29 November 2010
More techniques: NovAliX and Graffinity combine MS and SPR
We’ve written previously about Graffinity, which uses surface plasmon resonance (SPR) to find fragments, and NovAliX, which initially focused on mass spectrometry. The two companies have been collaborating since last year, and this has apparently been a fruitful partnership: this month NovAlix acquired a majority ownership stake in Graffinity (click here for press release). Earlier this year NovAlix also purchased an NMR-focused company, and they have already added crystallography expertise. Finding fragments effectively requires using a range of orthogonal technologies, and this latest move gives NovAlix a full suite of biophysical techniques.
12 November 2010
Fragments vs PDK1
Kinases have been a particularly productive target class for fragment-based drug discovery (and drug discovery in general), with nearly half of reported FBDD-derived clinical candidates targeting kinases. The latest dispatch from this field can be found in the November issue of ACS Medicinal Chemistry Letters.
In this paper, Jeffrey Axten and colleagues at GlaxoSmithKline describe their use of fragment screening to identify inhibitors of PDK1, a popular anti-cancer target. They started by assembling a library of fragments biased towards the purine-binding site of kinases, and tested 1065 of these in a biochemical screen at 400 micromolar concentration. Of these, 193 inhibited activity at least 60% and were further characterized; 89 had IC50 values better than 400 micromolar. A set of 36 of these, chosen on the basis of ligand efficiency and chemical tractability, were chosen for follow-up.
Saturation transfer difference (STD) NMR was used to confirm which fragments bound to PDK, which cut the number of hits in half. X-ray crystallography experiments were started before NMR and performed on 7 fragments; only the fragments that were confirmed by NMR gave interpretable data. One of these was the aminoindazole compound 8 (see figure).
A substructure search was conducted to find more elaborated molecules within the corporate screening collection, leading to compound 19, which has sub-micromolar potency. This compound also showed some signs of selectivity for PDK1 over other kinases. Although the paper stops here, Jeffrey Axten gave a nice presentation at FBLD 2010 in which he discussed subsequent medicinal chemistry that ultimately led to novel, high picomolar inhibitors of PDK1.
There are at least two lessons from this story. First, the significant attrition from the biochemical screen again emphasizes the need for orthogonal methods of fragment validation. Second, even though the fragment identified has been around the block with respect to kinases (as of last year, the aminoindazole substructure had appeared in over 70 kinase patents), skillful medicinal chemistry can still get you to novel compounds.
In this paper, Jeffrey Axten and colleagues at GlaxoSmithKline describe their use of fragment screening to identify inhibitors of PDK1, a popular anti-cancer target. They started by assembling a library of fragments biased towards the purine-binding site of kinases, and tested 1065 of these in a biochemical screen at 400 micromolar concentration. Of these, 193 inhibited activity at least 60% and were further characterized; 89 had IC50 values better than 400 micromolar. A set of 36 of these, chosen on the basis of ligand efficiency and chemical tractability, were chosen for follow-up.
Saturation transfer difference (STD) NMR was used to confirm which fragments bound to PDK, which cut the number of hits in half. X-ray crystallography experiments were started before NMR and performed on 7 fragments; only the fragments that were confirmed by NMR gave interpretable data. One of these was the aminoindazole compound 8 (see figure).
There are at least two lessons from this story. First, the significant attrition from the biochemical screen again emphasizes the need for orthogonal methods of fragment validation. Second, even though the fragment identified has been around the block with respect to kinases (as of last year, the aminoindazole substructure had appeared in over 70 kinase patents), skillful medicinal chemistry can still get you to novel compounds.
07 November 2010
Pin1 revisited
Earlier this year we highlighted a paper from Vernalis that described the use of NMR methods to discover inhibitors of the anti-cancer target Pin 1. In a recent issue of Bioorg. Med. Chem. Lett. the same team now reports a second series of compounds that inhibit this protein, also discovered and advanced through FBLD. The two papers together provide some interesting lessons.
Rather than using NMR, the researchers identified the second series of compounds with an inhibition assay. After screening 900 fragments at 2 mM, they obtained 40 hits, including 3 compounds previously discovered by NMR. Disturbingly though, follow-up NMR experiments confirmed binding for only 2 of the 37 new hits, suggesting that the remaining compounds may act through pathological mechanisms (see also here). Still, two hits are better than none, and the binding mode of one of the fragments (compound 3 in figure) was determined by X-ray crystallography. Several analogs of this were purchased and tested, and compound 5 was found to have an improved potency and ligand efficiency.
At this point chemistry entered the picture. The researchers synthesized several analogs of compound 5, guided by crystallography and modeling. This led to compound 10e and eventually to compound 20, with sub-micromolar biochemical activity and measurable cell activity.
