Today is the last day to submit a poster abstract for FBLD 2010, the first major fragment event on the east coast of the US (in Philadelphia, PA, from October 10-13). Registration will remain open for up to 250 attendees (with 177 coming so far) at $700 for industrial attendees and $350 for academic attendees. The hotel discount expires Sept 20, so book your room ASAP before they fill up.
I think this is the last fragment event this year, but if you know of anything else (or next year) please pass it on or leave a comment.
31 August 2010
30 August 2010
Evotec and BACE
Certain targets seem to be particularly popular with fragment-based methods, perhaps in part because they are recalcitrant to other approaches. One of these is the Alzheimer’s target BACE, as described in a post earlier this year on work from Schering-Plough (Merck). Now, in a recent issue of Bioorg. Med. Chem. Lett., James Madden and colleagues at Evotec describe their approach to this challenging protease.
The researchers started by screening their 20,000-fragment library in a functional assay at 1 mM, an endeavor that led to a number of hits, some of which were confirmed by surface plasmon resonance and crystallography. One of these, Compound 3 (see Figure), bore some resemblance to and bound in a similar fashion as a compound previously reported in the literature. The researchers were able to use the binding mode of this other compound to help them improve their fragment. After a couple cycles of synthesis, assays, and crystallography, the researchers arrived at compound 14.
The final molecule shows a 100-fold improvement in potency over the initial fragment and some cellular activity, and the researchers were able to maintain ligand efficiency throughout optimization, albeit at lower values than some previously reported molecules. However, the final compound is still relatively weak, and there is no information on brain penetration. Moreover, it shows activity against hERG, leading Evotec to deprioritize this series. Still, the paper is an easy read and a clear example of what has been called “fragment-assisted drug discovery,” in which traditional medicinal chemistry approaches (in this case borrowing from a competitor compound) are applied along with fragment methods to generate new molecules.
The researchers started by screening their 20,000-fragment library in a functional assay at 1 mM, an endeavor that led to a number of hits, some of which were confirmed by surface plasmon resonance and crystallography. One of these, Compound 3 (see Figure), bore some resemblance to and bound in a similar fashion as a compound previously reported in the literature. The researchers were able to use the binding mode of this other compound to help them improve their fragment. After a couple cycles of synthesis, assays, and crystallography, the researchers arrived at compound 14.
The final molecule shows a 100-fold improvement in potency over the initial fragment and some cellular activity, and the researchers were able to maintain ligand efficiency throughout optimization, albeit at lower values than some previously reported molecules. However, the final compound is still relatively weak, and there is no information on brain penetration. Moreover, it shows activity against hERG, leading Evotec to deprioritize this series. Still, the paper is an easy read and a clear example of what has been called “fragment-assisted drug discovery,” in which traditional medicinal chemistry approaches (in this case borrowing from a competitor compound) are applied along with fragment methods to generate new molecules.
25 August 2010
Thermodynamic and kinetic debate
Our friends over at FBDD-Lit have just pointed out an active discussion on the use of thermodynamic and kinetic parameters in medicinal chemistry going on at the Medicinal Chemistry and Drug Discovery LinkedIn group. This is a topic we’ve covered a couple times (here, here), and it’s nice to see a vigorous debate about, among other things, the usefulness of measuring enthalpy and entropy.
19 August 2010
Click here to link
Fragment linking is a topic we’ve discussed several times. One of the more interesting approaches is template-directed synthesis, in which a protein causes two fragments in close proximity to react with one another (see here for example). In a recent issue of Angew. Chem. Int. Ed., Beat Ernst and colleagues at the University of Basel provide a new variant of this theme, without requiring structural information about the protein.
The researchers were interested in a protein called myelin-associated glycoprotein, or MAG, which blocks axonal regrowth. They started with an NMR screen to determine which members of a fragment library bind to MAG as assessed by a phenomenon known as transverse magnetization decay; essentially, small molecules that bind to a protein behave like large molecules in showing a rapid decay in magnetization, so an increased magnetization decay of fragments in the presence of protein suggests binding. A number of fragments were identified as binders, but the site of binding was not determined.
To find molecules that could be linked, the researchers took a known ligand, the sialic acid derivative 1 (see figure – albeit larger than a fragment), and modified this to contain a spin-label. Spin-labels are small moieties that contain an unpaired electron and, just like large molecules, cause an increase in magnetization decay, but only to molecules within close proximity. The two effects are additive, and thus the researchers could determine which fragments bind to the protein in close proximity to the spin-label-containing derivative of compound 1. In fact, the distance dependence is so pronounced that different protons on the fragment can show different effects, thus indicating which portion of the fragment is close to the spin label (see here for a similar approach using ILOE). In this case, the researchers found that a nitroindole fragment (see figure) had its 5-membered ring positioned closer to the spin label than its 6-membered ring.
Knowing the relative positions of the two ligands, the researchers modified them so they could be linked together. They added functional groups with different linker lengths to create several analogs, replacing the spin label with an alkyne and adding an azide to the nitroindole fragment. They then incubated all the analogs together in the presence of the protein. Analysis of the reaction by HPLC-MS after three days at 37 degrees revealed one prominent product, with a mass consistent with compound 7. Two isomers of this product can be formed, with syn and anti configurations around the triazole, and the researchers synthesized both of them. Interestingly, the anti isomer (shown) had a Kd for MAG of 190 nM, while the syn isomer bound roughly 10-fold more weakly.
