74972 20250313085413.0 doi 10.3389/fmicb.2018.01659 sideral 107619 ART-2018-107619 eng García, M.T. Boldine-derived Alkaloids inhibit the activity of DNA topoisomerase I and growth of Mycobacterium tuberculosis 2018 Access copy available to the general public Unrestricted The spread of multidrug-resistant isolates of Mycobacterium tuberculosis requires the discovery of new drugs directed to new targets. In this study, we investigated the activity of two boldine-derived alkaloids, seconeolitsine (SCN) and N-methyl-seconeolitsine (N-SCN), against M. tuberculosis. These compounds have been shown to target DNA topoisomerase I enzyme and inhibit growth of Streptococcus pneumoniae. Both SCN and N-SCN inhibited M. tuberculosis growth at 1.95-15.6 µM, depending on the strain. In M. smegmatis this inhibitory effect correlated with the amount of topoisomerase I in the cell, hence demonstrating that this enzyme is the target for these alkaloids in mycobacteria. The gene coding for topoisomerase I of strain H37Rv (MtbTopoI) was cloned into pQE1 plasmid of Escherichia coli. MtbTopoI was overexpressed with an N-terminal 6-His-tag and purified by affinity chromatography. In vitro inhibition of MtbTopoI activity by SCN and N-SCN was tested using a plasmid relaxation assay. Both SCN and N-SCN inhibited 50% of the enzymatic activity at 5.6 and 8.4 µM, respectively. Cleavage of single-stranded DNA was also inhibited with SCN. The effects on DNA supercoiling were also evaluated in vivo in plasmid-containing cultures of M. tuberculosis. Plasmid supercoiling densities were -0.060 in cells untreated or treated with boldine, and -0.072 in 1 × MIC N-SCN treated cells, respectively, indicating that the plasmid became hypernegatively supercoiled in the presence of N-SCN. Altogether, these results demonstrate that the M. tuberculosis topoisomerase I enzyme is an attractive drug target, and that SCN and N-SCN are promising lead compounds for drug development. info:eu-repo/grantAgreement/EC/FP7/260872/EU/More Medicines for Tuberculosis/MM4TB info:eu-repo/grantAgreement/ES/MINECO/BIO2009-09405 info:eu-repo/grantAgreement/ES/MINECO/BIO2017-82951-R info:eu-repo/semantics/openAccess by http://creativecommons.org/licenses/by/3.0/es/ 4.259 2018 MICROBIOLOGY 32 / 133 = 0.241 2018 Q1 T1 1.633 2018 Microbiology (medical) 2018 Q1 Microbiology 2018 Q1 info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion Carreño, D. Tirado-Vélez, J.M. Ferrándiz, M.J. Rodrigues, L. Gracia, B. Universidad de Zaragoza (orcid)0000-0001-9233-5024 Amblar, M. Ainsa, J.A. Universidad de Zaragoza (orcid)0000-0003-2076-844X de la Campa, A.G. 1008 630 Universidad de Zaragoza Dpto. Microb.Med.Pr.,Sal.Públ. Área Microbiología 1008 X Universidad de Zaragoza Dpto. Microb.Med.Pr.,Sal.Públ. Proy. investigación HQA 9, JUL (2018), 1659 [9 pp] Front. microbiol. Frontiers in Microbiology 1664-302X 748389 http://zaguan.unizar.es/record/74972/files/texto_completo.pdf Versión publicada 28488 http://zaguan.unizar.es/record/74972/files/texto_completo.jpg?subformat=icon icon Versión publicada oai:zaguan.unizar.es:74972 articulos driver 2025-03-13-08:43:49 ARTICLE