Ultimately though, as in the previous Pin1 series, even this modest cellular potency was gained at the cost of unacceptable increases in size and hydrophobicity. This brings up an interesting question: at what point do you declare a target undruggable? The authors note that “the nature of the Pin1 active site makes it difficult to optimise hits into drug-like molecules.”
Fragment-based approaches can sometimes deliver inhibitors to challenging targets where HTS has failed. However, if the inhibitors can’t be transformed into drugs, is finding them actually a good thing? Researchers are getting better at improving potency at the same time as ligand efficiency for some targets, but ultimately getting to clinical candidates for harder targets may come down to how many resources you are willing to throw at a project: molecules such as ABT-263, though far from rule-of-5 compliant, are doing well in the clinic, but only after the investment of dozens if not hundreds of people-years.
Rather than using NMR, the researchers identified the second series of compounds with an inhibition assay. After screening 900 fragments at 2 mM, they obtained 40 hits, including 3 compounds previously discovered by NMR. Disturbingly though, follow-up NMR experiments confirmed binding for only 2 of the 37 new hits, suggesting that the remaining compounds may act through pathological mechanisms (see also here). Still, two hits are better than none, and the binding mode of one of the fragments (compound 3 in figure) was determined by X-ray crystallography. Several analogs of this were purchased and tested, and compound 5 was found to have an improved potency and ligand efficiency.
At this point chemistry entered the picture. The researchers synthesized several analogs of compound 5, guided by crystallography and modeling. This led to compound 10e and eventually to compound 20, with sub-micromolar biochemical activity and measurable cell activity.
Ultimately though, as in the previous Pin1 series, even this modest cellular potency was gained at the cost of unacceptable increases in size and hydrophobicity. This brings up an interesting question: at what point do you declare a target undruggable? The authors note that “the nature of the Pin1 active site makes it difficult to optimise hits into drug-like molecules.”
Fragment-based approaches can sometimes deliver inhibitors to challenging targets where HTS has failed. However, if the inhibitors can’t be transformed into drugs, is finding them actually a good thing? Researchers are getting better at improving potency at the same time as ligand efficiency for some targets, but ultimately getting to clinical candidates for harder targets may come down to how many resources you are willing to throw at a project: molecules such as ABT-263, though far from rule-of-5 compliant, are doing well in the clinic, but only after the investment of dozens if not hundreds of people-years.
03 November 2010
Ligand efficiency hot spots
Hot spots are regions of a protein with a particular predilection for binding to small molecules – thermodynamic sinkholes, so to speak. Discovering one of these can get you to potent molecules very quickly. In an effort to better understand hot spots, Iwan de Esch and colleagues at VU University in Amsterdam and collaborators at Beactica have deconstructed a potent ligand for nicotinic acetylcholine binding protein (AChBP), a model protein for ligand-gated ion channels, which are implicated in a variety of neurological diseases. They report their results in a recent issue of J. Med. Chem.
The researchers started with the previously reported quinuclidine compound 6 (see figure) and fragmented this into 20 analogs. They tested these in a surface plasmon resonance (SPR) assay as well as in a more conventional radioligand binding assay; the agreement between these very different assay formats was excellent, further validating the utility of SPR as a useful tool for discovering fragments.
Not surprisingly, some of the fragments have higher ligand efficiencies than the larger, more potent molecule, suggesting that there is a hot-spot that recognizes the core fragment 25 (which is structurally related to nicotine). This concept of “group efficiency” has been described previously, and can be useful for optimizing fragments. For example, compound 22, without a basic nitrogen atom, has the lowest ligand efficiency in the bunch; presumably, simply adding the nitrogen would give a sizable boost in potency.
However, one needs to be cautious. The researchers use computer docking to develop models of how each of these fragments bind, but as we have seen before, isolated fragments do not always recapitulate the binding modes of fully elaborated molecules. Still, particularly in the absence of structure (as is the case with many ion channels), exercises such as this could provide useful ideas for what to do with fragment hits.
The researchers started with the previously reported quinuclidine compound 6 (see figure) and fragmented this into 20 analogs. They tested these in a surface plasmon resonance (SPR) assay as well as in a more conventional radioligand binding assay; the agreement between these very different assay formats was excellent, further validating the utility of SPR as a useful tool for discovering fragments.
Not surprisingly, some of the fragments have higher ligand efficiencies than the larger, more potent molecule, suggesting that there is a hot-spot that recognizes the core fragment 25 (which is structurally related to nicotine). This concept of “group efficiency” has been described previously, and can be useful for optimizing fragments. For example, compound 22, without a basic nitrogen atom, has the lowest ligand efficiency in the bunch; presumably, simply adding the nitrogen would give a sizable boost in potency.
However, one needs to be cautious. The researchers use computer docking to develop models of how each of these fragments bind, but as we have seen before, isolated fragments do not always recapitulate the binding modes of fully elaborated molecules. Still, particularly in the absence of structure (as is the case with many ion channels), exercises such as this could provide useful ideas for what to do with fragment hits.