Biologics are a rapidly growing class of therapeutics with many advantages over traditional small molecule drugs. A major obstacle to their development is that proteins and peptides are easily ...destroyed by proteases and, thus, typically have prohibitively short half-lives in human gut, plasma, and cells. One of the most effective ways to prevent degradation is to engineer analogs from dextrorotary (D)-amino acids, with up to 10⁵-fold improvements in potency reported. We here propose a general peptide-engineering platform that overcomes limitations of previous methods. By creating a mirror image of every structure in the Protein Data Bank (PDB), we generate a database of ∼2.8 million D-peptides. To obtain a D-analog of a given peptide, we search the (D)-PDB for similar configurations of its critical—“hotspot”—residues. As a proof of concept, we apply our method to two peptides that are Food and Drug Administration approved as therapeutics for diabetes and osteoporosis, respectively. We obtain D-analogs that activate the GLP1 and PTH1 receptors with the same efficacy as their natural counterparts and show greatly increased half-life.
Peptides with a combination of high positive charge and high hydrophobicity have high antimicrobial activity, as epitomized by peptide venoms, which are designed by nature as disruptors of host ...membranes yet also display significant efficacy against pathogens. To investigate this phenomenon systematically, here we focus on ponericin W1, a peptide venom isolated from Pachycondyla goeldii ants (WLGSALKIGAKLLPSVVGLFKKKKQ) to examine whether Lys positioning can be broadly applied to optimize the functional range of existing natural sequences. We prepared sets of ponericin W1 analogues, where Lys residues were either distributed in an amphipathic manner throughout the sequence (PonAmp), clustered at the N-terminus (PonN), or clustered at the C-terminus (PonC), along with their counterparts of reduced hydrophobicity through 2–4 Leu-to-Ala replacements. We found that wild-type ponericin W1 and all three variants displayed toxicity against human erythrocytes, but hemolysis was eliminated by the replacement of two or more Leu residues by Ala residues. As well, peptides containing up to 3 Leu-to-Ala replacements retained antimicrobial activity against E. coli bacteria. Biophysical analyses of peptide–membrane interaction patterns by circular dichroism spectroscopy revealed a novel mode of cluster-dependent peptide positioning vis-à-vis the water–membrane interface, where PonAmp and PonC peptides displayed full or partial helical structures, while PonN peptides were unstructured, likely due, in part, to dynamic interchange between aqueous and membrane surface environments. The overall findings suggest that the lower membrane penetration of N-terminal charge-clustered constructs coupled with moderate sequence hydrophobicity may be advantageous for conferring enhanced target selectivity for bacterial versus mammalian membranes.
Small multidrug resistance (SMR) pumps represent a minimal paradigm of proton-coupled membrane transport in bacteria, yet no high-resolution structure of an SMR protein is available. Here, ...atomic-resolution structures of the Escherichia coli efflux-multidrug resistance E (EmrE) multidrug transporter in ligand-bound form are refined using microsecond molecular dynamics simulations biased using low-resolution data from X-ray crystal-lography. The structures are compatible with existing mutagenesis data as well as NMR and biochemical experiments, including pKas of the catalytic glutamate residues and the dissociation constant (KD
) of the tetraphenylphosphonium⁺ cation. The refined structures show the arrangement of residue side chains in the EmrE active site occupied by two different ligands and in the absence of a ligand, illustrating how EmrE can adopt structurally diverse active site configurations. The structures also show a stable, well-packed binding interface between the helices H4 of the two monomers, which is believed to be crucial for EmrE dimerization. Guided by the atomic details of this interface, we design proteolysis-resistant stapled peptides that bind to helix H4 of an EmrE monomer. The peptides are expected to interfere with the dimerization and thereby inhibit drug transport. Optimal positions of the peptide staple were determined using free-energy simulations of peptide binding to monomeric EmrE. Three of the four top-scoring peptides selected for experimental testing resulted in significant inhibition of proton-driven ethidium efflux in live cells without nonspecific toxicity. The approach described here is expected to be of general use for the design of peptide therapeutics.
Natural α-helical cationic antimicrobial peptide (CAP) sequences are predominantly amphipathic, with only ca. 2% containing four or more consecutive positively charged amino acids (Lys/Arg). We have ...designed synthetic CAPs that deviate from these natural sequences, as typified by the charge-clustered peptide KKKKKKAAFAAWAAFAA-NH2, (termed 6K-F17), which displays high antimicrobial activity with no toxicity to mammalian cells. We created a series of peptides varying in charge patterning, increasing the amphipathic character of 6K-F17 to mimic the design of natural CAPs (e.g., KAAKKFAKAWAKAFAA-NH2). Amphipathic sequences displayed increased antimicrobial activity against bacteria but were significantly more toxic to mammalian cells and more susceptible to protease degradation than their corresponding charge-clustered variants, suggesting that amphipathic sequences may be desirable in nature to allow for more versatile functions (i.e., antibacterial, antifungal, antipredator) and rapid clearance from vulnerable host cells. Our approach to clustering of charges may therefore allow for specialization against bacteria, in concert with prolonged peptide half-life.
