Structural Investigations of Prodrug Activation by Human Cytochrome P450 Enzymes

Some human cytochrome P450 enzymes are selectively expressed in tumor tissues, providing an opportunity for targeted, P450‐mediated activation of cancer prodrugs. P450 enzymes CYP1A1 and CYP2W1, expressed in the lung and in colon cancer cells respectively, selectively activate analogs of the natural product duocarmycin into extremely potent cytotoxins. Alterations of the duocarmycin scaffold modulate the selectivity of these compounds for CYP1A1 or CYP2W1 and determine the metabolites formed, but structural information on binding of active and inactive analogs is needed to guide further optimization. Thus, duocarmycin analogs yielding both active and inactive metabolites were used to probe binding to human CYP1A1 and CYP2W1. ICT2726 is a benzofuran‐based duocarmycin yielding only non‐toxic metabolites upon metabolism by CYP1A1 and CYP2W1. This makes ICT2726 valuable as a biomarker of tissue CYP1A1 and CYP2W1 expression. Both ICT2726 enantiomers demonstrated classical substrate‐like (type I) binding to CYP1A1 with sub‐micomolar binding affinities. While CYP2W1 bound ( S )‐ICT2726 with similar spectral changes, the affinity was lower at ~1 mM. However, ( R )‐ICT2726 did not cause significant spectral changes, indicating that either CYP2W1 is stereospecific for the ( S ) enantiomer or the ( R ) enantiomer binding mode is distinct and does not perturb the active site heme‐bound water typically associated with type I spectra. An X‐ray structure of CYP1A1 revealed an ( S )‐ICT2726 orientation consistent with the generation of a non‐toxic metabolite on the fluoroindole ring. While the overall structure was similar to previous 1A1 structures, this new structure revealed substantial remodeling of a key section of the active site roof. Instead of a break in the F helix normally observed for CYP1 enzymes, the region is helical with a halogen bond between F‐helix Asn222 and the ligand chlorine. In contrast, ICT2700 is an indole‐based duocarmycin activated into a cytotoxic metabolite by CYP1A1 and CYP2W1 hydroxylation on the indole benzene ring. Both ( S ) and ( R )‐ICT2700 interact with CYP1A1 yielding unusual spectral characteristics, including an absorbance maximum of 413 nm and minimum at 434 nm, and similar sub‐micromolar affinities. In contrast CYP2W1 interacts with both ( S ) and ( R )‐ICT2700 in a type II binding mode, suggestive of nitrogen coordination by the indole moiety. Further directions are to determine a structure of CYP1A1 with ICT2700 and any structure of CYP2W1, as each would further our understanding of selective duocarmycin activation. Overall, understanding the binding modes and structural determinants of duocarmycin interactions with the human P450 enzymes CYP1A1 and CYP2W1 will facilitate the development of pro‐drugs that can be activated in a tissue‐selective manner, decreasing off‐target effects in the treatment of lung and colon cancers. Support or Funding Information National Institutes of Health R37 GM076343, T32 GM08545 Dynamic Aspects of Chemical Biology This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .