There is a provocative Perspective piece in a recent Nature titled "Too many roads not taken". In it, the authors summarize some more extensive work (posted on Arxiv) and others showing that in several major fields of protein research, the biological community seems to be stuck on a few old favorites, with insufficient attention to other key players.
In the Nature piece, the focus is on protein kinases and nuclear hormone receptors, both key druggable classes. In the kinase area, they have a graph (the Arxiv site leads to the actual data) showing that the graph of attention (measured in bibliographic citations) given to kinases prior to 2003 is generally predictive of the attention given them in 2009; only a handful of kinases with little attention in the first time period are hot areas now. Of course, if the rest of the kinome were boring this would be justifiable, but they point to several papers which indicate that many of the unstudied kinases (for example, this series of shRNA experiments)
A very striking conclusion is underlined by their plot of NHR citations by gene, which is expanded upon significantly in the Arxiv preprint. Their contention is that the availability of chemical probes drives analysis in this family, and in their rank-ordered histogram of activity the proteins with chemical ligands are all at the top of the list. Granted, at the breakpoint the bars are nearly equally tiny on either side of the divide, but it does suggest a strong correlation. Easy to build universal approaches such as RNAi are very powerful, but especially for any kind of in vivo study are suboptimal -- not only are they hard to deliver, but utterly wiping out a protein is not a subtle way to probe a system. The Arxiv piece goes into more detail and posits that you really need good compounds, ones with good bioavailability and such. In other words, drug-like compounds.
So, they plead that we need to invest more in getting such compounds into the research community. Some of this could take the form of funding screening efforts in the public sphere, which like sequencing the human genome isn't the sexiest project from a scientific perspective -- just like the foundation is never the sexiest part of a great building. But foundations matter!
An alternative, suggested by the Arxiv paper, is that compounds may exist in the patent literature but are not widely commercially available. So, a public effort might focus on mining the patent literature, synthesizing various claimed compounds and verifying their activity. Of course, some (many?) compounds in the patents might not stand up to scrutiny. Another alternative would be to attempt to devise incentives for industry to release some of the inhibitors they have in house; obviously this would be a challenge as such compounds might prove to be the starting points for valuable drugs -- but of course they never will until someone proves the underlying kinases valueable. Some companies have generated unbelievable databases on kinase inhibitors (for example, this recent study) which have the potential to fill in the missing spots on the kinase tool compound grid.
I can't help thinking of the new translational medicine institute going in at the NIH. Trying to move research findings into the clinic to benefit patients is hard to argue against, but perhaps the NIH needs to also pour some resources into the humble task of finding basic tools.
Showing posts with label kinases. Show all posts
Showing posts with label kinases. Show all posts
Thursday, February 17, 2011
Friday, September 28, 2007
Casting a beady eye on kinases
A huge area of drug discovery is targeting protein kinases. There are about 500 protein kinase-like proteins in the human proteome. Some of these are probably not active (pseudokinases; review [paid]) and periodically there are claims of protein kinase activity in novel proteins, but that's the ballpark. An increasing number of drugs target these, with Gleevec as perhaps the best known but a large parade of others coming forward.
Most kinase-targeting drugs compete with ATP in the active site. The ATP-binding site of kinases shows a lot of conservation, and so cross-reactivity is a big topic in the field. What is desired for specificity depends on the target & disease & one's tolerance for risk. Gleevec was originally touted as being laser-focused on BCR-ABL, but it actually hits a number of kinases and many of these have yielded new markets, such as KIT for gastrointestinal stromal tumors. Being 'dirty' may be useful in oncology, where many kinases may be contributing to the tumor's growth & survival. On the other hand, in chronic diseases one probably wants a really focused drug (or at least can't tolerate one that isn't).
I got involved a little in kinase screening back at MLNM. The workhorse in the industry are in vitro assays using purified kinases. These are useful and can be run en masse, but everyone knows in vitro isn't always predictive of in vivo. Furthermore, despite diligent efforts by a number of vendors, not every kinase is available. So, the field is ripe for development of new approaches, especially ones which explore the compound in vivo.
In an ideal world, there would be a complete panel of biomarkers specific for each kinase which could be used to measure the impact of a compound on every kinase-regulated pathway in the cell. That's a long, long ways off -- only a few kinases really have good, reliable assays & many are essentially uncharacterized.
