To grow or to link: why not both?

What can you do with fragments? The idea of linking a couple together, while successfully demonstrated in the first SAR by NMR paper, generally seems to be more difficult than gradually growing one fragment. Now a new paper in Angewandte Chemie from Chris Abell and colleagues at the University of Cambridge presents a lovely comparison of these two strategies applied to a single target.

The researchers were interested in the M. tuberculosis enzyme pantothenate synthetase (PS) as a potential therapy for TB. Using a number of biophysical techniques including thermal shifts, NMR, and isothermal titration calorimetry, Abell and colleagues identified indole fragment 1 as a low-affinity binder from a library of about 1300 fragments (see figure below). X-ray crystallography revealed that the fragment binds in the ATP-binding site. An attempt to partially mimic the triphosphate by introducing negatively charged moieties led to modest improvements in potency (compounds 1a, 1b, and 2). Compound 2 bound in a similar position as compound 1, with the advantage that the methyl group off the sulfonamide is nicely positioned for further growing the molecule. Replacing this methyl group with a methylpyridine produced compound 4, increasing the affinity by about two orders of magnitude while maintaining ligand efficiency, and crystallography revealed that this moiety binds in the P2 pocket. Thus, the fragment growing approach began with an indole of low millimolar affinity and produced a molecule with low micromolar affinity after several iterations.



At the same time, the researchers also identified benzofuran fragment 5 (see figure below) and discovered that it binds in the P1 pocket some distance from the indole fragment 1, suggesting the two could be linked. In fact, a crystal structure revealed that the two fragments are able to bind to PS simultaneously. Linking these together through the acylsulfonamide linker employed above led to compound 8, with a potency similar to that obtained from fragment growing. Compounds 4 and 8 structurally resemble each other, but although the indole fragment of each binds in the same location, the terminal fragments (the methylpyridine in compound 4 and the benzofuran fragment in compound 8) bind in different locations, the former in the P2 pocket with the later in the P1 pocket. However, the benzofuran is somewhat twisted relative to the binding mode it adopts as a free fragment.



As the researchers observe, the ligand efficiency of compound 8 derived from fragment linking is lower than those derived from fragment growing, though even the molecules developed from growing have lower ligand efficiencies than the initial fragments.

The researchers conclude:

The two strategies resulted in similar compounds with similar potencies. This outcome obscures the fact that although the linking strategy appears more elegant, the limited repertoire of linkers is likely to compromise the binding of the original fragments. In comparison, the fragment-growing strategy provides more freedom for development at each stage and allows more room for further optimization.

True. But, the fragment linking strategy does provide a clear starting point for further optimization. The researchers did not describe how they selected the methylpyridyl fragment in compound 4 or how many other moieties they tested; 5-methylpyridine-2-sulfonamide does not seem like the first reagent one would grab from the shelf. However, the methylpyridine fragment is not dissimilar to the benzofuran fragment: swap the (hydrogen-bond accepting) oxygen for the (hydrogen-bond accepting) nitrogen, and the methyl would sit in a similar position as the phenyl ring (see figure above). In other words, medicinal chemistry on compound 8 could lead quite naturally to compound 4.

Infarmatik In-3D Library

In what we hope is a new series bringing the latest in Fragment Science up for discussion, we present today to you a discussion of Infarmatik's In-3D Library. We look forward to this discussion, and hopefully, many more.

Fragment based drug discovery has been shown to provide a rapid means for transforming low affinity “hits” to optimized leads. However, most currently available fragment libraries are limited in usefulness, mainly because over 90% of the molecules are planar and thus do not fit well into 3-dimensional receptor protein binding sites. InFarmatik realized [Ed: and others] that “real 3-D” structures offer a better fit within the uneven binding surfaces of protein hot-spots (business sites) than do planar compounds. To address this issue, we have developed a specific series of novel and diverse 3-D fragments, which are not available from any other commercial sources. The structure types of the first release contain 2,3, and 4 member non-aromatic ring systems, with various attachment points, including spiro and 1,2 anellation, 10 electron systems connected to saturated ring systems, saturated bis-heterocyclics and rod shaped compounds. We believe these compounds will exhibit the ability to bind to a wide array of protein targets. In addition, we can offer another 435 structures from existing stock, which conform to Ro3 and are quite “fragment-like”.

Most of the compounds have soft scaffold structures: meaning they were designed to have low reactivity centers to avoid non-specific binding, while preserving the ease of chemically coupling them to each other or to other fragments. The attachment points in the molecules in many cases are useful for regiospecific reactions.

