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Fragment linking is a topic we’ve discussed several times. One of the more interesting approaches is template-directed synthesis, in which a protein causes two fragments in close proximity to react with one another (see here for example). In a recent issue of Angew. Chem. Int. Ed., Beat Ernst and colleagues at the University of Basel provide a new variant of this theme, without requiring structural information about the protein.

The researchers were interested in a protein called myelin-associated glycoprotein, or MAG, which blocks axonal regrowth. They started with an NMR screen to determine which members of a fragment library bind to MAG as assessed by a phenomenon known as transverse magnetization decay; essentially, small molecules that bind to a protein behave like large molecules in showing a rapid decay in magnetization, so an increased magnetization decay of fragments in the presence of protein suggests binding. A number of fragments were identified as binders, but the site of binding was not determined.

To find molecules that could be linked, the researchers took a known ligand, the sialic acid derivative 1 (see figure – albeit larger than a fragment), and modified this to contain a spin-label. Spin-labels are small moieties that contain an unpaired electron and, just like large molecules, cause an increase in magnetization decay, but only to molecules within close proximity. The two effects are additive, and thus the researchers could determine which fragments bind to the protein in close proximity to the spin-label-containing derivative of compound 1. In fact, the distance dependence is so pronounced that different protons on the fragment can show different effects, thus indicating which portion of the fragment is close to the spin label (see here for a similar approach using ILOE). In this case, the researchers found that a nitroindole fragment (see figure) had its 5-membered ring positioned closer to the spin label than its 6-membered ring.



Knowing the relative positions of the two ligands, the researchers modified them so they could be linked together. They added functional groups with different linker lengths to create several analogs, replacing the spin label with an alkyne and adding an azide to the nitroindole fragment. They then incubated all the analogs together in the presence of the protein. Analysis of the reaction by HPLC-MS after three days at 37 degrees revealed one prominent product, with a mass consistent with compound 7. Two isomers of this product can be formed, with syn and anti configurations around the triazole, and the researchers synthesized both of them. Interestingly, the anti isomer (shown) had a Kd for MAG of 190 nM, while the syn isomer bound roughly 10-fold more weakly.

Although the ligand efficiency of the final compound is low, sugar-based molecules typically have low ligand efficiencies, and maintaining the same efficiency as starting compound 1 is impressive. However, the ligand efficiency of the final molecule is probably lower than the second-site ligand: the researchers don’t report its affinity, but since it would likely need to be 10 mM or better to be detected its ligand efficiency is probably at least 0.23 kcal/mol/atom.

Still, the final product is sufficiently potent that it could make a useful biological probe. Moreover, the approach is notable in not requiring structure of the protein – a rare and attractive feature for fragment linking.

Fragment specificity

A frequent topic in fragment roundtable discussions concerns specificity: do fragments hit lots of targets, or just a few? Isabelle Krimm and colleagues at the Université de Lyon in France studied this question experimentally and report their results in a recent issue of J. Med. Chem. The paper provides data for the ongoing debate of whether and how much specificity a fragment should exhibit before being pursued for further lead development.

The researchers assembled a diverse set of 150 fragments and used NMR techniques to determine whether they bind to five different proteins. Three of the proteins, Bcl-xL, Bcl-w, and Mcl-1 are related members of the Bcl-2 family of antiapoptotic proteins, and at least the first of these has been successfully targeted using fragment-based methods. The fourth protein, PRDX5, has proven to be much less yielding to inhibitor discovery, while the fifth, human serum albumin (HSA), binds a wide variety of small molecules.

After applying 1D-NMR techniques (WaterLOGSY and STD) to all of their fragments against each of the five proteins, the researchers used more rigorous but less sensitive 2D-NMR (HSQC) to determine the binding sites of the hits. (This later study revealed, in agreement with previous results from the same lab, that the fragments all bind in the “hot spots” or active sites of the proteins.)

More than two-thirds of the fragments bound to at least one protein, a rather high hit rate. However, the hit rates for each protein varied considerably, with only 7 hits for PRDX5 and 72 for HSA (with a close second of 71 for Bcl-xL). Within the Bcl-2 family there was little specificity observed: Mcl-1, with 29 hits, shared all but one hit with either Bcl-xL or Bcl-2 or both; such non-specificity among related proteins has been discussed previously. In the case of HSA and Bcl-xL, although both proteins had similar numbers of hits, just over half of these were in common, demonstrating that fragment specificity is not difficult even with small-molecule sponges such as HSA. That said, many fragments were remarkably nonspecific, with 22 hitting four of the 5 proteins. Amazingly, all 7 of the hits against PRDX5 also hit all four other proteins.

The physicochemical properties of the fragments that hit one or more proteins were compared with those of the library as a whole, and although most of the parameters were similar, the ClogP values (a measure of hydrophobicity) were considerably higher for hits, and highest of all for the non-specific hits.

These findings are more evidence that, as predicted almost a decade ago, fragments can bind to more proteins than can larger, more complex molecules. The follow-up question, how much does this matter, is still up for debate. There are plenty of examples of developing specific inhibitors from non-specific starting points during the course of fragment optimization. But how non-specific is too non-specific? Would you feel comfortable pursuing any of the fragments that hit all of the proteins?

FBDD and structural biology

The rise of fragment-based drug discovery has largely depended on the success of structural biology. FBDD began in earnest with NMR techniques in the mid 1990s, soon followed by high-throughput crystallography techniques in the early part of this century. In an article published online in Current Opinion in Structural Biology, Christopher Murray of Astex and Tom Blundell of the University of Cambridge discuss this reliance on structure.

The review describes several cases where structural biology played pivotal roles in advancing fragments to leads or drug candidates. Many have been discussed in Practical Fragments, including AT7519 and AT9283 from Astex, the JAK-2 program from SGX, DG-051 from deCODE, HSP90 inhibitors from Vernalis/Novartis and Evotec, Schering-Plough’s BACE inhibitors, and Plexxikon's indeglitazar.

The researchers also discuss the potential of fragment methods for generating inhibitors of antimicrobial targets, such as enzymes in the organisms that cause tuberculosis and sleeping sickness. In these cases too, structural biology played critical roles.

Structural biology is so important, the authors conclude, that “it is only through the expert use of structure-based drug design that FBDD can be expected to fulfill its promise of delivering candidates with the improved physical properties (lower molecular weight and lipophilicity) which it is hoped will lead to reduced attrition in clinical trials.”

But is dependence on structure truly inevitable? The authors themselves highlight one case in which a new antimicrobial agent with animal efficacy was developed using fragment-based methods in the absence of direct structural information. If this success could be generalized, it would open the potential of fragment methods to a much wider range of practitioners.