The latest Nature Biotech has a nice paper from CellZome on a proteomic approach to the problem & an accompanying News & Views item from a top mass spec person (either link prior requires Nat Biotech subscription, but Cellzome has the paper for free also).
The strategy is to derivatize beads with promiscuous kinase-binding compounds and use these to pull down bound kinases for identification by Mass Spec. Such has been described previously, particularly by the company which Cellzome acquired which had been doing this work. Big advances in this paper is to use iTRAQ labeling reagents to enable accurate quantification of the bound proteins and to use a competitive-binding format to assay test compounds.
The competitive binding angle is clever. Past efforts tried to derivatize the compound of interest to link it to the beads. This has many undesirable properties: the linkage may change the binding properties, each compound must be worked out separately. etc. The new work identified a set of standard promiscuous-binders which can be used to get a large fraction of the kinase repertoire. This same set can be used with just about any compound. By using these in a competitive format, where what is measured is how much the compound of interest disturbs the binding profile of the kinobeads, quantitative measurements can be made. Entire binding curves can be pulled out from a few experiments -- and binding curves for every kinase reliably pulled down by the beads. Slick!
Their coverage of the kinase world is quite good, though not perfect. In a set of experiments they pulled down 307 kinases. While that isn't everything, it's a lot -- and some of the rest may be pseudokinases or not expressed in any of the tissues they looked at. However, some probably just aren't bound well by the reference compound set. Whole small subtrees are missing from their Figure 2 -- examples of the missing include all the GPCR kinases (BARK, GRKs, etc), the two TLKS1, 2/4 polos, the WNKS, none of the 3 Akts, a number of miscellaneous cell cycle kinases (CDC7, BubR1&R2, etc. Some of those are mighty interesting, but of course the solution is to find more compounds to put on the beads.
What's also nice is that the assay isn't limited to kinases -- lots of other stuff comes down too. Initially it was apparently clogged with heat shock proteins (using a n ATP analog as the probe). But, in the current format they found a good sampling of non-kinases, and for Gleevec identified a candidate off-target with some known biology.
So what's the catch? Well, there are a few caveats. First, it is a binding assay, so hits need to be followed up to demonstrate actual inhibition. Second, it is going to be ATP-site specific. That covers most current kinase inhibitors, but there are probably many out there that work outside the ATP site, and this method will be blind to that (or to off-targets bound not by ATP-mimicry). Third, many of things being pulled down won't have much known about them -- do you worry about it or not?
As described in the M&M, the assay requires 5 mL of cell lysate -- not tiny, but not whopping. So this probably wouldn't be applied to every compound coming out of medicinal chemistry, but perhaps to either characterize representative members of particular lead families and to characterize compounds that are quite a ways down the med chem path.
One other interesting bit at the end: instead of adding the test compounds to lysates, they added the compounds & waited a few hours. As they note, many kinase inhibitors have slow off-rates -- they stick to their target rather fiercely. So, the assay can be run after a delay. The peptide mixtures could then be subjected to phosphopeptide enrichment & identification, enabling the phosphorylation state of downstream kinases to be probed & examined in relation to the concentration of compound used.
Most kinase-targeting drugs compete with ATP in the active site. The ATP-binding site of kinases shows a lot of conservation, and so cross-reactivity is a big topic in the field. What is desired for specificity depends on the target & disease & one's tolerance for risk. Gleevec was originally touted as being laser-focused on BCR-ABL, but it actually hits a number of kinases and many of these have yielded new markets, such as KIT for gastrointestinal stromal tumors. Being 'dirty' may be useful in oncology, where many kinases may be contributing to the tumor's growth & survival. On the other hand, in chronic diseases one probably wants a really focused drug (or at least can't tolerate one that isn't).
I got involved a little in kinase screening back at MLNM. The workhorse in the industry are in vitro assays using purified kinases. These are useful and can be run en masse, but everyone knows in vitro isn't always predictive of in vivo. Furthermore, despite diligent efforts by a number of vendors, not every kinase is available. So, the field is ripe for development of new approaches, especially ones which explore the compound in vivo.
