31 December 2009
Half the papers cover various forms of fragment screening: the Medical Structural Genomics of Pathogenic Protozoa Consortium describes their crystallographic approach, Helena Danielson discusses the use of SPR, and Gregg Siegal and Johan Hollander present their Target Immobilized NMR Screening (TINS) methodology.
Charles Reynolds and colleagues review metrics, such as ligand efficiency and fit quality, for evaluating fragment hits.
Finally, in the longest article in the collection, Vicki Nienaber discusses how fragment-based methods may be particularly useful for discovering compounds that will cross the blood-brain-barrier to target the central nervous system.
As 2009 comes to a close, Practical Fragments would like to thank our readers, old and new. May you all have a happy and productive 2010!
21 December 2009
February 3-5: CHI’s Molecular Medicine Tri-Conference will be held in my beautiful city of San Francisco, with a program on medicinal chemistry that includes a fragment track, 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.”
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. And if you missed the short course in San Francisco, you have another chance on April 26.
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, standing alone in the second half of the year, Fragment-based Lead Discovery 2010 will be held in Philadelphia, PA. This is the third in a popular series of conferences that started with FBLD 2008 in San Diego and continued this year in York, UK. An emphasis next 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. A web site will be available shortly where further details will be posted. We anticipate a call for oral and poster contributions during the Spring of 2010. 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.
15 December 2009
Simocyclinone D8 (SD8) is a dumbbell shaped molecule isolated several years ago from that ultimate micro-pharma, Streptomyces. Although SD8 blocks the action of bacterial DNA gyrase, which is also the target of fluoroquinolones such as ciprofloxacin and aminocoumarins such as novobiocin, it is mechanistically distinct from these older antibiotics. To understand why, Anthony Maxwell and colleagues at the John Innes Centre in Norwich, UK, solved the co-crystal structure of SD8 bound to GyrA. The structure reveals that the protein forms a dimer of dimers, with four molecules of SD8 bound to the four subunits of GyrA. Weirdly, each molecule of SD8 cross-links two separate subunits of GyrA, although mass-spectrometry, analytical ultracentrifugation, and modeling suggest that a single molecule of SD8 could also bind to a single GyrA subunit. The crystal structure shows that SD8 binds near – but not at – the fluoroquinolone binding site, blocking the DNA-binding portion of GyrA.
What caught my eye is the fact that both halves of the molecule are active by themselves, albeit with a loss in potency (see figure). The linker is over one nanometer long and doesn’t appear to make significant interactions with the protein; it would be fun to know how something like this evolved.
Antibiotics gleefully seem to ignore the Rule of 5, but it wouldn’t hurt to get a smaller, less complicated analog. Replacing either of the two ends with smaller fragments may be a productive approach, as would optimizing the individual “fragments” themselves.
12 December 2009
Together with Warren’s family, Axel and Jim are also organizing a commemorative fund:
The Warren L. DeLano Memorial Award for Computational Biosciences
This award shall be given to a top computational bioscientist in recognition of the contributions made by Warren L. DeLano to creating powerful visualization tools for three dimensional structures and making them freely accessible. The award, accompanying lecture, and honorium will be given annually in the context of a national bioscience meeting or a Bay Area gathering of computational bioscientists at Stanford, UCSF or UC Berkeley. For the award special emphasis will be given for Open Source developments and service to the bioscience community.
For the award selection, a committee will be formed consisting of experts in the computational and biological sciences. Submission for nominations will be open to everybody.
Tax deductible donations can be made by check to the address below or by PayPal.
Silicon Valley Community Foundation
memo: Warren L. DeLano Memorial Fund
2440 West El Camino Real, Suite 300
Mountain View, CA 94040
To endow this in perpetuity would require about $100,000, and the fund is off to a good start, with $23,000 contributed and another $30,000 pledged so far.
03 December 2009
The researchers used human carbonic anhydrase (hCA II), a venerable work-horse of biophysical studies. Benzenesulfonamide (compound 1, below) is a known binder, and the researchers studied the thermodynamics of 20 derivatives of this molecule using isothermal titration calorimetry (ITC), taking care to generate high-quality binding data. Adding a fluorine group to the 2-position of benzenesulfonamide (compound 2) improves the potency almost three-fold but lowers the ligand efficiency. In contrast, adding a fluorine to the 3-position (compound 3) improves the potency by seven-fold and also improves the ligand efficiency.
If you were choosing between these fragments solely on the basis of affinity or ligand efficiency, it would be reasonable to choose compound 3, and in fact a search of the literature turned up 15 carbonic anhydrase inhibitors that contained the 3-fluorobenzenesulfonamide substructure and none that contained the 2-flurobenzenesulfonamide substructure. However, a look at the thermodynamic parameters reveals that the affinity of compound 2 is driven by a sizable improvement in enthalpic binding, partially offset by lowered entropy. In contrast, compound 3 has a similar enthalpy of binding as compound 1 but increased entropy. What’s going on?
The researchers determined high-resolution crystal structures for all three of these molecules bound to hCA II. Interestingly, the structure of compound 2 shows a specific interaction between the fluorine atom and a main-chain NH of the protein. In compound 3, the fluorine points towards the hydrophobic wall of the protein.
