Abstract
Off-target binding of hydrophobic drugs can lead to unwanted side effects, either through specific or non-specific binding to unintended membrane protein targets. However, distinguishing the binding of drugs to membrane proteins from that of detergents, lipids and cofactors is challenging. Here, we use high-resolution mass spectrometry to study the effects of HIV protease inhibitors on the human zinc metalloprotease ZMPSTE24. This intramembrane protease plays a major role in converting prelamin A to mature lamin A. We monitored the proteolysis of farnesylated prelamin A peptide by ZMPSTE24 and unexpectedly found retention of the C-terminal peptide product with the enzyme. We also resolved binding of zinc, lipids and HIV protease inhibitors and showed that drug binding blocked prelamin A peptide cleavage and conferred stability to ZMPSTE24. Our results not only have relevance for the progeria-like side effects of certain HIV protease inhibitor drugs, but also highlight new approaches for documenting off-target drug binding.
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Acknowledgements
This work is supported by programme grants from the Medical Research Council (98101), the National Institutes of Health (NIH) and an ERC Advanced Investigator Award IMPRESS (26851). C.V.R. has a Wellcome Trust Investigator Award. E.P.C. and A.Q. are funded by the Structural Genomics Consortium, a registered charity (no. 1097737) that receives funds from AbbVie, Bayer, Boehringer Ingelheim, the Canada Foundation for Innovation, the Canadian Institutes for Health Research, Genome Canada, GlaxoSmithKline, Janssen, Lilly Canada, the Novartis Research Foundation, the Ontario Ministry of Economic Development and Innovation, Pfizer, Takeda and the Wellcome Trust (092809/Z/10/Z). E.P.C. and A.Q. are also funded by a Medical Research Council grant number MR/L017458/1. S.Mi. was funded for this work by NIH grant R01 GM041223. S.G.Y. was supported by NIH grants AG035626-10 and HL126551. The authors thank S. Wang and B. Rotty for purification of three of the protein samples used in this study. J.G. is a Junior Research Fellow at Queen's College, Oxford.
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S.Me. and C.V.R. designed the research with assistance from A.Q. and E.P.C. A.Q. and E.P.C. purified ZMPSTE24. S.Me. and J.G. carried out the mass spectrometry experiments. J.M. initiated preliminary mass spectrometry experiment. S.Mi. helped with new reagents. S.G.Y. performed the western blot analysis. S.Me. and C.V.R. wrote the manuscript with contributions from all authors.
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C.V.R. is a consultant to Omass Technologies Ltd, a spinout company from the Department of Chemistry at the University of Oxford.
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Mehmood, S., Marcoux, J., Gault, J. et al. Mass spectrometry captures off-target drug binding and provides mechanistic insights into the human metalloprotease ZMPSTE24. Nature Chem 8, 1152–1158 (2016). https://doi.org/10.1038/nchem.2591
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DOI: https://doi.org/10.1038/nchem.2591
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