Identification of the target protein of agelasine D, a marine sponge diterpene alkaloid, as an anti-dormant mycobacterial substance.

One of the major reasons for the wide epidemicity of tuberculosis and for the necessity for extensive chemotherapeutic regimens is that the causative agent, Mycobacterium tuberculosis, has an ability to become dormant. Therefore, new lead compounds that are anti-bacterial against M. tuberculosis in both active and dormant states are urgently needed. Marine sponge diterpene alkaloids, agelasines B, C, and D, from an Indonesian marine sponge of the genus Agelas were rediscovered as anti-dormant-mycobacterial substances. Based on the concept that the transformants over-expressing targets of antimicrobial substances confer drug resistance, strains resistant to agelasine D were screened from Mycobacterium smegmatis transformed with a genomic DNA library of Mycobacterium bovis BCG. Sequence analysis of the cosmids isolated from resistant transformants revealed that the responsible gene was located in the genome region between 3475.051 and 3502.901 kb. Further analysis of the transformants over-expressing the individual gene contained in this region indicated that BCG3185c (possibly a dioxygenase) might be a target of the molecule. Moreover, agelasine D was found to bind directly to recombinant BCG3185c protein (KD 2.42 μm), based on surface plasmon resonance (SPR). This evidence strongly suggests that the BCG3185c protein is the major target of agelasine D, and that the latter is the anti-mycobacterial substance against dormant bacilli.