In an ideal world, there would be a complete panel of biomarkers specific for each kinase which could be used to measure the impact of a compound on every kinase-regulated pathway in the cell. That's a long, long ways off -- only a few kinases really have good, reliable assays & many are essentially uncharacterized.
The latest Nature Biotech has a nice paper from CellZome on a proteomic approach to the problem & an accompanying News & Views item from a top mass spec person (either link prior requires Nat Biotech subscription, but Cellzome has the paper for free also).
The strategy is to derivatize beads with promiscuous kinase-binding compounds and use these to pull down bound kinases for identification by Mass Spec. Such has been described previously, particularly by the company which Cellzome acquired which had been doing this work. Big advances in this paper is to use iTRAQ labeling reagents to enable accurate quantification of the bound proteins and to use a competitive-binding format to assay test compounds.
The competitive binding angle is clever. Past efforts tried to derivatize the compound of interest to link it to the beads. This has many undesirable properties: the linkage may change the binding properties, each compound must be worked out separately. etc. The new work identified a set of standard promiscuous-binders which can be used to get a large fraction of the kinase repertoire. This same set can be used with just about any compound. By using these in a competitive format, where what is measured is how much the compound of interest disturbs the binding profile of the kinobeads, quantitative measurements can be made. Entire binding curves can be pulled out from a few experiments -- and binding curves for every kinase reliably pulled down by the beads. Slick!
Their coverage of the kinase world is quite good, though not perfect. In a set of experiments they pulled down 307 kinases. While that isn't everything, it's a lot -- and some of the rest may be pseudokinases or not expressed in any of the tissues they looked at. However, some probably just aren't bound well by the reference compound set. Whole small subtrees are missing from their Figure 2 -- examples of the missing include all the GPCR kinases (BARK, GRKs, etc), the two TLKS1, 2/4 polos, the WNKS, none of the 3 Akts, a number of miscellaneous cell cycle kinases (CDC7, BubR1&R2, etc. Some of those are mighty interesting, but of course the solution is to find more compounds to put on the beads.
What's also nice is that the assay isn't limited to kinases -- lots of other stuff comes down too. Initially it was apparently clogged with heat shock proteins (using a n ATP analog as the probe). But, in the current format they found a good sampling of non-kinases, and for Gleevec identified a candidate off-target with some known biology.
So what's the catch? Well, there are a few caveats. First, it is a binding assay, so hits need to be followed up to demonstrate actual inhibition. Second, it is going to be ATP-site specific. That covers most current kinase inhibitors, but there are probably many out there that work outside the ATP site, and this method will be blind to that (or to off-targets bound not by ATP-mimicry). Third, many of things being pulled down won't have much known about them -- do you worry about it or not?
As described in the M&M, the assay requires 5 mL of cell lysate -- not tiny, but not whopping. So this probably wouldn't be applied to every compound coming out of medicinal chemistry, but perhaps to either characterize representative members of particular lead families and to characterize compounds that are quite a ways down the med chem path.
One other interesting bit at the end: instead of adding the test compounds to lysates, they added the compounds & waited a few hours. As they note, many kinase inhibitors have slow off-rates -- they stick to their target rather fiercely. So, the assay can be run after a delay. The peptide mixtures could then be subjected to phosphopeptide enrichment & identification, enabling the phosphorylation state of downstream kinases to be probed & examined in relation to the concentration of compound used.
Thursday, February 15, 2007
A Novel Wooden Shoe
It is increasingly clear that many pathogens do not simply assault our bodies, but truly attempt to infiltrate them and co-opt normal cellular processes to their ends. A number of recent papers have described kinase-based strategies employed by Plasmodium and Toxoplasma.
Protein kinases are critical players in cellular signal transduction. By attaching a phosphate group to a serine, threonine or tyrosine (and occasionally, at least known to date, histidine) they can radically change the local structure and charge distribution of the substrate protein. This can in turn trigger intramolecular rearrangements or alter protein-protein interactions, which can cascade in numerous ways. Tapping into this network is potentially a devastating way to sabotage the host's cellular machinery.
