title: Mass spectrometry captures off-target drug binding and provides mechanistic insights into the human metalloprotease ZMPSTE24.
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.
authors.affiliation: Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK.
authors.forename: Shahid
authors.initials: S
authors.lastname: Mehmood
authors.affiliation: Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK.
authors.forename: Julien
authors.initials: J
authors.lastname: Marcoux
authors.affiliation: Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK.
authors.forename: Joseph
authors.identifier: 0000-0001-7686-2983
authors.initials: J
authors.lastname: Gault
authors.affiliation: Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Quigley
authors.affiliation: Department of Cell Biology, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: Susan
authors.initials: S
authors.lastname: Michaelis
authors.affiliation: Departments of Medicine and Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA.
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Young
authors.affiliation: Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK.
authors.forename: Elisabeth P
authors.initials: EP
authors.lastname: Carpenter
authors.affiliation: Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK.
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
mesh_terms: D017320:HIV Protease Inhibitors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D034904:Lamin Type A / Q000378:metabolism; D008055:Lipids / Q000737:chemistry; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D004364:Pharmaceutical Preparations / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017368:Protein Prenylation; D013379:Substrate Specificity; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
chemical_list: D017320:HIV Protease Inhibitors; D034904:Lamin Type A; D008055:Lipids; D008565:Membrane Proteins; D004364:Pharmaceutical Preparations; C090764:prelamin A; D008666:Metalloendopeptidases; C120130:ZMPSTE24 protein, human; D015032:Zinc