Virtual phosphate fragments

Phosphate groups are handy little things: easy for enzymes to put on and take off, they pack a lot of charge in a small volume, thereby providing plenty of binding energy for electrostatic interactions. Not surprisingly, they are ubiquitous in biology. Unfortunately, the same things that make them attractive for an organism make them problematic for drugs: they are easily removed, and their highly negative charge gives molecules containing phosphates a real problem getting across membranes. What’s a chemist to do?

This was the dilemma faced by Ruth Brenk, Ian Gilbert, and colleagues at the University of Dundee. They were interested in inhibiting the enzyme 6-phosphogluconate dehydrogenase (6PGDH) from the parasite that causes sleeping sickness. (See here for previous work from the same group using fragment methods to discover inhibitors against a different enzyme from the same organism.) The enzyme 6PGDH, as its name suggests, binds phosphate-containing substrates and has a very polar active site. Nanomolar inhibitors have been reported in the literature, but these contain phosphates and are not active in cell assays.

As reported in a recent issue of Bioorganic and Medicinal Chemistry, the researchers computationally filtered a set of commercially available compounds to find those that were less than 320 Da and were negatively charged, thereby potentially mimicking a phosphate. They then used DOCK 3.5.54 to see which of the resulting 64,000 molecules might bind in the active site of 6PGDH, resulting in 5836 possible hits. Subsequent triaging led to the purchase of 71 compounds. These were tested for inhibition of the enzyme at 200 micromolar concentration. Ten of these compounds inhibited the enzyme more than 80% at this concentration, of which 3 gave clean IC50 curves. These three molecules are all 5-membered carboxylic-acid-containing heterocycles, and although the IC50s are modest (ranging from 28 to 45 micromolar), they have good ligand efficiencies (up to 0.66 (kcal/mol)/atom). A computational search for analogs resulted in a few more active molecules with similar properties.

Whether these fragments can be advanced remains to be seen. The calculated solubilites, Log P, total polar surface area, and intestinal absorption parameters are more attractive than previous inhibitors, but the history of phosphate mimics is not encouraging. Most prominently, the protein PTP-1B, which recognizes phosphotyrosine residues, was once one of the hottest drug targets around, spawning a cottage industry of groups developing phosphotyrosine mimetics. Fragment methods were particularly effective, and numerous potent small molecules were published. But none of them were sufficiently drug-like, and to my knowledge none are in the clinic. Still, it is worth trying: 6PGHD may be more druggable, and approaches like this are likely to provide an answer.

ANCHORing fragments

Protein-protein interactions are intriguing though challenging targets for lead discovery, and fragment-based approaches have often been used to tackle them (for example here, here and here). One of the difficulties is trying to figure out which of the often many residues in a large contact surface are really important. To make this easier, Lidio Meireles, Alexander Dömling, and Carlos Camacho at University of Pittsburgh have unveiled a free web-based tool, described in a recent issue of Nucleic Acids Research.

The tool, called ANCHOR, is both a server and a database of protein-protein interactions. The database contains over 30,000 entries taken from the protein data bank (PDB). Each of these entries has been analyzed computationally. ANCHOR examines bound and free (as computationally isolated from the complex) forms of each protein, focusing on side chains that, depending on protein state, are either buried within the partner protein or exposed to solvent. The change in solvent-accessible surface area is calculated for every residue in the protein-protein contact area. ANCHOR also estimates each residue’s contribution to the binding free energy, using both electrostatic and solvation terms in the calculation.

While the absolute numbers should probably be taken with a grain of salt, the relative values could help identify “anchoring” residues most likely to be useful as initial fragments. This means you can enter a pdb number and rapidly find the residues likely to be most important. You can also do more complex queries across the entire database, for example searching for buried tryptophan residues for oncology targets. If your protein is not already in the database, you also have the option of uploading a structure for custom analysis.

What makes ANCHOR particularly appealing is its powerful graphical interface, which shows which residues are selected and allows significant customization. The whole system is quite intuitive and easy to use. Try it on your favorite protein-protein interaction and tell us what you think!

So Long, and Thanks for all the Fish

I am writing this to say thank you to everyone who reads this blog, and those who have contributed to this blog. When Dan and I started this, it was after meeting at a FBDD conference in San Diego. We decided that this field needed something like this. There are now LinkedIn groups, Facebook groups, other blogs about FBDD (all linked to the right). As many of you know, my current position does not involve FBDD. FBDD has been a passion of mine since I got involved in 2001. I appreciate my boss at the time Mike Shapiro for giving me the chance to set up and lead the FBDD efforts at Lilly. It was a fantastic experience and very successful. I also want to thank Mike for co-editing the book with me (please buy a copy, every copy earns me something close to three cents ;-)). I want to acknowledge all the great friends I have made through the years, including Dan who has picked up the onus of publishing this blog and has done and will continue to do a fantastic job.
I have always said that research carries a two year shelf-life. It's been almost two years since the book came out, that means I am done. I have nothing new to add to the field (and probably haven't for longer than two years). This means no more embarassing questions when I give talks on FBDD about what I doing now (which is nothing in FBDD). It is exciting to follow the field, but it is also really tough not being a part of it.
I wish you all the best of luck and ask that you don't be strangers. I will be speaking on NMR in the upcoming months, and am always happy to add more NMR speaking engagements (hint hint).

Metallophilic fragments

A post earlier this month mentioned matrix-metalloproteinases (MMPs). Now, a recent issue of J. Am. Chem. Soc. carries a Communication about fragment-libraries designed for zinc proteases, of which MMPs are a subset.

Seth Cohen and coworkers at University of California San Diego and the Weizmann Institute of Science in Israel designed two libraries based on known zinc chelators: quinoline sulfonamides (QSL) and benzimidazole sulfonamides (BISL) (see Figure). They rapidly assembled 40 of the former and 37 of the later using microwave chemistry and tested these against a handful of different MMPs.

Both libraries produced hits against MMP-2, MMP-3, MMP-8, and MMP-9. Control compounds designed not to chelate zinc showed no activity, and X-ray adsorption fine structure spectroscopy experiments suggest that the molecules are indeed binding to the catalytic zinc. Selectivity is often an issue in targeting metalloproteinases, and it was thus gratifying to find that at least one fragment inhibited MMP-2 with low micromolar activity while showing no activity against the other MMPs. Molecular modeling provides some rationale for this selectivity.

One could argue that many of the library members do not meet conventional definitions of fragments, and could be seen as more scaffold-like (or worse – one has a molecular weight pushing 600 Daltons!) And of course, it is not clear that either scaffold will be suitable for drug development or even tool compounds – it is possible their propensity for zinc binding will be a problem inside cells. Still, the notion of creating custom-made fragment libraries for various classes of targets certainly makes sense; folks have done this for kinases and even RNA, and it is reasonable to see this approach extended to metalloproteinases. Cohen and colleagues described a fragment library consisting of more conventional metal chelators earlier this year in ChemMedChem.

