Abstract
Mammalian carboxylesterases cleave the anticancer prodrug CPT-11 (Irinotecan) into SN-38, a potent topoisomerase I poison, and 4-piperidino-piperidine (4PP). We present the 2.5 Å crystal structure of rabbit liver carboxylesterase (rCE), the most efficient enzyme known to activate CPT-11 in this manner, in complex with the leaving group 4PP. 4PP is observed bound adjacent to a high-mannose Asn-linked glycosylation site on the surface of rCE. This product-binding site is separated from the catalytic gorge by a thin wall of amino acid side chains, suggesting that 4PP may be released through this secondary product exit pore. The crystallographic observation of a leaving group bound on the surface of rCE supports the 'back door' product exit site proposed for the acetylcholinesterases. These results may facilitate the design of improved anticancer drugs or enzymes for use in viral-directed cancer cotherapies.
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Acknowledgements
The authors wish to thank R. Watkins, J. Chrencik, T. Thieu, Y. Xue, E. Collins, L. Betts and the members of the Redinbo Laboratory for discussions and experimental assistance. We also thank G. Pielak, D. Erie and A. Tripathy for assistance with CD thermal denaturation studies. Supported by a Burroughs Wellcome Career Award in the Biomedical Sciences (M.R.R.) and by the NIH and American Lebanese Syrian Associated Charities (P.M.P.).
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Bencharit, S., Morton, C., Howard-Williams, E. et al. Structural insights into CPT-11 activation by mammalian carboxylesterases. Nat Struct Mol Biol 9, 337–342 (2002). https://doi.org/10.1038/nsb790
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DOI: https://doi.org/10.1038/nsb790
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