Who I'm Reading Today

https://www.science.org/content/blog-post/mirror-imaging-your-way-through

I found this to be an interesting paper, and it uses an idea that’s not always easy to realize. There are a lot of time when we’d like to be able to use small proteins and peptides as drugs, but they often have poor pharmacokinetics (absorption, membrane penetration, and most especially metabolic liability). In addition, some of these small-protein ideas can end up being immunogenic, since your body can react to them like the foreign substances they are, with your immune system taken them as signs of some sort of viral or bacterial attack.

As an aside, the current vogue for “peptides” (very loosely defined) among wellness types, bodybuilders, and other such groups is grimly amusing from a medicinal chemistry point of view. Most of these people have no actual idea of what they’re talking about, and “peptide” gets used as a synonym for “cool dietary supplement known to guru insiders” with no further thought. So asking someone if they take peptides just gets a blank stare from anyone who knows biochemistry, since your body is swimming with tens/hundreds of thousands of different short protein sequences that fit inside that name. And the number of different possible peptides, even reasonably short ones, is just beyond human comprehension. It’s not a very useful term when thrown around like this!

Anyway, what the new paper above is discussing is more in the “miniprotein” class - that is, long enough to fold itself into a defined three-dimensional structure. In this case, they’re about forty amino acids long. And the authors are looking to compare hit rates against a difficult target (PD-1/PD-L1) versus shorter sequences I blogged just the other day about a quick computational approach that came up empty against this one, and it’s really no wonder - that interface features two rather flat beta-sheet surfaces that don’t give small structures very much to grab on to, as far as anyone’s been able to see.

And they’re also interested in making these miniprotein candidates out of D-amino acids. Those are of course the mirror-image forms of the ones used normally in living systems, and the idea there is that a peptide or protein made out of such enantiomers will probably not be a substrate for hydrolase enzymes (and may be less immunogenic as well). But you’re not going to be able to take a sequence that you know binds to you target, synthesize it with all the amino acids flipped into the D stereochemistry, and expect it to bind. You’ve made a different compound entirely! So the D-proteins you get out of such a screening/synthesis exercise would not be expected to look much like sequences you’re seen before - you’re just using this non-natural stereochemistry to put the side chains and functional groups into the right position to hit the target, one way or another.

But how do you do that? There are, as mentioned, ridiculously huge numbers of potential protein candidates available, and the best ways to produce and evaluate huge mixed libraries of them rely on living-system-derived techniques like phage display (subject of the 2018 Chemistry Nobel, I might add). But the phages and bacteria you’d use for such library preparation are of course all using the good ol’ L amino acids that we’ve stuck with for a few billion years now. Well, here’s a way out: if you can go to the trouble of making a supply of your target protein (the one that you’re screening for binders to) as an all-D protein, then you can screen against whatever normal phage libraries you want. If you identify a binder, then all you have to do is synthesize that hit out of D amino acids so it can bind the natural L-protein target! This mirror-image screening was pioneered back in the 1990s and has been used in a number of applications since for people interested in D-protein screening hits.

Now, making any substantial sized protein out of all D amino acids is not enjoyable, but it’s gotten more feasible over the years with advances in organic-chemistry routes to protein synthesis. And that’s what this paper did, making a 146-amino acid construct of the PD-1 extracellular domain as the screening target. (It was assembled from seven shorter sequences by various chemical ligation reactions - not the work of a moment, but certainly doable if you have the time, skill, and patience. Oh, and the money. That too.

Most of the commercial screening libraries of various-length peptides didn’t yield much of interest. But their miniprotein library, which had been targeted towards a different purpose entirely (SUMO binding) from another project, actually yielded a micromolar binder. They used that sequence to prepare a new phage library with several hundred million proteins randomized around this one. The twenty or so best survivors from four successive rounds of screening and enrichment turned out to be pretty good when tested as individual compounds - many of them were sub-micromolar and one of them came in with an IC50 of 60 nanomolar (but was about a hundredfold less potent in the cell assay, which is certainly not unheard of either, unfortunately). NMR evidence seems to confirm direct binding to the PD-1 protein, albeit without many structural details.

This comes under the heading of “an interesting start”, and it certainly seems to demonstrate that miniproteins can be ligands for such difficult targets. Turning D proteins of any sort into drugs is something that hasn’t quite crossed the finish line yet, although there are two such candidates in clinical trials against other targets. It seems that the advantages of greater stability and lower immunogenicity can be realized with these things, although you certainly don’t want to take either one of those as given without proving it for yourself. There are though, an insane number of possible miniprotein scaffolds and designs, each of which can partake in the equally insane variety of protein sequences, so you’d have to think that there are drugs in there somewhere!

https://www.science.org/content/blog-post/mirror-imaging-your-way-through