Although the ligand efficiency of the final compound is low, sugar-based molecules typically have low ligand efficiencies, and maintaining the same efficiency as starting compound 1 is impressive. However, the ligand efficiency of the final molecule is probably lower than the second-site ligand: the researchers don’t report its affinity, but since it would likely need to be 10 mM or better to be detected its ligand efficiency is probably at least 0.23 kcal/mol/atom.
Still, the final product is sufficiently potent that it could make a useful biological probe. Moreover, the approach is notable in not requiring structure of the protein – a rare and attractive feature for fragment linking.
The researchers were interested in a protein called myelin-associated glycoprotein, or MAG, which blocks axonal regrowth. They started with an NMR screen to determine which members of a fragment library bind to MAG as assessed by a phenomenon known as transverse magnetization decay; essentially, small molecules that bind to a protein behave like large molecules in showing a rapid decay in magnetization, so an increased magnetization decay of fragments in the presence of protein suggests binding. A number of fragments were identified as binders, but the site of binding was not determined.
To find molecules that could be linked, the researchers took a known ligand, the sialic acid derivative 1 (see figure – albeit larger than a fragment), and modified this to contain a spin-label. Spin-labels are small moieties that contain an unpaired electron and, just like large molecules, cause an increase in magnetization decay, but only to molecules within close proximity. The two effects are additive, and thus the researchers could determine which fragments bind to the protein in close proximity to the spin-label-containing derivative of compound 1. In fact, the distance dependence is so pronounced that different protons on the fragment can show different effects, thus indicating which portion of the fragment is close to the spin label (see here for a similar approach using ILOE). In this case, the researchers found that a nitroindole fragment (see figure) had its 5-membered ring positioned closer to the spin label than its 6-membered ring.
Knowing the relative positions of the two ligands, the researchers modified them so they could be linked together. They added functional groups with different linker lengths to create several analogs, replacing the spin label with an alkyne and adding an azide to the nitroindole fragment. They then incubated all the analogs together in the presence of the protein. Analysis of the reaction by HPLC-MS after three days at 37 degrees revealed one prominent product, with a mass consistent with compound 7. Two isomers of this product can be formed, with syn and anti configurations around the triazole, and the researchers synthesized both of them. Interestingly, the anti isomer (shown) had a Kd for MAG of 190 nM, while the syn isomer bound roughly 10-fold more weakly.
Although the ligand efficiency of the final compound is low, sugar-based molecules typically have low ligand efficiencies, and maintaining the same efficiency as starting compound 1 is impressive. However, the ligand efficiency of the final molecule is probably lower than the second-site ligand: the researchers don’t report its affinity, but since it would likely need to be 10 mM or better to be detected its ligand efficiency is probably at least 0.23 kcal/mol/atom.
Still, the final product is sufficiently potent that it could make a useful biological probe. Moreover, the approach is notable in not requiring structure of the protein – a rare and attractive feature for fragment linking.
09 August 2010
Fragment specificity
A frequent topic in fragment roundtable discussions concerns specificity: do fragments hit lots of targets, or just a few? Isabelle Krimm and colleagues at the Université de Lyon in France studied this question experimentally and report their results in a recent issue of J. Med. Chem. The paper provides data for the ongoing debate of whether and how much specificity a fragment should exhibit before being pursued for further lead development.
The researchers assembled a diverse set of 150 fragments and used NMR techniques to determine whether they bind to five different proteins. Three of the proteins, Bcl-xL, Bcl-w, and Mcl-1 are related members of the Bcl-2 family of antiapoptotic proteins, and at least the first of these has been successfully targeted using fragment-based methods. The fourth protein, PRDX5, has proven to be much less yielding to inhibitor discovery, while the fifth, human serum albumin (HSA), binds a wide variety of small molecules.
After applying 1D-NMR techniques (WaterLOGSY and STD) to all of their fragments against each of the five proteins, the researchers used more rigorous but less sensitive 2D-NMR (HSQC) to determine the binding sites of the hits. (This later study revealed, in agreement with previous results from the same lab, that the fragments all bind in the “hot spots” or active sites of the proteins.)
More than two-thirds of the fragments bound to at least one protein, a rather high hit rate. However, the hit rates for each protein varied considerably, with only 7 hits for PRDX5 and 72 for HSA (with a close second of 71 for Bcl-xL). Within the Bcl-2 family there was little specificity observed: Mcl-1, with 29 hits, shared all but one hit with either Bcl-xL or Bcl-2 or both; such non-specificity among related proteins has been discussed previously. In the case of HSA and Bcl-xL, although both proteins had similar numbers of hits, just over half of these were in common, demonstrating that fragment specificity is not difficult even with small-molecule sponges such as HSA. That said, many fragments were remarkably nonspecific, with 22 hitting four of the 5 proteins. Amazingly, all 7 of the hits against PRDX5 also hit all four other proteins.
The physicochemical properties of the fragments that hit one or more proteins were compared with those of the library as a whole, and although most of the parameters were similar, the ClogP values (a measure of hydrophobicity) were considerably higher for hits, and highest of all for the non-specific hits.
These findings are more evidence that, as predicted almost a decade ago, fragments can bind to more proteins than can larger, more complex molecules. The follow-up question, how much does this matter, is still up for debate. There are plenty of examples of developing specific inhibitors from non-specific starting points during the course of fragment optimization. But how non-specific is too non-specific? Would you feel comfortable pursuing any of the fragments that hit all of the proteins?