This publication also confirms the results of our poll that fragment-based approaches are catching on in academia. But industry is already in the sandbox: at least two companies, AnCore and Viamet, are using similar strategies to target metalloproteins.

Poll results: academia/industry

Just a quick summary of our poll last month. The results (below and to right) show that just over half of you who responded (51%) are in industry and just under half (46%) are in academia, government, or other non-profit organizations. Also, over three-quarters of you (77%) are active practitioners of FBDD.

Thanks to everyone (over four score) who took the time to respond!

Fragment linking: how much is it worth?

Fragment linking is a topic we’ve discussed a few times. One of its great appeals is that, all other things being equal, the entropic cost of binding one linked molecule is less than the cost of binding two separate molecules. Thus, linking two fragments should give more than an additive increase in binding energy. As the late William Jencks noted, for two fragments A and B:

Kd(AB) = Kd(A) * Kd(B) * E
Where
Kd(AB) is the dissociation constant for the linked molecule AB
Kd(A) is the dissociation constant for fragment A
Kd(B) is the dissociation constant for fragment B
E is a “linking coefficient”, reflecting the costs and benefits of linking

The lower the Kd the better, so ideally E < 1, though in practice finding a suitable linker can be tricky and all too often E > 1 (sometimes >> 1). But how low can E go? How much of a boost can you get by linking two fragments? Claudio Luchinat and colleagues at the University of Florence looked at this question experimentally in a recent paper in J. Med. Chem.

The researchers took PMAHA, a known inhibitor of the matrix metalloproteinase MMP-12, and dissected it into two fragments, AHA and PMS, by conceptually “cleaving” the bond connecting them (see figure). This simplifies analysis: since the two fragments are almost identical to the linked molecule, there are no concerns that atoms in the linker interact with the protein.


The crystal structure of PMAHA bound to MMP-12 had been previously reported, but Luchinat and co-workers solved the co-crystal structure of AHA and PMS bound simultaneously to MMP-12. The two fragments overlay fairly well with the parent molecule: AHA binds to the catalytic zinc, while PMS binds in the S1’ pocket. The AHA fragment is rotated with respect to its position in PMAHA, though it makes the same interactions in both structures.

Thermodynamic binding parameters for the three molecules were determined (see figure). As expected, PMAHA binds considerably more tightly than the product of the affinities of the two fragments: E << 1 (in fact, about 0.0021). And in nice accord with theory, this enhanced affinity is entropic: both fragments bind with favorable enthalpy and unfavorable entropy, while the linked molecule has both favorable enthalpy and entropy. In other words, the salutary effect of linking these two fragments does seem to come entirely from entropic effects.

One of the more interesting lessons from this paper is a sense of how much of a boost in potency you can expect if fragment linking goes well: about 500-fold. In theory you could do better, but in practice you should expect much more modest benefits: a prominent success of SAR by NMR on a different metalloproteinase reported a 14-fold boost in affinity. But just like the lottery, the hope of a big payout will continue to attract people to the linking game.

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!

Fifth Annual Fragment-Based Drug Discovery

The first of two conferences in 2010 exclusively devoted to fragment-based drug discovery concluded in San Diego this week, and I thought I’d jot down some observations while my memories are still fresh.

The pre-conference short courses were quite successful (and I’m hopefully only slightly biased by the fact that Teddy and I were both instructors). Participants included folks with considerable experience in fragments, which allowed good discussion.

One talk from the conference that stands out in my mind was by Sandy Farmer of Boehringer Ingelheim. BI is a relative late-comer to fragment-based methods, really only starting in late 2004. Sandy described how fragment efforts are run in parallel with HTS. They use an intentionally modest fragment library of 2000 diverse compounds; increasing the size of this library tended to overwhelm downstream efforts. Fragments are confirmed using multiple assays, including SPR and size-exclusion chromatography coupled with mass spectrometry, with crystallography playing a pivotal role in determining which fragments to advance. Part of the challenge at BI has been getting chemists to accept FBS, or “faith-based synthesis,” particularly where initial fragments have low affinities. A focus on ligand efficiency helps, as do organizational strategies such as establishing a dedicated group of chemists focused on fragment projects.

Often at conferences you hear about success stories, but sometimes the continuing challenges are more instructive, as when Ravi Kurumbail at Pfizer discussed his efforts to discover drug-like inhibitors of the serine protease Factor XIa. One of the sobering findings was that, although a functional assay of 2500 fragments yielded a 6.5% hit rate, adding 0.01% detergent eliminated activity and revealed most ‘hits’ as false positives. Even one crystallographically characterized fragment with an apparent IC50 of 75 micromolar turned out to be an artifact after subsequent analysis – a reminder to always be vigilant at higher concentrations.

But back to success stories: Daniel Wyss gave an update on the BACE program from Merck (legacy Schering-Plough, which has run more than 30 fragment screens on various targets). We highlighted a couple publications resulting from this effort earlier this year. It turns out there are now three molecules from this program in early clinical trials – a clear indication of the importance of this target and the utility of fragment screening.

Finally, Rick Artis, formerly of Plexxikon (now Elan) gave an update on the PLX4032 Raf kinase program. This project demonstrates the potential for fragment-based efforts to move quickly: it was started in February 2005, the clinical candidate was identified in January 2006, the IND was filed in September, and the first patient was dosed in November of that year. The molecule has continued to move at warp speed through the clinic: it is now partnered with Roche in Phase III testing for metastatic melanoma, and was recently profiled in the New York Times. This lengthy but excellent article is well worth reading for a bit of perspective when life in the lab gets you down.

These are just a few of many nice talks and breakout discussions. I know that at least some readers of this blog were there – what were your impressions?

Hot spots for fragments

Although most people try to advance fragments to more potent molecules, some have taken the reverse approach: starting with potent binders and deconstructing them into fragments (see for example here, here, and here). A recent, thorough example in J. Med. Chem. shows how isolated fragments do not necessarily bind in the same manner as they do in fully elaborated molecules.

In this paper, Isabelle Krimm and colleagues at the Université de Lyon in France applied “fragment-based deconstruction” to inhibitors of the anti-cancer target Bcl-xL. This protein is one of the great success stories in fragment-based drug discovery: ABT-263, which is in multiple clinical trials, was discovered by researchers at Abbott using SAR-by-NMR. In that work, fragments were identified binding near each other on the protein (site 1 and site 2) and subsequently linked together. Very extensive medicinal chemistry eventually led to the picomolar inhibitor now in clinical testing.

