Fragments in the Caribbean

Last week saw the inaugural Zing FBDD conference in Punta Cana, Dominican Republic. Zing has been around only since 2007, and seems to focus on small conferences in exotic locales. The benefit is that they are able to attract high-profile speakers, as illustrated by the group photo below. However, in an era of shrinking travel budgets, getting approval to attend a conference at a resort is becoming a bit more challenging. That said, participants enjoyed nearly 30 presentations and great discussion – think of a Gordon Conference without the dorms, and breaks on the beach.

My favorite “equation” from the conference comes from Mike Serrano-Wu of the Broad Institute:

Undruggable = Undone

This was supported by some nice work on the anti-cancer target MCL-1, which makes a protein-protein interaction that was widely consider undruggable just a few years ago. An ¹⁹F NMR fragment screen gave a hit-rate of around 10%, leading eventually to low nanomolar leads. Fragment optimization was facilitated by a new crystal form of the protein that allowed the team to rapidly generate over a dozen protein-ligand co-crystal structures. Rumor has it that more details on this will be disclosed at FBLD 2014 in Basel in September (there are still a few openings available, but register soon.)

MCL-1 also figured heavily in talks by Andrew Petros (AbbVie, see also here) and Steve Fesik (Vanderbilt, see also here), who described cell-permeable molecules with high picomolar activity in biochemical assays. Steve also discussed programs against Ras and RPA, both also using SAR by NMR. As Mike Shapiro (Pfizer) pointed out in his opening presentation, one of the breakthrough ideas of SAR by NMR was to screen a library more than once per target, the second time in the presence of a first ligand to identify another. It is nice to see this strategy continuing to deliver against difficult targets, though preliminary results of our current poll (right hand side of page) indicate that linking is not necessarily easy.

One of the payoffs of doing fragment screens for many years on dozens of targets is a rich internal dataset. Chris Murray (Astex) mentioned that company researchers have solved close to 7000 protein crystal structures, more than a third of them with fragment ligands. A cross-target analysis found that hits tended to be more planar (ie, less “three-dimensional”, with apologies to Pete Kenny) than non-hits. This was particularly true for kinases; for six protein-protein interactions (PPIs) there was no correlation between shape and hit rate. Although defining complexity is difficult, Chris provided evidence that 3D fragments tend to be both larger and more complex.

Rod Hubbard (University of York and Vernalis) mentioned that Vernalis has determined more than 4000 protein crystal structures. Since 2002, 2050 fragments have been screened against more than 30 targets. Based on “sphericality” – the distance from the rod-sphere principle component axis – hits against kinases are marginally less spherical, while PPI hits reflect the shape of the overall library. So, despite the current push for more three-dimensional fragments, it remains to be seen whether this will be useful.

Jonathan Mason (Heptares) described how successful fragment approaches can be against membrane proteins such as GPCRs. Anyone who has worked on these targets will know that the SAR can be razor sharp, and their surfeit of structures is helping to explain this. For example, although many of the protein-ligand interactions appear merely hydrophobic, some displace high-energy water molecules, which can be revealed by crystal structures of both the free and bound forms of the protein. Displacement of high energy water molecules also helps to explain some “magic methyl” effects.

Fragment-finding methods were not neglected. Jonathan mentioned that, for the A2A receptor, SPR identified only orthosteric ligands, while TINS identified only allosteric ligands – the orthosteric ligands were actually too potent to be detected by this technique. John Quinn (Takeda, formerly SensiQ) and Aaron Martin (SensiQ) also discussed SPR, and in particular how variable temperature SPR analyses could be used to rank ligands based on their enthalpic binding, though as Chris Murray warned, this information can be difficult to use prospectively.

I also learned that a selective BCL-2 inhibitor from Vernalis and Servier has just entered into Phase 1 clinical trials. This has been the result of a long-running collaboration that has required creativity on the part of the scientists and patience on the part of management.

There is much more to tell – for example Teddy's extended metaphor of the Silk Road (this one, not this one!) – but in the interest of space I’ll stop here. Feel free to comment if you were there (or even if you weren’t!)