The researchers assembled a diverse set of 150 fragments and used NMR techniques to determine whether they bind to five different proteins. Three of the proteins, Bcl-xL, Bcl-w, and Mcl-1 are related members of the Bcl-2 family of antiapoptotic proteins, and at least the first of these has been successfully targeted using fragment-based methods. The fourth protein, PRDX5, has proven to be much less yielding to inhibitor discovery, while the fifth, human serum albumin (HSA), binds a wide variety of small molecules.
After applying 1D-NMR techniques (WaterLOGSY and STD) to all of their fragments against each of the five proteins, the researchers used more rigorous but less sensitive 2D-NMR (HSQC) to determine the binding sites of the hits. (This later study revealed, in agreement with previous results from the same lab, that the fragments all bind in the “hot spots” or active sites of the proteins.)
More than two-thirds of the fragments bound to at least one protein, a rather high hit rate. However, the hit rates for each protein varied considerably, with only 7 hits for PRDX5 and 72 for HSA (with a close second of 71 for Bcl-xL). Within the Bcl-2 family there was little specificity observed: Mcl-1, with 29 hits, shared all but one hit with either Bcl-xL or Bcl-2 or both; such non-specificity among related proteins has been discussed previously. In the case of HSA and Bcl-xL, although both proteins had similar numbers of hits, just over half of these were in common, demonstrating that fragment specificity is not difficult even with small-molecule sponges such as HSA. That said, many fragments were remarkably nonspecific, with 22 hitting four of the 5 proteins. Amazingly, all 7 of the hits against PRDX5 also hit all four other proteins.
The physicochemical properties of the fragments that hit one or more proteins were compared with those of the library as a whole, and although most of the parameters were similar, the ClogP values (a measure of hydrophobicity) were considerably higher for hits, and highest of all for the non-specific hits.
These findings are more evidence that, as predicted almost a decade ago, fragments can bind to more proteins than can larger, more complex molecules. The follow-up question, how much does this matter, is still up for debate. There are plenty of examples of developing specific inhibitors from non-specific starting points during the course of fragment optimization. But how non-specific is too non-specific? Would you feel comfortable pursuing any of the fragments that hit all of the proteins?
26 July 2010
FBDD and structural biology
The rise of fragment-based drug discovery has largely depended on the success of structural biology. FBDD began in earnest with NMR techniques in the mid 1990s, soon followed by high-throughput crystallography techniques in the early part of this century. In an article published online in Current Opinion in Structural Biology, Christopher Murray of Astex and Tom Blundell of the University of Cambridge discuss this reliance on structure.
The review describes several cases where structural biology played pivotal roles in advancing fragments to leads or drug candidates. Many have been discussed in Practical Fragments, including AT7519 and AT9283 from Astex, the JAK-2 program from SGX, DG-051 from deCODE, HSP90 inhibitors from Vernalis/Novartis and Evotec, Schering-Plough’s BACE inhibitors, and Plexxikon's indeglitazar.
The researchers also discuss the potential of fragment methods for generating inhibitors of antimicrobial targets, such as enzymes in the organisms that cause tuberculosis and sleeping sickness. In these cases too, structural biology played critical roles.
Structural biology is so important, the authors conclude, that “it is only through the expert use of structure-based drug design that FBDD can be expected to fulfill its promise of delivering candidates with the improved physical properties (lower molecular weight and lipophilicity) which it is hoped will lead to reduced attrition in clinical trials.”
But is dependence on structure truly inevitable? The authors themselves highlight one case in which a new antimicrobial agent with animal efficacy was developed using fragment-based methods in the absence of direct structural information. If this success could be generalized, it would open the potential of fragment methods to a much wider range of practitioners.
The review describes several cases where structural biology played pivotal roles in advancing fragments to leads or drug candidates. Many have been discussed in Practical Fragments, including AT7519 and AT9283 from Astex, the JAK-2 program from SGX, DG-051 from deCODE, HSP90 inhibitors from Vernalis/Novartis and Evotec, Schering-Plough’s BACE inhibitors, and Plexxikon's indeglitazar.
The researchers also discuss the potential of fragment methods for generating inhibitors of antimicrobial targets, such as enzymes in the organisms that cause tuberculosis and sleeping sickness. In these cases too, structural biology played critical roles.
Structural biology is so important, the authors conclude, that “it is only through the expert use of structure-based drug design that FBDD can be expected to fulfill its promise of delivering candidates with the improved physical properties (lower molecular weight and lipophilicity) which it is hoped will lead to reduced attrition in clinical trials.”
But is dependence on structure truly inevitable? The authors themselves highlight one case in which a new antimicrobial agent with animal efficacy was developed using fragment-based methods in the absence of direct structural information. If this success could be generalized, it would open the potential of fragment methods to a much wider range of practitioners.
21 July 2010
Virtual phosphate fragments
Phosphate groups are handy little things: easy for enzymes to put on and take off, they pack a lot of charge in a small volume, thereby providing plenty of binding energy for electrostatic interactions. Not surprisingly, they are ubiquitous in biology. Unfortunately, the same things that make them attractive for an organism make them problematic for drugs: they are easily removed, and their highly negative charge gives molecules containing phosphates a real problem getting across membranes. What’s a chemist to do?