Adding a 4-benzylamide substituent onto each of these molecules led to improvements in activity. However, this was a relatively modest boost for the more entropic compound 3 to compound 20, but considerably larger for the enthaplically-driven compound 2 to compound 19. Compound 19 shows a highly favorable binding enthalpy, and is the most potent and ligand-efficient of any of the three elaborated molecules.
Obtaining thermodynamic parameters for small-molecule protein interactions has historically been challenging, but in recent years miniaturization and improvements in technology have brought ITC into more non-specialist labs. If you have the resources, it may be worthwhile characterizing the thermodynamic profiles of your fragment hits, and – perhaps – looking more closely at those that show enthalpically-driven binding.
22 November 2009
The first paper, published by Timothy Ritchie and Simon Macdonald of GlaxoSmithKline in this month’s Drug Discovery Today, correlates the number of aromatic rings with several metrics associated with success in drug development. For this analysis, each ring in a fused system is counted separately, so indole is counted as having two aromatic rings. The researchers conclude that more than three aromatic rings correlates with an increased risk of compound attrition during drug development:
The fewer the number of aromatic rings contained in an oral drug candidate, the more developable that candidate is likely to be.This is not surprising, but with their access to a vast internal data set the researchers provide considerable supporting evidence. For example, the mean aromatic ring count declines from 3.3 to 2.3 as GSK compounds move from preclinical candidate selection to proof-of-concept in humans. Measured (kinetic) solubility decreases dramatically with increasing ring count: even two aromatic rings leads to many low solubility compounds, and with four aromatic rings the median solubility is only 0.012 mg/ml. Both c log P and log D increase with increasing ring count, as do serum albumin binding, P450 3A4 inhibition, and hERG inhibition – all factors one usually wants to decrease in drug development.
One caveat is that the authors do not control for size. As aromatic rings are added, molecular weight is likely to increase, and thus many of the properties could simply reflect the pharmaceutical liabilities of larger molecules. This is where the second paper comes in. In J. Med. Chem., Frank Lovering and colleagues at Pfizer (nee Wyeth) analyze the effect of aromaticity itself by defining a simple metric:
Fsp3 = number of sp3 hybridized carbons / total carbon count
The smaller the number, the more aromatic the compound; the larger the number, the less aromatic. Besides being a straightforward measure of saturation, the formula inherently controls for molecular size.
When the researchers examined published data sets, they found that the mean Fsp3 increases from 0.36 for 2.2 million molecules in discovery to 0.47 for 1179 approved drugs. They also investigated measured solubility and found a strong correlation: 104 molecules with a log S of -6 (quite insoluble) had an average Fsp3 of 0.31, while 194 molecules with a log S of 0 (very soluble) had an average Fsp3 of 0.56. The effect is even more striking with melting points, which negatively correlate with solubility: 1153 molecules with a melting point of 125 deg. C had an average Fsp3 of 0.31, while 375 molecules with a melting point of 275 deg. C had an average Fsp3 of 0.18.
OK, so let’s say we accept the premise that increasing aromatic character in a molecule leads to lower solubility and worse properties overall. The easiest solution might be to reduce the number of aromatics in a screening collection, but would this really be wise? Ritchie and MacDonald note that aromatics, with their rigid structures, are likely to have increased potency relative to unsaturated molecules. And particularly for fragment libraries, you want all the binding energy you can get.
An interesting study would be to correlate the hit rate for fragments with their aromatic character. Does the hit rate increase with decreasing Fsp3? These data must exist in companies that have been doing FBDD for years. Indeed, at FBLD 2009, Ijen Chen of Vernalis presented a nice analysis of hits against 12 targets, in which she noted that roughly 2/3 of the fragment library members didn’t hit any of the targets. I don’t think she mentioned aromaticity specifically, but she did note that the hits tended to be slightly more rigid and hydrophobic than the non-hits – just what you would expect for low-Fsp3 molecules.
So by all means avoid having too many aromatics, but don’t go to extremes: it’s finding the right balance of binding energy and pharmaceutical properties that makes drug discovery such a tricky business.
16 November 2009
As I read the paper, I wondered why fluorine-labeled fragments are not used more widely; Dalvit’s group published another paper about this approach recently in JACS. Fluorine has a strong NMR signal and is very sensitive to the local environment, so when a fluorine-containing fragment binds to a protein this can be easily detected. In fact, the dynamic range for this type of assay is so great that fragment binding can be detected at concentrations several orders of magnitude lower than their dissociation binding constants.
This seems like a very powerful approach, but I haven’t seen many other people using it. Are folks concerned about the need for fluorine in every fragment (although many are commercially available) or is there something else I’m missing?
12 November 2009
In the first, Australia’s Biota has agreed to acquire UK-based Prolysis Limited, which previously published some nice work on using FBLD to discover new antibiotic leads (see here and here). The price? Just $10.8 million. However, Biota did say it plans to invest up to $25 million over the next three years on programs Prolysis started.
At the same time, UK-based Astex Therapeutics announced a new partnership with GlaxoSmithKline. The deal is for multiple targets in multiple therapeutic areas, with Astex focused on fragment screening and lead discovery and GSK focused on optimization of the resulting leads as well as preclinical and clinical development. The price? $33 million in up-front cash and equity, with a total potential of more than $500 million (BioBucks).
