García Esteban, María TeresaCarreño, DavidTirado Vélez, José ManuelFerrándiz, María JoséRodrigues, LilianaGracia, BegoñaAmblar, MónicaAinsa, José A.Gonzalez de la Campa, Adela2024-12-112024-12-112018-07-24García MT, Carreño D, Tirado-Vélez JM, Ferrándiz MJ, Rodriguez L, Gracia B, Mónica A, Ainsa JA, De la Campa AG. Boldine-Derived Alkaloids Inhibit the Activity of DNA Topoisomerase I and Growth of Mycobacterium tuberculosis. Front. Microbiol. 2018. 9 (1659)): 1-9.1664-302X10.3389/fmicb.2018.01659https://hdl.handle.net/20.500.14352/112403The 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.engAttribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/journal articlehttps://doi.org/10.3389/fmicb.2018.0165930087665https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2018.01659/fullopen access579.61616.24-002.5616.9577.1577.2615.012Mycobacterium tuberculosisDNA topoisomerase I inhibitorDNA supercoilingN-methylseconeolitsineSeconeolitsineAntituberculosis activityDrug discoveryMicrobiología (Biología)Biología molecular (Biología)Enfermedades infecciosasMedicamentos2414 Microbiología2403 Bioquímica2415 Biología Molecular3201.03 Microbiología Clínica3205.05 Enfermedades Infecciosas3209.08 Preparación de Medicamentos