Cell-based assays:Layout 1 14/1/10 19:58 Page 67
Assays
about tying a particular assay, whether it’s cell- assay has got to remain as a validation step. And
based or not, as a predictor of what’s biologically the second aspect, is and even one of each is insuf-
relevant. I think the whole concept of biological ficient nowadays, given the repertoire of behav-
relevance is still ‘out there’. In terms of what’s a iours of different ligands have been found to
predictor, no matter what we do, or what’s going engender especially as a consequence of function-
to be the desired therapeutic endpoint, I think in al cell-based assays. Cell-based assays as Richard
99.9% of the cases we can’t answer that. pointed out provide context, but the context can
change from cell to cell. And on top of what we
Richard Eglen: I think if you look at biochemical know now especially in some GPCRs, which have
versus cell-based assays, I think historically bio- always been known to have lots of different possi-
chemical assays as mentioned in terms of ligand ble coupling partners which can change with the
binding to a membrane that has the GPCR for cell type, that can do different things. So a series
example, gave very robust straightforward infor- of ligands acting on the same receptor even in the
mation. The problem was, to Arthur’s point, that same cell type can bias different types of signals.
the cellular context was lost. And you lost a lot of And so you have no choice other than to use mul-
the functionality in terms of the cell signalling tiple functional endpoints when trying to decon-
pathway. And probably cell-based assays have volute those sorts of compounds. Because we
started to merge, because you’ve got the whole don’t know which is the right one. Is it the one
functionality of the cell as it relates to the function that gives you arista, or is it the one that causes G-
of the target. Now that gives you some advantage protein coupling to channel that’s going to be ther-
in terms of amplifying the signal to measure it and apeutically relevant?
you can get efficacy of the ligand versus the ability
to block the receptor. David Marks: Another issue with cell-based assays
But it’s also opened up some problems. To is that you also have to be careful that you have the
Arthur’s point, what’s the correct cell type to give correct coupling of the G-protein, especially with
you the right response? And how does this func- GPCRs. We’ve seen instances where we have used
tionality mimic the physiological or the disease sit- promiscuous proteins to couple to a calcium
uation as it relates to drug discovery? David, I response and then we find out that the compounds
don’t know if you want to answer that…? we’ve identified do not work when it’s correctly cou-
pled. So that’s an issue too. You have to be careful.
David Marks: Yes, there’s always the issue of
whether an in vitro cell-based assay will truly Arthur Christopoulos: So how many people are
mimic the in vivo response. I think, especially in using native cell expressors to screen, then?
the neuroscience area, they often have that prob- Endogenous expressors … that’s still not that com-
lem – the fact that you get a response in that cell mon, is it?
line doesn’t necessarily give you an in vivo
response. But back to what Robert was asking ear- Martin Valler: No. We’ve had the same experience.
lier, I think ideally you probably want to have a The same target mated by two different coupling
combination of an in vitro cell-based assay and a mechanisms and we’ve seen completely different
biochemical assay just to confirm that whatever results. But just to come back about the point
you identify in a cell-based assay is really acting via about why we’re really doing assays in the first
the receptor and not some secondary response in place, I think there are an increasing proportion of
an intracellular location. And alternatively, a pure assays that just can’t be addressed sensibly by bind-
biochemical binding assay may not work for cer- ing – and they can’t be confirmed either by bind-
tain G-coupled protein receptors, because for ing, because with the allosteric modulator class of
example, if you’re looking for an agonist response, targets, you’re lucky to get a displacement of the
a binding assay doesn’t really help you. I mean, ligands through such a binding. Maybe you might
you don’t know if you have an agonist or an antag- but in most cases you won’t. You’ll simply see a
onist. So you may need the combination of both. modulation of the functional activity, and the true
ligand binding will be completely unaffected.
Arthur Christopoulos: I think there’s no way That’s increasingly our direction, our rationale
around it now, in the sense that, for starters, the for doing more cell-based assays – projects that
industry people can correct me if I’m wrong, but we’re interested in functional readouts like
the cell-based assay has probably emerged up- allosteric modulators, positive or negative, and are
front now. But the binding or the biochemical less interested in straight inhibition.
Drug Discovery World Winter 2009/10 67
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