Kinases are opposed in their action by protein phosphatases, which knock the phosphates off the proteins. In most cases, kinase and phosphatase can easily interchange the states of the protein; there can be infinite back-and-forth so long as the protein sticks around. A few cases have been found where re-phosphorylation is presented by glycosylation of a residue -- the same side chain oxygen can't have both at once. But glycosylation can also be reversed, so this so-called yin-yang regulation simply adds a third possible state. A quick PubMed search reveals at least one reported example of host phosphatases being manipulated by a pathogen, albeit indirectly.
The newest Science adds a new pathogen sabot to those uncovered previously. Instead of merely stripping the phosphate off and leaving an exposed hydroxyl for re-phosphorylation, in this example the Shigella protein is a phosphothreonine lyase, an enzyme which actually modifies the side chain, abstracting both the phosphate and a hydrogen and leaving behind a double bond (see Figure 3D). Hence, the kinases so altered cannot be re-phosphorylated -- they are now permanently disabled. Since such dead proteins have the ability to tie up functional binding partners, acting as a dominant negative, this is potentially a very effective strategy. Also striking is the fact that a phospho-amino acid lyase activity had not described previously. It is difficult to believe this is the last novel biochemical strategem we will find in a pathogen's playbook.
Protein kinases are critical players in cellular signal transduction. By attaching a phosphate group to a serine, threonine or tyrosine (and occasionally, at least known to date, histidine) they can radically change the local structure and charge distribution of the substrate protein. This can in turn trigger intramolecular rearrangements or alter protein-protein interactions, which can cascade in numerous ways. Tapping into this network is potentially a devastating way to sabotage the host's cellular machinery.
Kinases are opposed in their action by protein phosphatases, which knock the phosphates off the proteins. In most cases, kinase and phosphatase can easily interchange the states of the protein; there can be infinite back-and-forth so long as the protein sticks around. A few cases have been found where re-phosphorylation is presented by glycosylation of a residue -- the same side chain oxygen can't have both at once. But glycosylation can also be reversed, so this so-called yin-yang regulation simply adds a third possible state. A quick PubMed search reveals at least one reported example of host phosphatases being manipulated by a pathogen, albeit indirectly.
The newest Science adds a new pathogen sabot to those uncovered previously. Instead of merely stripping the phosphate off and leaving an exposed hydroxyl for re-phosphorylation, in this example the Shigella protein is a phosphothreonine lyase, an enzyme which actually modifies the side chain, abstracting both the phosphate and a hydrogen and leaving behind a double bond (see Figure 3D). Hence, the kinases so altered cannot be re-phosphorylated -- they are now permanently disabled. Since such dead proteins have the ability to tie up functional binding partners, acting as a dominant negative, this is potentially a very effective strategy. Also striking is the fact that a phospho-amino acid lyase activity had not described previously. It is difficult to believe this is the last novel biochemical strategem we will find in a pathogen's playbook.
Friday, November 03, 2006
Phosphopallooza.
Protein phosphorylation is a hot topic in signal transduction research. Kinases can add phosphate groups to serines, threonines & tyrosines (and very rarely histidines), and phosphatases can take them off. These phosphorylations can shift the shape of the protein directly, or create (or destroy) binding sites for other proteins. Such bindings can in turn cause the assembly/disassembly of protein complexes, trigger the transport of a protein to another part of the cell, or lead to the protein being destroyed (or prevent such) by the proteasome. This is hardly a comprehensive list of what can happen.
Furthermore, a large (by some estimates 1/4 to 1/5) amount of the pharmaceutical industries efforts, including those at my (soon to be ex-) employer Millennium, are targeting protein kinases. If you wish to drug kinases, you really want to know what the downstream biology is and that starts with what does your kinase phosphorylate, when does it do it, and what events do those phosphorylations trigger.
A large number of methods have been published for finding phosphorylation sites on proteins, but by far the most productive have been mass spectrometric ones (MS for short). Using various sample workup strategies, cleverer-and-cleverer instrument designs, and better software, the MS folks keep pushing the envelope in an impressive manner.
The latest Cell has the latest leap forward: a paper describing 6,600 phosphorylation sites (on 2,244 proteins). To put this in perspective, the total number of previously published human phosphorylation sites (by my count) was around 12,000 -- this paper has found 50% as many as were previously known! Some prior papers (such as these two examples) had found close to 2,000 sites.
Now some of this depth came from many MS runs -- but that in itself illustrates how this task is getting simpler; otherwise so many runs wouldn't be practical. The multiple runs also were used to gather more data: looking at phosphorylation changes (quantitatively!) over a timecourse.
