Drug discovery folks spend a good amount of time and effort dealing with cell membranes. Our drug candidates stick to them, get imbedded in them, might have to slip through them to get to their target proteins, or may target proteins that are localized in them, can get actively transported through them or actively pumped back out. . .there are a lot of possibilities. One of the simplest possible interactions is that first one on the list, when a compound just seems to bind nonspecifically. But it turns out that it isn’t always as simple as it looks.
That’s the message of this paper, anyway. The authors (a team from Queensland) are looking at a particular cyclotide protein, kalata B1. Cyclotides are interesting beasts. They’re natural products produced by a number of plant species, multicyclic proteins that feature six cysteine residues linked into a distinctive cystine-knot form. They’re known to target the cell membrane, and have a distinct polar region that interacts with the charged phosphates in the lipid bilayer, while a hydrophobic region buries down into the lipid chains in the middle. So far, so good.
The odd thing, though, is that if you make the enantiomeric form of kalata B1, the one with all D-amino acids, it’s less potent across all its biological activities (which include hemolysis, insecticidal and antiviral effects, etc.) “Well sure”, many people will be saying. “Why wouldn’t it be? Proteins are chiral and that’s part of their structure when they bind to their targets”. But think about that: we’re used to “targets” in that context meaning “other proteins”, and sure, those are chiral too and the enantiomer of your active agent is going to be a mismatch with those. But when your target is just the cell membrane? A lipid bilayer? Why should you see such an effect there?
One possibility is that kalata B1 is in fact targeting some (unidentified) membrane protein, and thus running into chiral interactions that way. That’s an easy way out, and a pretty plausible one. But this paper investigated the weirder possibility. Glycerol itself has a plane of symmetry, but glyceride esters can be chiral as you start decorating that core with phosphates, phosphocholines, and different fatty acid esters. The authors actually synthesized enantiomeric model lipid bilayers and checked to see if kalata B1 recognizes the intrinsic chirality of the membrane. And it does.
The polar region of the protein doesn’t seem to care; it binds to the phosphotidylcholine head groups just through good old polar interactions. But the second part of the binding process, the insertion of the hydrophobic part of the cyclotide into the lipid bilayer, that one prefers the natural chirality. The team, as mentioned, already had both enantiomers of kalata B1 and a number of mutant forms of the various regions of the protein, and they were able to show all the match/mismatch possibilities: the unnatural protein has greater affinity for the unnatural membrane. When matched up, the proteins both bind better and perform their membrane-disrupting activities better. Meanwhile, when either enantiomer was tested in an achiral lipid-like model membrane, their activities were identical.
So we’re going to have to get used to paying attention to the chirality of the phopholipids themselves. It’s not clear how many other compounds recognize these sorts of interactions, but you can’t rule anything out. These effects could well be one of the subtle factors that go into the broad category of membrane permeability. It’s going to be of particular relevance to bioactive peptides, not least because the D-forms of those are often used in control experiments. The authors point out that early studies on helican antimicrobial peptides showed basically identical activity, and the assumption since then has been that membrane-targeting mechanisms don’t have to worry about this sort of thing. But there are outlier data points in the literature, which were generally explained away as some sort of off-target effect (just like the D- and L-kalata B1 results could have been). It ain’t that simple.