Biomedical Engineering Reference
In-Depth Information
15.5.3 Which Cell Type Is Inhibited?
One question that is not often asked during drug discovery is “Does the lead
compound target the correct cell type?” One reason that this question is not often
asked is that it is difficult tomeasure the effects of a compound on a particular cell type
when that cell type is mixed with many others. One of the best attributes of the flow
cytometry platform is that it can be used to identify cellular subsets present in patient
samples like peripheral blood (e.g., T cells, B cells, monocytes, and neutrophils).
Well-characterized cellular markers are available to identify these cell subsets and
by coupling these markers to phospho-specific antibodies, we are able to measure
intracellular signaling pathways directly in complex tissue samples such as themurine
spleen, lymph nodes, and peripheral blood [20, 21]. This enables us to answer
the question of whether a compound inhibits the target pathway in the target cell type
when the cell type is a part of a mixture of cell types.
A compound displays one of four general patterns of selectivity when analyzed in a
mixture of cells: (1) It inhibits all signaling in all cell types. (2) It inhibits one signaling
pathway, but in all cell types. (3) It inhibits all signaling in one cell type. (4) It inhibits
one signaling pathway in one cell type (Figure 15.5). The last option provides the ideal
drug candidate, as it would presumably have minimal side effects and optimal efficacy.
However, as we began to examine the effects of some of the lead compounds from
our screen of natural products, as well as commercially available Jak-Stat inhibitors,
we quickly discovered that inhibiting one pathway in one cell type was not common.
What we foundwere compounds that displayed strong patterns of selectivity for certain
signaling pathways, and one compound that showed potent selectivity toward signaling
in only one cell type (Figure 15.5). However, none of the compounds tested showed
selectivity for only one pathway in one cell type. Indeed, it has proven difficult to
incorporate moieties into drug leads that guide the molecule to a particular cell type.
Therefore, hybrid drugs, such as antibody-targeted therapeutics, might be the best
approach to achieving the ideal one target, one cell type drug. Importantly, phosphoflow
provides a unique platform that is capable of quantitatively measuring the inhibitory
effects of molecules directly in complex, heterogeneous populations of cells.
15.5.4 Painting a Druggable Landscape
Perhaps more important than understanding any one drug in our screening experiments
was the knowledge that we gained about the immune cell signaling network when we
clustered the inhibition profiles of all the compounds simultaneously. We were able to
identify signaling pathways and cell types that were inhibited at low doses by all the
compounds. These patterns of pathway and cell type “druggability” were apparent
even with the small number of compounds that we tested. We plan to expand our
screening efforts to more fully elucidate this druggable landscape. In our primary cell
screens, we performed dose-response experiments with each compound on 24
signaling pathways in 5 cell types [16]. Of the 120 cell type-pathway combinations
possible, 27 showed signaling changes greater than twofold upon induction. For these
27 cell type-pathway combinations, we obtained IC
50
values and were able to
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