Thiosemicarbazones Functioning as Zinc Metallochaperones to Reactivate Mutant p53.
Small-molecule restoration of wild-type structure and function to mutant p53 (so-called mutant reactivation) is a highly sought-after goal in cancer drug development. We previously discovered that small-molecule zinc chelators calledzinc metallochaperones(ZMCs) reactivate mutant p53 by restoring zinc binding to zinc-deficient p53 mutants. The lead compound identified from the NCI-60 human tumor cell lines screen, NSC319726 (ZMC1), belongs to the thiosemicarbazone (TSC) class of metal ion chelators that bind iron, copper, magnesium, zinc, and other transition metals. Here, we have investigated the other TSCs, NSC319725 and NSC328784, identified in the same screen, as well as the more well studied TSC, 3-AP (Triapine), to determine whether they function as ZMCs. We measured the zincKdzinc ionophore activity, ability to restore zinc to purified p53 DNA binding domain (DBD), and ability to restore site-specific DNA binding to purified R175H-DBD in vitro. We tested all four TSCs in a number of cell-based assays to examine mutant p53 reactivation and the generation of reactive oxygen species (ROS). We found that NSC319725 and NSC328784 behave similarly to ZMC1 in both biophysical and cell-based assays and are heretofore named ZMC2 (NSC319725) and ZMC3 (NSC328784). 3-AP generates a ROS signal similar to ZMC1-3, but it fails to function as a ZMC both in vitro and in cells and ultimately does not reactivate p53. These findings indicate that not all TSCs function as ZMCs, and much of their activity can be predicted by their affinity for zinc.
Rutgers Cancer Institute of New Jersey (X.Y., A.T.T., S.Z., Y.L., D.R.C.), Department of Surgery, Rutgers Robert Wood Johnson Medical School (X.Y., A.T.T, Y.L., D.R.C.), Rutgers Translational Sciences, Department of Chemistry and Chemical Biology (S.D.K.), Department of Medicinal Chemistry, Rutgers Ernest Mario School of Pharmacy (J.G., A.F.B., S.D.K.), Rutgers University, New Brunswick, New Jersey; Department of Biochemistry and Molecular Biology, State University of New York (SUNY) Upstate Medical University, Syracuse, New York (A.B., S.N.L.); and Mount Sinai St. Luke's Roosevelt General Surgery Residency Program, New York, New York (A.T.T.).
Xin
X
Yu
Rutgers Cancer Institute of New Jersey (X.Y., A.T.T., S.Z., Y.L., D.R.C.), Department of Surgery, Rutgers Robert Wood Johnson Medical School (X.Y., A.T.T, Y.L., D.R.C.), Rutgers Translational Sciences, Department of Chemistry and Chemical Biology (S.D.K.), Department of Medicinal Chemistry, Rutgers Ernest Mario School of Pharmacy (J.G., A.F.B., S.D.K.), Rutgers University, New Brunswick, New Jersey; Department of Biochemistry and Molecular Biology, State University of New York (SUNY) Upstate Medical University, Syracuse, New York (A.B., S.N.L.); and Mount Sinai St. Luke's Roosevelt General Surgery Residency Program, New York, New York (A.T.T.).
Adam
A
Blanden
Rutgers Cancer Institute of New Jersey (X.Y., A.T.T., S.Z., Y.L., D.R.C.), Department of Surgery, Rutgers Robert Wood Johnson Medical School (X.Y., A.T.T, Y.L., D.R.C.), Rutgers Translational Sciences, Department of Chemistry and Chemical Biology (S.D.K.), Department of Medicinal Chemistry, Rutgers Ernest Mario School of Pharmacy (J.G., A.F.B., S.D.K.), Rutgers University, New Brunswick, New Jersey; Department of Biochemistry and Molecular Biology, State University of New York (SUNY) Upstate Medical University, Syracuse, New York (A.B., S.N.L.); and Mount Sinai St. Luke's Roosevelt General Surgery Residency Program, New York, New York (A.T.T.).
Ashley T
AT
Tsang
Rutgers Cancer Institute of New Jersey (X.Y., A.T.T., S.Z., Y.L., D.R.C.), Department of Surgery, Rutgers Robert Wood Johnson Medical School (X.Y., A.T.T, Y.L., D.R.C.), Rutgers Translational Sciences, Department of Chemistry and Chemical Biology (S.D.K.), Department of Medicinal Chemistry, Rutgers Ernest Mario School of Pharmacy (J.G., A.F.B., S.D.K.), Rutgers University, New Brunswick, New Jersey; Department of Biochemistry and Molecular Biology, State University of New York (SUNY) Upstate Medical University, Syracuse, New York (A.B., S.N.L.); and Mount Sinai St. Luke's Roosevelt General Surgery Residency Program, New York, New York (A.T.T.).
