The use of small molecule probes to study spatially separated stimulus-induced signaling pathways

Abstract

Simultaneous activation of signaling pathways requires dynamic assembly of higher-order protein complexes at the cytoplasmic domains of membrane-associated receptors in a stimulus-specific manner. Here, using the paradigm of cellular activation through cytokine and innate immune receptors, we demonstrate the proof-of-principle application of small molecule probes for the dissection of receptor-proximal signaling processes, such as activation of the transcription factor NF-κB and the protein kinase p38.

Figure 1

NP1 acts as a specific modulator of NF- B signaling. (a) Generic structures of bisphenyl (BP)- and naphthyl (NP)-based members of the “credit card” library. (b) Western blot analysis of I B p and their phosphorylated forms (p-I B and p-p38, respectively) in extracts prepared from bone-marrow derived macrophages (BMDMs) after treatment with LPS alone or in combination with BP1, BP2 or NP1 (50 M of each). (c) Western blot analysis of IKK , I B , RelA and their phosphorylated forms in BMDM extracts after treatment with TNF and LPS for 5 min in the absence or presence of NP1 as indicated. (d) The profiles of IKK and p38 activities as well as I B protein concentration and RelA phosphorylation in BMDMs stimulated with LPS, TNF or in combination with NP1 as indicated. Quantitated and normalized results from three independent experiments are shown. (e) Northern blot analysis of total RNA prepared from BMDMs monitors the effect of NP1 on the mRNA expression levels of I B and MCP-1 genes induced by LPS or TNF.

Figure 2

NP1 selectively impairs IL-1R/TLR-mediated activation of the NF- B pathway. (a) The scheme shows recruitment of MyD88 and TRIF adaptor proteins to IL-1R and TLRs in response to receptor-specific ligands, such as LPS, IL-1, HKSA (heat-killed Staphylococcus aureus), pI:pC (polyI:polyC) and CpG (DNA oligonucleotide containing unmethylated CpG motifs). (b and c) Western blot analysis of total protein extracts prepared from BMDMs monitors the effect of NP1 on I B degradation and resynthesis as well as I B phosphorylation induced by IL-1 and HKSA (b) or pI:pC and CpG (c) as indicated. (d) The profiles of IKK and p38 activities in BMDMs stimulated with the indicated stimuli alone or in combination with NP1. Quantitated and normalized results from three experiments are shown.

Figure 3

Inhibitory effects of NP1-based scaffolds depend on their chemical reactivity. (a) Chemical structures of NP1 and its analogs. (b) The profiles of IKK and p38 activities and RelA phosphorylation in BMDMs stimulated with LPS alone or in combination with NP1 or its analogs as indicated. Quantitated and normalized results from three experiments are shown. (c) Western blot analysis of total protein extracts prepared from BMDMs monitors the effect of NP1 and its analogs on I B degradation and resynthesis as well as I B phosphorylation induced by LPS (left panel) and TNF (right panel) as indicated.