Identificación de compuestos con afinidad de unión por la proteína Lon en Acinetobacter baumannii por medio de un análisis in silico

Acinetobacter baumannii hace parte de la lista de bacterias que necesitan con urgencia nuevos antibióticos, junto a otras bacterias se encuentran como prioridad número 1 (crítica) para investigación y descubrimiento de nuevos antibióticos, además hace parte de las infecciones asociadas a la atención...

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Autores:
Lara Sanabria, David Orlando
Chicuasuque Pérez, Natalia
Tipo de recurso:
Trabajo de grado de pregrado
Fecha de publicación:
2021
Institución:
Colegio Mayor de Cundinamarca
Repositorio:
Repositorio Colegio Mayor de Cundinamarca
Idioma:
spa
OAI Identifier:
oai:repositorio.unicolmayor.edu.co:unicolmayor/5551
Acceso en línea:
https://repositorio.unicolmayor.edu.co/handle/unicolmayor/5551
Palabra clave:
Targets of action
Multidrug resistance
HAI
Pathogens
Acinetobacter baumannii
Lon protein
Serine proteases
ATP-dependent serine protease
Rights
closedAccess
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Derechos Reservados - Universidad Colegio Mayor de Cundinamarca, 2021
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oai_identifier_str oai:repositorio.unicolmayor.edu.co:unicolmayor/5551
network_acronym_str UCOLMAYOR2
network_name_str Repositorio Colegio Mayor de Cundinamarca
repository_id_str
dc.title.spa.fl_str_mv Identificación de compuestos con afinidad de unión por la proteína Lon en Acinetobacter baumannii por medio de un análisis in silico
title Identificación de compuestos con afinidad de unión por la proteína Lon en Acinetobacter baumannii por medio de un análisis in silico
spellingShingle Identificación de compuestos con afinidad de unión por la proteína Lon en Acinetobacter baumannii por medio de un análisis in silico
Targets of action
Multidrug resistance
HAI
Pathogens
Acinetobacter baumannii
Lon protein
Serine proteases
ATP-dependent serine protease
title_short Identificación de compuestos con afinidad de unión por la proteína Lon en Acinetobacter baumannii por medio de un análisis in silico
title_full Identificación de compuestos con afinidad de unión por la proteína Lon en Acinetobacter baumannii por medio de un análisis in silico
title_fullStr Identificación de compuestos con afinidad de unión por la proteína Lon en Acinetobacter baumannii por medio de un análisis in silico
title_full_unstemmed Identificación de compuestos con afinidad de unión por la proteína Lon en Acinetobacter baumannii por medio de un análisis in silico
title_sort Identificación de compuestos con afinidad de unión por la proteína Lon en Acinetobacter baumannii por medio de un análisis in silico
dc.creator.fl_str_mv Lara Sanabria, David Orlando
Chicuasuque Pérez, Natalia
dc.contributor.advisor.none.fl_str_mv Sánchez Mora, Ruth Mélida
Romero Calderón, Ibeth Cristina
dc.contributor.author.none.fl_str_mv Lara Sanabria, David Orlando
Chicuasuque Pérez, Natalia
dc.subject.lemb.none.fl_str_mv Targets of action
Multidrug resistance
HAI
Pathogens
topic Targets of action
Multidrug resistance
HAI
Pathogens
Acinetobacter baumannii
Lon protein
Serine proteases
ATP-dependent serine protease
dc.subject.proposal.spa.fl_str_mv Acinetobacter baumannii
dc.subject.proposal.eng.fl_str_mv Lon protein
Serine proteases
ATP-dependent serine protease
description Acinetobacter baumannii hace parte de la lista de bacterias que necesitan con urgencia nuevos antibióticos, junto a otras bacterias se encuentran como prioridad número 1 (crítica) para investigación y descubrimiento de nuevos antibióticos, además hace parte de las infecciones asociadas a la atención en salud (IAAS). Para el control de la enfermedad causada por A. baumannii es indispensable la búsqueda de nuevos blancos terapéuticos alternativos para implementar nuevos tratamientos, por ello se investigó a fondo las características de la proteasa Lon, una proteína que cumple diversas funciones muy importantes en Acinetobacter baumannii entre ellas la degradación de proteínas. Es por ello que en el presente trabajo se realizó una caracterización in silico de la proteína Lon de A. baumannii, así como también una identificación de compuestos con afinidad por la proteína mediante el uso de herramientas y bases de datos bioinformáticas de libre acceso. Lo cual permitió demostrar que A. baumannii posee un gen que codifica para la proteína Lon, la cual presenta todos los motivos y dominios importantes para la actividad catalítica que han sido descritos en la familia de proteasas Lon en bacterias Gram negativas. Mediante el uso de la herramienta SWISS-MODEL se obtuvo el modelo tridimensional de la proteína a partir de una plantilla de la proteasa Lon de E. coli, el cual presentó una topología correcta y alta calidad con puntajes típicamente encontrados en proteínas nativas. El acoplamiento molecular y las predicciones farmacocinéticas ADMET permitieron identificar compuestos con alta afinidad por la proteína Lon, dentro de las cuales se destacan ZINC000003435531, ZINC000005006669, ZINC000034924974, ZINC000000426267 y ZINC000041721989 como candidatos potenciales para estudios in vitro y el posible desarrollo a futuro de una terapia alternativa y selectiva como tratamiento de infecciones asociadas a A. baumannii.
