Sch-52900

Verticillins are a group of epipolythiodioxopiperazine alkaloids that have displayed potent cytotoxicity. To evaluate their potential further, a larger supply of these compounds was needed for both in vivo studies and analogue development via semisynthesis. To optimize the biosynthesis of these secondary metabolites, their production was analyzed in two different fungal strains (MSX59553 and MSX79542) under a suite of fermentation conditions. These studies were facilitated by the use of the droplet-liquid microjunction-surface sampling probe (droplet probe), which enables chemical analysis in situ directly from the surface of the cultures. These experiments showed that the production of verticillins was greatly affected by growth conditions; a significantly higher quantity of these alkaloids was noted when the fungal strains were grown on an oatmeal-based medium. Using these technologies to select the best among the tested growth conditions, the production of the verticillin analogues was increased while concomitantly decreasing the time required for fermentations from 5 weeks to about 11 days. Importantly, where we could previously supply 5–10 mg every 6 weeks, we are now able to supply 50–150 mg quantities of key analogues per month via laboratory scale fermentation.

Effective and rapid dereplication is a hallmark of present-day drug discovery from natural sources. This project strove to both decrease the time and expand the structural diversity associated with dereplication methodologies. A 5 min liquid chromatographic run time employing heated electrospray ionization (HESI) was evaluated to determine whether it could be used as a faster alternative over the 10 min ESI method we reported previously. Results revealed that the 5 min method was as sensitive as the 10 min method and, obviously, was twice as fast. To facilitate dereplication, the retention times, UV absorption maxima, full-scan HRMS and MS/MS were cross-referenced with an in-house database of over 300 fungal secondary metabolites. However, this strategy was dependent upon the makeup of the screening in-house database. Thus, mass defect filtering (MDF) was explored as an additional targeted screening strategy to permit identification of structurally related components. The use of a dereplication platform incorporating the 5 min chromatographic method together with MDF facilitated rapid and effective identification of known compounds and detection of structurally related analogs in extracts of fungal cultures.