In this sense, membrane proteins have been considered interesting targets due to their biological implication and for being highly accessible to active compounds. Particularly, P-type ATPases membrane transporters are interesting targets due to their implication in ionic homeostasis and mycobacterial viability. This work was oriented to CtpF, a calcium P-type ATPase, related to a broad number of biological conditions associated to processes of infection such as oxidative stress, adaptation of tubercle bacilli to anaerobic conditions, hypoxia and latency. Due to that, the main objective of this doctoral Thesis was to determine, through in silico and in vitro analysis, the potential of P-type ATPases of Mtb, especially the calcium transporter CtpF, as a target for the rational design of anti-TB compounds. Initially, a 3D homology model of CtpF was generated, which was employed for identified key pharmacophoric features of the CtpF-cyclopiazonic acid (CPA) complex, a well-known inhibitor of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA1a), from which its 3D structure is known experimentally and was used as a template in the construction of the model. By using a repertoire of experimental techniques, it was evaluated and found that CPA causes inhibition of the Ca2+-ATPase activity of CtpF, as well as mycobactericidal activity. The analysis of the transcriptional response of P2 ATPases to treatment with CPA showed a specific response of ctpF in comparison with other P-type ATPases. These initial results provide evidence that CtpF is a molecular target for the design of compounds with anti-TB potential. Thereupon, with the CtpF-CPA pharmacophoric features, a pharmacophore-based virtual screening was performed using the ZINC database in order to select candidate molecules to inhibitors of CtpF. Molecular docking-based virtual screening and binding free energy calculations (MM-GBSA) of selected candidates allowed identifying six compounds with the best relative binding energies to be evaluated in vitro. The compounds selected displayed in vitro antimycobacterial activity, showing a minimum inhibitory concentrations (MIC) ranging from 50 -100 μg/mL, and growth inhibitions of 29.5 - 64.0 % on Mtb. Likewise, they causes inhibition of Ca2+-ATPase activity in Mtb membrane vesicles (IC50) ranging from 4.1 - 35.8 μM. Finally, the activity of the compounds with the best biological activity was evaluated in a macrophage infection model, as an approach to evaluate the effect of compounds once the infection has occurred. The compound ZINC63908257 was the best candidate by displaying a MIC of 50 μg/mL, a Ca2+ P-type ATPase inhibition with IC50 = 4.4 μM and 81 % decrease in Mtb replication within macrophage. This compound showed cytotoxic activity of 12.9 % in MH-S cells and hemolysis of 2 % of human erythrocytes, thus, this compound shows a good pharmacokinetic profile (drug-like). Overall, the results presented here shows the importance of the P-type ATPases of Mtb for the mycobacteria survival during infection, and identify the CtpF as a key molecular target for the design of new antituberculous compounds. |
| publishDate |
2020 |
| dc.date.accessioned.spa.fl_str_mv |
2020-08-05T09:35:00Z |
| dc.date.available.spa.fl_str_mv |
2020-08-05T09:35:00Z |
| dc.date.issued.spa.fl_str_mv |
2020-02-14 |
| dc.type.spa.fl_str_mv |
Documento de trabajo |
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info:eu-repo/semantics/workingPaper |
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info:eu-repo/semantics/publishedVersion |
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http://purl.org/coar/resource_type/c_8042 |
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http://purl.org/coar/version/c_970fb48d4fbd8a85 |
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ATPasas tipo P de Mycobacterium tuberculosis como dianas para el diseño racional de compuestos antituberculosos |
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https://repositorio.unal.edu.co/handle/unal/77933 |
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ATPasas tipo P de Mycobacterium tuberculosis como dianas para el diseño racional de compuestos antituberculosos |
