title: Efflux by small multidrug resistance proteins is inhibited by membrane-interactive helix-stapled peptides.
abstract: Bacterial cell membranes contain several protein pumps that resist the toxic effects of drugs by efficiently extruding them. One family of these pumps, the small multidrug resistance proteins (SMRs), consists of proteins of about 110 residues that need to oligomerize to form a structural pathway for substrate extrusion. As such, SMR oligomerization sites should constitute viable targets for efflux inhibition, by disrupting protein-protein interactions between helical segments. To explore this proposition, we are using Hsmr, an SMR from Halobacter salinarum that dimerizes to extrude toxicants. Our previous work established that (i) Hsmr dimerization is mediated by a helix-helix interface in Hsmr transmembrane (TM) helix 4 (residues (90)GLALIVAGV(98)); and (ii) a peptide comprised of the full TM4(85-105) sequence inhibits Hsmr-mediated ethidium bromide efflux from bacterial cells. Here we define the minimal linear sequence for inhibitor activity (determined as TM4(88-100), and then "staple" this sequence via Grubbs metathesis to produce peptides typified by acetyl-A-(Sar)3-(88)VVGLXLIZXGVVV(100)-KKK-NH2 (X = 2-(4'-pentenyl)alanine at positions 92 and 96; Z = Val, Gly, or Asn at position 95)). The Asn(95) peptide displayed specific efflux inhibition and resensitization of Hsmr-expressing cells to ethidium bromide; and was non-hemolytic to human red blood cells. Stapling essentially prevented peptide degradation in blood plasma and liver homogenates versus an unstapled counterpart. The overall results confirm that the stapled analog of TM4(88-100) retains the structural complementarity required to disrupt the Hsmr TM4-TM4 locus in Hsmr, and portend the general validity of stapled peptides as therapeutics for the disruption of functional protein-protein interactions in membranes.
authors.affiliation: From the Division of Molecular Structure and Function, Research Institute, The Hospital for Sick Children, Toronto, Ontario M5G 0A4 and.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Bellmann-Sickert
authors.affiliation: From the Division of Molecular Structure and Function, Research Institute, The Hospital for Sick Children, Toronto, Ontario M5G 0A4 and the Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Tracy A
authors.initials: TA
authors.lastname: Stone
authors.affiliation: From the Division of Molecular Structure and Function, Research Institute, The Hospital for Sick Children, Toronto, Ontario M5G 0A4 and the Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Bradley E
authors.initials: BE
authors.lastname: Poulsen
authors.affiliation: From the Division of Molecular Structure and Function, Research Institute, The Hospital for Sick Children, Toronto, Ontario M5G 0A4 and the Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada deber@sickkids.ca.
authors.forename: Charles M
authors.initials: CM
authors.lastname: Deber
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002942:Circular Dichroism; D004912:Erythrocytes / Q000187:drug effects; D004996:Ethidium / Q000737:chemistry; D006217:Halobacterium / Q000378:metabolism; D006461:Hemolysis; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry; D008099:Liver / Q000187:drug effects; D008826:Microbial Sensitivity Tests; D010455:Peptides / Q000737:chemistry*; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D017433:Protein Structure, Secondary
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D008051:Lipid Bilayers; D010455:Peptides; D004996:Ethidium
keywords: Cell-penetrating Peptide (CPP); Membrane Bilayer; Membrane Protein; Multidrug Transporter; Peptide Chemical Synthesis; Peptide Conformation; Protein Drug Interaction