Here are the relevant properties of the 3-D fragment library:
Size: 119 3-D Fragments
Average MW=230 Da
average logP value (calculated) =1.88
confirmed minimum water solubility of at least 0.1% in 2% aqueous DMSO.
Solubility data available for all compounds
Highly diverse, as shown by 3-D Diversity Analysis using ChemAxon supplied tools
Here are the relevant properties of the new standard fragment set
· Size: 435 Fragments
· Average MW=237.8 Da
· Average LogP value (calculated) =2.27

Genentech’s affinity for Graffinity

Heidelberg-based Graffinity today announced that they would be collaborating with the Genentech division of Roche. Graffinity will apply its surface plasmon resonance (SPR) fragment-based technology to several Genentech targets. Financial details and specific targets have not been released, though Graffinity CEO Kristina Schmidt is quoted as saying that they plan “to explore drug targets that would remain white spaces on the map of drug discovery” with conventional high-throughput screening.

SPR is rapidly becoming a workhorse in the stable of FBDD techniques. Although it provides less information than NMR or X-ray approaches, SPR is faster, and can rapidly distinguish true hits from bad-acting artifacts. Typically a protein is immobilized on a gold surface, and fragments are allowed to flow past to detect those that bind. Graffinity reverses this process: they have a collection of about 110,000 small molecules, just over a fifth of which are fragments, immobilized in microarrays which can be screened against proteins (see here for full description).

Genentech is no stranger to SPR; one of the highlights of the recent FBLD 2009 meeting was a talk by Tony Giannetti on the use of this technology at Genentech against roughly 40 target proteins. The collaboration further validates the use of SPR for FBDD, and suggests that Graffinity has an interesting – and useful – angle.

Fragment-based events in 2009 and 2010 (and calls for abstracts)

We’re in the last quarter of 2009, and I know of just one more event this year involving fragments:

October 13: The Life Science Regional Technology Symposium will be held in Somerset, NJ, and Dr. Teddy Z. will be one of several excellent speakers.

2010

Next year is starting to take shape nicely, and two events have put out calls for abstracts, so if you have something interesting to present, now’s your chance!

February 3-5: Cambridge Healthtech Institute’s 17th International Molecular Medicine Tri-Conference will be held in my beautiful city of San Francisco, with a track on medicinal chemistry that will have some fragment talks, and a short course on “Fragment-Inspired Medicinal Chemistry” on February 2.

March 21-25: The spring ACS meeting will also be held in San Francisco. There will be a symposium on “Fragment Based Drug Design: Novel Approaches and Success Stories,” and Rachelle Bienstock at the FBDD LinkedIn site has put out a call for abstracts, due October 19.

April 20-25: The Keystone Symposium on computer-aided drug design will take place in brisk Whistler, British Columbia. Although not exclusively devoted to fragments, the schedule shows several talks on the topic.

April 27-28: Cambridge Healthtech Institute’s Fifth Annual Fragment-Based Drug Discovery will be held in summery San Diego. This conference has also put out a call for speakers, with a deadline of October 16.

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

Looks can be deceiving: Getting misled by crystal structures - part 2

Last year we highlighted a paper that touched on some of the ways crystal structures can mislead, and a theme of FBLD 2009 was how dubious data can derail modeling efforts. Now, Jens Erik Nielsen and colleagues at University College Dublin add to the discussion by showing how the crystal lattice can potentially distort protein-ligand interactions. Their paper in J. Med. Chem. provides an analysis of the prevalence of two common structural artifacts, plus a practical tool for detecting them.

The first problem the authors consider is that some ligands make “crystal contacts.” Because a crystal is made up of a three-dimensional lattice of proteins packed together, a ligand bound near the surface of one protein may be in close contact with another protein in the crystal (a nonbiological “symmetry mate”); this contact occurs only in the context of a crystal and could distort how the ligand binds to its (true) partner protein.

The second, related problem is that water molecules that appear in the crystal structure can form bridges between a ligand and its nonbiological symmetry mate.

The authors examined a set of 1300 protein-ligand crystal structures with noncovalently bound ligands and experimentally measured binding affinities (PDBbind Database). Of these, 36% of ligands showed crystal contacts, and a similar number (37%) had crystal-related water bridges.

This doesn’t mean that all of these structures are misleading: the researchers note that “it is entirely possible that crystal contacts in some cases do not perturb the geometry of a protein-ligand complex whatsoever.” However, removing these structures before running docking experiments did improve the results.

The tricky thing about these structural artifacts is that they are often invisible, even when suspected. Most non-crystallographers focus on just on a single protein-ligand complex and don’t consider the crystal lattice when examining a crystal structure. Happily, Nielsen and colleagues have constructed a simple online tool (LIGCRYST) that can evaluate structures from the pdb to search for these types of problems. Although I’m not a crystallographer, I found it quite easy to use.

Hopefully modelers will increasingly take crystal contacts into account, and the next time you examine a structure from the pdb, you may want to give it a quick run through LIGCRYST.