title: Synthetic metallochaperone ZMC1 rescues mutant p53 conformation by transporting zinc into cells as an ionophore.
abstract: p53 is a Zn(2+)-dependent tumor suppressor inactivated in >50% of human cancers. The most common mutation, R175H, inactivates p53 by reducing its affinity for the essential zinc ion, leaving the mutant protein unable to bind the metal in the low [Zn(2+)]free environment of the cell. The exploratory cancer drug zinc metallochaperone-1 (ZMC1) was previously demonstrated to reactivate this and other Zn(2+)-binding mutants by binding Zn(2+) and buffering it to a level such that Zn(2+) can repopulate the defective binding site, but how it accomplishes this in the context of living cells and organisms is unclear. In this study, we demonstrated that ZMC1 increases intracellular [Zn(2+)]free by functioning as a Zn(2+) ionophore, binding Zn(2+) in the extracellular environment, diffusing across the plasma membrane, and releasing it intracellularly. It raises intracellular [Zn(2+)]free in cancer (TOV112D) and noncancer human embryonic kidney cell line 293 to 15.8 and 18.1 nM, respectively, with half-times of 2-3 minutes. These [Zn(2+)]free levels are predicted to result in ∼90% saturation of p53-R175H, thus accounting for its observed reactivation. This mechanism is supported by the X-ray crystal structure of the [Zn(ZMC1)2] complex, which demonstrates structural and chemical features consistent with those of known metal ionophores. These findings provide a physical mechanism linking zinc metallochaperone-1 in both in vitro and in vivo activities and define the remaining critical parameter necessary for developing synthetic metallochaperones for clinical use.
authors.affiliation: Department of Biochemistry and Molecular Biology (A.R.B.,S.N.L.) and Department of Neuroscience and Physiology (R.S.O., E.C.O.), State University of New York Upstate Medical University, Syracuse, New York; Rutgers Cancer Institute of New Jersey (X.Y., D.R.C.), Department of Surgery, Rutgers Robert Wood Johnson Medical School (X.Y., D.R.C.), Office of Translational Sciences (J.A.G., D.J.A., S.D.K.), and Department of Chemistry and Chemical Biology (T.J.E.), Rutgers University, New Brunswick, New Jersey; and Department of Physics, Syracuse University, Syracuse, New York (A.J.W., L.M.).
authors.forename: Adam R
authors.initials: AR
authors.lastname: Blanden
authors.affiliation: Department of Biochemistry and Molecular Biology (A.R.B.,S.N.L.) and Department of Neuroscience and Physiology (R.S.O., E.C.O.), State University of New York Upstate Medical University, Syracuse, New York; Rutgers Cancer Institute of New Jersey (X.Y., D.R.C.), Department of Surgery, Rutgers Robert Wood Johnson Medical School (X.Y., D.R.C.), Office of Translational Sciences (J.A.G., D.J.A., S.D.K.), and Department of Chemistry and Chemical Biology (T.J.E.), Rutgers University, New Brunswick, New Jersey; and Department of Physics, Syracuse University, Syracuse, New York (A.J.W., L.M.).
authors.forename: Xin
authors.initials: X
authors.lastname: Yu
authors.affiliation: Department of Biochemistry and Molecular Biology (A.R.B.,S.N.L.) and Department of Neuroscience and Physiology (R.S.O., E.C.O.), State University of New York Upstate Medical University, Syracuse, New York; Rutgers Cancer Institute of New Jersey (X.Y., D.R.C.), Department of Surgery, Rutgers Robert Wood Johnson Medical School (X.Y., D.R.C.), Office of Translational Sciences (J.A.G., D.J.A., S.D.K.), and Department of Chemistry and Chemical Biology (T.J.E.), Rutgers University, New Brunswick, New Jersey; and Department of Physics, Syracuse University, Syracuse, New York (A.J.W., L.M.).
authors.forename: Aaron J
authors.initials: AJ
authors.lastname: Wolfe
authors.affiliation: Department of Biochemistry and Molecular Biology (A.R.B.,S.N.L.) and Department of Neuroscience and Physiology (R.S.O., E.C.O.), State University of New York Upstate Medical University, Syracuse, New York; Rutgers Cancer Institute of New Jersey (X.Y., D.R.C.), Department of Surgery, Rutgers Robert Wood Johnson Medical School (X.Y., D.R.C.), Office of Translational Sciences (J.A.G., D.J.A., S.D.K.), and Department of Chemistry and Chemical Biology (T.J.E.), Rutgers University, New Brunswick, New Jersey; and Department of Physics, Syracuse University, Syracuse, New York (A.J.W., L.M.).
authors.forename: John A
authors.initials: JA
authors.lastname: Gilleran
authors.affiliation: Department of Biochemistry and Molecular Biology (A.R.B.,S.N.L.) and Department of Neuroscience and Physiology (R.S.O., E.C.O.), State University of New York Upstate Medical University, Syracuse, New York; Rutgers Cancer Institute of New Jersey (X.Y., D.R.C.), Department of Surgery, Rutgers Robert Wood Johnson Medical School (X.Y., D.R.C.), Office of Translational Sciences (J.A.G., D.J.A., S.D.K.), and Department of Chemistry and Chemical Biology (T.J.E.), Rutgers University, New Brunswick, New Jersey; and Department of Physics, Syracuse University, Syracuse, New York (A.J.W., L.M.).