This was the dilemma faced by Ruth Brenk, Ian Gilbert, and colleagues at the University of Dundee. They were interested in inhibiting the enzyme 6-phosphogluconate dehydrogenase (6PGDH) from the parasite that causes sleeping sickness. (See here for previous work from the same group using fragment methods to discover inhibitors against a different enzyme from the same organism.) The enzyme 6PGDH, as its name suggests, binds phosphate-containing substrates and has a very polar active site. Nanomolar inhibitors have been reported in the literature, but these contain phosphates and are not active in cell assays.
As reported in a recent issue of Bioorganic and Medicinal Chemistry, the researchers computationally filtered a set of commercially available compounds to find those that were less than 320 Da and were negatively charged, thereby potentially mimicking a phosphate. They then used DOCK 3.5.54 to see which of the resulting 64,000 molecules might bind in the active site of 6PGDH, resulting in 5836 possible hits. Subsequent triaging led to the purchase of 71 compounds. These were tested for inhibition of the enzyme at 200 micromolar concentration. Ten of these compounds inhibited the enzyme more than 80% at this concentration, of which 3 gave clean IC50 curves. These three molecules are all 5-membered carboxylic-acid-containing heterocycles, and although the IC50s are modest (ranging from 28 to 45 micromolar), they have good ligand efficiencies (up to 0.66 (kcal/mol)/atom). A computational search for analogs resulted in a few more active molecules with similar properties.
Whether these fragments can be advanced remains to be seen. The calculated solubilites, Log P, total polar surface area, and intestinal absorption parameters are more attractive than previous inhibitors, but the history of phosphate mimics is not encouraging. Most prominently, the protein PTP-1B, which recognizes phosphotyrosine residues, was once one of the hottest drug targets around, spawning a cottage industry of groups developing phosphotyrosine mimetics. Fragment methods were particularly effective, and numerous potent small molecules were published. But none of them were sufficiently drug-like, and to my knowledge none are in the clinic. Still, it is worth trying: 6PGHD may be more druggable, and approaches like this are likely to provide an answer.
This was the dilemma faced by Ruth Brenk, Ian Gilbert, and colleagues at the University of Dundee. They were interested in inhibiting the enzyme 6-phosphogluconate dehydrogenase (6PGDH) from the parasite that causes sleeping sickness. (See here for previous work from the same group using fragment methods to discover inhibitors against a different enzyme from the same organism.) The enzyme 6PGDH, as its name suggests, binds phosphate-containing substrates and has a very polar active site. Nanomolar inhibitors have been reported in the literature, but these contain phosphates and are not active in cell assays.
As reported in a recent issue of Bioorganic and Medicinal Chemistry, the researchers computationally filtered a set of commercially available compounds to find those that were less than 320 Da and were negatively charged, thereby potentially mimicking a phosphate. They then used DOCK 3.5.54 to see which of the resulting 64,000 molecules might bind in the active site of 6PGDH, resulting in 5836 possible hits. Subsequent triaging led to the purchase of 71 compounds. These were tested for inhibition of the enzyme at 200 micromolar concentration. Ten of these compounds inhibited the enzyme more than 80% at this concentration, of which 3 gave clean IC50 curves. These three molecules are all 5-membered carboxylic-acid-containing heterocycles, and although the IC50s are modest (ranging from 28 to 45 micromolar), they have good ligand efficiencies (up to 0.66 (kcal/mol)/atom). A computational search for analogs resulted in a few more active molecules with similar properties.
Whether these fragments can be advanced remains to be seen. The calculated solubilites, Log P, total polar surface area, and intestinal absorption parameters are more attractive than previous inhibitors, but the history of phosphate mimics is not encouraging. Most prominently, the protein PTP-1B, which recognizes phosphotyrosine residues, was once one of the hottest drug targets around, spawning a cottage industry of groups developing phosphotyrosine mimetics. Fragment methods were particularly effective, and numerous potent small molecules were published. But none of them were sufficiently drug-like, and to my knowledge none are in the clinic. Still, it is worth trying: 6PGHD may be more druggable, and approaches like this are likely to provide an answer.
17 July 2010
ANCHORing fragments
Protein-protein interactions are intriguing though challenging targets for lead discovery, and fragment-based approaches have often been used to tackle them (for example here, here and here). One of the difficulties is trying to figure out which of the often many residues in a large contact surface are really important. To make this easier, Lidio Meireles, Alexander Dömling, and Carlos Camacho at University of Pittsburgh have unveiled a free web-based tool, described in a recent issue of Nucleic Acids Research.
The tool, called ANCHOR, is both a server and a database of protein-protein interactions. The database contains over 30,000 entries taken from the protein data bank (PDB). Each of these entries has been analyzed computationally. ANCHOR examines bound and free (as computationally isolated from the complex) forms of each protein, focusing on side chains that, depending on protein state, are either buried within the partner protein or exposed to solvent. The change in solvent-accessible surface area is calculated for every residue in the protein-protein contact area. ANCHOR also estimates each residue’s contribution to the binding free energy, using both electrostatic and solvation terms in the calculation.
While the absolute numbers should probably be taken with a grain of salt, the relative values could help identify “anchoring” residues most likely to be useful as initial fragments. This means you can enter a pdb number and rapidly find the residues likely to be most important. You can also do more complex queries across the entire database, for example searching for buried tryptophan residues for oncology targets. If your protein is not already in the database, you also have the option of uploading a structure for custom analysis.