Membrane proteins play the central roles in a variety of cellular processes, ranging from nutrient uptake and signalling, to cell-cell communication. Their biological functions are directly related ...to how they fold and assemble; defects often lead to disease. Protein–protein interactions (PPIs) within the membrane are therefore of great interest as therapeutic targets. Here we review the progress in the application of membrane–insertable peptides for the disruption or stabilization of membrane–based PPIs. We describe the design and preparation of transmembrane peptide mimics; and of several categories of peptidomimetics used for study, including d-enantiomers, non–natural amino acids, peptoids, and β-peptides. Further aspects of the review describe modifications to membrane–insertable peptides, including lipidation and cyclization via hydrocarbon stapling. These approaches provide a pathway toward the development of metabolically stable, non-toxic, and efficacious peptide modulators of membrane–based PPIs. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.
Display omitted
•Advances in peptide chemistry have made peptides a feasible therapeutic option.•Transmembrane peptides may be used to target membrane protein-protein interactions.•Peptidomimetics, lipidation and cyclization improve peptide stability and efficacy.•Membrane protein–protein interactions are now ‘druggable’ targets.
With the increasing recognition of biofilms in human disease, the development of novel antimicrobial therapies is of critical importance. For example, in patients with cystic fibrosis (CF), the ...acquisition of host-adapted, chronic Pseudomonas aeruginosa infection is associated with a decline in lung function and increased mortality. Our objective was to test the in vitro efficacy of a membrane-active antimicrobial peptide we designed, termed 6K-F17 (sequence: KKKKKK-AAFAAWAAFAA-NH
), against multidrug resistant P. aeruginosa biofilms. This peptide displays high antimicrobial activity against a range of pathogenic bacteria, yet is non-hemolytic to human erythrocytes and non-toxic to human bronchial epithelial cells. In the present work, P. aeruginosa strain PAO1, and four multidrug resistant (MDR) isolates from chronically infected CF individuals, were grown as 48-hour biofilms in a static biofilm slide chamber model. These biofilms were then exposed to varying concentrations of 6K-F17 alone, or in the presence of tobramycin, prior to confocal imaging. Biofilm biovolume and viability were assessed. 6K-F17 was able to kill biofilms - even in the presence of sputum - and greatly reduce biofilm biovolume in PAO1 and MDR isolates. Strikingly, when used in conjunction with tobramycin, low doses of 6K-F17 significantly potentiated tobramycin killing, leading to biofilm destruction.
Bacteria have acquired multiple mechanisms to evade the lethal effects of current therapeutics, hindering treatment of bacterial infections, such as those caused by the pathogen
, which is ...responsible for nosocomial and cystic fibrosis lung infections. One resistance mechanism involves membrane-embedded multidrug efflux pumps that can effectively extrude an array of substrates, including common antibiotics, dyes, and biocides. Among these is a small multidrug resistance (SMR) efflux protein, consisting of four transmembrane (TM) helices, that functions as an antiparallel dimer. TM helices 1 to 3 (TM1 to TM3) comprise the substrate binding pocket, while TM4 contains a GG7 heptad sequence motif that mediates the SMR TM4-TM4 dimerization. In the present work, we synthesized a series of peptides containing the residues centered on the TM4-TM4 binding interface found in the
SMR (PAsmr), typified by Ac-Ala-(Sar)
-LL
G
IA
LV-KKK-NH
(helix-helix interaction residues are underlined). Here, the acetylated N-terminal sarcosine (
-methyl-Gly) tag Ac-Ala-(Sar)
promotes membrane penetration, while the C-terminal Lys tag promotes selectivity for the negatively charged bacterial membranes. This peptide was observed to competitively disrupt PAsmr-mediated efflux, as measured by efflux inhibition of the fluorescent dye ethidium bromide, while having no effect on cell membrane integrity. Alternatively, a corresponding peptide in which the TM4 binding motif is scrambled was inactive in this assay. In addition, when
cells expressing PAsmr were combined with sublethal concentrations of several biocides, growth was significantly inhibited when peptide was added, notably, by up to 95% with the disinfectant benzylalkonium chloride. These results demonstrate promise for an efflux pump inhibitor to address the increasing threat of antibiotic-resistant bacteria.