publishDate 2021
dc.date.issued.none.fl_str_mv 2021-09
dc.date.accessioned.none.fl_str_mv 2022-05-10T02:31:22Z
dc.date.available.none.fl_str_mv 2022-05-10T02:31:22Z
dc.type.spa.fl_str_mv Trabajo de grado - Pregrado
dc.type.coar.spa.fl_str_mv http://purl.org/coar/resource_type/c_7a1f
dc.type.coarversion.spa.fl_str_mv http://purl.org/coar/version/c_970fb48d4fbd8a85
dc.type.content.spa.fl_str_mv Text
dc.type.driver.spa.fl_str_mv info:eu-repo/semantics/bachelorThesis
dc.type.redcol.spa.fl_str_mv https://purl.org/redcol/resource_type/TP
dc.type.version.spa.fl_str_mv info:eu-repo/semantics/publishedVersion
format http://purl.org/coar/resource_type/c_7a1f
status_str publishedVersion
dc.identifier.uri.none.fl_str_mv https://repositorio.unicolmayor.edu.co/handle/unicolmayor/5551
url https://repositorio.unicolmayor.edu.co/handle/unicolmayor/5551
dc.language.iso.spa.fl_str_mv spa
language spa
dc.relation.references.spa.fl_str_mv Joly-Guillou M. Clinical impact and pathogenicity of Acinetobacter. Clinical Microbiology and Infection. [Internet]. 2005. [cited 18 Dec 2020]. Available in: https://www.sciencedirect.com/science/article/pii/S1198743X14626008
Antunes L, Visca P, Towner K. Acinetobacter baumannii: evolution of a global pathogen. Pathogens and Disease. [Internet]. 2014. [cited 18 Dec 2020]. Available in: https://academic.oup.com/femspd/article/71/3/292/475786
La OMS publica la lista de las bacterias para las que se necesitan urgentemente nuevos antibióticos [Internet]. Who.int. 2017 [citado 20 septiembre 2020]. Disponible en: https://www.who.int/es/news-room/detail/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-needed
Cooper M, Shlaes D. Fix the antibiotics pipeline. Nature. [Internet]. 2011. [cited 18 Dec 2020]. Available in: https://www.nature.com/articles/472032a
Breidenstein E, Janot L, Strehmel J, Fernández L, Taylor PK, Kukavica-Ibrulj I, et al. The Lon Protease Is Essential for Full Virulence in Pseudomonas aeruginosa. PLoS One. [Internet]. 2012. [cited 18 Dec 2020]. Available in: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0049123
Rogers A et al. The LonA Protease Regulates Biofilm Formation, Motility, Virulence, and the Type VI Secretion System in Vibrio cholerae. Journal of bacteriology. [Internet]. 2016. [cited 18 Dec 2020]. 198(6), 973–985. Available in: https://journals.asm.org/doi/10.1128/JB.00741-15
Marr AK, Overhage J, Bains M, Hancock REW. The Lon protease of Pseudomonas aeruginosa is induced by aminoglycosides and is involved in biofilm formation and motility. Microbiology. [Internet]. 2007. [cited 18 Dec 2020]. 1;153(2):474–82. Available in: https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.20 06/002519-0#tab2
Asif M, Alvi I, Rehman S. Insight into Acinetobacter baumannii: pathogenesis, global resistance, mechanisms of resistance, treatment options, and alternative modalities. Infection and Drug Resistance. [Internet]. 2018. [cited 17 Dec 2020]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6110297/
Boletín Epidemiológico Semanal [Internet]. Instituto nacional de salud. 2020 [citado 20 septiembre 2020]. Disponible en: https://www.ins.gov.co/buscador-eventos/BoletinEpidemiologico/2020_Boletin_epidemiologico_semana_10.pdf#search=Acinetobacter%20baumannii
Khan H, Baig F, Mehboob R. Nosocomial infections: Epidemiology, prevention, control and surveillance. Asian Pacific Journal of Tropical Biomedicine. [Internet]. 2017. [cited 16 Dec 2021]. 7(5), 478-482. Available in: https://www.sciencedirect.com/science/article/pii/S2221169116309509?via%3Dihub
BROTE EPIDÉMICO POR ACINETOBACTER BAUMANII PRODUCTOR DE CARBAPENAMASA OXA-23 LIKE EN UNA UNIDAD DE HOSPITALIZACIÓN. Rev Esp Salud Pública. [Internet]. 2021 [citado 10 Jun 2021]. Disponible en: https://www.mscbs.gob.es/biblioPublic/publicaciones/recursos_propios/resp/revista_cdrom/VOL95/O_BREVES/RS95C_202102027.pdf
Haque M, Sartelli M, McKimm J, Bakar M. Health care-associated infections – an overview. Infection and Drug Resistance. [Internet]. 2018. [cited 17 Dec 2020]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6245375/
Mulani MS, Kamble EE, Kumkar SN, Tawre MS, Pardesi KR. Emerging Strategies to Combat ESKAPE Pathogens in the Era of Antimicrobial Resistance: A Review. Front Microbiol [Internet]. 2019 [cited 2021 Jun 10]. Available from: https://www.frontiersin.org/articles/10.3389/fmicb.2019.00539/pdf
Rodríguez C, Nastro M, Famiglietti A. Carbapenemases in Acinetobacter baumannii. Review of their dissemination in Latin America. Revista Argentina de Microbiología. [Internet]. 2018. [cited 11 jun 2021]. 50(3), 327-333. Available in: https://www.sciencedirect.com/science/article/pii/S0325754117301785?via%3Dihub
Urrutia Gómez JA, Rueda Riaño AM, Rojas Páez CA, Silva Rodríguez MA, Méndez Faniño YR. Eficacia de la colisina en el tratamiento de pacientes adultos con infecciones severas por Acinetobacter baummannii XDR en cuidados intensivos. Univ. Med. [Internet]. 15 de julio de 2016 [citado 11 de junio de 2021];57(2):212-25. Disponible en: https://revistas.javeriana.edu.co/index.php/vnimedica/article/view/17036
Varón Fabio Andres, Uribe Ana Maria, Palacios Jonathan Orlando, Sánchez Efraín Guillermo, Gutiérrez Deimer, Carvajal Katherine et al. Mortalidad y desenlaces clínicos en pacientes críticamente enfermos con infecciones por bacterias productoras de carbapenemasas en un hospital de alta complejidad en Bogotá, Colombia. Infect. [Internet]. 2021 Mar [cited 2021 June 11] ; 25 (1): 16-21. Available from: http://www.scielo.org.co/scielo.php?script=sci_arttext&pid=S0123-93922021000100016&lng=en.
Lemos EV, De la Hoz Restrepo F, Alvis N, Quevedo E, Cañon O, León Y. Mortalidad por Acinetobacter baumannii en unidades de cuidados intensivos en Colombia. Rev Panam Salud Publica. [Internet]. 2011. [citado 18 oct 2020];30:287–94. Disponible en::https://www.scielosp.org/article/rpsp/2011.v30n4/287-294/#ModalArticles
He L, Luo D, Yang F, Li C, Zhang X, Deng H, Zhang J. Multiple domains of bacterial and human Lon proteases define substrate selectivity. Emerging microbes & infections. [Internet]. 2018. [cited 14 oct 2020];7(1), 149. Available in: https://www.tandfonline.com/doi/full/10.1038/s41426-018-0148-4
Hua C, Wang T, Shao X, Xie Y, Huang H, Liu J. Pseudomonas syringae dual‐function protein Lon switches between virulence and metabolism by acting as both DNA ‐binding transcriptional regulator and protease in different environments. Environmental Microbiology. [Internet]. 2020. [cited 14 oct 2020]. Available in: https://sfamjournals.onlinelibrary.wiley.com/doi/abs/10.1111/1462-2920.15067