Saif
S
Zaman
Rutgers Cancer Institute of New Jersey (X.Y., A.T.T., S.Z., Y.L., D.R.C.), Department of Surgery, Rutgers Robert Wood Johnson Medical School (X.Y., A.T.T, Y.L., D.R.C.), Rutgers Translational Sciences, Department of Chemistry and Chemical Biology (S.D.K.), Department of Medicinal Chemistry, Rutgers Ernest Mario School of Pharmacy (J.G., A.F.B., S.D.K.), Rutgers University, New Brunswick, New Jersey; Department of Biochemistry and Molecular Biology, State University of New York (SUNY) Upstate Medical University, Syracuse, New York (A.B., S.N.L.); and Mount Sinai St. Luke's Roosevelt General Surgery Residency Program, New York, New York (A.T.T.).
Yue
Y
Liu
Rutgers Cancer Institute of New Jersey (X.Y., A.T.T., S.Z., Y.L., D.R.C.), Department of Surgery, Rutgers Robert Wood Johnson Medical School (X.Y., A.T.T, Y.L., D.R.C.), Rutgers Translational Sciences, Department of Chemistry and Chemical Biology (S.D.K.), Department of Medicinal Chemistry, Rutgers Ernest Mario School of Pharmacy (J.G., A.F.B., S.D.K.), Rutgers University, New Brunswick, New Jersey; Department of Biochemistry and Molecular Biology, State University of New York (SUNY) Upstate Medical University, Syracuse, New York (A.B., S.N.L.); and Mount Sinai St. Luke's Roosevelt General Surgery Residency Program, New York, New York (A.T.T.).
John
J
Gilleran
Rutgers Cancer Institute of New Jersey (X.Y., A.T.T., S.Z., Y.L., D.R.C.), Department of Surgery, Rutgers Robert Wood Johnson Medical School (X.Y., A.T.T, Y.L., D.R.C.), Rutgers Translational Sciences, Department of Chemistry and Chemical Biology (S.D.K.), Department of Medicinal Chemistry, Rutgers Ernest Mario School of Pharmacy (J.G., A.F.B., S.D.K.), Rutgers University, New Brunswick, New Jersey; Department of Biochemistry and Molecular Biology, State University of New York (SUNY) Upstate Medical University, Syracuse, New York (A.B., S.N.L.); and Mount Sinai St. Luke's Roosevelt General Surgery Residency Program, New York, New York (A.T.T.).
Anthony F
AF
Bencivenga
Rutgers Cancer Institute of New Jersey (X.Y., A.T.T., S.Z., Y.L., D.R.C.), Department of Surgery, Rutgers Robert Wood Johnson Medical School (X.Y., A.T.T, Y.L., D.R.C.), Rutgers Translational Sciences, Department of Chemistry and Chemical Biology (S.D.K.), Department of Medicinal Chemistry, Rutgers Ernest Mario School of Pharmacy (J.G., A.F.B., S.D.K.), Rutgers University, New Brunswick, New Jersey; Department of Biochemistry and Molecular Biology, State University of New York (SUNY) Upstate Medical University, Syracuse, New York (A.B., S.N.L.); and Mount Sinai St. Luke's Roosevelt General Surgery Residency Program, New York, New York (A.T.T.).
S David
SD
Kimball
Rutgers Cancer Institute of New Jersey (X.Y., A.T.T., S.Z., Y.L., D.R.C.), Department of Surgery, Rutgers Robert Wood Johnson Medical School (X.Y., A.T.T, Y.L., D.R.C.), Rutgers Translational Sciences, Department of Chemistry and Chemical Biology (S.D.K.), Department of Medicinal Chemistry, Rutgers Ernest Mario School of Pharmacy (J.G., A.F.B., S.D.K.), Rutgers University, New Brunswick, New Jersey; Department of Biochemistry and Molecular Biology, State University of New York (SUNY) Upstate Medical University, Syracuse, New York (A.B., S.N.L.); and Mount Sinai St. Luke's Roosevelt General Surgery Residency Program, New York, New York (A.T.T.) carpizdr@cinj.rutgers.edu lohs@upstate.edu.
Stewart N
SN
Loh
Rutgers Cancer Institute of New Jersey (X.Y., A.T.T., S.Z., Y.L., D.R.C.), Department of Surgery, Rutgers Robert Wood Johnson Medical School (X.Y., A.T.T, Y.L., D.R.C.), Rutgers Translational Sciences, Department of Chemistry and Chemical Biology (S.D.K.), Department of Medicinal Chemistry, Rutgers Ernest Mario School of Pharmacy (J.G., A.F.B., S.D.K.), Rutgers University, New Brunswick, New Jersey; Department of Biochemistry and Molecular Biology, State University of New York (SUNY) Upstate Medical University, Syracuse, New York (A.B., S.N.L.); and Mount Sinai St. Luke's Roosevelt General Surgery Residency Program, New York, New York (A.T.T.) carpizdr@cinj.rutgers.edu lohs@upstate.edu.
Darren R
DR
Carpizo
D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D006131:Growth Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D056511:Metallochaperones / Q000378:metabolism*; D009154:Mutation / Q000187:drug effects / Q000502:physiology*; D013882:Thiosemicarbazones / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
D006131:Growth Inhibitors; D056511:Metallochaperones; D013882:Thiosemicarbazones; D016159:Tumor Suppressor Protein p53; D015032:Zinc