Aram J. Krauson

Gain-of-Function Analogues of the Pore-Forming Peptide Melittin Selected by Orthogonal High-Throughput Screening

We recently developed an orthogonal, high-throughput assay to identify peptides that self-assemble into potent, equilibrium pores in synthetic lipid bilayers. Here, we use this assay as a high-throughput screen to select highly potent pore-forming peptides from a 7776-member rational combinatorial peptide library based on the sequence of the natural pore-forming peptide toxin melittin. In the library we varied ten critical residues in the melittin sequence, chosen to test specific structural hypotheses about the mechanism of pore formation. Using the new high-throughput assay, we screened the library for gain-of-function sequences at a peptide to lipid ratio of 1:1000 where native melittin is not active. More than 99% of the library sequences were also inactive under these conditions. A small number of library members (0.1%) were highly active. From these we identified 14 potent, gain-of-function, pore-forming sequences. These sequences differed from melittin in only 2-6 amino acids out of 26. Some native residues were highly conserved and others were consistently changed. The two factors that were essential for gain-of-function were the preservation of melittins proline-dependent break in the middle of the helix and the improvement and extension the amphipathic nature of the α-helix. In particular the highly cationic carboxyl-terminal sequence of melittin, is consistently changed in the gain-of-function variants to a sequence that it is capable of participating in an extended amphipathic α-helix. The most potent variants reside in a membrane-spanning orientation, in contrast to the parent melittin, which is predominantly surface bound. This structural information, taken together with the high-throughput tools developed for this work, enable the identification, refinement and optimization of pore-forming peptides for many potential applications.

Interactions of membrane active peptides with planar supported bilayers: an impedance spectroscopy study.

Membrane active peptides exert their biological effects by interacting directly with a cells lipid bilayer membrane. These therapeutically promising peptides have demonstrated a variety of activities including antimicrobial, cytolytic, membrane translocating, and cell penetrating activities. Here, we use electrochemical impedance spectroscopy (EIS) on polymer-cushioned supported lipid bilayers constructed on single crystal silicon to study two pairs of closely related membrane active peptides selected from rationally designed, combinatorial libraries to have different activities in lipid bilayers: translocation, permeabilization, or no activity. Using EIS, we observed that binding of a membrane translocating peptide to the lipid bilayer resulted in a small decrease in membrane resistance followed by a recovery back to the original value. The recovery may be directly attributable to peptide translocation. A nontranslocating peptide did not decrease the resistance. The other pair, two membrane permeabilizing peptides, caused an exponential decrease of membrane resistance in a concentration-dependent manner. This permeabilization of the supported bilayer occurs at peptide to lipid ratios as much as 1000-fold lower than that needed to observe effects in vesicle leakage assays and gives new insights into the fundamental peptide-bilayer interactions involved in membrane permeabilization.

Synthetic Molecular Evolution of Pore-Forming Peptides by Iterative Combinatorial Library Screening

We previously reported the de novo design of a combinatorial peptide library that was subjected to high-throughput screening to identify membrane-permeabilizing antimicrobial peptides that have β-sheet-like secondary structure. Those peptides do not form discrete pores in membranes but instead partition into membrane interfaces and cause transient permeabilization by membrane disruption, but only when present at high concentration. In this work, we used a consensus sequence from that initial screen as a template to design an iterative, second generation library. In the 24-26-residue, 16,200-member second generation library we varied six residues. Two diad repeat motifs of alternating polar and nonpolar amino acids were preserved to maintain a propensity for non-helical secondary structure. We used a new high-throughput assay to identify members that self-assemble into equilibrium pores in synthetic lipid bilayers. This screen was done at a very stringent peptide to lipid ratio of 1:1000 where most known membrane-permeabilizing peptides, including the template peptide, are not active. In a screen of 10,000 library members we identified 16 (~0.2%) that are equilibrium pore-formers at this high stringency. These rare and highly active peptides, which share a common sequence motif, are as potent as the most active pore-forming peptides known. Furthermore, they are not α-helical, which makes them unusual, as most of the highly potent pore-forming peptides are amphipathic α-helices. Here we demonstrate that this synthetic molecular evolution-based approach, taken together with the new high-throughput tools we have developed, enables the identification, refinement, and optimization of unique membrane active peptides.

Toward the de novo design of antimicrobial peptides: Lack of correlation between peptide permeabilization of lipid vesicles and antimicrobial, cytolytic, or cytotoxic activity in living cells

We previously performed a lipid vesicle‐based, high‐throughput screen on a 26‐residue combinatorial peptide library that was designed de novo to yield membrane‐permeabilizing peptides that fold into β‐sheets. The most active and soluble library members that were identified permeabilized lipid vesicles detectably, but not with high potency. Nonetheless, they were broad‐spectrum, membrane‐permeabilizing antibiotics with minimum sterilizing activity at low µM concentrations. In an expansion of that work, we recently performed an iterative screen in which an active consensus sequence from that first‐generation library was used as a template to design a second‐generation library which was then screened against lipid vesicles at very high stringency. Compared to the consensus sequence from the first library, the most active second‐generation peptides are highly potent, equilibrium pore‐formers in synthetic lipid vesicles. Here, we use these first‐ and second‐generation families of peptides to test the hypothesis that a large increase in potency in bacteria‐like lipid vesicles will correlate with a large improvement in antimicrobial activity. The results do not support the hypothesis. Despite a 20‐fold increase in potency against bacteria‐like lipid vesicles, the second‐generation peptides are only slightly more active against bacteria, and at the same time, are also more toxic against mammalian cells. The results suggest that a “pipeline” strategy toward the optimization of antimicrobial peptides could begin with a vesicle‐based screen for identifying families with broad‐spectrum activity, but will also need to include screening or optimization steps that are done under conditions that are more directly relevant to possible therapeutic applications.