Muselius B. Iron Limitation in Klebsiella pneumoniae Defines New Roles for Lon Protease in Homeostasis and Degradation by Quantitative Proteomics. frontiers in Microbiology. [Internet]. 2020. [cited 14 oct 2020
McVicker G, Hollingshead S, Pilla G, Tang CM. Maintenance of the virulence plasmid in Shigella flexneri is influenced by Lon and two functional partitioning systems. Molecular Microbiology. [Internet]. 2019. [cited 14 oct 2020]. 111 (5), 1355-1366. Available in: https://onlinelibrary.wiley.com/doi/full/10.1111/mmi.14225
Pei J, Yan J, Jiang Y. Characterization of the ATP-Dependent Lon-Like Protease in Methanobrevibacter smithii. Archaea. [Internet]. 2016 [cited 25 jun 2020]. Available in: https://www.hindawi.com/journals/archaea/2016/5759765
Gur E et al. Regulated proteolysis in Gram-negative bacteria — how and when?. Nature Reviews Microbiology. [Internet]. 2011 [cited 28 oct 2020]. Volume 9(12), 839–848. Available in: https://www.nature.com/articles/nrmicro2669
Raju R, Goldberg A, Rubin E. Bacterial proteolytic complexes as therapeutic targets. nature reviews drug discovery. [Internet]. 2012 [cited 28 oct 2020]; Volume (11): 777-789. Available in: https://www.nature.com/articles/nrd3846
Bayot A, Basse N, Lee I, Gareil M, Pirotte B, Bulteau A, Friguet B, Reboud-Ravaux, M. Towards the control of intracellular protein turnover: Mitochondrial Lon protease inhibitors versus proteasome inhibitors. Biochimie. [Internet]. 2008 [cited 26 oct 2020]. Available in: https://www.sciencedirect.com/science/article/abs/pii/S0300908407003045?via%3Dihub
Hosseingholi E, Zarrini G, Pashazadeh M, Mohammad S, Hayat G, Molavi G. In Silico Identification of Probable Drug and Vaccine Candidates Against Antibiotic-Resistant Acinetobacter baumannii. Microbial Drug Resistance. [Internet]. 2020. [cited 14 oct 2020]. Available in: https://www.liebertpub.com/doi/10.1089/mdr.2019.0236?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmed
Culp E, Wright GD. Bacterial proteases, untapped antimicrobial drug targets. J Antibiot (Tokyo). [Internet]. 2017. [cited 10 nov 2020];70(4):366-377. Available in: https://www.nature.com/articles/ja2016138
Moubarek C, Halat D. Insights into Acinetobacter baumannii: A Review of Microbiological, Virulence, and Resistance Traits in a Threatening Nosocomial Pathogen. Antibiotics. [Internet]. 2020. [cited 18 Dec 2020]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7148516/
Chaari A, Mnif B, Bahloul M, Mahjoubi F, Chtara K, Turki O, et al. Acinetobacter baumannii ventilator-associated pneumonia: epidemiology, clinical characteristics, and prognosis factors. Int J Infect Dis. [Internet].2013. [cited 10 Dec 2020];17(12):e1225-8. Available in: https://www.ijidonline.com/article/S1201-9712(13)00249-X/fulltext
Cillóniz C, Dominedò C, Torres A. Multidrug Resistant Gram-Negative Bacteria in Community-Acquired Pneumonia. Annual Update in Intensive Care and Emergency Medicine. [Internet]. 2019. [cited 10 Dec 2020]. 459–475. Available in: https://link.springer.com/chapter/10.1007%2F978-3-030-06067-1_36
Emanuele Durante-Mangoni et al. Colistin and Rifampicin Compared With Colistin Alone for the Treatment of Serious Infections Due to Extensively Drug-Resistant Acinetobacter baumannii: A Multicenter, Randomized Clinical Trial. Clinical Infectious Diseases.[Internet]. 2013. [cited 15 Dec 2020]. 349–358. Available in: https://academic.oup.com/cid/article/57/3/349/461315
Shi H, Lee J, Park S, Ko Y, Eom J. Colistin Plus Carbapenem versus Colistin Monotherapy in the Treatment of Carbapenem-Resistant Acinetobacter baumannii Pneumonia. Infection and drug resistance. [Internet]. 2019. [cited 18 Dec 2020]. 12, 3925–3934. Available in: https://www.dovepress.com/colistin-plus-carbapenem-versus-colistin-monotherapy-in-the-treatment--peer-reviewed-fulltext-article-IDR
Poulikakos P, Tansarli G, Falagas M. Combination antibiotic treatment versus monotherapy for multidrug-resistant, extensively drug-resistant, and pandrug-resistant Acinetobacter infections: a systematic review. European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology. [Internet]. 2014. [cited 18 Dec 2020].33(10), 1675–1685. Available in: https://link.springer.com/article/10.1007%2Fs10096-014-2124-9
Baran G, Erbay A, Bodur H, Onguru P, Akinci E et al. Risk factors for nosocomial imipenem-resistant Acinetobacter baumannii infections. International Journal of Infectious Diseases. [Internet]. 2008. [cited 18 Dec 2020]. Available in: https://www.ijidonline.com/article/S1201-9712(07)00068-9/fulltext
Freire M, Oliveira D, Garcia P, Hoff P, Pierrotti L, Abdala E. Bloodstream infection caused by extensively drug-resistant Acinetobacter baumannii in cancer patients: high mortality associated with delayed treatment rather than with the degree of neutropenia. Clinical Microbiology and Infection. [Internet]. 2008. [cited 18 Dec 2020]. Available in: https://www.clinicalmicrobiologyandinfection.com/article/S1198-743X(15)01036-8/fulltext
Davis K, Moran K, McAllister C, Gray P. Multidrug-Resistant Acinetobacter Extremity Infections in Soldiers. Emerging Infectious Diseases. [Internet]. 2005. [cited 18 Dec 2020]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3320488/
Alekshun M, Levy S. Molecular Mechanisms of Antibacterial Multidrug Resistance. Cell. [Internet]. 2007. [cited 18 Dec 2020]. Available in: https://www.sciencedirect.com/science/article/pii/S009286740700311X
Tacconelli E, Carrara E, Savoldi A, Harbarth S, Mendelson M, Monnet D et al. Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. The Lancet Infectious Diseases. [Internet]. 2018. [cited 18 Dec 2020]. Available in: https://www.thelancet.com/journals/laninf/article/PIIS1473-3099(17)30753-3/fulltext
Central Asian and Eastern European Surveillance of Antimicrobial Resistance [Internet]. Euro.who.int. 2017 [cited 18 December 2020]. Available in: https://www.euro.who.int/__data/assets/pdf_file/0005/354434/WHO_CAESAR_AnnualReport_2017.pdf?ua=1
Zhanel, G.G., Golden, A.R., Zelenitsky, S. et al. Cefiderocol: A Siderophore Cephalosporin with Activity Against Carbapenem-Resistant and Multidrug-Resistant Gram-Negative Bacilli. Drugs 79. [Internet]. 2019. [cited 18 Dec 2020]. 271–289. Available in: https://link.springer.com/article/10.1007%2Fs40265-019-1055-2
Isler B, Doi Y, Bonomo R, Paterson D. New Treatment Options against Carbapenem-Resistant Acinetobacter baumannii Infections. Antimicrobial Agents and Chemotherapy. [Internet]. 2018. [cited 18 Dec 2020]. Available in: https://aac.asm.org/content/63/1/e01110-18