Clinically relevant multidrug-resistant bacteria often arise due to overproduction of membrane-embedded efflux proteins that are capable of pumping antibiotics out of the bacterial cell before the ...drugs can exert their intended toxic effect. The Escherichia coli membrane protein AcrB is the archetypal protein utilized for bacterial efflux study because it can extrude a diverse range of antibiotic substrates and has close homologues in many Gram-negative pathogens. Three AcrB subunits, each of which contains 12 transmembrane (TM) helices, are known to trimerize to form the minimal functional unit, stabilized noncovalently by helix–helix interactions between TM1 and TM8. To inhibit the efflux activity of AcrB, we have rationally designed synthetic peptides aimed at destabilizing the AcrB trimerization interface by outcompeting the subunit interaction sites within the membrane. Here we report that peptides mimicking TM1 or TM8, with flanking N-terminal peptoid tags, and C-terminal lysine tags that aid in directing the peptides to their membrane-embedded target, decrease the AcrB-mediated efflux of the fluorescent substrate Nile red and potentiate the effect of the antimicrobials chloramphenicol and ethidium bromide. To further characterize the motif encompassing the interaction between TM1 and TM8, we used Förster resonance energy transfer to demonstrate dimerization. Using the TM1 and TM8 peptides, in conjunction with several selected mutant peptides, we highlight residues that may increase the potency and specificity of the peptide drug candidates. In targeting membrane-embedded protein–protein interactions, this work represents a novel approach to AcrB inhibition and, more broadly, a potential route to a new category of efflux pump inhibitors.
Display omitted
•Designed linear and hydrocarbon-stapled CAPs display similar antimicrobial activity.•Hydrocarbon stapling increases membrane interactions, but non-selectively.•Polar substitutions ...restore selectivity for bacterial membranes.•Linear and stapled analogs do not function through detergent-like mechanisms.
Cyclization has been recognized as a valuable technique for increasing the efficacy of small molecule and peptide therapeutics. Here we report the application of a hydrocarbon staple to a rationally-designed cationic antimicrobial peptide (CAP) that acquires increased membrane targeting and interaction vs. its linear counterpart. The previously-described CAP, 6K-F17 (KKKKKK-AAFAAWAAFAA-NH2) was used as the backbone for incorporation of an i to i + 4 helical hydrocarbon staple through olefin ring closing metathesis. Stapled versions of 6K-F17 showed an increase in non-selective membrane interaction, where the staple itself enhances the degree of membrane interaction and rate of cell death while maintaining high potency against bacterial membranes. However, the higher averaged hydrophobicity imparted by the staple also significantly increases toxicity to mammalian cells. This deleterious effect is countered through stepwise reduction of the stapled 6K-F17’s backbone hydrophobicity through polar amino acid substitutions. Circular dichroism assessment of secondary structure in various bacterial membrane mimetics reveals that a helical structure may improve – but is not an absolute requirement for – antimicrobial activity of 6K-F17. Further, phosphorus-31 static solid state NMR spectra revealed that both non-toxic stapled and linear peptides bind bacterial membranes in a similar manner that does not involve a detergent-like mechanism of lipid removal. The overall results suggest that the technique of hydrocarbon stapling can be readily applied to membrane-interactive CAPs to modulate how they interact and target biological membranes.
Chronic infection and inflammation are the primary causes of declining lung function in Cystic Fibrosis (CF) patients. ORKAMBI
(Lumacaftor-Ivacaftor) is an approved combination therapy for Cystic ...Fibrosis (CF) patients bearing the most common mutation, F508del, in the cystic fibrosis conductance regulator (CFTR) protein. It has been previously shown that ORKAMBI
-mediated rescue of CFTR is reduced by a pre-existing
infection. Here, we show that the infection of F508del-CFTR human bronchial epithelial (HBE) cells with lab strain and four different clinical strains of
isolated from the lung sputum of CF patients, decreases CFTR function in a strain-specific manner by 48 to 88%. The treatment of infected cells with antibiotic tobramycin or cationic antimicrobial peptide 6K-F17 was found to decrease clinical strain bacterial growth on HBE cells and restore ORKAMBI
-mediated rescue of F508del-CFTR function. Further, 6K-F17 was found to downregulate the expression of pro-inflammatory cytokines, interleukin (IL)-8, IL-6, and tumor necrosis factor-α in infected HBE cells. The results provide strong evidence for a combination therapy approach involving CFTR modulators and anti-infectives (i.e., tobramycin and/or 6K-F17) to improve their overall efficacy in CF patients.
PDF
