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!
14 May 2010
30 April 2010
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?
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?
25 April 2010
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.
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.
11 April 2010
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.
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.
01 April 2010
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:
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.
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.
29 March 2010
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.
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.
26 March 2010
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.
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.
20 March 2010
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.
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.
19 March 2010
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 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.
12 March 2010
“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?
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?
07 March 2010
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.
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.
21 February 2010
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.
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.
17 February 2010
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.
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.
15 February 2010
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.
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.
06 February 2010
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.”
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.”
30 January 2010
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?
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?
24 January 2010
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.
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.
11 January 2010
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.
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.
06 January 2010
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.
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.
31 December 2009
Current Topics in Medicinal Chemistry Special FBDD Issue
This year ends with an entire issue of Current Topics in Medicinal Chemistry devoted to FBDD. Rob van Montfort and Ian Collins provide a brief editorial overview of the six papers. Collins and colleagues also describe their application of fragment-based methods to develop inhibitors of the anti-cancer target protein kinase B (AKT).
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!
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
Fragment-based conferences in 2010
There have been some great conferences this past year (see summaries of some of them here and here), and 2010 is shaping up nicely, particularly with the announcement of FBLD 2010 (below).
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.
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
Natural linking – though not of fragments
We’ve previously discussed the appeal and challenges of fragment linking. A new paper in Science describes how a naturally occurring antibiotic makes use of a linking strategy, albeit using rather Brobdingnagian fragments.
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.
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
Warren DeLano Memorial Award
We wrote last month about Warren DeLano, and in the December issue of Nature Structural and Molecular Biology Axel Brunger and Jim Wells have written a beautiful obituary.
Together with Warren’s family, Axel and Jim are also organizing a commemorative fund:
The Warren L. DeLano Memorial Award for Computational Biosciences
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.
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
tel: 650.450.5400
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
Enthalpy versus entropy
The earliest stages of lead discovery usually focus on obtaining a molecule with decent affinity for a given target. Affinity, or binding energy, can be dissected into two components: enthalpy and entropy. On a (very) simplistic level, enthalpic binding comes via specific molecular interactions, such as hydrogen bonds, while entropic binding results from nonspecific hydrophobic interactions. Optimizing enthalpy is usually more difficult than optimizing entropy: engineering a polar interaction requires more precision than adding a bit of grease. In a new paper in ChemMedChem, Andrew Scott and colleagues at Pfizer show how fragments that owe more of their binding affinity to enthalpy make better starting points for optimization than do fragments whose binding is more entropic, even if the entropic fragment is more potent.
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.
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
Too many aromatics stink
A recent discussion centered on whether fragment libraries should be designed to include more “3-dimensional” molecules and reduce the number of flat, aromatic compounds. Two new papers suggest that doing so may improve pharmaceutical properties. What effect this would have on screening success is still unclear.
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:
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.
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
NMR vs other methods
Fragment-based lead discovery owes much of its popularity to NMR: the SAR by NMR papers published by Abbott in the mid 1990s demonstrated both the power and the practicality of the approach. Recently SPR has also come into its own as a means for screening fragments, and in a paper in this month’s issue of Drug Discovery Today Claudio Dalvit of Novartis and the Italian Institute of Technology compares these two techniques, along with fluorescence spectroscopy. Not surprisingly given the author’s longstanding research interest, NMR comes out favorably, though with the recommendation that the techniques are complementary, so researchers should combine techniques rather than simply selecting one over another.
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?
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
A tale of two deals
Two deals involving companies in the fragment space were announced today. I don’t have inside information on either of these, but superficially they are strikingly different.
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.
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
Remembering Warren Delano
For those of you who haven’t heard, Warren Delano, author of PyMOL, died earlier this week. His sister Jen has established a blog for people to record their memories.
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.
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
To grow or to link: why not both?
What can you do with fragments? The idea of linking a couple together, while successfully demonstrated in the first SAR by NMR paper, generally seems to be more difficult than gradually growing one fragment. Now a new paper in Angewandte Chemie from Chris Abell and colleagues at the University of Cambridge presents a lovely comparison of these two strategies applied to a single target.