Spellberg B, Bonomo R. Combination Therapy for Extreme Drug Resistant (XDR) Acinetobacter baumannii: Ready for Prime-Time?. Critical Care Medicine. [Internet]. 2015. [cited 18 Dec 2020]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4620434/
Khanh N, Riordan D, Hoang T, Thanh D, Thwaites G, Huong N et al. The induction and identification of novel Colistin resistance mutations in Acinetobacter baumannii and their implications. Scientific Reports. [Internet]. 2016. [cited 18 Dec 2020]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4916428/
Hua Y, Luo T, Yang Y, Dong D, Wang R, Wang J. Phage Therapy as a Promising New Treatment for Lung Infection Caused by Carbapenem-Resistant Acinetobacter baumannii in Mice. Frontiers in Microbiology. [Internet]. 2018. [cited 18 Dec 2020]. Available in: https://www.frontiersin.org/articles/10.3389/fmicb.2017.02659/full
Sauer R, Baker T. AAA+ Proteases: ATP-Fueled Machines of Protein Destruction. Annual Review of Biochemistry. [Internet]. 2011 [cited 14 oct 2020].(80): 587–612 Available in: https://www.annualreviews.org/doi/10.1146/annurev-biochem-060408-172623?url_ver=Z39.88-2003&rfr_id=ori%3Arid%3Acrossref.org&rfr_dat=cr_pub++0pubmed
Ching C, Yang B, Onwubueke C, Lazinski D, Camilli A, Godoy V. Lon Protease Has Multifaceted Biological Functions in Acinetobacter baumannii. J Bacteriol. [Internet]. 2018 [cited 25 jun 2020]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6304660/
Meng X, Zhang H, Mezei M, Cui M. Molecular docking: a powerful approach for structure-based drug discovery. Current computer-aided drug design. [Internet]. 2011. [cited 24 jul 2020]. 7(2), 146–157. Available in: https://doi.org/10.2174/157340911795677602
Silakari O, Singh P. Chapter 6 - Molecular docking analysis: Basic technique to predict drug-receptor interactions. Concepts and Experimental Protocols of Modelling and Informatics in Drug Design. [Internet]. 2021. [cited 24 jul 2020]. Pages 131-155. Available in: https://www.sciencedirect.com/science/article/pii/B9780128205464000064
Kitchen D, Decornez H, Furr J et al. Docking and scoring in virtual screening for drug discovery: methods and applications. Nat Rev Drug Discov 3. [Internet]. 2004. [cited 24 jul 2020]. 935–949. Available in: https://www.nature.com/articles/nrd1549
Lantz M. Are bacterial proteases important virulence factors?. J Periodontal. [Internet]. 2006. [cited 25 jul 2020]; Volume (32):126-132. Available in: https://pubmed.ncbi.nlm.nih.gov/9085222/
García-González G, González G, Palma-Nicolás J. Las proteasas de serina bacterianas y su implicación en la fisiopatología de la infección. Rev Laboratorio Clínico. [Internet]. 2019 [citado 28 junio 2020]; Volumen (12): 137-146. Disponible en: https://www.elsevier.es/es-revista-revista-del-laboratorio-clinico-282-articulo-las-proteasas-serina-bacterianas-su-S1888400819300303
Spyrakis F, Santucci M, Maso L et al. Virtual screening identifies broad-spectrum β-lactamase inhibitors with activity on clinically relevant serine- and metallo-carbapenemases. nature scientific reports. [Internet]. 2020. [cited 28 oct 2020]. Available in: https://www.nature.com/articles/s41598-020-69431-y#citeas
Kaur G, Pandey B, Kumar A, Garewal N, Grover A, Kaur J. Drug targeted virtual screening and molecular dynamics of LipU protein of Mycobacterium tuberculosis and Mycobacterium leprae. Journal of Biomolecular Structure and Dynamics. [Internet]. 2019. [cited 28 oct 2020]. Available in: https://www.tandfonline.com/action/showCitFormats?doi=10.1080%2F07391102.2018.1454852
Nitulescu G, Nicorescu IM, Olaru OT, et al. Molecular Docking and Screening Studies of New Natural Sortase A Inhibitors. Int J Mol Sci. [Internet]. 2017. [cited 28 oct 2020]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666896/
Skariyachan S, Manjunath M, Bachappanavar N. Screening of potential lead molecules against prioritised targets of multi-drug-resistant-Acinetobacter baumannii - insights from molecular docking, molecular dynamic simulations and in vitro assays. Journal of Biomolecular Structure and Dynamics. [Internet]. 2017. [cited 28 oct 2020]. Available in: https://pubmed.ncbi.nlm.nih.gov/29529934/
Verma P, Tiwari M, Tiwari V. In silico high-throughput virtual screening and molecular dynamics simulation study to identify inhibitor for AdeABC efflux pump of Acinetobacter baumannii. Journal of Biomolecular Structure and Dynamics. [Internet]. 2017. [cited 28 oct 2020]. Available in: https://www.tandfonline.com/doi/full/10.1080/07391102.2017.1317025?src=recsys
Skariyachan S, Gopal D, Kadam S, Muddebihalkar A, Uttarkar A, Niranjan V. Carbon fullerene acts as a potential lead molecule against prospective molecular targets of biofilm-producing multidrug-resistant Acinetobacter baumannii and Pseudomonas aeruginosa: computational modeling and MD simulation studies. Journal of Biomolecular Structure and Dynamics. [Internet]. 2020. [cited 28 oct 2020]. Available in: https://www.tandfonline.com/doi/full/10.1080/07391102.2020.1726821?src=recsys
RESOLUCIÓN NÚMERO 8430 DE 1993 [Internet]. Hospitalsanpedro.org. 1993 [cited 20 jan 2021]. Available from: https://www.hospitalsanpedro.org/images/Comite_Investigacion/Resolucion_8430_de_1993.pdf
Benkert P, Biasini M, Schwede T. Toward the estimation of the absolute quality of individual protein structure models. Bioinformatics (Oxford, England), 27(3), 343–350. [Internet]. 2011. [cited 24 may 2021]. Available in: https://academic.oup.com/bioinformatics/article/27/3/343/320383
Markus Wiederstein, Manfred J. Sippl, ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Research, Volume 35, Issue suppl_2, 1 July 2007, Pages W407–W410. [Internet]. 2007. [cited 25 may 2021]. Available in: https://academic.oup.com/nar/article/35/suppl_2/W407/2920938
Laskowski A, MacArthur M, Moss D, Thornton J. PROCHECK: a program to check the stereochemical quality of protein structures. Journal of Applied Crystallography, 26(2), 283–291. [Internet]. 1993. [cited 25 mar 2021]. Available in: https://onlinelibrary.wiley.com/doi/epdf/10.1107/S0021889892009944
Pundir S, Magrane M, Martin MJ, O'Donovan C. Searching and Navigating UniProt Databases. Curr Protoc Bioinformatics. [Internet]. 2016. [cited 15 feb 2021]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4522465/
Rotanova T, Botos I, Melnikov E, Rasulova F, Gustchina A, Maurizi M, Wlodawer A. Slicing a protease: Structural features of the ATP-dependent Lon proteases gleaned from investigations of isolated domains. Protein Science. [Internet]. 2006. [cited 24 jul 2021]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2242575/
