Fragments vs Abl: antagonists and agonists

A common concern with using biophysical techniques to identify fragments is that the functional implications of identified binders are not always clear, an issue we’ve discussed previously. In a new paper in J. Am. Chem. Soc., Wolfgang Jahnke (co-editor of the first book on FBDD) and colleagues at Novartis describe a clever NMR approach to address this problem and identify both agonists and antagonists that bind to an allosteric site on the protein tyrosine kinase Abl.

Abl is less well known than its famous cousin, Bcr-Abl, an oncogenic fusion protein in which the kinase activity is always turned on. Bcr-Abl is targeted by imatinib and a number of other kinase inhibitors; indeed, the success of imatinib against certain types of cancer has been largely responsible for the rush to develop drugs targeting kinases.

Most kinase-targeted drugs (including imatinib) bind in or near the conserved ATP-binding site. However, Abl offers another binding site, a pocket that can be filled by the fatty-acid myristic acid. This interaction causes conformational changes in the protein, stabilizing an inactive state. Indeed, previous research had identified molecules that bind in this pocket and block activity. Jahnke and colleagues used NMR screening of a 500-fragment library to try to identify new chemical scaffolds.

Several fragments were identified, some of which bound relatively tightly as judged by NMR and ITC. However, these fragments did not inhibit kinase activity. Crystallographic analysis of the fragments bound to Abl revealed that, although the fragments do bind in the myristate pocket, their binding modes are incompatible with the conformational changes needed to inhibit the kinase. Realizing that a specific valine residue is structurally disordered in the absence of myristate, the researchers established an NMR assay using Abl in which valine had been isotopically labeled to assess which molecules bind in a similar fashion to myristate (and thus block activity).

But what of the molecules that bind in the myristate pocket without causing conformational changes? Some of these can actually activate the kinase by competing with endogenous myristoyl groups. Fragment-based discovery of agonists is not unprecedented (see for example here and here), but it is rare. Assays such as the one described here to distinguish between different conformations of a protein could be practical complements to approaches that focus on binding alone. The paper is also a useful reminder that binders are not necessarily inhibitors, and can in fact be just the opposite.

A HERD of hydrogen-bonding fragments

The amino acids histidine (H or His), glutamic acid (E or Glu), arginine (R or Arg) and aspartic acid (D or Asp) are often found in ligand-binding sites in proteins. As such, finding fragments that preferentially interact with these amino acids could be useful for fragment-based ligand discovery. David Selwood and colleagues at University College London have combed the Protein Data Bank (PDB) to do just this; they report their results in a recent issue of J. Med. Chem.

The researchers analyzed over 8000 high-resolution protein-ligand structures in which the ligands formed hydrogen bonds with the side chains of His, Glu, Arg, or Asp. They defined fragments as “the largest ring assembly containing the atoms involved in hydrogen bonding.” This excludes functional groups linked to aliphatic chains, but given the importance of rings in most drug molecules this limitation seems reasonable. A total of 462 fragments were found; the number of fragments making hydrogen bonds with each amino acid was broadly similar, with a low of 130 for Arg and a high of 159 for Asp.

The diversity of fragments that interact with the acidic side chains of Asp and Glu is lower than that of fragments interacting with the basic side chains of His and Arg. Not surprisingly, amidines represent a large fraction of the former; these form two hydrogen bonds with either Glu or, more frequently, Asp. Cyclic diols are also common double hydrogen-bonding fragments for Glu and Asp, while cyclic aliphatic amines are perhaps less common than one might expect.

Among fragments that interact with Arg, 124 (91%) do so through an oxygen atom, with only 7 (5%) interacting through nitrogen, and a handful interacting through halogens (5) or sulfur (1 example). The His residue also shows a preference for oxygen-containing fragments, though at 63% this is less pronounced than the much more basic Arg.

One of the attractive features of this work is that, by focusing on fragments that form hydrogen bonds, the free energy of binding is likely to be dominated by enthalpic rather than entropic terms. As has been discussed (here, here, and here) this has some potential advantages for drugs.

This sort of analysis always leads one to ask whether the identified fragments represent limits on what could bind, or, as the researchers also speculate, “the limited variability of currently available chemical libraries from which drugs are derived.” There is clearly some justification for thinking the latter: the preponderance of amidines is likely due to the number of serine proteases that have been targeted with this functional group. Nonetheless, the overall set of fragments could be quite useful for computational screening. In fact, many of them could also be incorporated into physical fragment libraries. Perhaps one of the enterprising commercial fragment library suppliers will put together a sub-library based on this work.

Updated again: Fragment-based conferences in 2010

The year is not even half over, but most of the fragment events are behind us (see summaries here, here, and here). You still have a couple more opportunities for some great conferences.

June 6-9: The 32nd National Medicinal Chemistry Symposium will be held in Minneapolis, Minnesota, and Dave Rees is organizing a session on fragments on June 9. Looks like a great lineup, with top speakers from Astex, Plexxikon, Novartis, Abbott, and UC Berkeley.

October 10-13: Fragment-based Lead Discovery 2010 is the first major fragment event on the east coast of the US (in Philadelphia, PA). There are lots of excellent speakers, and the conference is still accepting abstracts for talks until May 31. Early bird-registration also ends that day. If you haven’t made it to any other fragment events this year, don’t miss FBLD 2010!

Know of anything else? Organizing a fragment event? Let us know and we’ll get the word out.

Poll: academia/industry

It’s been a while since we’ve done a poll, but the latest post at FBDD-Lit on fragments in academia, combined with an earlier post on this site, leads us to wonder how many of our readers are from academia and how many are from industry. (For purposes of this poll, let’s lump government and other non-profit organizations with academia).

Please respond by clicking on the right-hand side of the page – we’d like to know more about you!