Krimm and colleagues dissected 9 inhibitors of Bcl-2, including ABT-263, into 22 different fragments and studied their binding by NMR. They first used ligand-observed NMR (WaterLOGSY and saturation transfer difference, or STD) and found that 19 fragments interacted with the protein. When they then turned to protein-observed NMR (proton-15N heteronuclear single quantum correlation, or HSQC), only 13 fragments caused changes to the spectra of Bcl-xL, suggesting that the other six bound too weakly to detect. In fact, the most potent fragment has an affinity of just 2.7 mM, so it is not surprising that some of the fragments were undetectable.

The nice thing about protein-observed NMR is that it can provide insight into where on the protein the fragments bind, and in this case the researchers found that 12 of the 13 fragments that caused NMR shifts in the protein bind to site 1, despite the fact that structures and modeling suggest that some of these fragments should be binding in other sub-sites. (The 13th fragment appears to bind to multiple sites on the protein surface.) In other words, the binding modes of the isolated fragments are not the same as the binding modes of the fragments when assembled.

The authors conclude that fragments “will interact with their preferred binding site, which can be different from the site they occupy when they are included in the larger molecule.”

Interestingly, one of the fragments studied by Krimm (2,3-dihydroxynapthalene) was also tested at Abbott, but found to bind in site 2. The reason? In the Abbott study, this fragment (and a number of others) were tested in the presence of a fragment that binds to site 1. It seems that site 1 is a thermodynamic sink, or hot spot. Unless this site is filled, other fragments will bind there, even if they could also bind elsewhere on the protein. The implication is that, if you want to find fragments that bind to a new site on your protein, it may be worth screening in the presence of a fragment known to bind to an existing site.

Getting misled by NMR: ILOE artifacts

We’ve pointed out potential pitfalls with crystallography (here, here, and here) as well as with biochemical screening, but NMR has escaped attention– until now.

NMR has of course been a mainstay of fragment discovery methods since the original SAR by NMR paper. There have been plenty of developments since, but one that is particularly intriguing relies on the “interligand nuclear Overhauser effect,” or ILOE. In the “SAR by ILOE” approach, a 2D NMR experiment is used to detect when two small molecule ligands bind to a protein next to one another. There are some attractive features of this method. First, only ligands that bind in relatively close proximity to each other will generate a signal, thereby allowing researchers to identify fragments close enough to allow productive linking. Second, the technique can be applied to proteins that are too large to study by other NMR methods. In fact, it can be used even in the complete absence of structure. So what’s the problem?

In a new paper in J. Am. Chem. Soc., Chris Abell and colleagues at the University of Cambridge applied the approach to pantothenate synthetase (PtS) from M. tuberculosis. They previously did rigorous fragment screening followed by both linking and growing on this enzyme, which we discussed last year. Initial NMR experiments with compounds 1 and 2 (see figure) in the presence of PtS showed strong ILOE signals; the problem was that signals were seen between all the protons of compound 1 and all the protons of compound 2. This suggests non-specific binding: if the two molecules were binding next to each other in a single orientation you would expect that some protons from compound 1 would be closer to some protons in compound 2 than others, and there would thus be differences in signal intensities.

Adding a methyl group to compound 1 to give compound 4 didn’t help. In fact, there were ILOE signals from both the methyl groups of compound 4 to all the aromatic protons of compound 2, again suggesting non-specific binding. Even more damning, adding the substrates ATP and pantoate failed to significantly diminish the ILOE signals as expected; because crystallography showed these fragments bind in the active site, they should have been readily displaced by substrates.


Reasoning that the hydrophobic nature of compound 4 might be causing it to aggregate at high concentrations, the researchers appended a carboxyl group to give compound 5. NMR experiments with this compound in the presence of compound 2 and the protein now revealed specific ILOE signals between the 2-methyl group of compound 5 and H2 of compound 2. Moreover, this signal could be competed by adding ATP and pantoate.

Happily, linking these two fragments together resulted in compound 6, which bound to the enzyme three orders of magnitude more tightly than either of the starting fragments. The compound was also well-behaved mechanistically, showing competitive inhibition with ATP, and a crystal structure revealed that it binds as expected given the structures of the individual fragments.

Overall then this is a success story. However, it does suggest that the ILOE method may be more prone to aggregation artifacts than other biophysical methods. In particular, had the researchers not been able to do competition experiments (if, for example, they did not have another small molecule inhibitor available) they would have had a harder time sorting things out. Also, the researchers actually had crystal structures of both compounds 1 and 2 bound to PtS, so it is not clear how valuable the ILOE data really were for linking. Still, the potential advantages of an NMR-based method that doesn’t require structure are appealing. Hopefully we will see more applications of SAR-by-ILOE, now that people are more aware of the dangers.

The Rule of 1

Everyone is familiar with the Rule of 5, Lipinski’s famous set of guidelines for orally active small molecule drugs. Most folks working with fragments are also familiar with the Rule of 3, proposed by Astex researchers to guide fragment selection so as to avoid starting with something too large. On the assumption that if small is good, tiny is superlative, scientists at Lilliput Pharmaceuticals have proposed the Rule of 1:

MW < 100 Daltons
<= 1 Hydrogen bond donor
<= 1 Hydrogen bond acceptor
ClogP <= 1

By limiting themselves to molecules with less than 8 heavy atoms, Lilliput reckons it can purchase or synthesize just about every stable molecule; according to Reymond’s GDB database there are only a few tens of thousands of possibilities. “The rules of 5 and 3 are for the lily-livered,” says CEO I. M. Lyttle, Jr. “We aim for total coverage of chemical space.” Of course, finding fragments this small is bound to be a challenge, but anything they detect is likely to have killer ligand efficiency.

Native Mass Spectrometry

We’ve recently blogged on the biophysical techniques surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). This post discusses native mass spectrometry (MS), which was reviewed in Future Med Chem earlier this year. (Thanks to author Denis Zeyer for pointing this out).

Mass spectrometry involves ionizing a molecule, measuring its mass-to-charge ratio, and using that ratio to determine molecular weight. Since the process occurs in a vacuum under high electric fields, biomolecules such as proteins are usually denatured. However, under careful conditions, not only can biomolecules be kept in their native state, but complexes of multiple molecules can be kept together; by measuring the weights of these complexes, the individual components can be determined.

In the case of protein-small molecule complexes, the technique can be used to determine binding and stoichiometry (how many small molecules are bound to a given protein), and the authors discuss a number of papers in the field (including some seminal RNA-fragment examples).