One this this study wasn't designed to do is clearly assign the sites to kinases. Bioinformatic methods can be used to make guesses, but without some really painful work you can't really make a strong case. And if the site shouldn't look like any pattern for a known kinase -- good luck! There really aren't great methods for solving this (not to say there aren't some really clever tries).
Also interesting in this study is the low degree of overlap with previous studies. While the reference set they used is probably quite a bit lower than the 12K estimate I give, it is still quite large -- and most sites in the new paper weren't found in the older ones. There are in excess of 20 million Ser/Thr/Tyr in the proteome and many are probably not phosphorylated, but certainly a reasonable estimate would be north of 20K are.
For drug discovery, the sort of timecourse data in this paper is another proof-of-concept of the idea of discovering biomarkers for your kinase using high-throughput MS approaches (another case can be found in another paper). By pushing for so many sites, the number of candidates goes up substantially, since many sites found aren't modulated in an interesting way, at least in terms of pursuing a biomarker. This is noted in Figure 3 -- for the same protein, the temporal dynamics of phosphorylation at different sites can be quite different.
However, it remains to be seen how far into the process these MS approaches can be pushed. Most likely, the sites of interest will need to probed with immunologic assays, as previously discussed.
Protein phosphorylation is a hot topic in signal transduction research. Kinases can add phosphate groups to serines, threonines & tyrosines (and very rarely histidines), and phosphatases can take them off. These phosphorylations can shift the shape of the protein directly, or create (or destroy) binding sites for other proteins. Such bindings can in turn cause the assembly/disassembly of protein complexes, trigger the transport of a protein to another part of the cell, or lead to the protein being destroyed (or prevent such) by the proteasome. This is hardly a comprehensive list of what can happen.
Furthermore, a large (by some estimates 1/4 to 1/5) amount of the pharmaceutical industries efforts, including those at my (soon to be ex-) employer Millennium, are targeting protein kinases. If you wish to drug kinases, you really want to know what the downstream biology is and that starts with what does your kinase phosphorylate, when does it do it, and what events do those phosphorylations trigger.
A large number of methods have been published for finding phosphorylation sites on proteins, but by far the most productive have been mass spectrometric ones (MS for short). Using various sample workup strategies, cleverer-and-cleverer instrument designs, and better software, the MS folks keep pushing the envelope in an impressive manner.
The latest Cell has the latest leap forward: a paper describing 6,600 phosphorylation sites (on 2,244 proteins). To put this in perspective, the total number of previously published human phosphorylation sites (by my count) was around 12,000 -- this paper has found 50% as many as were previously known! Some prior papers (such as these two examples) had found close to 2,000 sites.
Now some of this depth came from many MS runs -- but that in itself illustrates how this task is getting simpler; otherwise so many runs wouldn't be practical. The multiple runs also were used to gather more data: looking at phosphorylation changes (quantitatively!) over a timecourse.
One this this study wasn't designed to do is clearly assign the sites to kinases. Bioinformatic methods can be used to make guesses, but without some really painful work you can't really make a strong case. And if the site shouldn't look like any pattern for a known kinase -- good luck! There really aren't great methods for solving this (not to say there aren't some really clever tries).
Also interesting in this study is the low degree of overlap with previous studies. While the reference set they used is probably quite a bit lower than the 12K estimate I give, it is still quite large -- and most sites in the new paper weren't found in the older ones. There are in excess of 20 million Ser/Thr/Tyr in the proteome and many are probably not phosphorylated, but certainly a reasonable estimate would be north of 20K are.
For drug discovery, the sort of timecourse data in this paper is another proof-of-concept of the idea of discovering biomarkers for your kinase using high-throughput MS approaches (another case can be found in another paper). By pushing for so many sites, the number of candidates goes up substantially, since many sites found aren't modulated in an interesting way, at least in terms of pursuing a biomarker. This is noted in Figure 3 -- for the same protein, the temporal dynamics of phosphorylation at different sites can be quite different.
However, it remains to be seen how far into the process these MS approaches can be pushed. Most likely, the sites of interest will need to probed with immunologic assays, as previously discussed.
Subscribe to:
Posts (Atom)