Slow-Binding Inhibition of <i>Mycobacterium tuberculosis</i> Shikimate Kinase by Manzamine Alkaloids

Tuberculosis represents a significant public health crisis. There is an urgent need for novel molecular scaffolds against this pathogen. We screened a small library of marine-derived compounds against shikimate kinase from <i>Mycobacterium tuberculosis</i> (<i>Mt</i>SK), a promising target for antitubercular drug development. Six manzamines previously shown to be active against <i>M. tuberculosis</i> were characterized as <i>Mt</i>SK inhibitors: manzamine A (<b>1</b>), 8-hydroxymanzamine A (<b>2</b>), manzamine E (<b>3</b>), manzamine F (<b>4</b>), 6-deoxymanzamine X (<b>5</b>), and 6-cyclohexamidomanzamine A (<b>6</b>). All six showed mixed noncompetitive inhibition of <i>Mt</i>SK. The lowest <i>K</i>_I values were obtained for <b>6</b> across all <i>Mt</i>SK–substrate complexes. Time-dependent analyses revealed two-step, slow-binding inhibition. The behavior of <b>1</b> was typical; initial formation of an enzyme–inhibitor complex (EI) obeyed an apparent <i>K</i>_I of ∼30 μM with forward (<i>k</i>₅) and reverse (<i>k</i>₆) rate constants for isomerization to an EI* complex of 0.18 and 0.08 min^–1, respectively. In contrast, <b>6</b> showed a lower <i>K</i>_I for the initial encounter complex (∼1.5 μM), substantially faster isomerization to EI* (<i>k</i>₅ = 0.91 min^–1), and slower back conversion of EI* to EI (<i>k</i>₆ = 0.04 min^–1). Thus, the overall inhibition constants, <i>K</i>_I*, for <b>1</b> and <b>6</b> were 10 and 0.06 μM, respectively. These findings were consistent with docking predictions of a favorable binding mode and a second, less tightly bound pose for <b>6</b> at <i>Mt</i>SK. Our results suggest that manzamines, in particular <b>6</b>, constitute a new scaffold from which drug candidates with novel mechanisms of action could be designed for the treatment of tuberculosis by targeting <i>Mt</i>SK