Cell-based assays:Layout 1 14/1/10 19:58 Page 69
Assays
Richard Eglen: So you mean it’s function? It’s a David Marks: Because that was easy to do. A lot of
transducer. chemists are not really focused on allosteric kinases.
A biologist would be more interested in that. And
Arthur Christopoulos: Yes, I mean it’s function. actually now there’s been more and more of that.
GPCRs, they’re promiscuous. They need partners that
can change, a lot. And the coupling partners, even in Arthur Christopoulos: Actually, that’s a good point.
the given cell type can vary a lot, and the same GPCR Let me ask this question. Is that then because of the
can simultaneously couple here, there, and every- switch in the way people screen for kinases?
where. And also in terms of the ligands that they
recognise, I think other than maybe drug transporters, David Marks: Yes.
GPCRs probably recognise a larger diversity of ligands
than any other type of protein. Moreso than kinases. Richard Eglen: I think the way we see it as an assay
So there are extra challenges there as well, associated provider is that there is definitely the thinking that
with that promiscuity, both on the outside, and on the assays for GPCRs are like looking into the lamp-
inside. These things then move along. post – that is, you do the assay that works because
that’s where the light is, as opposed to maybe that’s
David Marks: I will challenge that, naturally, since what the GPCR actually does physiologically. If
kinases have the same promiscuity if you look you consider to Arthur’s point that GPCRs are
inside the cell. Kinase pathways branch off and probably part of a signal cell, as part of a func-
overlap a lot. So in terms of signalling of the kinase tioning network, then probably what needs to be
in the cell, and how it behaves in different cell developed are systems that would relate to other
types, it is also different. parts of this network. And you need a cellular con-
text to do that. So beta arrestin, beta arrestin inde-
Arthur Christopoulos: Oh, I agree, I’m not denying pendent signalling, for example, GPCRs signalling
that. If you look in fact at the genome, there are through kinases. I think that the kinases are start-
more kinases than GPCRs. Am I wrong? ing to be worked out more quickly, because people
are putting them in the intracellular context.
Richard Eglen: There are 118 kinases.
David Marks: That’s true, if you look at a map of
Arthur Christopoulos: Are there? So about the the kinase.
same. So, they are prevalent, and probably they are
coming up as drug targets, and maybe they’re up Arthur Christopoulos: What’s the substrate for a
there with GPCRs. kinase?
David Marks: But actually there are many more David Marks: There are at least two substrates for
GPCR drugs developed. Based on GPCRs more a kinase.
than kinases. So kinases are actually early, even
though it seems like it’s very well developed in Arthur Christopoulos: That’s why I’m asking. I’m
terms of its assays. trying to get us on the same...
Arthur Christopoulos: Yes, but there were all those Guido Zaman: A protein and an ATP.
… I mean that recent Hopkins work that was sug-
gesting kinases are along the road. But in terms of Arthur Christopoulos: A protein and an ATP. So ATP
the level of function, kinases are serving a well- doesn’t vary. But the protein varies dramatically.
defined purpose. Whereas GPCRs, we’re still try-
ing to find out what these things are doing. And in David Marks: The ATP concentration in a cell
terms of targeting – put it this way: do we think doesn’t vary, that’s true. But the context can vary a
there will be more allosteric ligands for kinases, or lot, depending on the concentration of substrate
GPCRs? They both in theory should be both and other accessory matrix proteins that often the
allosterically targetable. kinase can bind to. It could modulate its activity
quite a bit.
Guido Zaman: I think in GPCRs there are more exam-
ples, but in kinases there are now more and more Richard Eglen: So I guess the point is that intra-
examples appearing. I think the traditional kinase cellularly, or even on the tyrosine kinase receptor
technologies, we fished out more ATP competitors. of the membrane, there are a lot of proteins that
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