Fragments vs FEN1: A chemical probe

Synthetic lethality is a relatively new approach to treating cancer by targeting proteins whose inhibition is lethal to cancer cells that have specific mutations. Disruption of flap endonuclease 1 (FEN1), an enzyme important for DNA replication and repair, becomes synthetic lethal combined with BRCA1 and BRCA2 loss of function mutations. However, although a few inhibitors have previously been reported, these had poor cell activity and physicochemical properties. In a new J. Med. Chem. paper, Sam Mann and collaborators at Artios Pharma, Merck KGaA, and several other organizations describe a chemical probe.

 

FEN1 is a member of the RAD2 nuclease family, all of which contain two magnesium atoms in the active site. Thus, the researchers set out to build a metal-chelating library, a strategy we’ve written about previously. Noting that a bivalent metal chelator requires at least three hydrogen bond acceptors, the researchers included fragments that were not strictly rule-of-three compliant. More than 300 fragments were screened in a biochemical assay against FEN1 and three related proteins, EXO1, GEN1, and XPG. Hits were selected based on potency, ligand efficiency, and selectivity. Two related fragments came out on top, one of which was characterized crystallographically bound to FEN1, confirming engagement with the catalytic magnesium ions

 

 

Compound 6 was trimmed back to the chelating core compound 7 before attempts were made to grow the molecule in several directions, leading eventually to compound 21, the first molecule to show target engagement in cells. Modeling suggesting the presence of a high-energy water that might be displaced, which was attempted by expanding the core to include a morpholine moiety. Further modulation of properties ultimately led to MSC778, with modest oral bioavailability in mice, rats, and dogs. The paper describes some nice medicinal chemistry that goes beyond the scope of this post. For example, there was a correlation between cellular target engagement and off-rates as determined by surface plasmon resonance (SPR). One wonders if a covalent inhibitor, with essentially no off-rate, could be even more effective.

 

MSC778 is at least 65-fold selective for FEN2 over related RAD2 family members. It is also clean in a panel of off-target safety assays. The molecule is cytotoxic to a cancer cell line in which the BRCA2 gene had been knocked out but less so to the same cell line carrying wild-type BRCA2. Surprisingly though, no tumor growth inhibition was seen in a mouse xenograft model using the mutant BRCA2 cell line at the highest tolerable dose. However, tumor stasis was seen when the compound was dosed in combination with niraparib, a PARP inhibitor, consistent with earlier cell experiments suggesting that PARP inhibitors could synergize with FEN1 inhibitors.

 

The lack of single agent activity seen with MSC778 was undoubtedly disappointing, though the researchers note that it is unclear whether this is “due to insufficient target coverage, or an unexpected disconnect between the phenotypic consequences of FEN1 inhibition in vitro and in vivo.” Nonetheless, MSC778 looks to be a useful chemical probe for further understanding the biology of FEN1. This paper is also a nice application of building and screening a metal-chelating fragment library, which could be useful for targeting additional metalloproteins.

Democratizing computational FBLD with BMaps

Computational approaches to FBLD continue to gain in power. For the most part, they require significant knowledge and installation of expensive, customized software. To remedy this, John Kulp, III and colleagues at Conifer Point Pharmaceuticals have introduced a new web-based application, BMaps, which they describe in a recent J. Chem. Inf. Mod. paper.

 

As the researchers note (and appropriately reference), there are more than a dozen virtual fragment-based design tools and another dozen web-based tools. BMaps (for Boltzmann Maps) aims to provide a full range of functions, from visualizing proteins, finding hot spots, docking fragments, and growing them. It also provides information on the energetics of bound water molecules, which as we’ve written can be crucial players in optimizing protein-ligand interactions.

 

Two key techniques used by BMaps are Grand Canonical Monte Carlo (GCMC) simulations and Simulated Annealing of Chemical Potential (SACP). The first entails comprehensive sampling of different fragment conformations on a protein of interest and assessing binding free energy. The second tool “forcefully inserts fragments into all the binding sites of the protein” and then removes them slowly to evaluate which are most difficult to remove, and thus most tightly bound. Together, GCMC-SACP can be used to evaluate fragment binding to any protein uploaded to the site from the protein data bank, AlphaFold, or any other source.

 

One nice feature of BMaps is a repository of several hundred proteins each with more than 100 fragment and water simulations. BMaps also contains a database of more than 4000 fragments, including MiniFrags. Users can import their own fragments or computationally deconstruct larger ligands. The paper itself is quite short, but the supporting information provides more guidance on how to use the software.

 

The researchers “aim to democratize the availability of accurate fragment and water maps,” a laudable goal. Most computational features are available with a free account, though with restrictions on the number of operations per month.

 

BMaps looks quite powerful and easy to use, but I do wish the researchers had included some full case studies, for example those used by the free FastGrow tool we highlighted last year. Try it out and let the community know what you think!

Spinach affects the Water

People often ask what a fragment is.  I like to paraphrase Justice Potter and say that it is like pornography; it is in the eye of the beholder.  I am not one for hard and fast rules as to what a fragment should be.  But, I also have a definite opinion what a fragment is NOT.  To me, what a fragment should be is easily described: relatively unadorned molecules.  I have a whole set of rules as to what the substituents should look like (coined the Zartler Optical Filter or ZOF by a cheeky comp chem friend).  In this paper, a group from Merck Serono decide to probe exactly what role the spinach on fragments play.  

Specifically, they deconstructed a TIE2 inhibitor (Figure 1) into its core hinge binding motif (Figure 2). 

Figure 1.  Crystal Structure of the Intact Inhibitor

This hinge binding motif has the advantage in that "decoration" can be introduced at the 4 or 8 position (Figure 2) as well as giving three donor/acceptor moieties. 

Figure 2. 4-Amino-8H-pyrido[2,3-d] pyrimidin-5-one (compound 1)
as core hinge binding motif.

They determined crystal structures for this molecule and four related fragments (Figure 3)

Figure 3.  Fragments for this study.

and then went to town on them with in silico methods to study the roles of water.  In one of those "gotta love it" moments, they classified the waters as "happy" or "unhappy", depending on whether they have positive or negative free energy, respectively.

So, what do we learn?  First, changes in the decoration leads to different binding modes.  In this case, they conclude that replacement of different water molecules leads to differences in binding modes.  Well, not surprising.  But, I think this is part of a trend, studying water and how fragments affect them, and vice versa.  In fact, the authors suggest using WaterMap could help to rationalize the roles of waters.  So, are we entering a brave new world of experimental verification of in silico predictions?