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.
29 March 2010
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.
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