Park S, Jia B, Yang J, Le Van D, Shao Y, Woo S et al. Oligomeric Structure of the ATP-dependent Protease La (Lon) of Escherichia coli. Molecules and Cells. [Internet]. 2006. [cited 24 jul 2021]. Available in: http://www.molcells.org/journal/view.html?year=2006&volume=21&number=1&spage=129#body01
Carr J, Arduino M, Swenson J. Acinetobacter baumannii. Centers for Disease Control and Prevention (CDC). [Internet]. 2004. [cited 21 jan 2021]. Available in: https://phil.cdc.gov/Details.aspx?pid=6498
Parker T. Colonial growth displayed by the Gram-negative bacterium Acinetobacter baumannii. Centers for Disease Control and Prevention (CDC). [Internet]. 2014. [cited 21 jan 2021]. Available in: https://phil.cdc.gov/Details.aspx?pid=17068
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spelling Sánchez Mora, Ruth Mélidaefe8f5fbfdf7ec7c24ba850634bfbb9aRomero Calderón, Ibeth Cristina1be6cd68f2562dc4b43f161eb1ab77a0Lara Sanabria, David Orlando879f7c2cdc900d5b206b31d37f49345cChicuasuque Pérez, Natalia1662bf984e05c82cdf48b0b07193d8962022-05-10T02:31:22Z2022-05-10T02:31:22Z2021-09https://repositorio.unicolmayor.edu.co/handle/unicolmayor/5551Acinetobacter baumannii hace parte de la lista de bacterias que necesitan con urgencia nuevos antibióticos, junto a otras bacterias se encuentran como prioridad número 1 (crítica) para investigación y descubrimiento de nuevos antibióticos, además hace parte de las infecciones asociadas a la atención en salud (IAAS). Para el control de la enfermedad causada por A. baumannii es indispensable la búsqueda de nuevos blancos terapéuticos alternativos para implementar nuevos tratamientos, por ello se investigó a fondo las características de la proteasa Lon, una proteína que cumple diversas funciones muy importantes en Acinetobacter baumannii entre ellas la degradación de proteínas. Es por ello que en el presente trabajo se realizó una caracterización in silico de la proteína Lon de A. baumannii, así como también una identificación de compuestos con afinidad por la proteína mediante el uso de herramientas y bases de datos bioinformáticas de libre acceso. Lo cual permitió demostrar que A. baumannii posee un gen que codifica para la proteína Lon, la cual presenta todos los motivos y dominios importantes para la actividad catalítica que han sido descritos en la familia de proteasas Lon en bacterias Gram negativas. Mediante el uso de la herramienta SWISS-MODEL se obtuvo el modelo tridimensional de la proteína a partir de una plantilla de la proteasa Lon de E. coli, el cual presentó una topología correcta y alta calidad con puntajes típicamente encontrados en proteínas nativas. El acoplamiento molecular y las predicciones farmacocinéticas ADMET permitieron identificar compuestos con alta afinidad por la proteína Lon, dentro de las cuales se destacan ZINC000003435531, ZINC000005006669, ZINC000034924974, ZINC000000426267 y ZINC000041721989 como candidatos potenciales para estudios in vitro y el posible desarrollo a futuro de una terapia alternativa y selectiva como tratamiento de infecciones asociadas a A. baumannii.Acinetobacter baumannii is part of the list of bacteria that urgently need new antibiotics, along with other bacteria are as priority number 1 (critical) for research and discovery of new antibiotics, it is also part of the healthcare associated infections (HAI). For the control of the disease caused by A. baumannii it is essential to search for a therapeutic target to implement new alternative treatments, therefore, the characteristics of the Lon protease, a protein that fulfills several very important functions in Acinetobacter baumannii, including protein degradation, were thoroughly investigated. For this reason, in the present work, an in silico characterization of the Lon protein of A. baumannii was carried out, as well as an identification of compounds with affinity for the protein by using freely available bioinformatics tools and databases. This allowed demonstrating that A. baumannii possesses a gene coding for the Lon protein, which presents all the motifs and domains important for catalytic activity that have been described in the Lon family of proteases in Gram-negative bacteria. Using the SWISS-MODEL tool, the three-dimensional model of the protein was obtained from a template of the E. coli Lon protease, which presented a correct topology and high quality with scores typically found in native proteins. Molecular docking and ADMET pharmacokinetic predictions allowed the identification of compounds with high affinity for the Lon protein, among which ZINC000003435531, ZINC000005006669, ZINC000034924974, ZINC000000426267 and ZINC000041721989 stand out as potential candidates for in vitro studies and the possible future development of an alternative and selective therapy for the treatment of infections associated with Acinetobacter baumannii.Resumen ……………………………………....……………………….………………….. 7 Abstract ………………………………………………………………….…………………. 8 Introducción ………………………………………………………………………………... 9 1.Objetivos ……………………………………………………………….……………….. 12 1.1 Objetivo general …………………………………………………….……………….. 12 1.2 Objetivos específicos ……………………………………………….……………….. 12 2. Marco referencial ……………………………………………………………………… 13 2.1. Antecedentes o estado del arte ………………………………………………....… 13 2.2. Marco teórico ………………………………………………………………………... 15 2.2.1. Acinetobacter baumannii: características generales ………………………..... 15 2.2.2. Enfermedades ocasionadas por A. baumannii ………………………………… 16 2.2.3. Tratamiento y resistencia ………………………………………………………… 16 2.2.4. Búsqueda de nuevos medicamentos para el control de A. baumannii ……... 20 2.2.5. Estudio de Proteasas Lon como posible blanco terapéutico ………………… 20 2.2.6. Proteasa Lon en Acinetobacter baumannii, función y clasificación …………. 23 2.2.7. Biología computacional en el diseño racional de fármacos…………….…….. 24 3. Bases Legales …………………………………………………………………………. 28 4. Materiales y métodos …………………………………………………………………. 28 5. Resultados ……………………………………………………………………………... 34 6. Discusión ……………………………………………………………………………….. 50 7. Conclusiones ………………………………………………………………………...… 52 8. Perspectivas …………………………………………………………………...….…… 53 9. Referencias bibliográficas ………………………………….…………………….…... 54 10. Anexos ……………………………………………………....………………………... 66PregradoBacteriólogo(a) y Laboratorista Clínico73p.application/pdfspaUniversidad Colegio Mayor de CundinamarcaFacultad de Ciencias de la SaludBogotáBacteriología y Laboratorio ClínicoDerechos Reservados - Universidad Colegio Mayor de Cundinamarca, 2021https://creativecommons.org/licenses/by-nc-sa/4.0/info:eu-repo/semantics/closedAccessAtribución-NoComercial-CompartirIgual 4.0 Internacional (CC BY-NC-SA 4.0)http://purl.org/coar/access_right/c_14cbIdentificación de compuestos con afinidad de unión por la proteína Lon en Acinetobacter baumannii por medio de un análisis in silicoTrabajo de grado - Pregradohttp://purl.org/coar/resource_type/c_7a1fhttp://purl.org/coar/version/c_970fb48d4fbd8a85Textinfo:eu-repo/semantics/bachelorThesishttps://purl.org/redcol/resource_type/TPinfo:eu-repo/semantics/publishedVersionJoly-Guillou M. Clinical impact and pathogenicity of Acinetobacter. Clinical Microbiology and Infection. [Internet]. 