Failing honorably and openly on PrP

Many of the posts on Practical Fragments – and indeed much of what appears in the literature – describe successes. This is obvious in the list of fragment-derived clinical compounds, and discoveries of high-affinity tool molecules or even advanceable fragment hits make up a large share of the 750+ posts on this blog. But of course, most of what we do in science fails, and such failures can also be informative. Eric Minikel and 25 collaborators from the Broad Institute and multiple other organizations have just published an illustrative case study on bioRxiv. (Eric also has a detailed and eloquent blog post of his own about the work.)

The researchers describe a five-year effort to find small-molecule binders of the prion protein, PrP, which misfolds and forms aggregates that lead to neurodegeneration. The hope was that binders could be turned into PrP stabilizers or perhaps even degraders. PrP has been studied for decades and there are plenty of literature reports of small molecules that seem to interact with the target, but none of these have been convincingly validated. Moreover, the crystallographic structure of the protein does not reveal attractive binding pockets.

Fragment-based methods have succeeded for other difficult targets, so the researchers performed STD and ¹⁹F NMR screens. Of 6630 pooled fragments, 238 initial hits were retested by STD NMR, leading to 80 hits that were then assessed by two-dimensional (TROSY) NMR. This led to a single hit, a substituted benzimidazole. Unfortunately the binding site could not be determined: chemical shift perturbations were spread across the protein. Differential scanning fluorimetry (DSF) showed the molecule caused a slight decrease in thermal stability. Both of these results suggest some sort of pathological mechanism, but the researchers did multiple experiments to rule out aggregation. A dose-response suggested a dissociation constant well above 1 mM, and none of 54 analogs tested proved any better. Soaking crystals of PrP with 20 of these was also unsuccessful.

Next, the researchers performed a thermal shift assay of just over 30,000 compounds (not necessarily fragments), which yielded both stabilizers and destabilizers. Unfortunately, none of 93 tested by two-dimensional NMR (HSQC) revealed any sign of binding. A DNA-encoded library (DEL) screen of 256,000 macrocycles also didn’t yield any confirmed hits (though more on that below), nor did a computational screen of just under 7 million molecules.

Given this experience, the researchers cautiously conclude that their “results may hint toward relative rarity of PrP binders in chemical space.” They do suggest several alternative approaches, such as screening the more biologically-relevant membrane-bound form of the protein. It may also be worth doing a high-concentration crystallographic screen. Finally, the researchers note that one of the DEL scaffolds “was judged to be a likely covalent binder and was not pursued further.” This decision may be worth revisiting. Indeed, covalent approaches have led to clinical compounds against another formerly undruggable target, KRAS.

According to the blog post, the researchers have pivoted to oligonucleotide therapeutics, where they seem to be making good progress. This makes sense, and I wish them luck. But I hope someone returns to PrP itself with new tools. If they do, the assays established and described here will prove invaluable.