title: Computational design and experimental characterization of peptides intended for pH-dependent membrane insertion and pore formation.
abstract: There are many opportunities to use macromolecules, such as peptides and oligonucleotides, for intracellular applications. Despite this, general methods for delivering these molecules to the cytosol in a safe and efficient manner are not available. Efforts to develop a variety of intracellular drug delivery systems such as viral vectors, lipoplexes, nanoparticles, and amphiphilic peptides have been made, but various challenges such as delivery efficiency, toxicity, and controllability remain. A central challenge is the ability to selectively perturb, not destroy, the membrane to facilitate cargo introduction. Herein, we describe our efforts to design and characterize peptides that form pores inside membranes at acidic pH, so-called pH-switchable pore formation (PSPF) peptides, as a potential means for facilitating cargo translocation through membranes. Consistent with pore formation, these peptides exhibit low-pH-triggered selective release of ATP and miRNA, but not hemoglobin, from red blood cells. Consistent with these observations, biophysical studies (tryptophan fluorescence, circular dichroism, size-exclusion chromatography, analytical ultracentrifugation, and attenuated total reflectance Fourier transformed infrared spectroscopy) show that decreased pH destabilizes the PSPF peptides in aqueous systems while promoting their membrane insertion. Together, these results suggest that reduced pH drives insertion of PSPF peptides into membranes, leading to target-specific escape through a proposed pore formation mechanism.
authors: †Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.
authors: Yao
authors: Y
authors: Zhang
authors: René
authors: R
authors: Bartz
authors: †Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.
authors: Gevorg
authors: G
authors: Grigoryan
authors: †Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.
authors: Michael
authors: M
authors: Bryant
authors: Jeff
authors: J
authors: Aaronson
authors: Stephen
authors: S
authors: Beck
authors: Nathalie
authors: N
authors: Innocent
authors: Lee
authors: L
authors: Klein
authors: William
authors: W
authors: Procopio
authors: Tom
authors: T
authors: Tucker
authors: Vasant
authors: V
authors: Jadhav
authors: David M
authors: DM
authors: Tellers
authors: §Department of Pharmaceutical Chemistry, University of California-San Francisco, San Francisco, California 94158, United States.
authors: William F
authors: WF
authors: DeGrado
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D002850:Chromatography, Gel; D002942:Circular Dichroism; D015195:Drug Design; D004912:Erythrocytes / Q000187:drug effects; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers / Q000378:metabolism; D035683:MicroRNAs / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000008:administration & dosage* / Q000737:chemistry* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D012995:Solubility; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
chemical_list: D008051:Lipid Bilayers; D035683:MicroRNAs; D010455:Peptides; D014364:Tryptophan; D000255:Adenosine Triphosphate
references: J Control Release. 2006 May 15;112(2):240-8
references: 16545884
references: Biochim Biophys Acta. 1999 Dec 15;1462(1-2):55-70
references: 10590302
references: J Med Chem. 2010 Nov 25;53(22):7887-901
references: 20806941
references: Bioconjug Chem. 2008 Apr;19(4):920-7
references: 18376855
references: FEBS J. 2010 Dec;277(23):4814-27
references: 21078116
references: Proc Natl Acad Sci U S A. 2001 Jul 31;98(16):9056-61
references: 11481472
references: Protein Sci. 2003 Aug;12(8):1732-40
references: 12876322
references: Biochemistry. 1997 Dec 16;36(50):15865-72
