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.

Keywords: Cell-penetrating Peptide (CPP); Membrane Bilayer; Membrane Protein; Multidrug Transporter; Peptide Chemical Synthesis; Peptide Conformation; Protein Drug Interaction.

FIGURE 1.

Inhibition of membrane-based protein-protein interactions by a stapled peptide. Wild type EmrE, an SMR homologue of Hsmr, relies on dimerization for function (depicted is EmrE antiparallel dimer from Protein Data Bank entry 3B5D) (36). Dimerization depends on interaction between TM4 of both monomers. A peptide resembling the interaction sequence of TM4 can compete out this helix-helix interaction, by inserting into the membrane and interacting at the target locus with TM4 of a given monomer, thereby preventing dimerization and consequently efflux of substrate. Introducing a staple renders the peptide into a preformed helical shape that enables optimal interaction of the hydrophobic staple moiety with both the target TM4-TM4 interaction motif and the lipid environment.

FIGURE 2.

Overview of synthesized peptides. Solubility tags: T₁, CH₃CO-Ala-Sar₃-; T₂, -Lys₃-NH₂; X, (S)-N-Fmoc-2-(4′-pentenyl)alanine; X′, (R)-N-Fmoc-2-(7′-octenyl)alanine.

FIGURE 3.

Circular dichroism spectra of selected linear and stapled Hsmr TM4 peptides. Spectra were obtained with 20 μm peptide in 20 mm SDS. See text for further discussion.

FIGURE 4.

Profiles of peptide biological activity. Four assays, ethidium efflux activity (Efflux), ER, AA, and hemolytic activity in human RBC are depicted for linear peptides TM4, TM4(90–98), and TM4(88–100) (left panels); and stapled peptides S-TM4(88–100), S-TM4(88–100)-G95, and S-TM4(88–100)-N95 (right panels). Efflux assays were run using samples of 1 μm peptide and 1 μg/ml of ethidium. Efflux rate constants were determined using first-order exponential decay and normalized to ethidium efflux in bacteria without addition of peptide (k_average = 2.4 ± 0.4 s⁻¹ with n = 11). Cells not expressing Hsmr have a relative efflux rate of 0.15 ± 0.02 (25). Values are given as mean ± S.E. of at least two independent experiments and significance values refer to comparison of all peptides to TM4(88–100)scr (****, p < 0.001; ***, p < 0.005; *, p < 0.05). Values for ER, AA, and RBCs are given as the minimal inhibitory or hemolytic concentration, which is the lowest concentration at which complete growth inhibition occurred or hemoglobin concentrations reached at least 10% of the positive control. Values for all assays were determined in at least two independent experiments.

FIGURE 5.

Metabolic stability of unstapled versus stapled peptide in human blood plasma and in bovine liver homogenates. 10 μm peptides (TAMRA-labeled TM4(88–100) and TAMRA-labeled S-TM4(88–100)-N95) were incubated in the respective medium for the indicated time periods. Cleavage products were separated by reversed phase HPLC at 565 nm and identified by MALDI-TOF mass spectrometry. Decay of the intact peptide (%) was determined by peak integration. See ”Materials and Methods“ for further details.