Cell-based assays:Layout 1 14/1/10 19:58 Page 68
Assays
David Marks: And I think it’s also very important not for ATP competitor compounds, but look
to have eventually a cell type that is the more directly for allosteric modulators.
appropriate cell type for the target. For example,
with metabolic diseases, we always make sure that Martina Bielefeld-Sevigny: So for enzymes, then
we use muscle or liver cells to check up on what- the other question for kinases, what’s the sub-
ever we use as a surrogate cell during the screen. strate? Most of the time you’re working with a
peptide. How relevant is this really now in the cell
Richard Eglen: If you look at the way kinases context? Are there other co-factors that are really
assays have gone, comparing and contrasting them needed that modulate the kinase or proteins? So
to GPCRs – where with kinase assays, biochemical there still may be differences so that you have…
assays have been relatively successful. They’ve got but you still need both of the approaches to get to
the crystal structures of the kinases, they can now a good answer.
pick up allosteric regulators of kinases, as well as
kinases interacting with other proteins. Is the fact David Marks: I agree we still need cell-based
that this is not yet here with GPCRs not a bio- assays ultimately to qualify your kinase inhibitors.
chemical problem, it’s that the technology is not And not only that, I mean, you mentioned about
here to do it biochemically with GPCRs. I mean selectivity – you can do all kinds of selectivity
the crystal structures are already starting to roll screening in vitro. But how relevant that selectivi-
through for GPCRs. It’s just that you cannot do it ty is in a cell setting is still questionable. Because
on a technical basis. it all depends on KM of ATP for your particular
enzyme. And that could change the real, true selec-
Guido Zaman: I think I agree with you. The major, tivity profile in a cell. Because if you look at a
the first thing about cell-based assays for GPCRs is compound that may have some activity at a cer-
they are resistant to purification. And radioligand tain ATP concentration in vitro, but in the cell the
binding assays have the disadvantage of creating ATP concentration may be much higher, and that
radioactive waste. So I think cell-based assays activity may not be relevant. Because ATP will
work well for GPCRs but kinases purified by bio- compete out the activity of your compound in
chemical assays is a prime platform. Then again, if vivo, inside the cell. So I think it’s still important
you have allosteric modulators which indeed can and we find that it’s very important to check to
only pick up the cellular assays, in the second line, make sure that your compound, I mean look at
you always want to do binding assays to confirm selectivity, after you do the in vitro panel you also
specificity of your compounds, either with the do some in vivo activity profiling of your com-
radio-labelled variant of your allosteric modulator, pound in the cell to look to see if it is actually
or you may want to do disassociation experiments inhibiting the potential non-selective activity that
with natural ligands to confirm that it’s allosteric. you are worried about in the cell. And we’ve seen
situations where we see good inhibition of a
David Marks: But we have also other binding kinase, a non-selective kinase activity in vitro, but
assays that can be used in place of a required radi- it really wasn’t a problem in the cell.
olabel… that can be labelled for direct binding, for
purified proteins. And actually for GPCRs that Arthur Christopoulos: I want to get back to
might not be too far away. So that might be anoth- Richard’s point, the whole kinases versus GPCRs
er method if you’re concerned about radioactivity, thing to point out a couple of things. Yes, I think in
and disposability. These binding assays even for the structural side of things, the kinases are ahead
GPCRs can be potentially developed quite soon. of the GPCRs, but as Richard said we’re seeing
structures starting to come out now. If you were to
Guido Zaman: I agree to a different path for bind- ask about an emerging trend, I would suggest that
ing, different tools. But also if you go back to we will be seeing structural-based design on GPCRs
kinases for instance, the traditional method is the in the next few years. There are actually companies
enzyme activity assay. I think another advantage of based around that now. The second thing is, if we
biochemical assays is that you can do very precise consider the targets, GPCRs versus kinases, kinases
selectivity determinations. I think binding maybe is are very well developed along a certain path –
the more traditional way of looking at GPCRs, but because they’re kinases. If you look at the GPCRs,
you can also look at binding with kinase assays. I could be wrong, but I think they’re far more
And if you start to think about this, you can go to promiscuous in what they do compared to kinases.
totally new concepts while you look for instance, I mean, a kinase phosphorylates proteins.
68 Drug Discovery World Winter 2009/10
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