title: In vitro selection of mutant HDM2 resistant to Nutlin inhibition.
abstract: HDM2 binds to the p53 tumour suppressor and targets it for proteosomal degradation. Presently in clinical trials, the small molecule Nutlin-3A competitively binds to HDM2 and abrogates its repressive function. Using a novel in vitro selection methodology, we simulated the emergence of resistance by evolving HDM2 mutants capable of binding p53 in the presence of Nutlin concentrations that inhibit the wild-type HDM2-p53 interaction. The in vitro phenotypes were recapitulated in ex vivo assays measuring both p53 transactivation function and the direct p53-HDM2 interaction in the presence of Nutlin. Mutations conferring drug resistance were not confined to the N-terminal p53/Nutlin-binding domain, and were additionally seen in the acidic, zinc finger and RING domains. Mechanistic insights gleaned from this broad spectrum of mutations will aid in future drug design and further our understanding of the complex p53-HDM2 interaction.
authors: p53Lab, Agency for Science, Technology and Research, Singapore, Singapore.
authors: Siau Jia
authors: SJ
authors: Wei
authors: Thomas
authors: T
authors: Joseph
authors: Adelene Y L
authors: AY
authors: Sim
authors: Larisa
authors: L
authors: Yurlova
authors: Kourosh
authors: K
authors: Zolghadr
authors: David
authors: D
authors: Lane
authors: Chandra
authors: C
authors: Verma
authors: Farid
authors: F
authors: Ghadessy
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry* / Q000494:pharmacology; D001665:Binding Sites; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D007093:Imidazoles / Q000737:chemistry* / Q000494:pharmacology; D051379:Mice; D062105:Molecular Docking Simulation; D009154:Mutation; D010879:Piperazines / Q000737:chemistry* / Q000494:pharmacology; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051736:Proto-Oncogene Proteins c-mdm2 / Q000737:chemistry* / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000235:genetics
chemical_list: D000970:Antineoplastic Agents; D007093:Imidazoles; D010879:Piperazines; D016159:Tumor Suppressor Protein p53; C482205:nutlin 3; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2
references: Lancet Oncol. 2012 Nov;13(11):1133-40
references: 23084521
references: J Am Chem Soc. 2007 Mar 7;129(9):2456-7
references: 17284038
references: Nature. 2010 Aug 12;466(7308):869-73
references: 20668451
references: Hum Mutat. 2003 Mar;21(3):176-81
references: 12619103
references: J Biol Chem. 2006 Nov 3;281(44):33030-5
references: 16905541
references: Proc Natl Acad Sci U S A. 2009 Dec 1;106(48):20411-6
references: 19915144
references: Cell Cycle. 2010 Mar 15;9(6):1167-81
references: 20190571
references: Proc Natl Acad Sci U S A. 2008 Feb 12;105(6):2070-5
references: 18227510
references: J Comput Chem. 2004 Jul 15;25(9):1157-74
references: 15116359
references: Science. 2001 Aug 3;293(5531):876-80
references: 11423618
references: Cancer Res. 2011 Jun 15;71(12):4205-13
references: 21515735
references: Cell. 2003 Mar 21;112(6):831-43
references: 12654249
references: Proc Natl Acad Sci U S A. 2010 Aug 10;107(32):14321-6
references: 20660730
references: Science. 1996 Nov 8;274(5289):948-53
references: 8875929
references: Biochemistry. 2006 Aug 1;45(30):9238-45
references: 16866370
references: Cell. 2004 Nov 24;119(5):591-602
references: 15550242
references: Cancer Res. 1991 Dec 1;51(23 Pt 1):6304-11
references: 1933891
references: PLoS One. 2011 Apr 29;6(4):e18934
references: 21572516
references: Science. 2004 Feb 6;303(5659):844-8
references: 14704432
references: Nature. 1995 Nov 9;378(6553):203-6
references: 7477326
references: Leukemia. 2000 Jan;14(1):61-7
references: 10637478
references: ACS Chem Biol. 2013 Mar 15;8(3):506-12
references: 23214419
references: J Am Chem Soc. 2008 May 21;130(20):6472-8
references: 18435534
references: Mol Cell Biol. 1994 Nov;14(11):7414-20
references: 7935455
references: Cell. 1992 Jun 26;69(7):1237-45
references: 1535557
references: Mol Cell Proteomics. 2008 Nov;7(11):2279-87
references: 18622019
references: Protein Sci. 2006 Feb;15(2):384-9
references: 16385008
references: Annu Rev Pharmacol Toxicol. 2009;49:223-41
references: 18834305
references: FEBS Lett. 1997 Dec 22;420(1):25-7
references: 9450543
references: J Biol Chem. 2009 Apr 24;284(17):11517-30
references: 19188367
references: J Mol Biol. 1999 Jan 15;285(2):775-83
references: 9878443
references: J Mol Biol. 2007 Aug 31;371(5):1238-48
references: 17610896
references: Int J Cancer. 2001 Jun 15;92(6):790-6
references: 11351297
references: Clin Cancer Res. 2009 Nov 15;15(22):6862-70
references: 19861442
references: Cancer Cell. 2003 Jun;3(6):577-87
references: 12842086
references: Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7491-5
references: 1323840
references: Cancer Res. 1992 Apr 15;52(8):2340-3
references: 1559236
references: Proc Natl Acad Sci U S A. 2003 Feb 18;100(4):1645-8
references: 12552135
references: PLoS One. 2012;7(8):e42642
references: 22912717
references: J Mol Biol. 2001 Nov 23;314(2):263-77
references: 11718560
references: Nat Genet. 2001 Sep;29(1):92-5
references: 11528400
references: Nature. 1997 May 15;387(6630):299-303
references: 9153396
references: J Chem Biol. 2009 Aug;2(3):113-29
references: 19568783
references: J Pathol. 1997 May;182(1):54-61
references: 9227342
references: Cancer Discov. 2012 Mar;2(3):214-26
references: 22585993
references: Nat Biotechnol. 1998 Jul;16(7):652-6
references: 9661199
references: J Med Chem. 2012 Jun 14;55(11):4936-54
references: 22524527
references: Mol Cell Biol. 1993 Jul;13(7):4107-14
references: 7686617
references: J Mol Biol. 2010 May 7;398(3):414-28
references: 20303977
references: Mol Cell Biol. 2007 Feb;27(3):1056-68
references: 17116689
references: Cancer Res. 2008 Oct 15;68(20):8260-8
references: 18922897
references: Oncogene. 2011 Nov 17;30(46):4678-86
references: 21643018
references: J Am Chem Soc. 2012 Jan 11;134(1):103-6
references: 22148351
references: PLoS Genet. 2009 May;5(5):e1000462
references: 19424414
references: Nature. 1997 May 15;387(6630):296-9
references: 9153395
references: Nat Protoc. 2010 May;5(5):883-97
references: 20431534
references: J Mol Biol. 2005 Jul 15;350(3):587-98
references: 15953616
references: Proc Natl Acad Sci U S A. 2006 Jan 31;103(5):1227-32
references: 16432196
references: Nature. 1992 Jul 2;358(6381):80-3
references: 1614537
references: Curr Pharm Des. 2011;17(6):569-77
references: 21391907
references: J Biol Chem. 2000 Mar 24;275(12):8945-51
references: 10722742