Astex of course is one of the few intact survivors of the first wave of fragment-based companies and has put several compounds into the clinic, including AT9283, AT7519, and others. It’s encouraging to see that deals of this size are still being done for what look to be fairly early stage collaborations.
06 November 2009
Many of you have used PyMOL for visualizing crystallographic and NMR structures. Even if you haven’t used the program directly, hardly a week goes by without papers appearing in Science, Nature, and other high-profile journals adorned with beautiful illustrations created with his software. Every protein-fragment structure I’ve looked at in detail has been through the lens of PyMOL.
Warren and I overlapped in Jim Wells’ group at Genentech, and Warren was one of the first people to join Sunesis, where he stayed until PyMOL became so successful that he left to devote himself to it full time. He was a brilliant programmer, a gifted scientist, and a valued friend.
Warren’s insights and creations illuminated macromolecular structures with the clarity of science and the grace of art. The world is darker without him.
28 October 2009
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.
22 October 2009
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
15 October 2009
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.
07 October 2009
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.
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.
04 October 2009
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.
27 September 2009
On the broad topic of why FBLD is useful, an interesting shift in emphasis seems to have occurred. A few years ago a key argument in favor of fragments was getting compounds to the clinic faster, but there is now a greater focus on quality over speed. In summarizing over a decade of fragment work at Abbott, Phil Hajduk noted that FBLD hits consistently bind more efficiently than those from HTS. Similarly, Chris Murray of Astex noted that, among their five clinical candidates (four of which target kinases), the average ClogP was 1.7 (vs 4.1 for a set of 45 reported orally active kinase inhibitors), while the average molecular weight was 390 (vs 457).
One theme that differentiated this meeting from others was a strong focus on modeling: an entire day was devoted to sessions on “fragments, scoring functions and docking” and “design from fragments.” This concluded with a lively round table discussion, chaired by Vernalis’ James Davidson, titled “Chemistry challenging modeling.” But challenges didn’t only come from chemists: one prominent modeler noted that there have been no fundamentally new approaches to modeling in the past two decades; another asked why, despite the number of interesting new chemistries out there, so many modelers restrict themselves to the same old standbys such as amide bonds.
Part of the problem with modeling, of course, is separating hits from noise: true hits often show up near – but not at – the top of a ranked list, so how does one decide what is worth pursuing? Phil Hajduk discussed the use of “Belief Theory”, in which the similarity of an unknown molecule to a known active is used to evaluate the unknown.
Another problem is the quality of primary data: As Hajduk noted, “no one takes experimental error into account” when predicting ligand binding, and a recent analysis suggests that over-fitting data is a substantial problem with many computational approaches. This is all the more problematic when the data are not just noisy but spurious; Practical Fragments has noted the problem of aggregation, and UCSF’s Brian Shoichet emphasized this point, noting that 85-95% of hits from a high-throughput screen could be artifacts, while 85-100% of what remains could also be bogus. He did note, though, that fragments are less problematic in this regard than larger molecules. And Genentech’s Tony Giannetti, Vernalis’ James Murray, and others illustrated how surface plasmon resonance is effective at weeding out bad actors.
Getting better data will clearly be essential to getting better models, but one essential category, the forces involved in protein-small molecule interactions, is still poorly understood. Gerhard Klebe of the University of Marburg presented a detailed and elegant set of experiments exploring the effects of chemical structure on the enthalpy and entropy of binding to the protein thrombin. He emphasized that desolvation of fragments from water is critical, and only possible if compensated by strong interactions with the protein. This also implies that you want fragments that have low desolvation penalties as well as high solubilities – a tricky balancing act.
FBLD 2009 was held barely six months after Fragments 2009, and it is a testament to the vibrancy of the field that both conferences managed to be so successful and exciting while sharing very few speakers in common.
For the other two hundred plus attendees at the conference, what were some of your impressions?
23 September 2009
Don Huddler from GSK will be talking about SPR in fragment screening.
Bill Metzler from BMS will be talking about the uses of biophysical methods and structural information for hit prioritization.
I will be talking about how to put together an integrated FBDD paradigm.
There is one more talk of the TBD variety, but I think it will be a very nice complement to these other three.
Please come out and see what the state of the art is.
17 September 2009
deCODE Chemistry and Biostructures
Structure Based Design
I’m sure there are plenty of omissions; put them in the comments and we’ll add them in the next update.
10 September 2009
This is also the second fragment deal that GSK has done in the past month; we already noted their collaboration with Vernalis.
What do you think? Does this flurry of new deals signify increasing use of FBDD?
09 September 2009
But don’t be put off by the name of the journal: with 14 articles covering close to 200 pages, there is something here for almost everyone, even for those whose interest in computers ends at using them to read this blog! A brief editorial outlines the challenges of FBDD, and a longer introductory piece gives an overview of the field. Several articles focus largely on specific targets such as p38alpha, heparanase, and Eg5, while one is devoted to assessing druggability.
Finally, two articles address the important topic of designing fragment libraries, one from the perspective of big pharma (nicely summarized here), the other from biotech.