The researchers were interested in the M. tuberculosis enzyme pantothenate synthetase (PS) as a potential therapy for TB. Using a number of biophysical techniques including thermal shifts, NMR, and isothermal titration calorimetry, Abell and colleagues identified indole fragment 1 as a low-affinity binder from a library of about 1300 fragments (see figure below). X-ray crystallography revealed that the fragment binds in the ATP-binding site. An attempt to partially mimic the triphosphate by introducing negatively charged moieties led to modest improvements in potency (compounds 1a, 1b, and 2). Compound 2 bound in a similar position as compound 1, with the advantage that the methyl group off the sulfonamide is nicely positioned for further growing the molecule. Replacing this methyl group with a methylpyridine produced compound 4, increasing the affinity by about two orders of magnitude while maintaining ligand efficiency, and crystallography revealed that this moiety binds in the P2 pocket. Thus, the fragment growing approach began with an indole of low millimolar affinity and produced a molecule with low micromolar affinity after several iterations.
At the same time, the researchers also identified benzofuran fragment 5 (see figure below) and discovered that it binds in the P1 pocket some distance from the indole fragment 1, suggesting the two could be linked. In fact, a crystal structure revealed that the two fragments are able to bind to PS simultaneously. Linking these together through the acylsulfonamide linker employed above led to compound 8, with a potency similar to that obtained from fragment growing. Compounds 4 and 8 structurally resemble each other, but although the indole fragment of each binds in the same location, the terminal fragments (the methylpyridine in compound 4 and the benzofuran fragment in compound 8) bind in different locations, the former in the P2 pocket with the later in the P1 pocket. However, the benzofuran is somewhat twisted relative to the binding mode it adopts as a free fragment.
As the researchers observe, the ligand efficiency of compound 8 derived from fragment linking is lower than those derived from fragment growing, though even the molecules developed from growing have lower ligand efficiencies than the initial fragments.
The researchers conclude:
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.
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
Infarmatik In-3D Library
In what we hope is a new series bringing the latest in Fragment Science up for discussion, we present today to you a discussion of Infarmatik's In-3D Library. We look forward to this discussion, and hopefully, many more.
Fragment based drug discovery has been shown to provide a rapid means for transforming low affinity “hits” to optimized leads. However, most currently available fragment libraries are limited in usefulness, mainly because over 90% of the molecules are planar and thus do not fit well into 3-dimensional receptor protein binding sites. InFarmatik realized [Ed: and others] that “real 3-D” structures offer a better fit within the uneven binding surfaces of protein hot-spots (business sites) than do planar compounds. To address this issue, we have developed a specific series of novel and diverse 3-D fragments, which are not available from any other commercial sources. The structure types of the first release contain 2,3, and 4 member non-aromatic ring systems, with various attachment points, including spiro and 1,2 anellation, 10 electron systems connected to saturated ring systems, saturated bis-heterocyclics and rod shaped compounds. We believe these compounds will exhibit the ability to bind to a wide array of protein targets. In addition, we can offer another 435 structures from existing stock, which conform to Ro3 and are quite “fragment-like”.
Most of the compounds have soft scaffold structures: meaning they were designed to have low reactivity centers to avoid non-specific binding, while preserving the ease of chemically coupling them to each other or to other fragments. The attachment points in the molecules in many cases are useful for regiospecific reactions.
Here are the relevant properties of the 3-D fragment library:
Size: 119 3-D Fragments
Average MW=230 Da
average logP value (calculated) =1.88
confirmed minimum water solubility of at least 0.1% in 2% aqueous DMSO.
Solubility data available for all compounds
Highly diverse, as shown by 3-D Diversity Analysis using ChemAxon supplied tools
Here are the relevant properties of the new standard fragment set
· Size: 435 Fragments
· Average MW=237.8 Da
· Average LogP value (calculated) =2.27
15 October 2009
Genentech’s affinity for Graffinity
Heidelberg-based Graffinity today announced that they would be collaborating with the Genentech division of Roche. Graffinity will apply its surface plasmon resonance (SPR) fragment-based technology to several Genentech targets. Financial details and specific targets have not been released, though Graffinity CEO Kristina Schmidt is quoted as saying that they plan “to explore drug targets that would remain white spaces on the map of drug discovery” with conventional high-throughput screening.
SPR is rapidly becoming a workhorse in the stable of FBDD techniques. Although it provides less information than NMR or X-ray approaches, SPR is faster, and can rapidly distinguish true hits from bad-acting artifacts. Typically a protein is immobilized on a gold surface, and fragments are allowed to flow past to detect those that bind. Graffinity reverses this process: they have a collection of about 110,000 small molecules, just over a fifth of which are fragments, immobilized in microarrays which can be screened against proteins (see here for full description).
Genentech is no stranger to SPR; one of the highlights of the recent FBLD 2009 meeting was a talk by Tony Giannetti on the use of this technology at Genentech against roughly 40 target proteins. The collaboration further validates the use of SPR for FBDD, and suggests that Graffinity has an interesting – and useful – angle.
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.
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