Molecular basis of inhibition of acid sensing ion channel 1A by diminazene

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation permeable ion channels expressed primarily in neurons. Here we employed site-directed mutagenesis and electrophysiology to investigate the mechanism of inhibition of ASIC1a by diminazene. This compound inhibits mouse ASIC1a with a half-maximal inhibitory concentration (IC50) of 2.4 μM. At first, we examined whether neutralizing mutations of Glu79 and Glu416 alter diminazene block. These residues form a hexagonal array in the lower palm domain that was previously shown to contribute to pore opening in response to extracellular acidification. Significantly, single Gln substitutions at positions 79 and 416 in ASIC1a reduced diminazene apparent affinity by 6–7 fold. This result suggests that diminazene inhibits ASIC1a in part by limiting conformational rearrangement in the lower palm domain. Because diminazene is charged at physiological pHs, we assessed whether it inhibits ASIC1a by blocking the ion channel pore. Consistent with the notion that diminazene binds to a site within the membrane electric field, diminazene block showed a strong dependence with the membrane potential. Moreover, a Gly to Ala mutation at position 438, in the ion conduction pathway of ASIC1a, increased diminazene IC50 by one order of magnitude and eliminated the voltage dependence of block. Taken together, our results indicate that the inhibition of ASIC1a by diminazene involves both allosteric modulation and blocking of ion flow through the conduction pathway. Our findings provide a foundation for the development of more selective and potent ASIC pore blockers.

Functional Coupling between the Finger and Thumb Domains of ASIC1A

Acid sensing ion channels (ASICs) are neuronal cation selective channels that respond to sudden drops in extracellular pH and desensitize in the continuous presence of protons. The mechanism that allows these channels to sense and respond to changes in extracellular pH is not well understood. Here we examined the contribution to channel activation of the finger and thumb domains of ASIC1a, two areas that reside in close proximity in the periphery of the extracellular region. Residues located at the interface of these two domains were individually mutated to Cys and the reactivity of the mutant channels toward the thiol reactive reagent MTSET was assessed using the two-electrode voltage clamp technique. We identified seven sites in the thumb domain, positions 325, 327, 344, 345, 348, 351 and 352, and two in the finger domain, positions 152 and 154, where MTSET treatment reduced the magnitude of the response to extracellular acidification. Residues 325, 327, 344, 345, 348 in the thumb domain are oriented toward residues 152 and 154 in the finger domain in the solved atomic structure of ASIC1 at low pH. Our results indicate that the finger and thumb domains experience a conformational change and become closer in response to extracellular acidification. To further assess the role of the finger-thumb interactions in ASIC1a proton activation, we generated channels with substitutions at neighboring positions in the finger and thumb domains. We found that the response to extracellular acidification after MTSET treatment of double mutant channels bearing Cys substitutions at positions 154 and 325 was significantly lower than the response of channels bearing individual mutations at these positions, consistent with functional coupling between the finger and thumb domain. Taken together, our results suggest that the finger and thumb domains contribute to ASIC1a activation.

High Throughput Screen of Combinatorial Peptide Library for Gain-of-Function and Loss-of-Function Changes to Melittin

Melittin, the main peptide component of European Honey Bee venom, is an amphipathic, 26-amino acid peptide that lyses bacterial and mammalian cells by forming transmembrane pores. Our research focuses on the various mechanisms of peptide permeation of membranes. We have designed orthogonal, fluorescence-based assays to characterize long-lived pore-forming peptides such as melittin. In these assays, peptides are incubated overnight with vesicles containing dye-labeled lipids and entrapped terbium. In the first measurement, the sum of the lytic activity is determined by measuring the terbium released from the vesicles. In the second measurement, we add a non membrane-permeable quencher of dye-labeled lipids. Using these assays we have observed a significant difference between melittin and other lytic peptides, such as alamethicin. Even at very high lipid concentrations (peptide:lipid < 1:2000) alamethicin forms long-lived pores, which release ∼100% of entrapped contents and give the quencher 100% access to the inside of a vesicle after overnight incubation. Melittins activity diminishes significantly at lipid concentrations larger than P:L = 1:500. To learn what factors modulate melittins activity, we have designed a 7,776-member, melittin-based combinatorial peptide library in which we vary critical residues in its natural sequence. We also incorporate self-associating motifs that are found in alpha-helical membrane proteins. Library members were screened using the orthogonal assays at very high and very low stringency. Selected positives from the highly stringent assay (i.e. gain in activity sequences) show a high frequency of alanine substitutions at specific polar and basic residues. Selected negatives from the low-stringency assay (i.e. loss of activity sequences) show two key nonpolar-to-glycine replacements as well as a substitution of the proline residue. Selected peptides from both screens have been contrasted to melittins activity by using biophysical techniques, antimicrobial and hemolytic assays.