authors.forename: David J
authors.initials: DJ
authors.lastname: Augeri
authors.affiliation: Department of Biochemistry and Molecular Biology (A.R.B.,S.N.L.) and Department of Neuroscience and Physiology (R.S.O., E.C.O.), State University of New York Upstate Medical University, Syracuse, New York; Rutgers Cancer Institute of New Jersey (X.Y., D.R.C.), Department of Surgery, Rutgers Robert Wood Johnson Medical School (X.Y., D.R.C.), Office of Translational Sciences (J.A.G., D.J.A., S.D.K.), and Department of Chemistry and Chemical Biology (T.J.E.), Rutgers University, New Brunswick, New Jersey; and Department of Physics, Syracuse University, Syracuse, New York (A.J.W., L.M.).
authors.forename: Ryan S
authors.initials: RS
authors.lastname: O'Dell
authors.affiliation: Department of Biochemistry and Molecular Biology (A.R.B.,S.N.L.) and Department of Neuroscience and Physiology (R.S.O., E.C.O.), State University of New York Upstate Medical University, Syracuse, New York; Rutgers Cancer Institute of New Jersey (X.Y., D.R.C.), Department of Surgery, Rutgers Robert Wood Johnson Medical School (X.Y., D.R.C.), Office of Translational Sciences (J.A.G., D.J.A., S.D.K.), and Department of Chemistry and Chemical Biology (T.J.E.), Rutgers University, New Brunswick, New Jersey; and Department of Physics, Syracuse University, Syracuse, New York (A.J.W., L.M.).
authors.forename: Eric C
authors.initials: EC
authors.lastname: Olson
authors.affiliation: Department of Biochemistry and Molecular Biology (A.R.B.,S.N.L.) and Department of Neuroscience and Physiology (R.S.O., E.C.O.), State University of New York Upstate Medical University, Syracuse, New York; Rutgers Cancer Institute of New Jersey (X.Y., D.R.C.), Department of Surgery, Rutgers Robert Wood Johnson Medical School (X.Y., D.R.C.), Office of Translational Sciences (J.A.G., D.J.A., S.D.K.), and Department of Chemistry and Chemical Biology (T.J.E.), Rutgers University, New Brunswick, New Jersey; and Department of Physics, Syracuse University, Syracuse, New York (A.J.W., L.M.).
authors.forename: S David
authors.initials: SD
authors.lastname: Kimball
authors.affiliation: Department of Biochemistry and Molecular Biology (A.R.B.,S.N.L.) and Department of Neuroscience and Physiology (R.S.O., E.C.O.), State University of New York Upstate Medical University, Syracuse, New York; Rutgers Cancer Institute of New Jersey (X.Y., D.R.C.), Department of Surgery, Rutgers Robert Wood Johnson Medical School (X.Y., D.R.C.), Office of Translational Sciences (J.A.G., D.J.A., S.D.K.), and Department of Chemistry and Chemical Biology (T.J.E.), Rutgers University, New Brunswick, New Jersey; and Department of Physics, Syracuse University, Syracuse, New York (A.J.W., L.M.).
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Emge
authors.affiliation: Department of Biochemistry and Molecular Biology (A.R.B.,S.N.L.) and Department of Neuroscience and Physiology (R.S.O., E.C.O.), State University of New York Upstate Medical University, Syracuse, New York; Rutgers Cancer Institute of New Jersey (X.Y., D.R.C.), Department of Surgery, Rutgers Robert Wood Johnson Medical School (X.Y., D.R.C.), Office of Translational Sciences (J.A.G., D.J.A., S.D.K.), and Department of Chemistry and Chemical Biology (T.J.E.), Rutgers University, New Brunswick, New Jersey; and Department of Physics, Syracuse University, Syracuse, New York (A.J.W., L.M.).
authors.forename: Liviu
authors.initials: L
authors.lastname: Movileanu
authors.affiliation: Department of Biochemistry and Molecular Biology (A.R.B.,S.N.L.) and Department of Neuroscience and Physiology (R.S.O., E.C.O.), State University of New York Upstate Medical University, Syracuse, New York; Rutgers Cancer Institute of New Jersey (X.Y., D.R.C.), Department of Surgery, Rutgers Robert Wood Johnson Medical School (X.Y., D.R.C.), Office of Translational Sciences (J.A.G., D.J.A., S.D.K.), and Department of Chemistry and Chemical Biology (T.J.E.), Rutgers University, New Brunswick, New Jersey; and Department of Physics, Syracuse University, Syracuse, New York (A.J.W., L.M.).
authors.forename: Darren R
authors.initials: DR
authors.lastname: Carpizo
authors.affiliation: Department of Biochemistry and Molecular Biology (A.R.B.,S.N.L.) and Department of Neuroscience and Physiology (R.S.O., E.C.O.), State University of New York Upstate Medical University, Syracuse, New York; Rutgers Cancer Institute of New Jersey (X.Y., D.R.C.), Department of Surgery, Rutgers Robert Wood Johnson Medical School (X.Y., D.R.C.), Office of Translational Sciences (J.A.G., D.J.A., S.D.K.), and Department of Chemistry and Chemical Biology (T.J.E.), Rutgers University, New Brunswick, New Jersey; and Department of Physics, Syracuse University, Syracuse, New York (A.J.W., L.M.) lohs@upstate.edu.
authors.forename: Stewart N
authors.initials: SN
authors.lastname: Loh
mesh_terms: D001665:Binding Sites; D001692:Biological Transport / Q000502:physiology*; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007476:Ionophores / Q000378:metabolism*; D056511:Metallochaperones / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D007476:Ionophores; D056511:Metallochaperones; D016159:Tumor Suppressor Protein p53; C017800:zinc-binding protein; D015032:Zinc