Protein-protein interactions have often been targeted by fragment efforts, partly I think out of desperation when all else fails. That said, there have been notable successes. Earlier this year we highlighted one example from Stephen Fesik’s group at Vanderbilt University. In a recent paper in J. Med. Chem., the same lab now reports progress on a different target.
Replication protein A (RPA) is important for DNA replication and repair, and is thus an intriguing anti-cancer target. RPA binds to single-stranded DNA as well as to various other proteins involved in the DNA-damage response, such as ATRIP. The site targeted here is the “basic cleft” of RPA that binds ATRIP.
The researchers used the venerable SAR by NMR approach, screening a library of 14,976 fragments against 15N-labeled protein using HMQC and looking for changes in chemical shifts. A total of 149 fragments produced significant and specific chemical shift differences at 0.8 mM concentration. One of the nice features of SAR by NMR is that not only do you get hits, you find out where they bind. In this case, most of the hits bind in the basic cleft. This region has two sub-sites; some fragments bind to one or the other, while many bind to both. Not surprisingly for a basic binding site, most of the fragments identified are negatively charged.
Although all the hits are relatively weak (the best have dissociation constants around 0.5 mM), some could be improved through various strategies to low micromolar inhibitors, the subject of a paper earlier this year.
In the current paper, the researchers used crystallography to further define the binding modes of select fragments. They found that fragment 2 and fragment 4 could bind to both subsites of the basic cleft, but that when co-crystallized together fragment 2 binds to one subsite while fragment 4 binds to the other. The two fragments come within a few Ångstroms of one another, suggesting that they could be linked.
Fragment linking doesn't always work as well as one might hope, and although the initial linked compound 7 is nearly 30-fold more potent than fragment 4, its ligand efficiency drops considerably. However, structure-based optimization to compound 8 was able to improve the affinity by another two orders of magnitude.
Teddy has argued that SAR by NMR is dangerous because of its reliance on labeled protein and because the initial application involved fragment linking, leading people to believe that these are necessary requirements for successful prosecution of fragments. There are now plenty of examples of using other methods to find and advance fragments, but this paper illustrates that SAR by NMR can still be incredibly powerful.
Of course, the final molecule reported here has warts, notably a thioamide, two carboxylic acids, a molecular weight over 600, and a ClogP>7. Indeed, the absence of reported cellular data is perhaps telling. And yet, Bcl inhibitors are also superficially unattractive but are in the clinic. Clearly more medicinal chemistry needs to be done on these molecules, if nothing else to improve potency, but that’s not to say there isn’t a path forward. It will be fun to watch this story progress.