Two of the authors are applying native MS to fragments at French company NovAliX. They describe screening their 350-compound fragment library against the anticancer target Hsp90 to identify 40 fragments that bind to the protein, and further characterized some of these crystallographically. The entire screen, in duplicate, required 2 milligrams of protein.

There are a few limiting issues with native mass spectrometry. First, the technique requires careful choice of buffers; in particular, detergents are not compatible. That can be a problem because omitting detergents sometimes leads to small-molecule aggregation, even with legitimate binders. The authors note that multiple binding is sometimes observed in native mass-spectrometry; it would be interesting to follow up on these observations with activity assays to determine how often these are truly non-specific or just appear so under the assay conditions.

Another issue is that the stability of protein-small molecule complexes in native mass spectrometry assays does not necessarily correlate with the (more relevant) solution-phase affinity. In the gas phase, polar interactions such as hydrogen bonds and electrostatic interactions are strengthened, while the hydrophobic effect is weakened. Intriguingly, a window into gas-phase affinity could actually be an advantage for fragment-based approaches. Polar interactions tend to be enthapically driven, while hydrophobic interactions contribute to overall affinity primarily through entropic effects. If it is true that fragments showing predominantly enthalpic binding are more attractive starting points than those whose major binding energy comes from entropy (as argued here), mass spectrometry may be a good way of finding these fragments. I don’t recall seeing a systematic study dissecting the free energies of binding of hits from native mass spectrometry into their enthalpic and entropic components. If you know of one, I would be interested to hear about it in the comments section.

ACS Spring Meeting 2010

The spring national meeting of the American Chemical Society has just concluded in (uncharacteristically sunny) San Francisco. The main fragment event was a full day session organized by Rachelle Bienstock of the NIH. The theme was “Fragment based drug discovery: success stories due to novel computational methods applications.” Rachelle is planning on getting some of the speakers to write chapters for a book, so I won’t do more than give a very brief overview here.

The session was very multinational, with speakers from France, Germany, Russia, and the UK, in addition to the US, and a good mix of companies and academics. On the computational corporate side John MacCuish from Mesa Analytics described the molecular shape fingerprints approach, Carsten Detering of BioSolveIT provided several examples of applying his company’s methods for fragment linking and scaffold hopping, and Francois Delfaud of MEDIT described mining the pdb for protein-fragment interactions and applying this to Eg5 inhibitors. On the computational academic side, Tobias Lippert of the Center for Bioinformatics in Hamburg discussed the Qsearch program, Vladimer Poroikov of the Institute of Biomedical Chemistry in Moscow discussed PASS, which relies on a large training set to predict actives and inactives, and Dima Kozakov of Boston University presented the FTMap approach for predicting fragment-binding pockets in protein-protein interactions.

Moving away from the purely computational, Yongjin Xu of Novartis described the application of virtual fragment screening to identify p38 and BRaf inhibitors, Vicki Nienaber of Zenobia described iterative fragment screening to identify potent and selective LRRK2 inhibitors, and I presented Carmot’s Chemotype Evolution approach. Finally, GPCRs appear to be increasingly amenable to FBLD; Richard Law of Evotec presented a number of applications of computational methods to various programs including histamine receptors, while Miles Congreve of Heptares presented their StaR Technology for generating stabilized GPCRs suitable for SPR, NMR, and crystallography and discussed applications to the adenosine A2A receptor and the beta-1 adrenoreceptor. In the later case, the researchers were able to obtain 9 co-crystal structures and found that agonists and antagonists bound somewhat differently.

There were also a few other relevant posters and talks throughout the conference. For example, I learned that Locus Pharmaceuticals has transformed itself into Ansaris; Fouzia Machrouhi presented a poster on developing nanomolar inhibitors of the protein kinase AMPK.

Finally, Andrew Woodhead presented an update on Astex’s CDK2 program. One of the earliest posts on Practical Fragments described Astex’s fragment-based discovery of AT7519, which is in clinical trials for cancer. However, with an oral bioavailability of less than 1%, this compound is administered intravenously. Extensive medicinal chemistry ultimately revealed that a relatively minor change – capping the secondary amine with a methyl sulfonamide – led to a molecule with dramatically improved oral bioavailabilty. This molecule, AT9311, also retains good activity in mouse xenograft models. This is a useful reminder that fragment-based methods are not a replacement for solid (and inevitably subsequent) medicinal chemistry.

Updated: Fragment-based conferences in 2010

The year is already off to a good start, with one fragment event behind us and several more ahead. Here’s an update, starting with a major event next week.

March 21-25: The spring ACS meeting is being held in San Francisco. There will be a full day symposium on March 24, “Fragment Based Drug Design: Novel Approaches and Success Stories,” as well as a number of other relevant talks and posters scattered throughout.

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

April 27-28: Cambridge Healthtech Institute’s Fifth Annual Fragment-Based Drug Discovery will be held in San Diego. Two pre-conference short courses are also devoted to the topic on April 26, and since both Teddy and I will be participating stop by and tell us what you think of the blog!

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: Finally, registration and calls for abstracts have opened for FBLD 2010 in Philadelphia, PA. This is the third in a popular series of conferences that started with FBLD 2008 in San Diego and continued last year in York, UK. An emphasis this year will be on biophysical methods - old and new - for fragment identification and characterization, as well as sessions on libraries, chemical strategies for fragment evolution, and success stories. As far as we know this is the first major fragment event on the east coast of the US, so don't miss it!

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

Fragments in silico find new sites in crystals

Last year we highlighted a study in which virtual screening identified a number of functionally active fragments and crystallography confirmed their binding modes. In a recent issue of Bioorg. Med. Chem. Lett. researchers from Sanofi-aventis report a more complicated case: fragments that bind not only in a manner different than predicted, but in a completely different site.

The team used the computational docking method Glide to select 200 compounds likely to bind in the active site of the cytokine MIF (migration inhibitory factor). Of these, 23 were tested in crystallographic soaking studies, resulting in 5 co-crystal structures. Three of these bound in the active site, but the other two bound in a hydrophobic “cryptic” site on the protein surface formed by the rotation of a tyrosine residue. Protein rearrangements are not uncommon; a similar example was reported last year in which fragments were found to bind differently than predicted due to unforeseen protein movements. The cryptic site does appear to be real: the authors crystallized a compound reported in the patent literature and found that it binds across both the active and cryptic sites.

This is the third in a recent series of papers featured on this site in which fragment approaches found new binding sites on proteins. However, like the HIV-protease example, there is no functional data presented; I’ll take this to mean that the compounds are probably weak, if they show any detectable activity. The question of what to do with a fragment remains challenging, though (to be somewhat self-promoting) we are working on practical solutions.

What to do with a fragment is also a theme of the upcoming FBLD 2010, so if you have a success story you can share, consider submitting an abstract.