07 September 2009
Both papers focus on the major histocompatibility complex (MHC) Class I proteins. These proteins bind 8-11 amino acid intracellular peptides and present them on the cell surface, allowing passing T cells to survey the contents of cells for viruses, bacteria, or other nasties and, when appropriate, eliminate the infected cells. As might be expected given their function, the MHC proteins are quite promiscuous in which peptides they bind to, frustrating a general understanding of the molecular recognition. Moreover, crystallography is complicated by the fact that MHC class I proteins do not crystallize in the absence of a bound ligand.
In the first paper, Anastassis Perrakis, Ton Schumacher, and colleagues at the Netherlands Cancer Institute designed a 9-amino acid "conditional" peptide ligand for MHC that contains two internal photosensitive nitrophenyl substituents. They were able to crystallize this in complex with MHC and solve the structure. When they exposed these crystals to UV-light, the nitrophenyl groups caused the peptide to break apart into into three pieces. Interestingly, structural characterization after this exposure revealed that while the central portion of the peptide was gone, the two end bits were still bound to MHC. However, the researchers were able to successfully replace these remnants with new, full length peptides derived from HIV and avian flu proteins by soaking the crystals for just a few hours in buffer containing the new peptides. The resulting structures were identical with previously determined structures, even revealing some side-chain movement. A second paper from Ton Schumacher, Huib Ovaa, and colleagues reports a similar strategy, this time using diol-containing peptides and mild chemical cleavage with sodium periodate rather than UV-light, although in this case the reaction is done in solution rather than in crystals.
This seems like an interesting approach for tackling peptide-binding proteins, and possibly even small-molecule binding proteins, though this would require more effort to design destructible ligands.
29 August 2009
The researchers were looking for noncovalent inhibitors of cruzain, a popular protease target for Chagas’ disease. After a virtual screen of commercial lead-like compounds, 17 molecules were purchased and tested in enzymatic assays, and compound 1 (below) inhibited cruzain, albeit weakly. However, the compound looked like the real deal: it showed no time-dependence; it was active in the presence of detergent; and Lineweaver-Burk plots revealed that it was mechanistically competitive.
The researchers thus turned to medicinal chemistry, replacing the ester group of compound 1 with an oxadiazole bioisostere and swapping the aryl group for a substituted pyrazole, ultimately arriving at molecules such as compound 21, more than two orders of magnitude more potent than the starting molecule.
So far, so standard: similar stories appear every week in J. Med. Chem., Bioorg. Med. Chem. Lett., ChemMedChem, and other journals, and it would not have been surprising to see this published with a title like “Discovery of a high affinity inhibitor of cruzain.” Only in this case, the researchers became suspicious: most of the molecules were not active against the targeted protozoa, and many of the dose-response curves had unusually steep Hill slopes, a tell-tale sign of aggregation. Looking more closely at their protocol, the researchers also realized that the concentration of non-ionic detergent in their assays was ten-fold lower than they had thought. D’oh!
A series of tests confirmed that, despite interpretable and rationalizable SAR, the series had been optimized for aggregation-based inhibition: compound 21, with an IC50 of 200 nM in buffer containing 0.001% of the detergent Triton X-100, showed no inhibition whatsoever in 0.01% Triton X-100. The compound also inhibited AmpC beta-lactamase, an enzyme particularly sensitive to aggregators, and this inhibition could be reversed with detergent. Finally, dynamic light scattering (DLS) revealed the presence of particles (or aggregates) in aqueous solutions of compound 21.
But the tale gets even more twisted. Some of the aggregators show legitimate, competitive binding to cruzain under high-detergent conditions, albeit at much higher concentrations (with IC50s above 40 micromolar). Conversely, compound 1 actually shows noncompetitive behavior in low-detergent conditions, though again only at fairly high concentrations. In other words, promiscuous inhibitors can behave legitimately under sufficiently stringent conditions, and legitimate inhibitors can behave promiscuously under less stringent conditions.
What’s especially sobering is how easy this promiscuity would have been to overlook: many molecules with good activity in biochemical assays don’t show any effects in cells, and it is easy to ignore steep slopes in inhibition assays. How many of those “Discovery of a high affinity inhibitor of Hot Target X” papers actually report promiscuous inhibitors? The authors, who have been researching this problem for a long time, end on a justifiably paranoid note:
The cautionary contribution of this study is to point out that even within a clear SAR series, one is never entirely free from the concern that non-stoichiometric, artifactual mechanisms are contributing to the inhibition one observes.This is a serious problem, both for the researchers doing the original work and for anyone trying to follow up on the results. But one can take precautions, summarized below and described more fully here:
- Add non-ionic detergent to the assay (Triton-X 100, Tween-20, CHAPS, others)
- Increase protein concentration – this should have no effect on genuine binders (within limits)
- Characterize the mechanism of inhibition (competitive, noncompetitive, or uncompetitive): competitive inhibitors are normally not promiscuous
- Centrifuge your samples and retest them – this can sometimes remove aggregators
- Examine your samples with DLS or flow cytometry – aggregators can sometimes be directly observed as 50-1000 nm particles
- Look closely at your dose-response curve - unusually steep slopes can signal aggregation
And of course, biophysical methods such as SPR, NMR, and X-ray crystallography can provide more information than biochemical assays and reveal stoichiometric (and – in the case of SPR – superstoichiometric) binding.