What makes ANCHOR particularly appealing is its powerful graphical interface, which shows which residues are selected and allows significant customization. The whole system is quite intuitive and easy to use. Try it on your favorite protein-protein interaction and tell us what you think!
The tool, called ANCHOR, is both a server and a database of protein-protein interactions. The database contains over 30,000 entries taken from the protein data bank (PDB). Each of these entries has been analyzed computationally. ANCHOR examines bound and free (as computationally isolated from the complex) forms of each protein, focusing on side chains that, depending on protein state, are either buried within the partner protein or exposed to solvent. The change in solvent-accessible surface area is calculated for every residue in the protein-protein contact area. ANCHOR also estimates each residue’s contribution to the binding free energy, using both electrostatic and solvation terms in the calculation.
While the absolute numbers should probably be taken with a grain of salt, the relative values could help identify “anchoring” residues most likely to be useful as initial fragments. This means you can enter a pdb number and rapidly find the residues likely to be most important. You can also do more complex queries across the entire database, for example searching for buried tryptophan residues for oncology targets. If your protein is not already in the database, you also have the option of uploading a structure for custom analysis.
What makes ANCHOR particularly appealing is its powerful graphical interface, which shows which residues are selected and allows significant customization. The whole system is quite intuitive and easy to use. Try it on your favorite protein-protein interaction and tell us what you think!
11 July 2010
So Long, and Thanks for all the Fish
I am writing this to say thank you to everyone who reads this blog, and those who have contributed to this blog. When Dan and I started this, it was after meeting at a FBDD conference in San Diego. We decided that this field needed something like this. There are now LinkedIn groups, Facebook groups, other blogs about FBDD (all linked to the right). As many of you know, my current position does not involve FBDD. FBDD has been a passion of mine since I got involved in 2001. I appreciate my boss at the time Mike Shapiro for giving me the chance to set up and lead the FBDD efforts at Lilly. It was a fantastic experience and very successful. I also want to thank Mike for co-editing the book with me (please buy a copy, every copy earns me something close to three cents ;-)). I want to acknowledge all the great friends I have made through the years, including Dan who has picked up the onus of publishing this blog and has done and will continue to do a fantastic job.
I have always said that research carries a two year shelf-life. It's been almost two years since the book came out, that means I am done. I have nothing new to add to the field (and probably haven't for longer than two years). This means no more embarassing questions when I give talks on FBDD about what I doing now (which is nothing in FBDD). It is exciting to follow the field, but it is also really tough not being a part of it.
I wish you all the best of luck and ask that you don't be strangers. I will be speaking on NMR in the upcoming months, and am always happy to add more NMR speaking engagements (hint hint).
I have always said that research carries a two year shelf-life. It's been almost two years since the book came out, that means I am done. I have nothing new to add to the field (and probably haven't for longer than two years). This means no more embarassing questions when I give talks on FBDD about what I doing now (which is nothing in FBDD). It is exciting to follow the field, but it is also really tough not being a part of it.
I wish you all the best of luck and ask that you don't be strangers. I will be speaking on NMR in the upcoming months, and am always happy to add more NMR speaking engagements (hint hint).
24 June 2010
Metallophilic fragments
A post earlier this month mentioned matrix-metalloproteinases (MMPs). Now, a recent issue of J. Am. Chem. Soc. carries a Communication about fragment-libraries designed for zinc proteases, of which MMPs are a subset.
Seth Cohen and coworkers at University of California San Diego and the Weizmann Institute of Science in Israel designed two libraries based on known zinc chelators: quinoline sulfonamides (QSL) and benzimidazole sulfonamides (BISL) (see Figure). They rapidly assembled 40 of the former and 37 of the later using microwave chemistry and tested these against a handful of different MMPs.
Both libraries produced hits against MMP-2, MMP-3, MMP-8, and MMP-9. Control compounds designed not to chelate zinc showed no activity, and X-ray adsorption fine structure spectroscopy experiments suggest that the molecules are indeed binding to the catalytic zinc. Selectivity is often an issue in targeting metalloproteinases, and it was thus gratifying to find that at least one fragment inhibited MMP-2 with low micromolar activity while showing no activity against the other MMPs. Molecular modeling provides some rationale for this selectivity.
One could argue that many of the library members do not meet conventional definitions of fragments, and could be seen as more scaffold-like (or worse – one has a molecular weight pushing 600 Daltons!) And of course, it is not clear that either scaffold will be suitable for drug development or even tool compounds – it is possible their propensity for zinc binding will be a problem inside cells. Still, the notion of creating custom-made fragment libraries for various classes of targets certainly makes sense; folks have done this for kinases and even RNA, and it is reasonable to see this approach extended to metalloproteinases. Cohen and colleagues described a fragment library consisting of more conventional metal chelators earlier this year in ChemMedChem.
This publication also confirms the results of our poll that fragment-based approaches are catching on in academia. But industry is already in the sandbox: at least two companies, AnCore and Viamet, are using similar strategies to target metalloproteins.
Seth Cohen and coworkers at University of California San Diego and the Weizmann Institute of Science in Israel designed two libraries based on known zinc chelators: quinoline sulfonamides (QSL) and benzimidazole sulfonamides (BISL) (see Figure). They rapidly assembled 40 of the former and 37 of the later using microwave chemistry and tested these against a handful of different MMPs.