2005. [cited 18 Dec 2020]. Available in: https://www.sciencedirect.com/science/article/pii/S1198743X14626008Antunes L, Visca P, Towner K. Acinetobacter baumannii: evolution of a global pathogen. Pathogens and Disease. [Internet]. 2014. [cited 18 Dec 2020]. Available in: https://academic.oup.com/femspd/article/71/3/292/475786La OMS publica la lista de las bacterias para las que se necesitan urgentemente nuevos antibióticos [Internet]. Who.int. 2017 [citado 20 septiembre 2020]. Disponible en: https://www.who.int/es/news-room/detail/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-neededCooper M, Shlaes D. Fix the antibiotics pipeline. Nature. [Internet]. 2011. [cited 18 Dec 2020]. Available in: https://www.nature.com/articles/472032aBreidenstein E, Janot L, Strehmel J, Fernández L, Taylor PK, Kukavica-Ibrulj I, et al. The Lon Protease Is Essential for Full Virulence in Pseudomonas aeruginosa. PLoS One. [Internet]. 2012. [cited 18 Dec 2020]. Available in: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0049123Rogers A et al. The LonA Protease Regulates Biofilm Formation, Motility, Virulence, and the Type VI Secretion System in Vibrio cholerae. Journal of bacteriology. [Internet]. 2016. [cited 18 Dec 2020]. 198(6), 973–985. Available in: https://journals.asm.org/doi/10.1128/JB.00741-15Marr AK, Overhage J, Bains M, Hancock REW. The Lon protease of Pseudomonas aeruginosa is induced by aminoglycosides and is involved in biofilm formation and motility. Microbiology. [Internet]. 2007. [cited 18 Dec 2020]. 1;153(2):474–82. Available in: https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.20 06/002519-0#tab2Asif M, Alvi I, Rehman S. Insight into Acinetobacter baumannii: pathogenesis, global resistance, mechanisms of resistance, treatment options, and alternative modalities. Infection and Drug Resistance. [Internet]. 2018. [cited 17 Dec 2020]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6110297/Boletín Epidemiológico Semanal [Internet]. Instituto nacional de salud. 2020 [citado 20 septiembre 2020]. Disponible en: https://www.ins.gov.co/buscador-eventos/BoletinEpidemiologico/2020_Boletin_epidemiologico_semana_10.pdf#search=Acinetobacter%20baumanniiKhan H, Baig F, Mehboob R. Nosocomial infections: Epidemiology, prevention, control and surveillance. Asian Pacific Journal of Tropical Biomedicine. [Internet]. 2017. [cited 16 Dec 2021]. 7(5), 478-482. Available in: https://www.sciencedirect.com/science/article/pii/S2221169116309509?via%3DihubBROTE EPIDÉMICO POR ACINETOBACTER BAUMANII PRODUCTOR DE CARBAPENAMASA OXA-23 LIKE EN UNA UNIDAD DE HOSPITALIZACIÓN. Rev Esp Salud Pública. [Internet]. 2021 [citado 10 Jun 2021]. Disponible en: https://www.mscbs.gob.es/biblioPublic/publicaciones/recursos_propios/resp/revista_cdrom/VOL95/O_BREVES/RS95C_202102027.pdfHaque M, Sartelli M, McKimm J, Bakar M. Health care-associated infections – an overview. Infection and Drug Resistance. [Internet]. 2018. [cited 17 Dec 2020]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6245375/Mulani MS, Kamble EE, Kumkar SN, Tawre MS, Pardesi KR. Emerging Strategies to Combat ESKAPE Pathogens in the Era of Antimicrobial Resistance: A Review. Front Microbiol [Internet]. 2019 [cited 2021 Jun 10]. Available from: https://www.frontiersin.org/articles/10.3389/fmicb.2019.00539/pdfRodríguez C, Nastro M, Famiglietti A. Carbapenemases in Acinetobacter baumannii. Review of their dissemination in Latin America. Revista Argentina de Microbiología. [Internet]. 2018. [cited 11 jun 2021]. 50(3), 327-333. Available in: https://www.sciencedirect.com/science/article/pii/S0325754117301785?via%3DihubUrrutia Gómez JA, Rueda Riaño AM, Rojas Páez CA, Silva Rodríguez MA, Méndez Faniño YR. Eficacia de la colisina en el tratamiento de pacientes adultos con infecciones severas por Acinetobacter baummannii XDR en cuidados intensivos. Univ. Med. [Internet]. 15 de julio de 2016 [citado 11 de junio de 2021];57(2):212-25. Disponible en: https://revistas.javeriana.edu.co/index.php/vnimedica/article/view/17036Varón Fabio Andres, Uribe Ana Maria, Palacios Jonathan Orlando, Sánchez Efraín Guillermo, Gutiérrez Deimer, Carvajal Katherine et al. Mortalidad y desenlaces clínicos en pacientes críticamente enfermos con infecciones por bacterias productoras de carbapenemasas en un hospital de alta complejidad en Bogotá, Colombia. Infect. [Internet]. 2021 Mar [cited 2021 June 11] ; 25 (1): 16-21. Available from: http://www.scielo.org.co/scielo.php?script=sci_arttext&pid=S0123-93922021000100016&lng=en.Lemos EV, De la Hoz Restrepo F, Alvis N, Quevedo E, Cañon O, León Y. Mortalidad por Acinetobacter baumannii en unidades de cuidados intensivos en Colombia. Rev Panam Salud Publica. [Internet]. 2011. [citado 18 oct 2020];30:287–94. Disponible en::https://www.scielosp.org/article/rpsp/2011.v30n4/287-294/#ModalArticlesHe L, Luo D, Yang F, Li C, Zhang X, Deng H, Zhang J. Multiple domains of bacterial and human Lon proteases define substrate selectivity. Emerging microbes & infections. [Internet]. 2018. [cited 14 oct 2020];7(1), 149. Available in: https://www.tandfonline.com/doi/full/10.1038/s41426-018-0148-4Hua C, Wang T, Shao X, Xie Y, Huang H, Liu J. Pseudomonas syringae dual‐function protein Lon switches between virulence and metabolism by acting as both DNA ‐binding transcriptional regulator and protease in different environments. Environmental Microbiology. [Internet]. 2020. [cited 14 oct 2020]. Available in: https://sfamjournals.onlinelibrary.wiley.com/doi/abs/10.1111/1462-2920.15067Muselius B. Iron Limitation in Klebsiella pneumoniae Defines New Roles for Lon Protease in Homeostasis and Degradation by Quantitative Proteomics. frontiers in Microbiology. [Internet]. 2020. [cited 14 oct 2020McVicker G, Hollingshead S, Pilla G, Tang CM. Maintenance of the virulence plasmid in Shigella flexneri is influenced by Lon and two functional partitioning systems. Molecular Microbiology. [Internet]. 2019. [cited 14 oct 2020]. 111 (5), 1355-1366. Available in: https://onlinelibrary.wiley.com/doi/full/10.1111/mmi.14225Pei J, Yan J, Jiang Y. Characterization of the ATP-Dependent Lon-Like Protease in Methanobrevibacter smithii. Archaea. [Internet]. 