Difficulty sorting true low-affinity binders from false positives stymied fragment-based approaches for decades, and in fact the nature of promiscuous inhibition caused by aggregation wasn’t even characterized until earlier this century. We now have techniques to sort deceptive aggregation from true but faint affinity. Let’s make sure these tools are consistently used.
23 August 2009
Put simply, DCC generates new molecules with some desired property by allowing smaller molecules to assemble reversibly under selection pressure. If the selection pressure is binding to a protein target and the molecules undergoing reactions are fragments, DCC can be used for FBDD. As we previously noted, Huc and Lehn published one of the earliest demonstrations of this. DCC has also been used at a few companies, including Astex and Sunesis, and even formed the basis of the (sadly) short-lived Therascope.
Rademann’s approach, "dynamic ligation screening", is based on labeling one fragment with a fluorescent probe and then screening it in a fluorescence polarization assay with other test fragments. If the labeled fragment binds competitively with a test fragment, this implies that the two fragments bind to the same site. However, if the fluorescence polarization signal increases in the presence of the test fragment (indicating increased binding of the labeled fragment), this suggests that the test fragment and the labeled fragment are binding cooperatively.
The researchers applied dynamic ligation screening to the protease caspase-3, a key mediator of apoptosis relevant for many diseases. As their labeled “fragment,” they chose a high-affinity tetrapeptide containing an alpha-ketoaldehyde: the ketone interacts covalently with the catalytic cysteine of the enzyme, while the aldehyde can form imines with amine-containing fragments. Interestingly, this strategy selects for fragments that bind in the S1’ subsite of the enzyme, which has not received as much attention as the tetrapeptide binding sites S1-S4.
A fluorescently labeled version of the tetrapeptide was screened against a library of 7,397 fragments, of which 4,019 contained primary amines. Of these, 78 fragments caused a decrease in the fluorescence polarization signal, suggesting that they compete with the tetrapeptide for binding. These were tested in an enzymatic assay: 21 of them were active at 10 micromolar concentrations, and four had Ki values from 3.1 to 5.5 micromolar; these four molecules have electrophilic carbons, making it likely that they bind to the catalytic cysteine residue.
Of greater interest, 176 fragments were cooperative, increasing the fluorescence polarization (FP) of the labeled tetrapeptide fragment by at least 20%. 50 of these were tested in an enzymatic assay, with the amine shown below emerging as the most potent FP enhancer and a Ki of 120 micromolar alone. A series of experiments guided by mathematical modeling suggested that the protein was templating the formation of an imine bond between the aldehyde of the tetrapeptide and the amine. Moreover, the reduced (amine) version of this conjugate exhibits a very high affinity for caspase-3, with a Ki of 80 picomolar.
Of course, affinity is not everything: with a molecular weight of 767 Da and a clearly peptidic nature, the pharmaceutical properties of this molecule, and even its cell activity, are questionable.
This study is reminiscent of some work we did at Sunesis, using caspase-3 to template the assembly of a non-peptidic inhibitor using Tethering. In that case we built molecules in the S1-S4 pockets, but did not do much work to extend into the S1’ pocket. It would be interesting to see if the fragment Rademann and colleagues discovered also boosts the potency of the molecules we identified.
For dynamic ligation screening to be general it needs to surmount at least two major potential limitations. First, it remains to be seen whether the technique will work with actual fragments, which are likely to have far lower affinities than the 25 nM tetrapeptide used in this study. Second, cooperative binding of the fragments does not translate to synergy in the final molecule: the conjugate has a lower ligand efficiency than either of the fragments, despite the apparent cooperativity of the two fragments binding to the target. This could be because the conjugate contains an amine, whereas the two fragments in solution presumably were linked by an imine; the differences in geometry and chemical nature between these two moieties are profound, and one could imagine that many amine-linked compounds would not be selected as imines, and vice versa.
Still, this is an interesting approach to tackle the long-standing challenge of linking fragments, and it will be fun to watch for new developments.
13 August 2009
The “fragments of life” (FOL) screening library consists of three sets of molecules:
- 218 “molecules of life,” which are known metabolites from some living organism
- 666 synthetic derivatives and isosteres of known metabolites
- 445 synthetic biaryl molecules, which mimic peptide turns (biaryls have also previously been reported to be privileged pharmacophores)
The fragment library was tested against Leukotriene A4 Hydrolase (LTA4H), an enzyme with two functions: it has an aminopeptidase activity whose biological relevance is unknown, and an epoxide hydrolase activity that converts leukotriene A4 to the inflammatory leukotriene B4, which is implicated in heart disease and inflammation. Both activities map to a single active-site, a long cleft containing a catalytic zinc.
About 200 of these fragments were screened by soaking crystals of LTA4H in pools containing 8 compounds. Although all compounds in a given pool were structurally diverse, in some cases electron density was ambiguous, necessitating subsequent soaks of individual fragments to confirm hits. Ultimately 13 fragments were found to bind LTA4H, a hit rate of 6%. These fragments were tested in functional assays and found to have IC50s as good as 178 nM for bestatin, though the next best was mid-micromolar. Most of the fragments bound in the active site, although one fragment bound on the surface of the enzyme. Considerable structural data are presented in the paper, and all the structures have been deposited in the protein data bank.