Poll: fragment linking and growing

A seminal paper in the fragment field is the 1996 SAR by NMR report in which two fragments were linked together. In theory, linking fragments can give a massive improvement in affinity beyond simple additivity, but in practice this is rare. The challenges of linking were not obvious in the early days, and led to much hair-pulling. Indeed, partially for this reason, Teddy has asserted that the 1996 paper is not just the most impactful paper in the field but also the most destructive.

Nonetheless, there are successful examples of linking, particularly for challenging targets (such as here and here). So how often does it really work?

Our latest poll has two questions: one on fragment linking, the other on fragment growing (see sidebars on right side of page). Tell us whether, in your experience, fragment linking didn’t work at all, worked marginally (ie, perhaps a modest boost in potency), worked OK (perhaps additivity), or worked well (synergy). You can vote multiple times, so if you’ve worked on multiple projects with different outcomes, please vote early and often. We’re asking the same questions for fragment growing since these two strategies are often compared.

Admittedly the categories are somewhat fungible: one person’s “OK” may be another person’s “well,” and some may see merging where others see linking. Still, hopefully we’ll get enough votes to discern some trends.

You Probably Already Knew This...

Academics can spend time and resources doing, and publishing, things that people in the industry already "know".  This keeps the grants, the students, the invitations to speak rolling in.  It also allows you to cite their work when proposing something.  This is key for the FBHG community.  There are many luminaries in the FBHG field, and we highlight their work here all the time. Sometimes, they work together as a supergroup.  Sometimes, Cream is the result.

Brian Shoichet and Gregg Siegal/ZoBio have combined to work together.  In this work, they propose to combine empirical screening (TINS and SPR) with in silico screening against AmpC (a well studied target).  They ran a portion of the ZoBio 1281 fragment library against AmpC.  They got a 3.2% active rate, 41 fragments bound.  6 of these were competitive in the active site against a known inhibitor.  35 of 41 NMR actives were studied by NMR; 19 could have Kds determined (0.4 to 5.8 mM).  13 fragments had weak, but uncharacterizable binding; 3 were true non-binders. That's a 90% confirmation rate.  34 of 35 were then tested in a biochemical assay.  9 fragments had Ki below 10 mM.  Of the 25 with Ki > 10mM, one was found to bind to target by X-ray, but 25A from the active site.  They then did an in silico screen with 300,000 fragments and tested 18 of the top ranked ones in a biochemical assay.  

So, what did they find? 

"The correspondence of the ZoBio inhibitor structures with the predicted docking poses was spotty. "  and "There was better correspondence between the crystal structures of the docking-derived fragments and their predicted poses."

So, this isn't shocking, but it is good to know.  This is also consistent with this comment.  So, the take home from this paper is that in silico screening can help explore chemical space that the experimentally much smaller libraries miss.  To that end, the authors then do a a virtual experiment to determine how big a fragment library you would need to cover the "biorelevant" fragment space [I'll save my ranting on this for some other forum].  Their answer is here [Link currently not working, so the answer is 32,000.]

Getting misled by crystal structures: part 4

A picture is worth a thousand words, but words can mislead as easily as inform. So it is with crystal structures, as Charles Reynolds discusses in the July issue of ACS Med. Chem. Lett. We’ve touched on this issue before (for example, here and here), but this is a nice update.

He starts with a cringe-worthy catalog of horrors found in the protein data bank (pdb):

Just to give a few examples: 1xqd contains three planar oxygens as part of a phosphate group; 1pme features a planar sulfur in the sulfoxide; 1tnk, a 1.8 Å resolution structure, contains a nonplanar tetrahedral aromatic carbon as part of a substituted aniline; and 4g93 contains an olefin that is twisted nearly 90° out of the plane.

Of course, with 100,000 structures, it is inevitable some dross will slip through, but Reynolds argues that around a quarter of all co-crystal structures contain errors so severe that they could lead to misinterpretations.

