Journal of Computer-Aided Molecular Design 2011 Special FBDD Issue

The most recent issue of J. Comput. Aided Mol. Des. is entirely devoted to fragment-based drug discovery. This is the second special issue they’ve dedicated to this topic, the first one being in 2009.

Associate Editor Wendy Warr starts by interviewing Sandy Farmer of Boehringer Ingelheim. There are many insights and tips here, and I strongly recommend it for a view of how fragment-based approaches are practiced at one large company. A few quotes give a sense of the flavor.

On corporate environment:

In most cases, the difference between success and failure has little to do with the process and supporting technologies (they work!), but rather much more to do with the organizational structure to support FBDD and the organizational mindset to accept the different risk profile and resource model behind FBDD.

On success rates:

We have found that FBDD has truly failed in only 2-3 targets out of over a dozen or so.

On cost:

FBDD must be viewed as an investment opportunity, not a manufacturing process. And the business decisions surrounding FBDD should factor that in. FBDD is more about the opportunity cost (of not doing it) than the “run” cost (of doing it).

On expertise:

Successful FBDD still requires a strong gut feeling.

On small companies:

In the end, FBDD will always have a lower barrier to entry than HTS for a small company wanting to get into the drug-discovery space.

The key to success for such companies is to identify or construct some technology platform.

There’s a lot of other really great content in the issue, much of which has been covered in previous posts on fragment library design, biolayer interferometry, LLEAT, and companies doing FBLD. The other articles are described briefly below.

Jean-Louis Reymond and colleagues have two articles for mining chemical structures, one analyzing their enumerated set of all compounds having up to 13 heavy atoms (GDB-13), the other focused on visualizing chemical space covered by molecules in PubChem. They have also put up a free web-based search tool (available here) for mining these databases.

Roland Bürli and colleagues at BioFocus describe their fragment library and its application to discover fragment hits against the kinase p38alpha. A range of techniques are used, with reasonably good correlation between them.

Finally, M. Catherine Johnson and colleagues present work they did at Pfizer on the anticancer target PDK1 (see here and here for other fragment-based approaches to this kinase). NMR screening provided a number of different fragment hits that were used to mine the corporate compound collection for more potent analogs, and crystallography-guided parallel chemistry ultimately led to low micromolar inhibitors.

Fragments vs. PDK1 again: into the adaptive pocket

PDK1 holds a certain appeal for fragment-based approaches. This kinase, an upstream member of the phosphatidylinositol-3 kinase (PI3K) signaling pathway, is an attractive anti-cancer target and has been successfully targeted using FBDD by researchers at Vernalis and GlaxoSmithKline (see here for a very recent publication). A series of allosteric activators was also discovered by researchers at Pfizer. The latest report on this target, an inhibitor which is particularly potent against the unphosphorylated form of PDK1, was just published online in Bioorg. Med. Chem. Lett.; it describes some of the work my colleagues at Sunesis and I did in collaboration with researchers at Biogen Idec.

To find inhibitors against PDK1, we used Tethering with extenders. This approach starts by covalently modifying a protein of interest with an “extender,” which is a fragment designed to bind in a desired site on a protein and to capture other fragments; it contains a protein-reactive functionality as well as a masked thiol. In this case, we used a generic fragment known to interact with the purine-binding pocket of kinases, a diaminopyrimidine (red in upper left of figure). The protein-extender complex was then screened against a library of several thousand disulfide-containing fragments under partially reducing conditions; only fragments with some affinity for the protein will remain covalently bound to the protein and thus be detectable by mass-spectrometry. The pyridinone (blue in figure) was one of the strongest hits.

We synthesized several molecules containing purine-pocket binding elements connected to pyridinones by flexible linkers and found compound 25 to be one of the most potent. The SAR revealed the importance of a hydrogen-bond donor-acceptor pair (green atoms in figure) a specific distance from the pyridinone fragment, and further medicinal chemistry led ultimately to compound 33. In addition to being quite potent, compound 33 was remarkably selective for PDK1: in a panel of 241 kinases screened at 10 micromolar concentration of compound 33, only PDK1 and one other kinase were inhibited by 80% or more.

The selectivity of compound 33 was partially explained by the X-ray crystallographic structure, which revealed that the pyridinone fragment discovered through Tethering binds in the so-called adaptive pocket with the activation loop in the “DFG-out” conformation. I believe this is the only series of inhibitors for which this binding mode has been observed for PDK1. Consistent with the structure, these inhibitors are more potent against the unphosphorylated (inactive) form of PDK1 than against the phosphorylated (active) form.

Compound 33 was effective at preventing phosphorylation of the PDK1 substrate Akt both in cells as well as in a xenograft model. Though I may be biased in thinking so, this is a nice application of a fragment-based approach to discover a strikingly specific kinase inhibitor. I hope that people will use compound 33 to further probe the biology of the PI3K pathway. Indeed, at least one completely independent team of researchers seems to already be doing so.

Fragments vs PDK1

Kinases have been a particularly productive target class for fragment-based drug discovery (and drug discovery in general), with nearly half of reported FBDD-derived clinical candidates targeting kinases. The latest dispatch from this field can be found in the November issue of ACS Medicinal Chemistry Letters.

In this paper, Jeffrey Axten and colleagues at GlaxoSmithKline describe their use of fragment screening to identify inhibitors of PDK1, a popular anti-cancer target. They started by assembling a library of fragments biased towards the purine-binding site of kinases, and tested 1065 of these in a biochemical screen at 400 micromolar concentration. Of these, 193 inhibited activity at least 60% and were further characterized; 89 had IC50 values better than 400 micromolar. A set of 36 of these, chosen on the basis of ligand efficiency and chemical tractability, were chosen for follow-up.

Saturation transfer difference (STD) NMR was used to confirm which fragments bound to PDK, which cut the number of hits in half. X-ray crystallography experiments were started before NMR and performed on 7 fragments; only the fragments that were confirmed by NMR gave interpretable data. One of these was the aminoindazole compound 8 (see figure).

A substructure search was conducted to find more elaborated molecules within the corporate screening collection, leading to compound 19, which has sub-micromolar potency. This compound also showed some signs of selectivity for PDK1 over other kinases. Although the paper stops here, Jeffrey Axten gave a nice presentation at FBLD 2010 in which he discussed subsequent medicinal chemistry that ultimately led to novel, high picomolar inhibitors of PDK1.

There are at least two lessons from this story. First, the significant attrition from the biochemical screen again emphasizes the need for orthogonal methods of fragment validation. Second, even though the fragment identified has been around the block with respect to kinases (as of last year, the aminoindazole substructure had appeared in over 70 kinase patents), skillful medicinal chemistry can still get you to novel compounds.