Engineering Selective Peptide Nanopores

Researchers have been fascinated by pore-forming peptides for quite a while because these peptides can selectively permeabilize membranes, offering huge potential for use in biotechnology, drug delivery, and biosensing. A particularly famous example is melittin, the main component of honeybee venom. Melittin is well-known for its strong ability to lyse or break apart cell membranes and this characteristic has spurred interest in using it for various medical applications including antimicrobial treatments, cancer therapies, and drug delivery systems. However, melittin has some notable drawbacks. For example, it tends to lack selectivity and can be quite toxic to human cells, which limits its practical use in treatments. The problem with melittin, and other similar peptides that can permeabilize membranes, is that they often attack a wide range of cell membranes, not just their intended targets. This broad effect can lead to unwanted side effects, as it harms healthy cells in addition to any intended bacterial or cancer cells. Because of this indiscriminate cytotoxicity, melittin has a narrow therapeutic window, meaning it’s challenging to use effectively without causing harm. Despite many efforts to modify melittin, creating versions that can selectively form pores in synthetic membranes without being toxic to human cells remains an ongoing struggle. This challenge has prompted researchers like Professors William Wimley, and Kalina Hristova and their students  to dig deeper. They set out to refine melittin-like peptides, eventually developing a new family of peptides known as macrolittins. These were designed using a process called synthetic molecular evolution, where they screened successive generations of peptide libraries for desired characteristics. Their main goal was to keep melittin’s pore-forming abilities while making it more selective and less toxic. Right now, the field still faces significant challenges. One major question is how to design these peptides so they can target specific membrane types, like synthetic lipid bilayers, while sparing healthy cells. Another hurdle is figuring out how to make stable nanopores large enough to allow macromolecules to pass through, even at low peptide concentrations. Most traditional membrane-permeabilizing peptides aren’t selective enough and can cause random membrane disruptions, which isn’t ideal for many biotechnological applications.

In light of these challenges, a recent study published in ACS Nano Journal, led by Professor William Wimley from Tulane University School of Medicine, along with researchers Leisheng Sun, Professor Kalina Hristova from Johns Hopkins University, and Professor Ana-Nicoleta Bondar from the University of Bucharest, took a closer look at how macrolittins interact with synthetic membranes to try understand better the molecular mechanisms that help stabilize the nanopores formed by these peptides. The team performed a series of advanced molecular dynamics simulations to study the structural features that allow macrolittins to form selective nanopores. Using atomistic simulations, they modeled how macrolittins interact with synthetic membranes made of 1-palmitoyl, 2-oleoyl-phosphatidylcholine (POPC) which is a lipid commonly used to mimic cell membranes. The authors found that macrolittins could form stable, membrane-spanning nanopores even at very low peptide concentrations and also the simulations indicated that the nanopores were held together by a robust network of hydrogen bonds involving the peptides’ charged and polar residues, water molecules, and the lipid headgroups.  According to the authors, it is this extensive network, which seem to span the entire membrane was essential for maintaining nanopore stability. To confirm these simulation findings, the researchers moved on to lab experiments to observe how macrolittins worked in synthetic membranes. To do this, they tested the peptides’ ability to permeabilize different types of lipid bilayers, including POPC, cholesterol-containing membranes, and thicker lipid compositions. Their results were striking: macrolittins selectively formed nanopores in POPC bilayers at very low concentrations, but they didn’t affect cholesterol-rich or thicker membranes. This finding aligned with what they saw in the simulations and also in experiments with cellular membranes, demonstrating that macrolittins work well with POPC bilayers but are less active in more complex membrane environments. By contrast, melittin and its variant, MelP5, permeabilized a wide range of membranes, including cellular membranes, showing much less selectivity.

In another key experiment, the team looked at how certain polar residues influenced macrolittin activity. They swapped specific polar amino acids (glutamate at positions 4 and 8, and glutamine at position 17) for nonpolar ones, similar to those in melittin, and then tested these modified peptides in synthetic membranes. The changes revealed that while the modified versions retained some activity, they were less potent and lost much of the selectivity seen in the unmodified macrolittins. The hydrogen bond network in these modified peptides was also less organized, which led to less stable nanopores. Indeed the experiment highlighted the importance of specific polar residues in maintaining both nanopore stability and membrane selectivity. To evaluate macrolittin toxicity, the authors performed tests on human cells, including HeLa cells and red blood cells. They compared the cytotoxicity of macrolittins, MelP5, and the peptide variants. Remarkably, macrolittins showed almost no toxicity to these cells even at high concentrations which suggest that they are quite safe in mammalian systems. On the other hand, MelP5 was highly toxic and killed cells at much lower concentrations. Interestingly, the modified macrolittins, with nonpolar substitutions, displayed intermediate toxicity, highlighting again the role of the polar residues in reducing toxicity and enhancing membrane selectivity. Additionally, the researchers investigated how macrolittins could cause membrane fusion, a sign of nanopore formation. They found that, even at very low concentrations, macrolittins induced vesicle fusion in synthetic POPC membranes—a process that usually indicates large nanopores have formed. MelP5, however, caused little to no fusion, even at higher concentrations, highlighting a unique ability of macrolittins to form macromolecule-sized pores that promote fusion. Adding cholesterol to the membranes significantly reduced macrolittin-induced fusion, which was consistent with their earlier findings that macrolittins were less effective in cholesterol-rich bilayers. The peptide variants also showed reduced fusion activity, further emphasizing the importance of specific polar residues in driving both nanopore formation and membrane fusion.

Wrapping things up, Professor William Wimley and his collaborators have really pushed the envelope in understanding macrolittins and how these peptides work at the molecular level. They’ve managed to break down the complexity of how macrolittins can be fine-tuned to be more selective and stable when they interact with cell membranes. This has been a tough nut to crack because it’s always been hard to balance how powerful these peptides are with how picky they can be about which membranes they target. One of the biggest takeaways from this research is the role of certain polar residues and the intricate hydrogen bond networks they form. These details might sound technical, but they’re actually the secret sauce that could help scientists design nanopore-forming peptides that are both safe and effective. What’s really exciting is how this could change the game for drug delivery. Imagine being able to use these macrolittins to create tiny pores in synthetic membranes that release medications exactly where they’re needed, without damaging healthy cells along the way. This could make treatments not only more effective but also way safer, with fewer side effects. On top of that, this research could lead to breakthroughs in developing new antimicrobial agents. A lot of the current options are a bit of a blunt tool—they don’t just target harmful bacteria; they often end up affecting healthy cells too. But since macrolittins can be more selective, they could inspire new treatments that focus on the bad guys without harming the good ones. That’s a big deal, especially with antibiotic resistance becoming a huge concern. Beyond healthcare, these findings could have ripple effects in other areas too. For example, in biotechnology, controlled membrane permeability is a big deal. Macrolittins could be the key to biosensors that need that kind of precision. They might also be useful in bioprocessing, helping with the separation or purification of large molecules, which is often a tricky task. The research also opens up interesting paths for future studies. The team found that cholesterol-rich membranes are more resistant to macrolittins, which hints at the possibility of tailoring the lipid environment to control how these peptides behave. It’s like being able to tweak the setting to suit different needs, potentially leading to customized nanopore systems for various applications. All in all, this work is a big step forward, not just in understanding these peptides, but in finding practical ways to use them.