Why is the situation so dire? Reynolds suggests a number of reasons. First, there’s the push for quantity over quality: fully refining a structure may not be as valued as solving a new one. Second, small molecules comprise only a small portion of the overall structure and thus make minimal contributions to the metrics crystallographers use to assess quality during refinement. Third, with the exception of very high resolution structures, the quality of the electron density maps are such that properly placing the small molecule requires a fair bit of modeling. This challenge is complicated by the fact that most crystallographers were not trained as chemists and thus may not immediately recoil from a tetrahedral aromatic carbon atom. Also, much of the off-the-shelf software used for refining structures is not optimized for small molecules.

Nonetheless, there is good software available that properly accounts for small molecules. Hopefully publicizing errors will encourage more crystallographers to use it. In the meantime, caveat viewor!

EthR revisited: fragment growing this time

A few months ago we described a fragment linking approach against the protein EthR, a transcriptional repressor from Mycobacterium tubercuolosis responsible for resistance to the second-line tuberculosis drug ethionamide. In a new paper in J. Med. Chem., a different team led by Benoit Deprez and Nicolas Willand (Université Lille Nord de France and Institut Pasteur) describe work on the same target using fragment growing and merging.

The researchers started with a fragment (compound 3) they had previously made as part of an in-situ click chemistry effort. A thermal shift assay revealed that this compound marginally stabilized EthR. More convincingly, it displayed mid-micromolar inhibition of EthR binding to DNA, with respectable ligand efficiency.

Interestingly, when compound 3 was cocrystallized with the protein, it bound at two different locations within the binding site. (In the work we highlighted previously this year, a different fragment also bound at two sites, and in that case the researchers linked fragments bound at each site to create a tighter binder.) In the current paper, the researchers focused on fragment growing.

Compound 3 is a sulfonamide that can be readily constructed from amines and sulfonyl chlorides, and the researchers started by constructing a 976-member virtual library of larger sulfonamides. These were then screened in silico against the protein, and many of the top-scoring hits resulted from an isopentylamine building block (such as compound 4). Ten of these were made and tested, and indeed, compounds 4 and 8 were more effective than compound 3 at stabilizing EthR in the thermal shift assay. Moreover, not only did compound 8 show low micromolar activity in the DNA-binding assay (IC₅₀ = 4.9 µM), it also showed low micromolar activity in sensitizing M. tubercuolosis to ethionamide (EC₅₀ = 5.7 µM).

Crystallography of compound 8 bound to EthR revealed that the isopentyl substituent was binding in a hydrophobic part of the pocket, and adding a few fluorine atoms (compound 17) gave a satisfying increase in potency as well as solubility. Replacing the sulfonamide with an amide (compound 19) further improved potency.

The researchers also made a couple compounds in which a second copy of compound 3 was merged with compound 19, and although this approach did produce a compound with nearly the same potency, it was also larger and less soluble.

This team has been pursuing EthR for some time, and they were able to use information from previous structures both in the computational screening as well as in the optimization. In that sense, this is an example of fragment-assisted drug discovery. It is also another nice example of fragment work in academia.

Halogen Hydrogen Bonding...Designable or Not?

The use of brominated fragments for X-ray screening is well known; it was the basis for former company SGX (now part of Lilly).  The purported advantage of brominated fragment is that you can identify the fragment unambiguously using anomolous dispersion.  In this paper, they are focused on using fragments to identify surface binding sites on HIV protease.  Prior work has focused on creating a new crystal form (complexed with TL-3, a known active site inhibitor) that has four solvent accesible sites: the exosite, the flap, and the two previous identified sites.  They took 68 brominated fragments and soaked these crystals: 23 fragments were found.  However, most of these actives were uninteresting.  Two compounds were found to be interesting, one bound in the exosite and one in the flap site. 

So, what's interesting in this paper?  Well, they (re)discover that brominated fragments can bind all over with a variety of affinities.  However, the bromine allows you to unambiguously identify those fragments through anomolous dispersion.  This is NOT interesting.  They discover that although it is a subject of much debate lately: specific interactions of the ligand with the target dominate the "bromine interaction".  This IS interesting.  They do not discuss this in much detail, but their grand extrapolations of this method to general applicability I don't buy. 

 I think the key take away from this paper is whether the halogen hydrogen bond undesignable and just a subject of serendipity?