Comprehensive Structure–Activity Profiling of Micheliolide and its Targeted Proteome in Leukemia Cells via Probe-Guided Late-Stage C–H Functionalization

The plant-derived
sesquiterpene lactone micheliolide was recently
found to possess promising antileukemic activity, including the ability
to target and kill leukemia stem cells. Efforts toward improving the
biological activity of micheliolide and investigating its mechanism
of action have been hindered by the paucity of preexisting functional
groups amenable for late-stage derivatization of this molecule. Here,
we report the implementation of a probe-based P450 fingerprinting
strategy to rapidly evolve engineered P450 catalysts useful for the
regio- and stereoselective hydroxylation of micheliolide at two previously
inaccessible aliphatic positions in this complex natural product.
Via P450-mediated chemoenzymatic synthesis, a broad panel of novel
micheliolide analogs could thus be obtained to gain structure–activity
insights into the effect of C2, C4, and C14 substitutions on the antileukemic
activity of micheliolide, ultimately leading to the discovery of “micheliologs”
with improved potency against acute myelogenic leukemia cells. These
late-stage C–H functionalization routes could be further leveraged
to generate a panel of affinity probes for conducting a comprehensive
analysis of the protein targeting profile of micheliolide in leukemia
cells via chemical proteomics analyses. These studies introduce new
micheliolide-based antileukemic agents and shed new light onto the
biomolecular targets and mechanism of action of micheliolide in leukemia
cells. More broadly, this work showcases the value of the present
P450-mediated C–H functionalization strategy for streamlining
the late-stage diversification and elucidation of the biomolecular
targets of a complex bioactive molecule.