Biomedical Engineering Reference
In-Depth Information
communication between cells. When cytokines bind to their cognate receptors on
the surface of a particular cell, they induce activation of Jaks, which in turn
phosphorylate Stats. The Stats act as transcription factors and lead to transcription
of target genes necessary for cell differentiation, maturation, or activation. In
signaling profiling studies, as well as in drug screening efforts, we measure the
phosphorylation state of the Stat proteinsasreadoutsofcytokineactivity.
Because the Stat proteins are relatively abundant transcription factors, signal-
to-noise and Z
0
characteristics of phospho flow assays for Stat phosphorylation are
excellent.
15.5.2 Analyzing Multiple Pathways Improves Selectivity
In our efforts to identify inhibitors of Jak-Stat signaling, we began by testing a
natural product compound library in a myeloid cell line [16]. In these cell line
assays, cells were treated with compounds, incubated, and then activated by adding
three different stimuli to the cells simultaneously. The stimuli were chosen to induce
three different signaling pathways: IFN
g
to induce Stat1, GM-CSF to induce Stat5,
and anisomycin to induce p38 MAP kinase. The goal of the screen was to find
compounds that modulated Stat1 and/or Stat5 signaling, but not p38 (i.e., we sought
compounds selective for Jak-Stat signaling relative to MAP kinase signaling
pathways).
What was striking from this initial screen was the importance of measuring more
than one signaling pathway. If only one pathway had been examined, rather than three
at once in individual cells, wewould have identified several potential lead compounds
that inhibited each of the three pathways. Yet, as we looked at the effects of these
inhibitors on the other pathways measured, the number of selective hits dwindled. For
instance, although eight compounds inhibited GM-CSF-induced Stat5 phosphoryla-
tion, only one of these compounds was selective for this pathway. Six compounds
inhibited IFN
g
-induced Stat1 phosphorylation, but none were specific to only this
pathway [16]. Therefore, by examining only three pathways simultaneously, we were
able to rapidly reduce the number of false leads and focus on compounds with desired
selectivity profiles. One can imagine that if the number of pathways examined were
expanded to 10 or 20, even fewer selective compounds would have been identified, but
each lead compound would have a good chance of success in preclinical and
toxicology testing.
These results highlight the difficulty of modulating signaling in the context of the
multitude of processes constantly occurring during cellular biochemistry. Therefore,
although inhibitors can be identified via assays with purified proteins, the complexity
of cellular metabolism cannot be appreciated until the compound is added directly to
cells. For example, many compounds bind nonselectively to critical enzymes in ATP
metabolism or glycolysis leading to toxicity. Clearly, the ideal situation is to find
compounds that are selective in in vitro assays and then to test these using phospho
flow to find compounds that are not only effective in the cellular context but also show
selectivity toward the pathway of interest.
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