“The Hidden Pool” revisited

Last year, in response to a post by Teddy on whether there is a “hidden pool” of FBDD practitioners being trained in academia, guest blogger Derren Begley suggested that for the most part fragment-based approaches are restricted to industry: in universities “there are ‘puddles’ of FBDD here and there, but not what I would call a vast resource.” I think this statement was true at the time, but may now be changing. For example, Practical Fragments' last four posts have all covered papers that came out of academia.

There also seems to be an increasing trend of industrial scientists moving to academia, driven by factors ranging from the decreasing number of jobs in industry to the increased freedom in academia. These moves span the gamut, from world-class scientists leading entire departments to folks coming in as assistant professors, staff scientists, and research associates. But they are bringing their interest in fragments with them. In fact, of the last four blog posts mentioned, at least two involved people with current or former industry ties.

Finally, there seems to be increasing academic interest in fragments. I’ve given a couple talks in the past two months at Carnegie Mellon – University of Pittsburgh and St. Jude Children’s Research Hospital, and Peter Kenny has spent the past year as an itinerant fragment evangelist at universities around the world. I know that St. Jude in particular is actively seeking someone with an interest in FBDD, and with resources comparable to what you would find in big-pharma, they make a pretty appealing destination.

What are you seeing? Is FBDD going ivory?

HIV protease vs fragments

HIV protease (HIV PR) is a well-known and successfully exploited Achilles heel (OK, maybe more of an Alexandrian sword) of the virus HIV. Although there is no shortage of successful drugs on the market that target this enzyme, resistance is an issue, and new approaches are always welcome. To this end, researchers led by C. David Stout at Scripps Research Institute have performed a fragment screen against HIV protease, the results of which are reported in the March issue of Chemical Biology and Drug Design.

This is really a crystallography paper, and gives a thorough, nuts-and-bolts description of doing a crystallographic fragment screen. The authors screened a library of 384 commercially available fragments with an average molecular weight of only 142 Da. Some of this work was done at Active Sight, and although I believe Active Sight has closed, some of the folks have moved to Zenobia, so I suspect their fragment library incorporates some of the same features.

The researchers used five different crystal forms of HIV PR and examined a total of 808 crystals, 507 of which were co-crystallization experiments and 301 of which had the fragments soaked into crystals that had previously been grown. In all, 378 data sets were collected. Most of these were done in the presence of an active site inhibitor, thus specifically targeting the search for fragments that bind outside of the active site. Three fragments were identified binding to two different sites, and these data have been deposited in the protein data bank. The authors argue that these fragments could be binding to allosteric sites that might keep the protease in its “closed,” inhibited conformation.

Like the recent p53 example, there is still a long way to go: it is not even clear that these fragments have functional activity. Still, the discovery of these small-molecule binding sites illustrates that fragment methods can reveal something new even about an enzyme as well-characterized as HIV protease.

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.

Isothermal titration calorimetry (ITC)

Our last post covered SPR. While we’re on the topic of biophysical methods, we should touch on isothermal titration calorimetry (ITC). A Perspective in last month’s issue of Nature Reviews Drug Discovery gives a very readable and concise summary of the technique, along with its applications for fragment-based drug discovery.

In ITC, two samples are mixed together, and the change in heat is precisely measured. If one solution contains a protein and the other a small molecule, one can determine the enthalpy (deltaH) as well as the overall free energy (deltaG) of binding (and thus the affinity), entropy (deltaS), and stoichiometry. In their article, John Ladbury, Gerhard Klebe, and Ernesto Freire, all long-time proponents of the technique, describe the importance of enthalpically-driven versus entropically-driven protein-ligand interactions.

It turns out that compounds derived from medicinal chemistry efforts have a greater entropic component to their affinities than do natural ligands, which rely more heavily on enthalpy. This is because it is easier to improve entropy than enthalpy: enthalpy is dependent on the number and strength of non-covalent bonds between a protein and its ligand, and as anyone who has tried to engineer a specific hydrogen bond can attest, this is easier said than done. Entropy, on the other hand, can often be increased just by making a compound more hydrophobic. However, increasing hydrophobicity too much decreases solubility and can cause other problems. The authors suggest that, while it may be easier to improve entropy than enthalpy, focusing on the later parameter will lead to better drugs. In fact, for statins and HIV protease inhibitors, first-in-class compounds were largely entropically-driven, while best-in-class compounds have their affinities dominated by enthalpy. Just as natural ligands have evolved to rely more on enthalpy than entropy, drug developers are also selecting for enthalpically driven binders as they optimize other parameters. But this selection has been indirect, and the authors suggest that researchers should deliberately select for enthalpic binders.

The authors acknowledge that commercially available ITC instruments are not sufficiently high-throughput for primary screening, and also that fragment interactions are sometimes so weak that dissociation constants may not be measurable with the technology. Nevertheless, it is possible to measure enthalpy of binding even for fragments, and, as we noted last year, this can lead to superior molecules.

Despite its power, ITC does not seem to be used often in fragment campaigns: at a roundtable discussion at the recent Tri-Conference, not one of the dozen or so participants had direct experience with the method. I suspect this has to do both with the availability of instruments as well as perceived difficulties with the experiments. Hopefully this will change, but whether the technique will become as popular as SPR remains to be seen.

Surface Plasmon Resonance (SPR)

Fragment-based drug discovery took off with NMR in the 1990s and went mainstream with X-ray crystallography in the 2000s. Now surface plasmon resonance (SPR) is becoming increasingly popular as a primary means of identifying hits. The technique has been mentioned more than a dozen times on Practical Fragments, but we’ve never devoted an entire post to it until now.

This post follows up on two recent publications. The first is an excellent summary of SPR by our friends at FBDD-Lit. Peter Kenny gives an overview of the technique and reports on a workshop given by SPR mavens Dave Myszka and Rebecca Rich. He also covers some of the seminal papers in the field.

The second report is in the brand new journal ACS Medicinal Chemistry Letters. In it, Iva Navratilova and Andrew Hopkins of the University of Dundee provide practical advice on using SPR for fragment-screening.

The authors describe their work on using SPR to identify fragments that bind to carbonic anhydrase II, a popular target for proof-of-concept studies. They screened a library of 656 fragments with molecular weights between 94 to 341 Da, with an average of 187 Da, or about 13 non-hydrogen atoms. The entire screen, which was done at three concentrations (16.6, 50, and 150 micromolar) took 4 weeks from assay development to hit confirmation on a Biacore T100, and consumed a total of 27 micrograms of protein.

