Abstract
Protein-protein interactions (PPI) mediate the formation of intermolecular networks that control biological signaling. For this reason, PPIs are of outstanding interest in pharmacology, as they display high specificity and may represent a vast pool of potentially druggable targets. However, the study of physiologic PPIs can be limited by conventional assays that often have large sample requirements and relatively low sensitivity. Here, we build on a novel method, immunoprecipitation detected by flow cytometry (IP-FCM), to assess PPI modulation during either signal transduction or pharmacologic inhibition by two different classes of small-molecule compounds. First, we showed that IP-FCM can detect statistically significant differences in samples possessing a defined PPI change as low as 10%. This sensitivity allowed IP-FCM to detect a PPI that increases transiently during T cell signaling, the antigen-inducible interaction between ZAP70 and the T cell antigen receptor (TCR)/CD3 complex. In contrast, IP-FCM detected no ZAP70 recruitment when T cells were stimulated with antigen in the presence of the src-family kinase inhibitor, PP2. Further, we tested whether IP-FCM possessed sufficient sensitivity to detect the effect of a second, rare class of compounds called SMIPPI (small-molecule inhibitor of PPI). We found that the first-generation non-optimized SMIPPI, Ro-26-4550, inhibited the IL-2:CD25 interaction detected by IP-FCM. This inhibition was detectable using either a recombinant CD25-Fc chimera or physiologic full-length CD25 captured from T cell lysates. Thus, we demonstrate that IP-FCM is a sensitive tool for measuring physiologic PPIs that are modulated by signal transduction and pharmacologic inhibition.
Highlights
Cell signaling pathways often involve numerous protein-protein interactions (PPI) in processes as diverse as receptor:ligand binding, signal transduction across physical barriers, and translocation of signals between different cellular compartments [1,2]
We report that immunoprecipitation detected by flow cytometry (IP-FCM) is able to measure the transient, inducible increase in ZAP70 association with the T cell antigen receptor (TCR)/CD3 complex upon physiologic TCR engagement, an event which can be blocked by the small molecule src-family kinase inhibitor, PP2
Our model multiprotein complex is ab T cell antigen receptor (TCR)/CD3: the TCR proper is composed of a disulfide-linked ab heterodimer, while CD3 is composed of subunits c, d, e, and f (Figure 1; Reference [22])
Summary
Cell signaling pathways often involve numerous protein-protein interactions (PPI) in processes as diverse as receptor:ligand binding, signal transduction across physical barriers, and translocation of signals between different cellular compartments [1,2] Together, these PPIs are thought to form a system with emergent network properties, integrating signals from a variety of inputs into coordinated responses. There is significant interest in the generation of biotechnological assays that would display sufficient sensitivity to detect subtle changes in PPIs, changes of a magnitude on scale with those that occur in response to distinct physiologic conditions Such high-sensitivity PPI assays could be useful in drug development, if they could be proved capable of detecting the effects on PPI that result from drug targeting. The prediction is that SMIPPI might provide increased on-target specificity and fewer side effects than drugs targeting enzymes; by targeting only a specific interaction of a given pair of proteins, those proteins might still perform other non-pathologic functions
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