I love this blog. But sometimes it drags. Finding new articles to blog about can be hard, there seems to be waves. So, I love it when someone points one out to me. This article was sent to me by someone associated with it. We don't always blog those articles people point out to us, but this one has all the hallmarks: high visibility journal, good target, and "innovative" in the approach. So, let's dive in and see what we have here.
Nicotinic receptors (nAChR) are pentameric, ligand-gated ion channels. nAChR mutations are implicated in a wide range of neurological disorders and are the targets for a wide range of current drugs. It is important to note that most of these drugs work through an allosteric site distant from the agonist binding site. Most high resolution, structural data is from homologous molluscan Acetylcholine binding proteins. The orthosteric site is located at an interface of a "principal" and "Complementary" subunit. Ligand-binding induces conformational changes in the ligand binding site which are coupled to the ion opening. There is little information on structural implications at allosteric sites. The authors decided to address this unmet need with a chimeric human α7 ligand binding domain and AChBP which has 71% sequence similarity to the native protein, compared to 33% for the most commonly used target (Aplysia).
The authors used SPR against a target with a blocked orthosteric site. They did this one of two ways: 1. pre-incubation with a high affinity orthosteric ligand or 2. mixing each fragment with an orthosteric ligand of lower affinity. This second approach (Figure 1) allows detection of fragments not competing for binding to the orthosteric site and were therefore potential allosteric ligands.
In screening 3000 fragments, they found 300 putative allosteric binders. Follow up, including dose-response, led to 24 fragments being selected for co-crystallization. Crystal trials were set up using blocked nAChR and fragments that were soluble at 5-10mM; yielding in the end 5 crystal structures. All five proved to be allosteric binders in three separate locations, including one never observed before (top pocket). The most potent of these fragments had a IC50 of 34 uM, while the least potent was 400 uM.
|Figure 2. Allosteric Binding Sites|
This is a really nice piece of work. I think the SPR assay is really clever and I would expect that many people will now be taking a similar approach to discovering allosteric sites in their targets.