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?
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