2016 [cited 25 jun 2020]. Available in: https://www.hindawi.com/journals/archaea/2016/5759765Gur E et al. Regulated proteolysis in Gram-negative bacteria — how and when?. Nature Reviews Microbiology. [Internet]. 2011 [cited 28 oct 2020]. Volume 9(12), 839–848. Available in: https://www.nature.com/articles/nrmicro2669Raju R, Goldberg A, Rubin E. Bacterial proteolytic complexes as therapeutic targets. nature reviews drug discovery. [Internet]. 2012 [cited 28 oct 2020]; Volume (11): 777-789. Available in: https://www.nature.com/articles/nrd3846Bayot A, Basse N, Lee I, Gareil M, Pirotte B, Bulteau A, Friguet B, Reboud-Ravaux, M. Towards the control of intracellular protein turnover: Mitochondrial Lon protease inhibitors versus proteasome inhibitors. Biochimie. [Internet]. 2008 [cited 26 oct 2020]. Available in: https://www.sciencedirect.com/science/article/abs/pii/S0300908407003045?via%3DihubHosseingholi E, Zarrini G, Pashazadeh M, Mohammad S, Hayat G, Molavi G. In Silico Identification of Probable Drug and Vaccine Candidates Against Antibiotic-Resistant Acinetobacter baumannii. Microbial Drug Resistance. [Internet]. 2020. [cited 14 oct 2020]. Available in: https://www.liebertpub.com/doi/10.1089/mdr.2019.0236?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmedCulp E, Wright GD. Bacterial proteases, untapped antimicrobial drug targets. J Antibiot (Tokyo). [Internet]. 2017. [cited 10 nov 2020];70(4):366-377. Available in: https://www.nature.com/articles/ja2016138Moubarek C, Halat D. Insights into Acinetobacter baumannii: A Review of Microbiological, Virulence, and Resistance Traits in a Threatening Nosocomial Pathogen. Antibiotics. [Internet]. 2020. [cited 18 Dec 2020]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7148516/Chaari A, Mnif B, Bahloul M, Mahjoubi F, Chtara K, Turki O, et al. Acinetobacter baumannii ventilator-associated pneumonia: epidemiology, clinical characteristics, and prognosis factors. Int J Infect Dis. [Internet].2013. [cited 10 Dec 2020];17(12):e1225-8. Available in: https://www.ijidonline.com/article/S1201-9712(13)00249-X/fulltextCillóniz C, Dominedò C, Torres A. Multidrug Resistant Gram-Negative Bacteria in Community-Acquired Pneumonia. Annual Update in Intensive Care and Emergency Medicine. [Internet]. 2019. [cited 10 Dec 2020]. 459–475. Available in: https://link.springer.com/chapter/10.1007%2F978-3-030-06067-1_36Emanuele Durante-Mangoni et al. Colistin and Rifampicin Compared With Colistin Alone for the Treatment of Serious Infections Due to Extensively Drug-Resistant Acinetobacter baumannii: A Multicenter, Randomized Clinical Trial. Clinical Infectious Diseases.[Internet]. 2013. [cited 15 Dec 2020]. 349–358. Available in: https://academic.oup.com/cid/article/57/3/349/461315Shi H, Lee J, Park S, Ko Y, Eom J. Colistin Plus Carbapenem versus Colistin Monotherapy in the Treatment of Carbapenem-Resistant Acinetobacter baumannii Pneumonia. Infection and drug resistance. [Internet]. 2019. [cited 18 Dec 2020]. 12, 3925–3934. Available in: https://www.dovepress.com/colistin-plus-carbapenem-versus-colistin-monotherapy-in-the-treatment--peer-reviewed-fulltext-article-IDRPoulikakos P, Tansarli G, Falagas M. Combination antibiotic treatment versus monotherapy for multidrug-resistant, extensively drug-resistant, and pandrug-resistant Acinetobacter infections: a systematic review. European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology. [Internet]. 2014. [cited 18 Dec 2020].33(10), 1675–1685. Available in: https://link.springer.com/article/10.1007%2Fs10096-014-2124-9Baran G, Erbay A, Bodur H, Onguru P, Akinci E et al. Risk factors for nosocomial imipenem-resistant Acinetobacter baumannii infections. International Journal of Infectious Diseases. [Internet]. 2008. [cited 18 Dec 2020]. Available in: https://www.ijidonline.com/article/S1201-9712(07)00068-9/fulltextFreire M, Oliveira D, Garcia P, Hoff P, Pierrotti L, Abdala E. Bloodstream infection caused by extensively drug-resistant Acinetobacter baumannii in cancer patients: high mortality associated with delayed treatment rather than with the degree of neutropenia. Clinical Microbiology and Infection. [Internet]. 2008. [cited 18 Dec 2020]. Available in: https://www.clinicalmicrobiologyandinfection.com/article/S1198-743X(15)01036-8/fulltextDavis K, Moran K, McAllister C, Gray P. Multidrug-Resistant Acinetobacter Extremity Infections in Soldiers. Emerging Infectious Diseases. [Internet]. 2005. [cited 18 Dec 2020]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3320488/Alekshun M, Levy S. Molecular Mechanisms of Antibacterial Multidrug Resistance. Cell. [Internet]. 2007. [cited 18 Dec 2020]. Available in: https://www.sciencedirect.com/science/article/pii/S009286740700311XTacconelli E, Carrara E, Savoldi A, Harbarth S, Mendelson M, Monnet D et al. Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. The Lancet Infectious Diseases. [Internet]. 2018. [cited 18 Dec 2020]. Available in: https://www.thelancet.com/journals/laninf/article/PIIS1473-3099(17)30753-3/fulltextCentral Asian and Eastern European Surveillance of Antimicrobial Resistance [Internet]. Euro.who.int. 2017 [cited 18 December 2020]. Available in: https://www.euro.who.int/__data/assets/pdf_file/0005/354434/WHO_CAESAR_AnnualReport_2017.pdf?ua=1Zhanel, G.G., Golden, A.R., Zelenitsky, S. et al. Cefiderocol: A Siderophore Cephalosporin with Activity Against Carbapenem-Resistant and Multidrug-Resistant Gram-Negative Bacilli. Drugs 79. [Internet]. 2019. [cited 18 Dec 2020]. 271–289. Available in: https://link.springer.com/article/10.1007%2Fs40265-019-1055-2Isler B, Doi Y, Bonomo R, Paterson D. New Treatment Options against Carbapenem-Resistant Acinetobacter baumannii Infections. Antimicrobial Agents and Chemotherapy. [Internet]. 2018. [cited 18 Dec 2020]. Available in: https://aac.asm.org/content/63/1/e01110-18Spellberg B, Bonomo R. Combination Therapy for Extreme Drug Resistant (XDR) Acinetobacter baumannii: Ready for Prime-Time?. Critical Care Medicine. [Internet]. 2015. [cited 18 Dec 2020]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4620434/Khanh N, Riordan D, Hoang T, Thanh D, Thwaites G, Huong N et al. The induction and identification of novel Colistin resistance mutations in Acinetobacter baumannii and their implications. Scientific Reports. [Internet]. 2016. [cited 18 Dec 2020]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4916428/Hua Y, Luo T, Yang Y, Dong D, Wang R, Wang J. Phage Therapy as a Promising New Treatment for Lung Infection Caused by Carbapenem-Resistant Acinetobacter baumannii in Mice. Frontiers in Microbiology. [Internet]. 