Importantly, Navratilova and Hopkins were keenly aware of the potential for false positives or nonspecific binders (of which there were 230 at the highest concentration!) One way they controlled for such artifacts was to include an unrelated reference protein; data could be corrected by subtracting the response to the reference protein from the response to the target protein. Another analytical method to reduce the number of false positives was to only consider compounds that exceeded a minimum threshold for ligand efficiency (a metric invented by Hopkins and co-workers), a decision justified here given the often high affinities observed for carbonic anhydrase inhibitors. After these filters, an examination of the stoichiometry of binding revealed a dozen specific binders and four non-specific binders, a hit rate of 1.8%.

My one reservation with this paper is that carbonic anhydrase is a particularly easy test case, unlikely to fairly represent many of targets that people screen. Indeed, the confirmed hits (all of which contain sulfonamides), have affinities from 0.13 to 14 micromolar – far better than a typical fragment screen, and comparable to many HTS screens. Still, the tools and analyses described should apply to more challenging targets.

Finally, it is worth noting that if you want access to SPR technology but don’t have the resources or expertise to do it yourself, at least a couple companies (Beactica and Graffinity) specialize in applying SPR to FBDD.

Molecular Medicine Tri-Conference 2010

The first event on our 2010 calendar, the Molecular Medicine Tri-Conference 2010, was held in San Francisco earlier this week. There were fragment talks and a roundtable, as well as a number of vendors selling fragment libraries – we’ve recently noted how rapidly this area has expanded.

Michael Hennig presented a nice overview of the history and development of fragment-screening at F. Hoffmann-La Roche (Basel). Work done there back in the late 1990s relied on NMR and crystallographic screening of a library of 300 fragments, described in the seminal “needle-screening” publication in J. Med. Chem. Today that library has grown to 6000 compounds following a relaxed rule-of-3 (allowing in particular more hydrogen-bond acceptors and higher lipophilicity) and requiring at least one hydrogen bond donor or acceptor and at least one ring. Also, the primary screening technique is now surface-plasmon resonance (SPR), with crystallographic follow-up; the entire collection can be screened on a single Biacore instrument in four weeks.

Hennig shared two case studies, one on BACE-1, the other on chymase. In the second case, a dozen fragments were successfully co-crystallized with the enzyme, and all but one of these bound in the S1 pocket, revealing the importance of this site for binding. In response to a question about how widely FBDD is used at Roche, Hennig said that it is applied to all targets that are technically feasible.

In another talk, James Madden described fragment-based discovery at Evotec. An increasingly stringent series of assays (from high-throughput high-concentration functional assays, through SPR and/or ligand-detected NMR, and finally crystallography and/or protein-detected NMR) helps keep the number of compounds manageable at each step. Madden also presented two cases studies, BACE-1 (clearly a popular target for FBDD, perhaps due to its intractability to many other approaches) and PDE10a.

A fun talk with relevance beyond FBDD was “Examples of X-ray Bloopers”, by Edward Kesicki of the Infectious Disease Research Institute (IDRI) in Seattle, WA. He described several cautionary tales from his own experience. In one case, a chemist provided the structure of the wrong enantiomer to a crystallographer, who duly refined the data, resulting in weeks of confusion and time-consuming follow-up experiments. In two others, crystallographers inadvertently omitted methylene units in fitting electron density. We’ve previously commented on the dangers of taking crystallographic data at face value, and Kesicki also mentioned an effort by Stephen Warren of Gonzaga University to comb through and correct structures in the protein data bank. He has a lot of work to do: of the 1000 structures examined thus far, roughly 20% have problems with the ligands.

Finally, in a panel discussion on “medicinal chemistry drivers,” someone asked about the role of fragment-based drug discovery. Consistent with the idea that fragment approaches are becoming increasingly integrated with other lead-finding activities, Hing Sham of Elan said that he was neither pro-fragment nor anti-fragment – “it’s just another tool in the toolbox.”

Commercial fragments – 2010 edition

A particularly active discussion on LinkedIn has prompted us to update the list of commercially available fragments - looks like a number of new additions since our last post. Note that we are restricting this list to suppliers specifically offering fragments, as opposed to general compound libraries (which of course will likely contain many fragments, but will make this list too unwieldy, and are probably less of a go-to source for people just entering the field).

Asinex
4500 Rule of 3 (RO3) compounds
1800 compounds MW < 250, solubility > 0.1 mM (PBS) and > 10 mM (DMSO)

ChemBridge Corporation
~5000 RO3 fragments

Edelris
~1900 fragments, expanding to "3-D" fragments

Enamine
1190 fragments w. strict Rule of 3
11,717 fragment extension set

InFarmatik
140 “3-D” fragments
198 diverse fragments

Iota Pharmaceuticals
Focused on fragment-based discovery
1500 fragments available for purchase
4000 additional fragments in collaboration with Vitas-M

Key Organics
6335 RO3 fragments

Life Chemicals
22,000 fragments w. MW < 300, clogP < 3
9000 fragments with rotatable bond, PSA, HBA limits

Maybridge (Thermo Fisher Scientific)
30,000 fragment library (MW < 350)
1000 RO3 fragments w. aqueous solubility > 1mM
1500 Br- and 5300 F- containing fragments

Otava
~3800 fragments, mostly RO3 (increased HB acceptors for kinases)
All have at least one ring; filtered to remove certain functionalities

Prestwick Chemical
720 fragments including known drugs, RO3 compliant

Pyxis (with Merachem)
317 RO3 fragments from drugs and natural products
Filtered to remove certain functionalities

Zenobia Therapeutics
352 very small fragments (Avg. MW 155)
Verified solubility at 200 mM in DMSO

I’ve also been told that BioFocus sells fragments, but can’t find this on their web-site.

Finally, Cambridge MedChem Consulting has a nice list with more detailed descriptions of many of these suppliers.

Are there companies we’re missing? Does anyone have any experience with any of these that you would like to share?

Pinning fragments on Pin1

Pin1, a potential anti-cancer target, catalyzes isomerization around phosphoserine-proline and phosphothreonine-proline bonds. Its binding site is relatively shallow, complicating efforts to discover small, non-peptidic inhibitors. In a recent paper in Bioorg. Med. Chem. Lett., Jonathan Moore and colleagues at Vernalis describe their fragment-based approach to tackling this problem.

The researchers used NMR-screening of roughly 1200 fragments to identify five that competed known inhibitors; Compound 4 (see figure) was the most potent, with an IC50 of 16 micromolar in a functional assay. NMR experiments showed a weaker binding interaction, on the order of 200 micromolar, and surface-plasmon resonance (SPR) experiments were even less conclusive: the compound showed super-stoichiometric binding, indicating that multiple molecules were interacting with the enzyme rather than sitting specifically in the binding site. However, the researchers were able to obtain a crystal structure showing that the molecule binds in a hydrophobic pocket at the active site. Although they don’t mention it in this paper, in public presentations the researchers have reported seeing additional molecules of Compound 4 pile on top of each other, essentially forming a stack on top of the protein. Perhaps, as the authors suggest in the supplementary material, this is an example of a particularly insidious aggregation phenomenon: a legitimate hit that can also form aggregates.