Interestingly, the researchers also found that some of the fragments only appeared to bind when they were soaked in the presence of another fragment, bestatin. Bestatin also caused the binding mode of another fragment to shift compared to its binding mode without bestatin.
Based on the crystal structures available, some of the fragments were elaborated to provide more potent inhibitors, increasing affinity by some four orders of magnitude, as well as improving ligand efficiency (see figure). Crystallography revealed that these more potent compounds bind in a similar fashion to the fragments.
Compounds 14 and 18 also bind in a similar manner to DG-051, which has recently completed phase IIa clinical trials. There is apparently another manuscript in the works focused exclusively on this molecule. We look forward to reading the full story.
11 August 2009
The fragment effort started with a library of fragments grouped into pools of 10-12 each and screened using three different NMR methods (saturation transfer difference, water-LOGSY, and T2 relaxation filtered 1D). Compounds were tested in the presence and absence of PU3, a compound known to bind to the ATP site of Hsp90. Of the 1351 fragments tested, 59 of them (4.4%) were confirmed in all three NMR experiments and competed with PU3. Interestingly, a further 158 compounds were found to bind to Hsp90 but could not be displaced by PU3, suggesting that they bind outside of the ATP binding site; these were not pursued.
Two of the fragments identified, along with their IC50s in a fluorescence polarization (FP) assay, are 10 and 11 (see figure – click to see larger image). These fragments were also characterized crystallographically. An interesting aside is that the binding mode of fragment 10 differed depending on whether it was soaked into Hsp90 crystals or co-crystallized with the protein. (This is reminiscent of the different binding modes, both productive, observed for two fragments using crystallography versus NMR on Hsp90 by researchers at Abbott.)
In addition to the fragment work on Hsp90, a virtual screen of 700,000 (non-fragment) compounds was conducted, leading to the purchase of 719 commercially available molecules that were tested in the FP assay. Two hits, 12 and 13, are shown in the figure and were also characterized crystallographically.
With these SAR and X-ray crystal structures in hand, the researchers added elements from the larger compounds (a phenyl from 12 and 13 or the amide from 13) to their fragments to generate the more potent fragments 14 and 15. Further structural examination led to the 2-amino-thienopyrimidine scaffold 16, which formed the basis for subsequent optimization.
Appending a phenyl group onto compound 16 led to the expected boost in potency, with the dichloro compound 21e having good biochemical as well as cell-based activity. Addition of a solubilizing group led ultimately to NVP-BEP800/VER-82576, which, in addition to potent biochemical and cell activity, also showed tumor regression in mice following once-daily oral dosing, along with pharmacodynamic biomarkers consistent with Hsp90 inhibition. This compound also showed good antiproliferative effects in a number of human cancer cell lines. Crystallographic characterization revealed that the molecule binds in a manner consistent with the previous structures (and with the co-crystallized structure of fragment 10).
This could be seen as a nice example of what has been dubbed fragment-assisted drug discovery: fragments were not the sole drivers of the project, but they did play an important role in guiding the overall strategy to develop optimal molecules.
In related news, Vernalis just announced a multimillion dollar collaboration with GlaxoSmithKline around an undisclosed cancer target – another indication that fragment-based approaches have not just scientific value, but monetary value as well.
06 August 2009
August 16-20: The fall ACS meeting this year will be held in sweltering Washington, DC. Although there are no sessions devoted exclusively to fragments, a number of FBLD talks and posters are sprinkled throughout the conference.
September 21-23: Much-anticipated FBLD 2009 will be held in historic York, UK. There will also be a one-day workshop on September 20 to provide an overview of fragment-based drug discovery for newcomers to the field. The draft schedule for the conference has just been released (pdf) – looks like a great lineup, so if you missed the earlier conferences this year, don’t miss this one!
February 3-5: CHI’s Molecular Medicine Tri-Conference is being held in the beautiful city of San Francisco, with a track on medicinal chemistry that will probably have some fragment talks, and a fragment workshop on February 2.
March 21-25: The spring ACS meeting will be in foggy (but beautiful) San Francisco. No schedule or link yet.
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 plenty of talks on FBLD.
April 26: The CHI fragment-conference will be held in summery San Diego. No web link yet, but I’ll have this when it becomes available.
As always, let us know if we’ve missed anything and we’ll get the word out!
01 August 2009
The researchers, mostly from Evotec, started with a high-concentration biochemical displacement assay to screen 20,000 fragments against Hsp90. A relatively potent aminopyrimidine (compound 1, below) was characterized crystallographically, and this structure was then used to run a virtual screen of 3.8 million commercially available molecules using the program GOLD 3.0.1. Some of the resulting hits were purchased and tested, including compound 3, which showed sub-micromolar biochemical activity but no cell-based activity. Subsequent modeling and medicinal chemistry led to compound 19, which, in addition to mid-nanomolar biochemical activity, also displayed submicromolar cell activity in A549 and HCT116 cancer cell lines.