2018. [cited 18 Dec 2020]. Available in: https://www.frontiersin.org/articles/10.3389/fmicb.2017.02659/fullSauer R, Baker T. AAA+ Proteases: ATP-Fueled Machines of Protein Destruction. Annual Review of Biochemistry. [Internet]. 2011 [cited 14 oct 2020].(80): 587–612 Available in: https://www.annualreviews.org/doi/10.1146/annurev-biochem-060408-172623?url_ver=Z39.88-2003&rfr_id=ori%3Arid%3Acrossref.org&rfr_dat=cr_pub++0pubmedChing C, Yang B, Onwubueke C, Lazinski D, Camilli A, Godoy V. Lon Protease Has Multifaceted Biological Functions in Acinetobacter baumannii. J Bacteriol. [Internet]. 2018 [cited 25 jun 2020]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6304660/Meng X, Zhang H, Mezei M, Cui M. Molecular docking: a powerful approach for structure-based drug discovery. Current computer-aided drug design. [Internet]. 2011. [cited 24 jul 2020]. 7(2), 146–157. Available in: https://doi.org/10.2174/157340911795677602Silakari O, Singh P. Chapter 6 - Molecular docking analysis: Basic technique to predict drug-receptor interactions. Concepts and Experimental Protocols of Modelling and Informatics in Drug Design. [Internet]. 2021. [cited 24 jul 2020]. Pages 131-155. Available in: https://www.sciencedirect.com/science/article/pii/B9780128205464000064Kitchen D, Decornez H, Furr J et al. Docking and scoring in virtual screening for drug discovery: methods and applications. Nat Rev Drug Discov 3. [Internet]. 2004. [cited 24 jul 2020]. 935–949. Available in: https://www.nature.com/articles/nrd1549Lantz M. Are bacterial proteases important virulence factors?. J Periodontal. [Internet]. 2006. [cited 25 jul 2020]; Volume (32):126-132. Available in: https://pubmed.ncbi.nlm.nih.gov/9085222/García-González G, González G, Palma-Nicolás J. Las proteasas de serina bacterianas y su implicación en la fisiopatología de la infección. Rev Laboratorio Clínico. [Internet]. 2019 [citado 28 junio 2020]; Volumen (12): 137-146. Disponible en: https://www.elsevier.es/es-revista-revista-del-laboratorio-clinico-282-articulo-las-proteasas-serina-bacterianas-su-S1888400819300303Spyrakis F, Santucci M, Maso L et al. Virtual screening identifies broad-spectrum β-lactamase inhibitors with activity on clinically relevant serine- and metallo-carbapenemases. nature scientific reports. [Internet]. 2020. [cited 28 oct 2020]. Available in: https://www.nature.com/articles/s41598-020-69431-y#citeasKaur G, Pandey B, Kumar A, Garewal N, Grover A, Kaur J. Drug targeted virtual screening and molecular dynamics of LipU protein of Mycobacterium tuberculosis and Mycobacterium leprae. Journal of Biomolecular Structure and Dynamics. [Internet]. 2019. [cited 28 oct 2020]. Available in: https://www.tandfonline.com/action/showCitFormats?doi=10.1080%2F07391102.2018.1454852Nitulescu G, Nicorescu IM, Olaru OT, et al. Molecular Docking and Screening Studies of New Natural Sortase A Inhibitors. Int J Mol Sci. [Internet]. 2017. [cited 28 oct 2020]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666896/Skariyachan S, Manjunath M, Bachappanavar N. Screening of potential lead molecules against prioritised targets of multi-drug-resistant-Acinetobacter baumannii - insights from molecular docking, molecular dynamic simulations and in vitro assays. Journal of Biomolecular Structure and Dynamics. [Internet]. 2017. [cited 28 oct 2020]. Available in: https://pubmed.ncbi.nlm.nih.gov/29529934/Verma P, Tiwari M, Tiwari V. In silico high-throughput virtual screening and molecular dynamics simulation study to identify inhibitor for AdeABC efflux pump of Acinetobacter baumannii. Journal of Biomolecular Structure and Dynamics. [Internet]. 2017. [cited 28 oct 2020]. Available in: https://www.tandfonline.com/doi/full/10.1080/07391102.2017.1317025?src=recsysSkariyachan S, Gopal D, Kadam S, Muddebihalkar A, Uttarkar A, Niranjan V. Carbon fullerene acts as a potential lead molecule against prospective molecular targets of biofilm-producing multidrug-resistant Acinetobacter baumannii and Pseudomonas aeruginosa: computational modeling and MD simulation studies. Journal of Biomolecular Structure and Dynamics. [Internet]. 2020. [cited 28 oct 2020]. Available in: https://www.tandfonline.com/doi/full/10.1080/07391102.2020.1726821?src=recsysRESOLUCIÓN NÚMERO 8430 DE 1993 [Internet]. Hospitalsanpedro.org. 1993 [cited 20 jan 2021]. Available from: https://www.hospitalsanpedro.org/images/Comite_Investigacion/Resolucion_8430_de_1993.pdfBenkert P, Biasini M, Schwede T. Toward the estimation of the absolute quality of individual protein structure models. Bioinformatics (Oxford, England), 27(3), 343–350. [Internet]. 2011. [cited 24 may 2021]. Available in: https://academic.oup.com/bioinformatics/article/27/3/343/320383Markus Wiederstein, Manfred J. Sippl, ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Research, Volume 35, Issue suppl_2, 1 July 2007, Pages W407–W410. [Internet]. 2007. [cited 25 may 2021]. Available in: https://academic.oup.com/nar/article/35/suppl_2/W407/2920938Laskowski A, MacArthur M, Moss D, Thornton J. PROCHECK: a program to check the stereochemical quality of protein structures. Journal of Applied Crystallography, 26(2), 283–291. [Internet]. 1993. [cited 25 mar 2021]. Available in: https://onlinelibrary.wiley.com/doi/epdf/10.1107/S0021889892009944Pundir S, Magrane M, Martin MJ, O'Donovan C. Searching and Navigating UniProt Databases. Curr Protoc Bioinformatics. [Internet]. 2016. [cited 15 feb 2021]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4522465/Rotanova T, Botos I, Melnikov E, Rasulova F, Gustchina A, Maurizi M, Wlodawer A. Slicing a protease: Structural features of the ATP-dependent Lon proteases gleaned from investigations of isolated domains. Protein Science. [Internet]. 2006. [cited 24 jul 2021]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2242575/Park S, Jia B, Yang J, Le Van D, Shao Y, Woo S et al. Oligomeric Structure of the ATP-dependent Protease La (Lon) of Escherichia coli. Molecules and Cells. [Internet]. 2006. [cited 24 jul 2021]. Available in: http://www.molcells.org/journal/view.html?year=2006&volume=21&number=1&spage=129#body01Carr J, Arduino M, Swenson J. Acinetobacter baumannii. Centers for Disease Control and Prevention (CDC). [Internet]. 2004. [cited 21 jan 2021]. Available in: https://phil.cdc.gov/Details.aspx?pid=6498Parker T. Colonial growth displayed by the Gram-negative bacterium Acinetobacter baumannii. Centers for Disease Control and Prevention (CDC). [Internet]. 2014. [cited 21 jan 2021]. 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Unicolmayorrepositorio@unicolmayor.edu.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