title: In vitro evolution of ligands to the membrane protein caveolin.
abstract: Membrane proteins comprise a third of the human genome, yet present challenging targets for reverse chemical genetics. For example, although implicated in numerous diseases including multiple myeloma, the membrane protein caveolin-1 appears to offer a poor target for the discovery of synthetic ligands due to its largely unknown structure and insolubility. To break this impasse and identify new classes of caveolae controlling lead compounds, we applied phage-based, reverse chemical genetics for the discovery of caveolin-1 ligands derived from the anti-HIV therapeutic T20. Substitution of homologous residues into the T20 sequence used a process analogous to medicinal chemistry for the affinity maturation to bind caveolin. The resultant caveolin-1 ligands bound with >1000-fold higher affinity than wild-type T20. Two types of ELISAs and isothermal titration calorimetry (ITC) measurements demonstrated high affinity binding to caveolin by the T20 variants with K(d) values in the 150 nM range. Microscopy experiments with the highest affinity caveolin ligands confirmed colocalization of the ligands with endogenous caveolin in NIH 3T3 cells. The results establish the foundation for targeting caveolin and caveolae formation in living cells.
authors: Department of Chemistry, University of California, Irvine, California 92697, USA.
authors: Sudipta
authors: S
authors: Majumdar
authors: Agnes
authors: A
authors: Hajduczki
authors: Rosemarie
authors: R
authors: Vithayathil
authors: Tivoli J
authors: TJ
authors: Olsen
authors: Ryan M
authors: RM
authors: Spitler
authors: Aaron S
authors: AS
authors: Mendez
authors: Travis D
authors: TD
authors: Thompson
authors: Gregory A
authors: GA
authors: Weiss
references: Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10339-43
references: 7479780
references: Proc Natl Acad Sci U S A. 1980 Mar;77(3):1496-500
references: 6929499
references: Am J Physiol Lung Cell Mol Physiol. 2006 Feb;290(2):L405-13
references: 16183667
references: J Biol Chem. 2005 Mar 25;280(12):11259-73
references: 15640162
references: Nat Cell Biol. 2009 Jul;11(7):807-14
references: 19525939
references: J Biol Chem. 1985 May 10;260(9):5787-96
references: 3988773
references: J Cell Biol. 1990 Dec;111(6 Pt 2):2931-8
references: 2148564
references: Nature. 2009 May 21;459(7245):344-6
references: 19458709
references: Proc Natl Acad Sci U S A. 1997 Sep 30;94(20):10693-8
references: 9380697
references: Clin Cancer Res. 1998 Aug;4(8):1873-80
references: 9717814
references: Cell. 1992 Feb 21;68(4):673-82
references: 1739974
references: Chembiochem. 2008 Nov 24;9(17):2846-52
references: 18973165
references: Nat Rev Mol Cell Biol. 2007 Mar;8(3):185-94
references: 17318224
references: Biochem Biophys Res Commun. 2005 Dec 23;338(3):1383-90
references: 16263077
references: Cancer Lett. 2006 Feb 20;233(1):10-5
references: 16473666
references: Anal Biochem. 1989 May 15;179(1):131-7
references: 2757186
references: Bioorg Med Chem Lett. 2008 Nov 15;18(22):5937-40
references: 18667306
references: J Cell Biol. 1993 Aug;122(4):789-807
references: 8349730
references: Bioconjug Chem. 2008 Jun;19(6):1113-8
references: 18498185
references: Protein Eng Des Sel. 2006 Jun;19(6):245-53
references: 16740523
references: Biochemistry. 2000 Feb 22;39(7):1634-42
references: 10677212
references: Immunity. 2004 Nov;21(5):617-27
references: 15539149
references: Traffic. 2000 Apr;1(4):326-41
references: 11208118
references: Chem Biol. 2003 Feb;10(2):161-8
references: 12618188
references: Methods Enzymol. 2009;455:127-55
references: 19289205
references: Methods Enzymol. 2000;328:333-63
references: 11075354
references: Neurobiol Aging. 2004 Jul;25(6):753-9
references: 15165700
references: FEBS Lett. 1998 Oct 9;436(3):403-10
references: 9801158
references: ACS Chem Biol. 2007 Jul 20;2(7):493-500
references: 17602618
references: Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):9770-4
references: 7937889
references: Nat Struct Biol. 2000 Nov;7 Suppl:928-30
references: 11103989
references: Physiol Rev. 2004 Oct;84(4):1341-79
references: 15383654
references: Cell. 2008 Jan 11;132(1):113-24
references: 18191225
references: J Biol Chem. 2007 Mar 2;282(9):6143-52
references: 17197700
references: J Biol Chem. 1997 Jan 24;272(4):2437-45
references: 8999956
references: Mol Biol Cell. 2000 Jan;11(1):325-37
references: 10637311
references: J Biol Chem. 2007 Mar 30;282(13):9612-9620
references: 17276993
references: J Biol Chem. 2004 Apr 2;279(14):14171-8
references: 14734557