Tenth Annual Fragment-based Drug Discovery Meeting...Teddy's Thoughts

Dan posted his thoughts here.  Like Dan, CHI put me to work: I chaired the first session in PPIs, co-taught (with Dan) our award winning FBDD course to 22 attendees (a new high which I think shows that interest in FBDD is still growing), moderated a breakfast roundtable on kinetics and thermodynamics,  judged posters.  All of this during weather which made the natives shiver, and me feel like spring is really here (64F and cloudy).  

First off, there was live tweeting of talks by me and a few others: @iceobar, @moleculesmith, and others.  Beware that the Dubai Diamond Conference was also going on that week.  

In the PPI track, just as last year, fragments were a key component to various projects.  Mark McCoy, Merck (and he taught me more about NMR than just about anyone, whether he will admit it or not) gave a great talk on HDM2-p53.  I took away a few things from his talk which I really liked.  Merck (legacy S-P) really relies on NMR structural information: HSQC-based screening, NMR-based Ki,  and NMR-driven docking.  I was particularly intrigued with the NMR-driven docking because they were able to generate 80+ models with a 75% success rate that was confirmed by X-ray (once that was enabled).  They were forced down this path because they went 2 years without a X-ray structure. 

Joe Patel of AZ talked on SOS-RAS.  What I liked was that AZ uses a modified Voldemort Rule (which Harren Jhoti incorrectly attributes to me; I am the Boswell to Rod Hubbard's Johnson): HAC less than/equal20, cLogP less than5, less than3 rings, less than5 rot bonds, less than 3 HBD, and less than 5 HBA.  Their initial X-ray screening ended up at a wall, so they went to a covalent approach. 

Troy Messick of the Wistar gave a nice talk on using fragments and SBDD to drug an "undruggable" target.  I think this is exactly this is exactly the kind of success that has led FBDD to be ubiquitous these days.  I have to admit I have and am working with the Wistar on the NMR component of their screening, so I may be biased. 

I won't go into the various talks from the FBDD track,  However, echoing Dan this is really a great conference.  My main take home themes is that FBDD is really mainstream.  It's no longer the red headed stepchild to other hit generation processes (apologies to my ginger friends).  Biophysics is also seeing a huge growth, having grown up with FBDD, but really finding a lot more uptake outside of that space.  Next week I will post summaries of roundtables and some useful information. 

Halogenated fragments stabilize mutant p53

Practical Fragments recently discussed using fluorinated fragments for ¹⁹F NMR, but there are other halogens out there – are these useful for constructing fragment libraries? SGX Pharmaceuticals had a collection of fragments enriched with bromine atoms, the thought being that this atom would facilitate crystallography. Halogens can also make productive interactions with proteins, including so-called “halogen bonds” to backbone carbonyl atoms or pi-systems. With this in mind, Andreas Joerger at Cambridge University and Frank Boeckler at Eberhard-Karls University and their colleagues have assembled and screened a “halogen-enriched fragment library.” Their results are reported in a recent issue of J. Am. Chem. Soc.

The library consists of 79 non-reactive, soluble aromatic compounds containing bromine or iodine. Because these elements are so large, the researchers used a modified rule of 3 – instead of a molecular weight limit of 300, they limited the fragments to no more than 22 heavy atoms (see also our recent post on this topic here). They then screened this library against the Y220C mutant form of p53, which contains a surface crevice that destabilizes the protein and contributes to cancer cell survival. Thermal shift assays were used as the primary screen, with hits being confirmed by 2D NMR and ITC. This resulted in the discovery of compound 3, which crystallography confirmed was making a halogen bond to a backbone carbonyl.

Modifying the amine substituent improved potency modestly, and building off the phenyl ring towards a nearby pocket improved the potency further, albeit at a cost in ligand efficiency. Still, this compound (PhiKan5196) does represent the most potent Y220C binder reported, and represents an order of magnitude improvement over previous work. Moreover, the molecule induces apoptosis in p53 Y220C containing human cancer cell lines but not in matched wild-type p53 cell lines. (Unfortunately the compound also appears to be generally cytotoxic.)

This library is an interesting approach in part because it is somewhat heretical: for various reasons most library designers exclude molecules containing bromine or, especially, iodine. That said, the thyroid hormones do contain iodine aplenty, and MEK kinase seems to have a predilection for bromine or iodine as well. What do you think? Are halogenated fragments a useful tool for certain targets, or an unproductive diversion?

Stabilizing p53 with a little help from fragments

The protein p53 is inactivated in a large fraction of cancer cells and has long been of interest for oncology. Mutations of the gene frequently lead to a destabilized form of the protein. For example, substitution of cysteine for tyrosine at position 220 causes the mutant protein to rapidly denature at body temperature and also opens a reasonably large and hydrophobic crevice on the surface of the protein at lower temperatures. If molecules could be identified that bind in this crevice, the protein might be stabilized, restoring its function. In a recent paper* in Chemistry and Biology, Alan Fersht and colleagues at Cambridge University have targeted this crevice using fragment screening.

The researchers assembled a fragment library of 1895 molecules from three commercial vendors (ChemBridge, Life Chemicals, and Maybridge). They then used two orthogonal screening methods, NMR (WaterLOGSY) and thermal denaturation scanning fluorimetry, to identify fragment hits. These were then confirmed using two-dimensional HSQC NMR. WaterLOGSY identified 70 confirmed hits, while thermal screening identified only 17; oddly, only three of these were in common. The authors suggest that fluorescence quenching may lead to a higher false negative rate for the thermal denaturation method, but it is also possible that the NMR method is identifying fragments that bind so weakly as to show no effect on protein stability.

Of the 84 hits, three fragments could subsequently be characterized bound to p53 crystallographically. They all fit in the Y220C crevice, though each sits in a somewhat different location.

There is still a long way to go for these molecules: the most potent fragment has a Kd of 105 micromolar. Still, with a ligand efficiency of 0.33 kcal/mol per atom, this compares favorably to the best molecule the authors had previously identified from an in silico screen of 2.7 million molecules (Kd roughly 150 micromolar, ligand efficiency 0.29 kcal/mol per atom).

Although it is still not clear that stabilizing mutant p53 will be a viable approach for treating cancer, the identification of a number of diverse fragments suggests that the Y220C site may be druggable. Moreover, the fragments themselves are potential starting points for developing more potent molecules.

*Thanks to Mauro Angiolini for bringing this publication to our attention on LinkedIn.