In addition to compound 1, a number of other fragments containing the aminopyrimidine substructure were also identified as hits. This moiety seems to be a privileged pharmacophore for Hsp90: for a fun read, check out this 2007 paper from researchers at Abbott, in which fragments are linked together in a couple different ways as well as grown. As in the more recent paper, the protein displays a remarkable degree of flexibility to accommodate small molecule binders.
28 July 2009
If there's a hidden pool of FBDD talent, it is most likely in industry, not in academia. Fragment-based approaches are occasionally mentioned in courses or lectures by professors who want to appear up-to-date. But the only folks actually doing fragment-based work (as distinct from structure-based methods) are in a few key labs, all led by PIs with prior industry experience.
If you look at the 2008 FBLD Conference poster session, almost half of the presenters were from a single academic lab. The rest were largely virtual docking, traditional medicinal chemistry, or people from industry. One of the conference organizers told me that there were less than a handful of us graduate students in attendance; compare that to attendees at any given Gordon Conference. So I believe there are "puddles" of FBDD here and there, but not what I would call a vast resource.
That said, it seems the skills one needs to do FBDD can be acquired by other means in academia (ie. structural biology PhD, synthetic chemistry training, etc.). But if companies are looking to hire PhDs well-versed in fragment-based methodologies, there is currently not a huge group being freshly minted each year at commencement.[Ed Note: I also think it is interesting that the industrial types that go to industry fair not well in terms of grants and such (my impression talking to the ones I know). But, I would be interested in hearing from those types also.]
26 July 2009
The first, from researchers at the VU University, Amsterdam, and IOTA Pharmaceuticals, discusses 23 examples. Many of these have been reviewed elsewhere, but the paper also describes some studies that are unpublished or just reported at meetings. It’s a nice, thorough introduction to the field, and the organization of the review, by institution, gives a flavor of the diversity of approaches.
The second review, from researchers at Astex Therapeutics, provides a historical perspective and clinical focus. There are also useful tables of commercial suppliers of fragments as well as FBDD-derived compounds that have made it into clinical development.
In an accompanying editorial, Mark Whittaker of Evotec asks whether fragment-based drug discovery (FBDD) should really be called fragment-assisted drug discovery (FADD):
This is more than just a difference in semantics, but is, in fact, a broader question of when and how to apply fragment approaches to lead generation, either on their own or in concert with other hit finding techniques.
He goes on to explain that although fragment-based methods can be used by themselves to generate leads, they can also be complementary to other approaches to assess target druggability or focus later hit-finding. This conclusion is consistent with Practical Fragments’ latest poll, in which 85% of respondents reported that, far from being a fad, FBDD (or, if you like, FADD) is integrated in the hit finding stage at their company.
21 July 2009
On June 22, FBDD researchers from 11 Japanese pharmas/biotechs got together in hot and humid Tokyo. This one-day meeting, hosted by a crystallography-based CRO PharmAxess (www.pharmaxess.com) and an in silico-based CRO PharmaDesign (www.pharmadesign.co.jp/eng), started in the morning with reviews of benefits and techniques of FBDD, and then culminated in the afternoon with enthusiastic discussions on a non-confidential basis. The aim of this unofficial meeting was to share experienced problem-and-solution cases while carrying out FBDD, rather than reporting success stories in a conference fashion. Finally, it was adopted unanimously that the meeting should be held periodically once or twice a year.
Daisuke is organizing the next meeting – we’ll post the date and location as soon as we know (and everyone please contact us about other upcoming fragment events).
It’s great to see the field becoming more collaborative and international. Although the symposium proposals for Pacifichem 2010 are already set, perhaps a FBDD track at Pacifichem 2015 will give the fragment community an excuse to get together in Hawaii!
20 July 2009
Ruth Brenk and colleagues at the University of Dundee were interested in pteridine reductase 1 (PTR1), an enzyme from Trypanosoma brucei, the protozoan that causes sleeping sickness. They used the program DOCK 3.5.54 (which has been successfully used for fragment-docking) to screen 26,084 commercially available fragments against the crystal structure of PTR1. After a variety of computational and manual filters were applied, the researchers purchased and tested 45 compounds in an enzymatic assay. Of these, 10 fragments inhibited PTR1 at least 30% at 100 micromolar concentration, the most potent of which was compound 4 (below).
Removing the chlorine atom to generate compound 5 resulted in a dramatic loss in activity, while adding the dichlorobenzyl moiety caused a similarly large boost in activity (compound 9). The researchers were able to characterize the binding mode of each of these molecules crystallographically, and it turns out that, despite sharing a common aminobenzimidazole core, they all bind in very different fashions.
The initial compound 4 binds in two orientations, one of which closely resembles the binding mode predicted from the computational screen, with hydrogen bonds between the fragment and the enzyme cofactor NADP+. Compound 5 makes indirect (water-mediated) hydrogen bonds with the cofactor, while compound 9 binds in a completely different manner some distance from the cofactor.
Brenk and colleagues observed a hydrophobic pocket near compound 9 which they exploited to generate the low nanomolar compound 12; crystallography confirmed this binds in a similar fashion to compound 9. This molecule also displayed impressive selectivity against the potential off-target dihydrofolate reductase. Unfortunately, despite the promising biochemical activity of compound 12, it displays only modest activity against T. brucei in cell culture.
