Medicinal chemists like it when one enantiomer of a compound binds to a target much more than the other one. That tells you that you’re getting real binding to a protein target, and the bigger the difference between the two, the more you can say about the actual binding site. But is this always true?
For the non-chemists, an enantiomer is a single right-handed or left-handed form of a drug; think of a glove or a shoe. Not every object (or drug) can come in those mirror-image forms, though – there’s no such thing, clearly, as a right-handed baseball bat or a left-handed drinking glass. The same with drugs – aspirin has no enantiomers, and neither does, say, Claritin (loratadine). When “handed” isomers (enantiomers) exist, though, they can have very different actions in the body, because all the proteins and carbohydrates in living cells are single-handed forms themselves. To add to the fun, some drugs that have enantiomers are sold as pure right- or left-handed forms, while others are sold as a fifty-fifty mix, which is called a racemic mixture (like ibuprofen – in its case, the body quickly turns the pure form back into the mixture anyway, so there’s no point).
Here’s a new paper that shows a particularly strong example of how enantiomers can end up performing the same way. The authors (from Sheffield and Syngenta) were looking at inhibitors of the plant enzyme imidazoleglycerolphosphate-dehydratase (IGPD) as potential new herbicides, and they describe work on triazole phosphonate compounds that mimic a key intermediate in the reaction. So far, so good – this is just the sort of thing that you do when you’re looking for enzyme inhibitors. Their lead compound was a racemate, and when they grew crystals of the IGPD protein in its presence, they obtained a bound structure down to 1.85A resolution.
Another explanation for non-chemists in the crowd: this refers to X-ray crystallography, which can determine (atom by atom) the structure of crystalline compounds. Protein crystals are notoriously tricky to grow, and you generally get somewhat fuzzier data back from them, but if you can get a structure of your target protein with your drug bound to the site of action, you can learn a great deal of information that’s otherwise very difficult to get or just unobtainable. 1.85 Angstrom is pretty decent resolution for a protein crystal, but not enough to really nail down the details of a small molecule – as will become apparent!
At that level, they could see the conformation of the protein chains well in the active site, and also got the triazole and the phosphonate ends of the molecule pretty sharply. But the middle part of the molecule, the part with the chiral center, was fuzzier and lacked electron density, which is a bit odd when the two ends are well-resolved. A different protein construct gave a much better-resolved structure (1.1A), but even this one, at first, looked wonky around the chiral center. Digging into the data, they found that a model with 60/40 occupancy for the S and R enantiomers, respectively, lands right on the electron densities: in other words, either enantiomer is capable of binding well to the target, with no change in the protein structure at all.
Sure enough, the resolved enantiomers were close to margin of error in the enzyme assay, centering around 20 nanomolar, and performed the same in greenhouse trials. Crystallizing either pure enantiomer in the enzyme gave the structures that they’d modeled from the racemate, with only a water molecule or two moving around a bit. There’s just enough room for the two enantiomers to get their phosphonates and triazoles in basically the same spot, while connecting the two differently (there’s a pseudo-mirror-plane arrangement).
When you get fuzzy electron density in a bound-inhibitor study, that can be just plain old disorder, and that’s how it’s often interpreted. But there’s disorder and there’s disorder – as the paper says, if they hadn’t gotten down to 1.1A resolution, they might not have ever figured out what was going on how (or how). Chirally promiscuous enzymes are probably more common than we think, so if you get a flattish ratio when you test your two enantiomers, don’t start to worry: there are more ways for compounds to bind than we think there are. . .