references: 9398319
references: J Struct Biol. 2002 Jan-Feb;137(1-2):41-53
references: 12064932
references: Biophys J. 2002 May;82(5):2720-36
references: 11964258
references: Proteins. 1990;7(1):1-15
references: 2184436
references: J Am Chem Soc. 2009 Aug 19;131(32):11341-3
references: 19722646
references: FEBS Lett. 2002 Oct 2;529(1):49-53
references: 12354612
references: J Biol Chem. 1987 May 15;262(14):6500-5
references: 3571268
references: Nat Rev Mol Cell Biol. 2003 Apr;4(4):309-19
references: 12671653
references: J Biol Chem. 2009 Mar 13;284(11):6972-81
references: 19129199
references: Mol Membr Biol. 1999 Jan-Mar;16(1):3-9
references: 10332732
references: Trends Biochem Sci. 1996 Oct;21(10):375-82
references: 8918191
references: Nat Rev Mol Cell Biol. 2006 Jul;7(7):495-504
references: 16773132
references: Proc Natl Acad Sci U S A. 2009 Mar 31;106(13):5105-10
references: 19289834
references: Biochemistry. 1991 Oct 22;30(42):10211-20
references: 1931950
references: Nanomedicine (Lond). 2010 Feb;5(2):193-208
references: 20148632
references: Biochemistry. 1997 Mar 4;36(9):2450-8
references: 9054549
references: Expert Opin Drug Deliv. 2011 Feb;8(2):171-90
references: 21226651
references: Proc Natl Acad Sci U S A. 2010 Nov 23;107(47):20246-50
references: 21048084
references: Science. 1990 Nov 2;250(4981):646-51
references: 2237415
references: Proc Natl Acad Sci U S A. 2006 Sep 12;103(37):13658-63
references: 16954199
references: Biomacromolecules. 2006 Aug;7(8):2407-14
references: 16903689
references: Annu Rev Biophys Biomol Struct. 1999;28:319-65
references: 10410805
references: Annu Rev Biochem. 1986;55:663-700
references: 2874766
references: Nature. 2006 May 4;441(7089):111-4
references: 16565705
references: Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8408-12
references: 7667303
references: Mol Ther. 2010 Jan;18(1):171-80
references: 19738601
references: Proteins. 2002 May 1;47(2):209-18
references: 11933067
references: Biotechnol J. 2009 Nov;4(11):1559-72
references: 19844918
references: Protein Sci. 1998 Jun;7(6):1404-14
references: 9655345
references: Science. 2011 May 27;332(6033):1071-6
references: 21617073
references: Science. 2007 Mar 30;315(5820):1817-22
references: 17395823
references: Proc Natl Acad Sci U S A. 2004 Feb 17;101(7):1828-33
references: 14766985
references: Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7297-301
references: 7638184
references: Biochemistry. 2007 Dec 4;46(48):13856-63
references: 17994771
references: Mol Biosyst. 2009 Nov;5(11):1306-10
references: 19823746
references: Int J Mol Sci. 2008 Jun;9(7):1276-320
references: 19325804
references: J Mol Biol. 2006 Feb 3;355(5):968-79
references: 16343527
references: Annu Rev Biomed Eng. 2006;8:343-75
references: 16834560
references: Proc Natl Acad Sci U S A. 2010 Feb 2;107(5):2213-8
references: 20133865
references: Curr Opin Struct Biol. 1998 Dec;8(6):778-84
references: 9914258
references: ACS Chem Biol. 2010 Aug 20;5(8):747-52
references: 20545362
references: J Mol Biol. 2011 Jan 28;405(4):1079-100
references: 20932976
references: Curr Opin Struct Biol. 2001 Aug;11(4):450-7
references: 11495738
references: Trends Microbiol. 2005 Feb;13(2):72-8
references: 15680766
references: Nat Struct Biol. 2000 Feb;7(2):161-6
references: 10655620
references: Exp Neurol. 2004 Nov;190(1):1-16
references: 15473976
references: Mol Pharm. 2010 Dec 6;7(6):1899-912
references: 20822168
references: J Am Chem Soc. 2004 Apr 28;126(16):5078-9
references: 15099085
references: Biochem J. 2011 Apr 15;435(2):475-87
references: 21265735
references: Proc Natl Acad Sci U S A. 2011 Mar 8;108(10):3958-63
references: 21321234
references: Nat Rev Microbiol. 2005 Mar;3(3):238-50
references: 15703760
references: Biochemistry. 1999 Sep 14;38(37):11905-13
references: 10508393