AUTHOR=García María T. , Carreño David , Tirado-Vélez José M. , Ferrándiz María J. , Rodrigues Liliana , Gracia Begoña , Amblar Mónica , Ainsa José A. , de la Campa Adela G. 

TITLE=Boldine-Derived Alkaloids Inhibit the Activity of DNA Topoisomerase I and Growth of Mycobacterium tuberculosis

JOURNAL=Frontiers in Microbiology

VOLUME=9

YEAR=2018

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2018.01659

DOI=10.3389/fmicb.2018.01659

ISSN=1664-302X

ABSTRACT=<p>The spread of multidrug-resistant isolates of <italic>Mycobacterium tuberculosis</italic> 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 <italic>N</italic>-methyl-seconeolitsine (<italic>N</italic>-SCN), against <italic>M. tuberculosis</italic>. These compounds have been shown to target DNA topoisomerase I enzyme and inhibit growth of <italic>Streptococcus pneumoniae</italic>. Both SCN and <italic>N</italic>-SCN inhibited <italic>M. tuberculosis</italic> growth at 1.95–15.6 μM, depending on the strain. In <italic>M. smegmatis</italic> 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 <italic>Escherichia coli</italic>. MtbTopoI was overexpressed with an N-terminal 6-His-tag and purified by affinity chromatography. <italic>In vitro</italic> inhibition of MtbTopoI activity by SCN and <italic>N</italic>-SCN was tested using a plasmid relaxation assay. Both SCN and <italic>N</italic>-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 <italic>in vivo</italic> in plasmid-containing cultures of <italic>M. tuberculosis</italic>. Plasmid supercoiling densities were −0.060 in cells untreated or treated with boldine, and −0.072 in 1 × MIC <italic>N</italic>-SCN treated cells, respectively, indicating that the plasmid became hypernegatively supercoiled in the presence of <italic>N</italic>-SCN. Altogether, these results demonstrate that the <italic>M. tuberculosis</italic> topoisomerase I enzyme is an attractive drug target, and that SCN and <italic>N</italic>-SCN are promising lead compounds for drug development.</p>