In order to access other parts of the protein, and reduce the propensity for aggregation, the researchers relied on analog screening and modeling to generate Compound 18a, which is a more “three-dimensional” molecule. Further elaboration led to a series of compounds such as 19e, with low nanomolar potency, as well as one-to-one binding in the SPR assay.

Despite their potency, these molecules were inactive in cell-based assays, likely due to high polar surface areas and their resulting low cell permeabilities. To fix this, the researchers replaced the benzimidazole fragment with a naphthyl group, which led to a decrease in biochemical potency but did lead to cell-active molecules such as Compound 23b. Moreover, a crystal structure revealed that this molecule binds in a similar manner to the original fragment 4.

This paper exemplifies another example of fragment-assisted lead discovery: the original fragment morphed from an indole to a benzimidazole to a napthyl group, yet the final molecule still owes a debt to the initial fragment.

There and back again: fragments and BACE-1

One of the many interesting talks at FBLD 2009 was by Daniel Wyss of Schering-Plough on how fragment-based screening was used to discover potent and selective inhibitors of the Alzheimer’s disease target BACE-1. Two papers published online in J. Med. Chem. now begin to tell the full story.

BACE-1 is an aspartyl protease, a class of enzymes that has proven to be druggable, as illustrated by the number of HIV-1 protease inhibitors on the market. However, BACE-1 has an unusually shallow, flexible, and hydrophilic active site, and its location in the brain means that candidate drugs need to be particularly small with a limited number of hydrogen bond donors.

The first paper discusses how Wang and colleagues used 15N-HSQC NMR screening of a library of ~10,000 compounds, about half of them fragments, against the BACE-1 catalytic domain. This resulted in 9 distinct classes of hits, some of which were as potent as 30 micromolar as judged by NMR-based dissociation constants. The compound most extensively pursued was compound 2 (see figure), an isothiourea. Some 200 analogs of this were present in the corporate collection (an advantage of working in big pharma!), and 15 of these showed activity in an enzymatic assay, of which compound 3 was the most potent. Extensive NMR analysis and an X-ray crystal structure revealed that the isothiourea makes hydrogen-bond contacts to both catalytic aspartates and extends towards the S1 pocket and S3 subpocket (S3sp).


Isothioureas are potentially toxic and unstable, so structure-based design was applied to replace this moiety. One outcome was a series of 2-aminopyridines such as compound 4. Unfortunately, although they showed measurable binding by NMR and some could even be characterized crystallographically, most had little or no activity in a BACE-1 functional assay.

That’s where the second paper by Zhu and colleagues comes in. Careful analysis of the crystal structure of compound 3 combined with parallel synthesis led to a series of iminohydantoins (or cyclic acylguanidines) such as compound 23. Interestingly, a close analog of this compound made similar contacts but flipped to another orientation. These different binding modes complicated medicinal chemistry efforts, requiring parallel chemistry and NMR-based screening (as most early compounds were at best only weakly active). Ultimately this effort yielded potent inhibitors such as compound 39, with nanomolar biochemical activity. However, the compound also has a clogP of 7.5, a molecular weight of more than 500 Da, and only modest bioavailability, so turning this into a brain-active drug could be problematic.

Strikingly, truncating a large portion of the molecule (to generate compound 40) yielded a much smaller compound that, despite its reduced biochemical potency, had an improved ligand efficiency, as well as measurable brain penetration. Further simplification led to compound 41, which, being rule-of-three compliant, can be considered a fragment. In other words, these two papers report the optimization of a low-affinity fragment to a high-affinity ligand and on to a medium affinity fragment. Given that the second paper is subtitled “Part 1”, we can look forward to reading further chapters.

Many FBLD publications report the rapid discovery of new leads against established targets such as kinases or Hsp90. BACE-1 is just the opposite: references suggest that the program had been in place since before 2004. These papers, along with previous papers (for example here and here) on BACE-1 from AstraZeneca and Astex, illustrate that FBLD is also powerful for discovering leads against difficult targets.

Fragments in the Clinic: DG-051

Last August we highlighted work from deCODE on their leukotriene A4 hydrolase (LTA4H) program. That paper described the construction of a fragment library based on naturally occurring compounds, crystallographic screening against LTA4H, and optimization of inhibitors for this cardiovascular disease target. In a new paper published in J. Med. Chem., the researchers provide a fuller description of the discovery of the resulting clinical compound, DG-051.

As noted in the previous paper, crystallographic screening of deCODE’s fragment library identified several hydrophobic hits such as Compound 6 (see figure). At the same time, the researchers were aware of research from Searle that had produced inhibitors such as Compound 5. Appending the pyrrolidine of this compound onto deCODE’s fragment led to a modest increase in potency (Compound 9), though the resultant compound was still orders of magnitude weaker than Compound 5. Crystallography suggested a couple bad interactions in Compound 9 compared to Compound 5, so the researchers modified Compound 5 to generate Compound 14, which was active in a whole blood assay but suffered from rapid metabolism. Replacing the central methylene with an oxygen and adding a chlorine (Compound 17) improved biochemical potency slightly while dramatically improving pharmacokinetics.



Several crystal structures of LTA4H showed an acetate ion bound to the catalytic zinc, and the researchers sought to combine this “fragment” with their existing series, generating clinical compound DG-051. This did not lead to an improvement in biochemical potency (and actually decreased ligand efficiency), but it did lead to a roughly ten-fold improvement in potency in the whole-blood assay, as well as improvements in solubility and DMPK parameters. Interestingly, both enantiomers were equipotent, and the S-enantiomer was ultimately chosen due to ease of synthesis.

This story could be seen as an example of what has been called “fragment-assisted drug discovery:” unlike AT9283 or Indeglitazar, the fragments identified (acetate aside) didn’t end up in the clinical compound, and it could be argued that the initial lead was taken from the literature. But information gleaned studying the fragments fed into the design of a molecule that was sufficiently active, stable, selective, and novel for development.

The article states that DG-051 entered phase 2 clinical trials “for the prevention of myocardial infarction and stroke”, although no reports of development appear in clinicaltrials.gov. Also, in what has been an all-too-common event over the past year, deCODE filed for Chapter 11 bankruptcy and announced that it planned to sell “substantially all of its assets.” Practical Fragments wishes the best of luck to all the folks there. Happily the structural biology and fragment-screening group has (re)gained independence as Emerald BioStructures.