This study illustrates two important points. First, it can be hazardous to assume that even very closely related molecules, such as 4 and 5, bind in the same manner. Second, because of this, one should not adhere too slavishly to models, even those based on crystal structures. The binding modes of compounds 4 and 5 would not accommodate the dichlorobenzyl moiety, and yet this addition provided a sizable boost in potency. Sometimes it pays to make substitutions even where you wouldn’t expect them to make sense, especially where the changes are easy to make.
29 June 2009
The new dataset, GDB-13, contains 977,468,314 molecules containing carbon, oxygen, and nitrogen atoms (as well as hydrogens, of course). Unlike its predecessor it excludes fluorine, but it happily adds chlorine as an aromatic substituent as well as sulfur in heterocycles or in sulfones, sulfonamides, or thioureas. To speed calculation (which still required the equivalent of 4.5 years of CPU time), a few other simplifications were made to limit the number of heteroatoms in a given structure.
The resulting collection, while huge, is thus obviously incomplete: about two-thirds of 619,675 molecules that contain up to 13 atoms and are reported in a variety of databases do not appear in GDB-13. And GDB-13 has many unconventional structures – over half of the molecules contain one or more three- or four-membered rings.
Still, there is lots of neat stuff here: for example, 804,153 structural isomers of aspirin, and 18,371,393 structural isomers of mexiletine! And since 45.1% of the new molecules are rule-of-three compliant, there are hundreds of millions of virgin fragments just waiting to be made – and tested.
27 June 2009
There are a few previous reports of discovering fragments that bind to RNA and their subsequent optimization; Ibis Therapeutics was particularly active in this field a few years back. In the current study, Fareed Aboul-ela and coworkers at Louisiana State University started by analyzing 120 known RNA-binding ligands and comparing these to known drugs and publicly available compounds. A variety of computationally derived physicochemical descriptors failed to differentiate the RNA binders from other molecules, but the authors note that:
This result does not preclude the likelihood that a finite set of chemical moieties constitute a “privileged” RNA binding set. The special properties of these functionalities may be too subtle or complex to detect using standard descriptors.
Following up on this hypothesis, the authors computationally “cleaved” their RNA binders to generate a set of fragments, and then purchased just over a hundred of these. These were then screened using four different NMR experiments to see if any bound to a 27-residue oligonucleotide derived from E. coli 16S rRNA, an important antibiotic target. Happily, five fragments were identified as binding to the RNA target, two of which had not previously been identified in the literature.
Whether these fragments can be advanced to high affinity binders, and whether the library will be generally useful against RNA, remain open questions. But one nice feature of this paper is the complete list of fragments tested provided in the supporting information. This list will allow other researchers to easily assemble their own screening set and test its utility. And if it proves useful, perhaps it will one day be sold by one of the commercial suppliers of fragments.
21 June 2009
Alexander Shekhtman and colleagues at SUNY Albany have developed a method they call “screening of small molecule interactor library by using in-cell NMR”, or SMILI-NMR. The process starts by overexpressing two proteins within cells (E. coli, in this case). If the proteins are sequentially expressed, one of them can be selectively labeled with NMR-active isotopes. To test their system, the researchers overexpressed the model proteins FKBP and FRB. These proteins interact only weakly by themselves, but in the presence of the small molecule rapamycin they form a high affinity complex. By performing NMR on the cells, the researchers could observe changes in NMR peaks corresponding to formation of the ternary complex inside the cells when rapamycin was added. They could also do competition studies: adding the small molecule ascomycin to this complex causes a change in the NMR peaks corresponding to the rapamycin being competed away by the ascomycin.
The next step was to look for new molecules that would modulate the interaction between FKBP and FRB, and the researchers chose a library of 289 dipeptides, which are actively transported into cells. The dipeptides were mostly fragment-sized, ranging from a low molecular weight of 132 (Gly-Gly) to a high of 390 (Trp-Trp). The dipeptides were screened in pools (organized in a matrix) and then deconvoluted to identify the most active molecules. Interestingly, none of the molecules caused discrete changes to the NMR spectra as observed with rapamycin or ascomycin, but several caused some of the NMR peaks to disappear and the remaining peaks to broaden dramatically. The most potent compound was Ala-Glu (MW 218), which caused this phenomenon at 5 mM concentration. The authors interpret this effect as being caused by the formation of a large complex consisting of many molecules of FKBP, FRB, and Ala-Glu. Interestingly, although ascomycin could reverse the effect of Ala-Glu, rapamycin could not.
The dipeptide Ala-Glu also behaved similarly to rapamycin in yeast cells: both molecules prevented growth by yeast expressing FKBP, while having no effect on yeast lacking FKBP. This was attributed to both molecules facilitating complex formation between FKBP and FRB within yeast.
The Ala-Glu “fragment” has some issues (ClogP = -4, for example); it would be interesting to see how some of the original FKBP fragments discovered at Abbott behave in this assay. And although not everyone has access to a 700 MHz NMR with a cryoprobe, this is an intriguing approach for studying protein-protein interactions in a very biologically relevant milieu.