Both libraries produced hits against MMP-2, MMP-3, MMP-8, and MMP-9. Control compounds designed not to chelate zinc showed no activity, and X-ray adsorption fine structure spectroscopy experiments suggest that the molecules are indeed binding to the catalytic zinc. Selectivity is often an issue in targeting metalloproteinases, and it was thus gratifying to find that at least one fragment inhibited MMP-2 with low micromolar activity while showing no activity against the other MMPs. Molecular modeling provides some rationale for this selectivity.
One could argue that many of the library members do not meet conventional definitions of fragments, and could be seen as more scaffold-like (or worse – one has a molecular weight pushing 600 Daltons!) And of course, it is not clear that either scaffold will be suitable for drug development or even tool compounds – it is possible their propensity for zinc binding will be a problem inside cells. Still, the notion of creating custom-made fragment libraries for various classes of targets certainly makes sense; folks have done this for kinases and even RNA, and it is reasonable to see this approach extended to metalloproteinases. Cohen and colleagues described a fragment library consisting of more conventional metal chelators earlier this year in ChemMedChem.
This publication also confirms the results of our poll that fragment-based approaches are catching on in academia. But industry is already in the sandbox: at least two companies, AnCore and Viamet, are using similar strategies to target metalloproteins.
23 June 2010
Poll results: academia/industry
Just a quick summary of our poll last month. The results (below and to right) show that just over half of you who responded (51%) are in industry and just under half (46%) are in academia, government, or other non-profit organizations. Also, over three-quarters of you (77%) are active practitioners of FBDD.
Thanks to everyone (over four score) who took the time to respond!
Thanks to everyone (over four score) who took the time to respond!
11 June 2010
Fragment linking: how much is it worth?
Fragment linking is a topic we’ve discussed a few times. One of its great appeals is that, all other things being equal, the entropic cost of binding one linked molecule is less than the cost of binding two separate molecules. Thus, linking two fragments should give more than an additive increase in binding energy. As the late William Jencks noted, for two fragments A and B:
The lower the Kd the better, so ideally E < 1, though in practice finding a suitable linker can be tricky and all too often E > 1 (sometimes >> 1). But how low can E go? How much of a boost can you get by linking two fragments? Claudio Luchinat and colleagues at the University of Florence looked at this question experimentally in a recent paper in J. Med. Chem.
The researchers took PMAHA, a known inhibitor of the matrix metalloproteinase MMP-12, and dissected it into two fragments, AHA and PMS, by conceptually “cleaving” the bond connecting them (see figure). This simplifies analysis: since the two fragments are almost identical to the linked molecule, there are no concerns that atoms in the linker interact with the protein.
The crystal structure of PMAHA bound to MMP-12 had been previously reported, but Luchinat and co-workers solved the co-crystal structure of AHA and PMS bound simultaneously to MMP-12. The two fragments overlay fairly well with the parent molecule: AHA binds to the catalytic zinc, while PMS binds in the S1’ pocket. The AHA fragment is rotated with respect to its position in PMAHA, though it makes the same interactions in both structures.
Thermodynamic binding parameters for the three molecules were determined (see figure). As expected, PMAHA binds considerably more tightly than the product of the affinities of the two fragments: E << 1 (in fact, about 0.0021). And in nice accord with theory, this enhanced affinity is entropic: both fragments bind with favorable enthalpy and unfavorable entropy, while the linked molecule has both favorable enthalpy and entropy. In other words, the salutary effect of linking these two fragments does seem to come entirely from entropic effects.
One of the more interesting lessons from this paper is a sense of how much of a boost in potency you can expect if fragment linking goes well: about 500-fold. In theory you could do better, but in practice you should expect much more modest benefits: a prominent success of SAR by NMR on a different metalloproteinase reported a 14-fold boost in affinity. But just like the lottery, the hope of a big payout will continue to attract people to the linking game.
Kd(AB) = Kd(A) * Kd(B) * E
Where
Kd(AB) is the dissociation constant for the linked molecule AB
Kd(A) is the dissociation constant for fragment A
Kd(B) is the dissociation constant for fragment B
E is a “linking coefficient”, reflecting the costs and benefits of linking
The lower the Kd the better, so ideally E < 1, though in practice finding a suitable linker can be tricky and all too often E > 1 (sometimes >> 1). But how low can E go? How much of a boost can you get by linking two fragments? Claudio Luchinat and colleagues at the University of Florence looked at this question experimentally in a recent paper in J. Med. Chem.
The researchers took PMAHA, a known inhibitor of the matrix metalloproteinase MMP-12, and dissected it into two fragments, AHA and PMS, by conceptually “cleaving” the bond connecting them (see figure). This simplifies analysis: since the two fragments are almost identical to the linked molecule, there are no concerns that atoms in the linker interact with the protein.
The crystal structure of PMAHA bound to MMP-12 had been previously reported, but Luchinat and co-workers solved the co-crystal structure of AHA and PMS bound simultaneously to MMP-12. The two fragments overlay fairly well with the parent molecule: AHA binds to the catalytic zinc, while PMS binds in the S1’ pocket. The AHA fragment is rotated with respect to its position in PMAHA, though it makes the same interactions in both structures.
Thermodynamic binding parameters for the three molecules were determined (see figure). As expected, PMAHA binds considerably more tightly than the product of the affinities of the two fragments: E << 1 (in fact, about 0.0021). And in nice accord with theory, this enhanced affinity is entropic: both fragments bind with favorable enthalpy and unfavorable entropy, while the linked molecule has both favorable enthalpy and entropy. In other words, the salutary effect of linking these two fragments does seem to come entirely from entropic effects.