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Atribución-NoComercial-SinDerivadas 4.0 InternacionalDerechos reservados - Universidad Nacional de ColombiaAcceso abiertohttp://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2Soto Ospina, Carlos Yesid4393e614-27e4-4a4f-9a3e-41bca2eeaf49-1López Vallejo, Fabián Harvey36affc04-e4da-4319-a025-df0499f0373b-1Santos Ruiz, Paola Andrea86c28970-ae0e-43b8-9ce1-02748c032a33Bioquímica y Biología Molecular de las Micobacterias2020-08-05T09:35:00Z2020-08-05T09:35:00Z2020-02-14ATPasas tipo P de Mycobacterium tuberculosis como dianas para el diseño racional de compuestos antituberculososhttps://repositorio.unal.edu.co/handle/unal/77933Tuberculosis (TB) is an infectious disease caused by the acid-fast bacillus Mycobacterium tuberculosis (Mtb), which is one of the most important public health problems worldwide. Furthermore, the emergence of resistant Mtb strains to current anti-TB drugs has increased the search for alternative therapeutic targets and methods for the rational design of new effective drugs. In this sense, membrane proteins have been considered interesting targets due to their biological implication and for being highly accessible to active compounds. Particularly, P-type ATPases membrane transporters are interesting targets due to their implication in ionic homeostasis and mycobacterial viability. This work was oriented to CtpF, a calcium P-type ATPase, related to a broad number of biological conditions associated to processes of infection such as oxidative stress, adaptation of tubercle bacilli to anaerobic conditions, hypoxia and latency. Due to that, the main objective of this doctoral Thesis was to determine, through in silico and in vitro analysis, the potential of P-type ATPases of Mtb, especially the calcium transporter CtpF, as a target for the rational design of anti-TB compounds. Initially, a 3D homology model of CtpF was generated, which was employed for identified key pharmacophoric features of the CtpF-cyclopiazonic acid (CPA) complex, a well-known inhibitor of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA1a), from which its 3D structure is known experimentally and was used as a template in the construction of the model. By using a repertoire of experimental techniques, it was evaluated and found that CPA causes inhibition of the Ca2+-ATPase activity of CtpF, as well as mycobactericidal activity. The analysis of the transcriptional response of P2 ATPases to treatment with CPA showed a specific response of ctpF in comparison with other P-type ATPases. These initial results provide evidence that CtpF is a molecular target for the design of compounds with anti-TB potential. Thereupon, with the CtpF-CPA pharmacophoric features, a pharmacophore-based virtual screening was performed using the ZINC database in order to select candidate molecules to inhibitors of CtpF. Molecular docking-based virtual screening and binding free energy calculations (MM-GBSA) of selected candidates allowed identifying six compounds with the best relative binding energies to be evaluated in vitro. The compounds selected displayed in vitro antimycobacterial activity, showing a minimum inhibitory concentrations (MIC) ranging from 50 -100 μg/mL, and growth inhibitions of 29.5 - 64.0 % on Mtb. Likewise, they causes inhibition of Ca2+-ATPase activity in Mtb membrane vesicles (IC50) ranging from 4.1 - 35.8 μM. Finally, the activity of the compounds with the best biological activity was evaluated in a macrophage infection model, as an approach to evaluate the effect of compounds once the infection has occurred. The compound ZINC63908257 was the best candidate by displaying a MIC of 50 μg/mL, a Ca2+ P-type ATPase inhibition with IC50 = 4.4 μM and 81 % decrease in Mtb replication within macrophage. This compound showed cytotoxic activity of 12.9 % in MH-S cells and hemolysis of 2 % of human erythrocytes, thus, this compound shows a good pharmacokinetic profile (drug-like). Overall, the results presented here shows the importance of the P-type ATPases of Mtb for the mycobacteria survival during infection, and identify the CtpF as a key molecular target for the design of new antituberculous compounds.La Tuberculosis (TB) es una enfermedad infectocontagiosa causada por el bacilo ácido-alcohol resistente Mycobacterium tuberculosis (Mtb). A su vez, la TB es un problema muy relevante de salud pública a nivel mundial. Por otra parte, la aparición de cepas de Mtb resistentes a los fármacos antituberculosos actualmente empleados, ha impulsado la búsqueda de dianas terapéuticas alternativas y metodologías para el diseño racional de nuevos fármacos efectivos. En ese sentido, las proteínas de membrana son considerados blancos de interés al ser mayormente accesibles a los compuestos activos. Particularmente los transportadores de membrana ATPasas tipo P son dianas interesantes por su implicación en la homeóstasis iónica y la viabilidad de las micobacterias. El presente trabajo se orientó en CtpF, una ATPasa tipo P de Mtb transportadora de Ca2+, relacionada con una gran cantidad de condiciones biológicas asociadas al proceso de infección tales como estrés oxidativo, la adaptación del bacilo tuberculoso a condiciones anaeróbicas, hipoxia y latencia. Por lo anterior, el objetivo principal de esta Tesis fue determinar mediante análisis in silico e in vitro el potencial de las ATPasas tipo P de Mtb, especialmente