16 December 2013

Fragment linking on RPA: another protein-protein interaction inhibitor

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.

3 comments:

Lewis Vidler said...

A property that I think is of relevance here is LLE. For a few of the compounds here it would look like LLE is < 0 suggesting that much of the binding is occurring through the fact that the compound does not want to be in solution. Identifying both compounds binding at different sites when the crystallography of the compounds separately places them in the same site is consistent with this. Not to say that this is not good work, this target is likely extremely challenging. For the ABT BCL family inhibitors the LogP is indeed high, but for some of them they are extremely potent (<10 pM) thus achieve a positive LLE. Personally, a cutoff of LLE < 0 is often a good metric for identifying promiscuous compounds (as always not to be used as a hard rule!).

Dan Erlanson said...

Lewis raises an excellent point about LLE. I've never quite figured out how to apply LLE to fragments, but I'd have to agree that LLE values < 0 sound a bit dubious.

On a related note, the LLEAT values are also very low, with all molecules having scores < 0.10.

Looking at the two fragments more closely reveals that they are both very lipophilic, with cLogP > 3. Although the experimentals state that the fragment library was chosen according to "criteria related to the commonly used 'rule of 3'", they go on to state (presumably erroneously) that "compounds were removed from consideration if they possessed... a cLogP less than 3.0" (italics mine). A telling slip?

Alex Waterson said...

I am one of the coauthors on this paper. I regularly read this blog, but do not regularly comment. Thanks for the writeup - you have correctly identified some of the challenges in this target and in the chemical matter that we have reported on in this paper. Lewis brings up an interesting point about the LLE above. Although I honestly did not really consider the possibility that the multiple binding modes for many of the fragments may be a indicator of desolvation energy playing a large role in the binding, it is quite clear to us that the hydrophobicity of the linked compounds is a major player in the binding affinity. We based most of this optimization work (and all of the fragment evaluations) on LE, but have incorporated LLE concepts into later optimizations (the LLE of this series is all pretty terrible, rarely going above 0).

Regarding the fragment library, ours is built around the Rule of 3, but is not completely Rule of three compliant. Because the stated goal of our lab is to apply fragment screening to difficult PPI targets, we elected to relax some of the Ro3 requirements for certain substructures that have been common among PPI inhibitors in the past. I will point out that the compound 4 is not a fragment from our library, but was from a fragment-inspired purchasing campaign to expand the SAR. We did not limit to Ro3 compliant molecules in this purchasing and it thus led to some real grease balls. We were confident that we could build this hydrophobicity out of them, but this simply did not work in most cases.

As Lewis mentioned above, this is a pretty difficult target. We are competing with an amphipathic helix that relies on considerable hydrophobic contacts for binding. We have worked on multiple series for this and the optimizations (even with LLE used as a metric and starting with fragments that are better than this from a clogp/LLE standpoint) keep coming back to highly hydrophobic molecules with acids. Balancing the physical properties with the binding affinity has been a significant challenge.

This work continues - we should have at least a couple more papers coming out on this work in the near future.