One of the more interesting lessons from this paper is a sense of how much of a boost in potency you can expect if fragment linking goes well: about 500-fold. In theory you could do better, but in practice you should expect much more modest benefits: a prominent success of SAR by NMR on a different metalloproteinase reported a 14-fold boost in affinity. But just like the lottery, the hope of a big payout will continue to attract people to the linking game.
31 May 2010
Fragments vs Abl: antagonists and agonists
A common concern with using biophysical techniques to identify fragments is that the functional implications of identified binders are not always clear, an issue we’ve discussed previously. In a new paper in J. Am. Chem. Soc., Wolfgang Jahnke (co-editor of the first book on FBDD) and colleagues at Novartis describe a clever NMR approach to address this problem and identify both agonists and antagonists that bind to an allosteric site on the protein tyrosine kinase Abl.
Abl is less well known than its famous cousin, Bcr-Abl, an oncogenic fusion protein in which the kinase activity is always turned on. Bcr-Abl is targeted by imatinib and a number of other kinase inhibitors; indeed, the success of imatinib against certain types of cancer has been largely responsible for the rush to develop drugs targeting kinases.
Most kinase-targeted drugs (including imatinib) bind in or near the conserved ATP-binding site. However, Abl offers another binding site, a pocket that can be filled by the fatty-acid myristic acid. This interaction causes conformational changes in the protein, stabilizing an inactive state. Indeed, previous research had identified molecules that bind in this pocket and block activity. Jahnke and colleagues used NMR screening of a 500-fragment library to try to identify new chemical scaffolds.
Several fragments were identified, some of which bound relatively tightly as judged by NMR and ITC. However, these fragments did not inhibit kinase activity. Crystallographic analysis of the fragments bound to Abl revealed that, although the fragments do bind in the myristate pocket, their binding modes are incompatible with the conformational changes needed to inhibit the kinase. Realizing that a specific valine residue is structurally disordered in the absence of myristate, the researchers established an NMR assay using Abl in which valine had been isotopically labeled to assess which molecules bind in a similar fashion to myristate (and thus block activity).
But what of the molecules that bind in the myristate pocket without causing conformational changes? Some of these can actually activate the kinase by competing with endogenous myristoyl groups. Fragment-based discovery of agonists is not unprecedented (see for example here and here), but it is rare. Assays such as the one described here to distinguish between different conformations of a protein could be practical complements to approaches that focus on binding alone. The paper is also a useful reminder that binders are not necessarily inhibitors, and can in fact be just the opposite.
Abl is less well known than its famous cousin, Bcr-Abl, an oncogenic fusion protein in which the kinase activity is always turned on. Bcr-Abl is targeted by imatinib and a number of other kinase inhibitors; indeed, the success of imatinib against certain types of cancer has been largely responsible for the rush to develop drugs targeting kinases.
Most kinase-targeted drugs (including imatinib) bind in or near the conserved ATP-binding site. However, Abl offers another binding site, a pocket that can be filled by the fatty-acid myristic acid. This interaction causes conformational changes in the protein, stabilizing an inactive state. Indeed, previous research had identified molecules that bind in this pocket and block activity. Jahnke and colleagues used NMR screening of a 500-fragment library to try to identify new chemical scaffolds.
Several fragments were identified, some of which bound relatively tightly as judged by NMR and ITC. However, these fragments did not inhibit kinase activity. Crystallographic analysis of the fragments bound to Abl revealed that, although the fragments do bind in the myristate pocket, their binding modes are incompatible with the conformational changes needed to inhibit the kinase. Realizing that a specific valine residue is structurally disordered in the absence of myristate, the researchers established an NMR assay using Abl in which valine had been isotopically labeled to assess which molecules bind in a similar fashion to myristate (and thus block activity).
But what of the molecules that bind in the myristate pocket without causing conformational changes? Some of these can actually activate the kinase by competing with endogenous myristoyl groups. Fragment-based discovery of agonists is not unprecedented (see for example here and here), but it is rare. Assays such as the one described here to distinguish between different conformations of a protein could be practical complements to approaches that focus on binding alone. The paper is also a useful reminder that binders are not necessarily inhibitors, and can in fact be just the opposite.
20 May 2010
A HERD of hydrogen-bonding fragments
The amino acids histidine (H or His), glutamic acid (E or Glu), arginine (R or Arg) and aspartic acid (D or Asp) are often found in ligand-binding sites in proteins. As such, finding fragments that preferentially interact with these amino acids could be useful for fragment-based ligand discovery. David Selwood and colleagues at University College London have combed the Protein Data Bank (PDB) to do just this; they report their results in a recent issue of J. Med. Chem.
The researchers analyzed over 8000 high-resolution protein-ligand structures in which the ligands formed hydrogen bonds with the side chains of His, Glu, Arg, or Asp. They defined fragments as “the largest ring assembly containing the atoms involved in hydrogen bonding.” This excludes functional groups linked to aliphatic chains, but given the importance of rings in most drug molecules this limitation seems reasonable. A total of 462 fragments were found; the number of fragments making hydrogen bonds with each amino acid was broadly similar, with a low of 130 for Arg and a high of 159 for Asp.