el trasportador de calcio CtpF, como diana para la búsqueda racional de compuestos con actividad antituberculosa. Inicialmente se generó un modelo 3D de CtpF por homología, el que fue empleado para identificar las características farmacofóricas del complejo CtpF-ácido ciclopiazónico (CPA), un inhibidor de la Ca2+-ATPasa de retículo sarco-endoplásmico (SERCA1a), de la que se conoce experimentalmente su estructura 3D, y fue usada como plantilla en la construcción del modelo. Utilizando un repertorio de técnicas experimentales, se evaluó y encontró que CPA causa inhibición de la actividad Ca2+-ATPasa de CtpF, así como actividad micobactericida. El análisis de la respuesta transcripcional de los genes de las ATPasas tipo P2 al tratamiento con CPA, mostró una respuesta específica de ctpF en comparación a otras ATPasas tipo P. Estos resultados iniciales permitieron sugerir a CtpF como una diana molecular para el diseño de compuestos con potencial anti-TB. A continuación, con las características farmacofóricas CtpF-CPA se realizó un cribado virtual basado en farmacóforo utilizando la base de datos ZINC, para seleccionar moléculas candidatas a inhibidores de CtpF. Estudios de acoplamiento molecular y cálculos de MM-GBSA de los candidatos permitieron la identificación de seis compuestos con la mejor energía libre de unión para ser evaluados in vitro. Los compuestos finalmente seleccionados demostraron tener actividad antimicobacteriana mostrando una concentración mínima inhibitoria (CMI) entre 50 - 100 μg/mL, e inhibición del crecimiento de Mtb entre el 29.5 - 64.0 %. De manera similar, causaron inhibición de la actividad Ca2+-ATPasa en vesículas de membrana de Mtb con un rango IC50 entre 4.1 - 35.8 μM. Finalmente se evaluó la actividad de los compuestos con mejor respuesta biológica, en un modelo de infección de macrófagos, como un acercamiento al efecto de los compuestos en la sobrevida de Mtb durante la infección. El compuesto ZINC63908257 fue seleccionado como el candidato más activo con una CMI de 50 μg/mL, inhibición de la actividad Ca2+-ATPasa con IC50 = 4.4 μM y disminución del 81 % de la replicación intracelular de Mtb en macrófagos una vez ocurrida la fagocitosis. Este compuesto demostró un efecto citotóxico del 12.9 % en células MH-S y hemólisis del 2 % de glóbulos rojos humanos, además de presentar propiedades farmacocinéticas adecuadas (drug-like). El conjunto de resultados obtenidos muestra la importancia de las ATPasas tipo P de Mtb para la supervivencia del bacilo durante la infección, e identifican la proteína CtpF como una diana molecular clave para el diseño de nuevos compuestos antituberculosos.Proyecto DIB_2016_Numero_35885 y Colciencias Programa Nacional en Ciencias Básicas-Cod. 110171250419Búsqueda racional de compuestos inhibidores de la actividad ATPasa tipo P de membrana plasmática y determinación de su actividad antimicobacteriana.Doctorado143application/pdfspa572 - Bioquímica614 - Medicina Forense; incidencia de lesiones, heridas, enfermedades; medicina preventiva públicaP-type ATPasestuberculosisantituberculous compoundscyclopiazonic acidmolecular dockingATPasas tipo Ptuberculosiscompuestos antituberculososácido ciclopiazónicoacoplamiento molecularATPasas tipo P de Mycobacterium tuberculosis como dianas para el diseño racional de compuestos antituberculososDocumento de trabajoinfo:eu-repo/semantics/workingPaperinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_8042http://purl.org/coar/version/c_970fb48d4fbd8a85Texthttp://purl.org/redcol/resource_type/WPBogotá - Ciencias - Doctorado en Ciencias - BioquímicaDepartamento de QuímicaUniversidad Nacional de Colombia - Sede BogotáAchard, M.E.S., Stafford, S.L., Bokil, N.J., Chartres, J., Bernhardt, P. 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Drug Discov. 12, 388–404. https://doi.org/10.1038/nrd4001ORIGINALTesis_DoctodoBioquímica_P.Santos_2020.pdfTesis_DoctodoBioquímica_P.Santos_2020.pdfapplication/pdf4276954https://repositorio.unal.edu.co/bitstream/unal/77933/1/Tesis_DoctodoBioqu%c3%admica_P.Santos_2020.pdf38a98443b715a1cd9a1687111ce6a387MD51LICENSElicense.txtlicense.txttext/plain; charset=utf-83991https://repositorio.unal.edu.co/bitstream/unal/77933/2/license.txt6f3f13b02594d02ad110b3ad534cd5dfMD52CC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-8811https://repositorio.unal.edu.co/bitstream/unal/77933/3/license_rdf217700a34da79ed616c2feb68d4c5e06MD53THUMBNAILTesis_DoctodoBioquímica_P.Santos_2020.pdf.jpgTesis_DoctodoBioquímica_P.Santos_2020.pdf.jpgGenerated Thumbnailimage/jpeg4350https://repositorio.unal.edu.co/bitstream/unal/77933/4/Tesis_DoctodoBioqu%c3%admica_P.Santos_2020.pdf.jpg33f0b6d1304f88c31245fb48c75dc9a3MD54unal/77933oai:repositorio.unal.edu.co:unal/779332024-07-18 23:10:20.286Repositorio 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