The diversity of fragments that interact with the acidic side chains of Asp and Glu is lower than that of fragments interacting with the basic side chains of His and Arg. Not surprisingly, amidines represent a large fraction of the former; these form two hydrogen bonds with either Glu or, more frequently, Asp. Cyclic diols are also common double hydrogen-bonding fragments for Glu and Asp, while cyclic aliphatic amines are perhaps less common than one might expect.
Among fragments that interact with Arg, 124 (91%) do so through an oxygen atom, with only 7 (5%) interacting through nitrogen, and a handful interacting through halogens (5) or sulfur (1 example). The His residue also shows a preference for oxygen-containing fragments, though at 63% this is less pronounced than the much more basic Arg.
One of the attractive features of this work is that, by focusing on fragments that form hydrogen bonds, the free energy of binding is likely to be dominated by enthalpic rather than entropic terms. As has been discussed (here, here, and here) this has some potential advantages for drugs.
This sort of analysis always leads one to ask whether the identified fragments represent limits on what could bind, or, as the researchers also speculate, “the limited variability of currently available chemical libraries from which drugs are derived.” There is clearly some justification for thinking the latter: the preponderance of amidines is likely due to the number of serine proteases that have been targeted with this functional group. Nonetheless, the overall set of fragments could be quite useful for computational screening. In fact, many of them could also be incorporated into physical fragment libraries. Perhaps one of the enterprising commercial fragment library suppliers will put together a sub-library based on this work.
The researchers analyzed over 8000 high-resolution protein-ligand structures in which the ligands formed hydrogen bonds with the side chains of His, Glu, Arg, or Asp. They defined fragments as “the largest ring assembly containing the atoms involved in hydrogen bonding.” This excludes functional groups linked to aliphatic chains, but given the importance of rings in most drug molecules this limitation seems reasonable. A total of 462 fragments were found; the number of fragments making hydrogen bonds with each amino acid was broadly similar, with a low of 130 for Arg and a high of 159 for Asp.
The diversity of fragments that interact with the acidic side chains of Asp and Glu is lower than that of fragments interacting with the basic side chains of His and Arg. Not surprisingly, amidines represent a large fraction of the former; these form two hydrogen bonds with either Glu or, more frequently, Asp. Cyclic diols are also common double hydrogen-bonding fragments for Glu and Asp, while cyclic aliphatic amines are perhaps less common than one might expect.
Among fragments that interact with Arg, 124 (91%) do so through an oxygen atom, with only 7 (5%) interacting through nitrogen, and a handful interacting through halogens (5) or sulfur (1 example). The His residue also shows a preference for oxygen-containing fragments, though at 63% this is less pronounced than the much more basic Arg.
One of the attractive features of this work is that, by focusing on fragments that form hydrogen bonds, the free energy of binding is likely to be dominated by enthalpic rather than entropic terms. As has been discussed (here, here, and here) this has some potential advantages for drugs.
This sort of analysis always leads one to ask whether the identified fragments represent limits on what could bind, or, as the researchers also speculate, “the limited variability of currently available chemical libraries from which drugs are derived.” There is clearly some justification for thinking the latter: the preponderance of amidines is likely due to the number of serine proteases that have been targeted with this functional group. Nonetheless, the overall set of fragments could be quite useful for computational screening. In fact, many of them could also be incorporated into physical fragment libraries. Perhaps one of the enterprising commercial fragment library suppliers will put together a sub-library based on this work.
19 May 2010
Updated again: Fragment-based conferences in 2010
The year is not even half over, but most of the fragment events are behind us (see summaries here, here, and here). You still have a couple more opportunities for some great conferences.
June 6-9: The 32nd National Medicinal Chemistry Symposium will be held in Minneapolis, Minnesota, and Dave Rees is organizing a session on fragments on June 9. Looks like a great lineup, with top speakers from Astex, Plexxikon, Novartis, Abbott, and UC Berkeley.
October 10-13: Fragment-based Lead Discovery 2010 is the first major fragment event on the east coast of the US (in Philadelphia, PA). There are lots of excellent speakers, and the conference is still accepting abstracts for talks until May 31. Early bird-registration also ends that day. If you haven’t made it to any other fragment events this year, don’t miss FBLD 2010!
Know of anything else? Organizing a fragment event? Let us know and we’ll get the word out.
June 6-9: The 32nd National Medicinal Chemistry Symposium will be held in Minneapolis, Minnesota, and Dave Rees is organizing a session on fragments on June 9. Looks like a great lineup, with top speakers from Astex, Plexxikon, Novartis, Abbott, and UC Berkeley.
October 10-13: Fragment-based Lead Discovery 2010 is the first major fragment event on the east coast of the US (in Philadelphia, PA). There are lots of excellent speakers, and the conference is still accepting abstracts for talks until May 31. Early bird-registration also ends that day. If you haven’t made it to any other fragment events this year, don’t miss FBLD 2010!
Know of anything else? Organizing a fragment event? Let us know and we’ll get the word out.
14 May 2010
Poll: academia/industry
It’s been a while since we’ve done a poll, but the latest post at FBDD-Lit on fragments in academia, combined with an earlier post on this site, leads us to wonder how many of our readers are from academia and how many are from industry. (For purposes of this poll, let’s lump government and other non-profit organizations with academia).
Please respond by clicking on the right-hand side of the page – we’d like to know more about you!
Please respond by clicking on the right-hand side of the page – we’d like to know more about you!
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.
Subscribe to:
Posts (Atom)
Subscribe To
Posts
All Comments