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

Article abstract of DOI:10.1016/j.bmcl.2012.01.024

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 cy

Full text of DOI:10.1016/j.bmcl.2012.01.024

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2043–2045
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The use of small molecule probes to study spatially separated
stimulus-induced signaling pathways
Vladimir V. Kravchenko a, Christian Gloeckner a,b,c,d, G. Neil Stowe a,b,c, Young J. Kang a, Peter S. Tobias a,
a,b,c,d,
John C. Mathison a, Richard J. Ulevitch a, Gunnar F. Kaufmann a,b, , Kim D. Janda
⇑
⇑
a Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, 92037 CA, USA
b Department of Chemistry, The Scripps Research Institute, La Jolla, 92037 CA, USA
c The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, 92037 CA, USA
d Worm Institute for Research and Medicine (WIRM), The Scripps Research Institute, La Jolla, 92037 CA, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Simultaneous activation of signaling pathways requires dynamic assembly of higher-order protein com-
plexes 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
Received 29 November 2011
Revised 5 January 2012
Accepted 9 January 2012
Available online 14 January 2012
signaling processes, such as activation of the transcription factor NF-jB and the protein kinase p38.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
NF-jB pathway
Toll-like-receptor
Selective inhibitor
Innate immunity
Tumor necrosis factor
An intricate network of protein–protein interactions (PPIs) is
involved in many essential cellular processes, such as receptor-
dependent signaling pathways.1,2 In order to understand the
mechanisms by which the same downstream signaling pathway
is activated through different receptors, selective inhibition of PPIs
involved in these processes could be a useful tool for basic research
as well as drug discovery.3 A recent development in the field of PPI
studies is the use of small molecules that exhibit specificity and
affinity to crucial dominant protein–protein interfaces, also known
as hot-spots.4–6 We examined this tact for the dissection of recep-
For example, bacterial lipopolysaccharide (LPS) activates the
IKK and MAPK cascades through the Toll-like receptor 4 (TLR4), a
member of the innate immune receptor family,12 while tumor
necrosis factor alpha (TNF) signals through the TNF receptor
(TNFR), member of the TNFR superfamily.13
These observations prompted our investigations into whether a
small molecule-based strategy might allow for dissection of the
receptor-proximal signaling processes induced by different NF-
jB-activating stimuli, namely LPS and TNF. As a starting point an
initial screening was undertaken using our non-peptidic small
tor-mediated signaling mechanisms with focus on the NF-
jB path-
molecule library, two scaffolds contained within the library are
way as model system of biomedical importance.7
depicted in Figure 1a.14 Thus, using a cell line carrying an NF-
jB re-
Upon engagement of cytokine and innate immune receptors,
activation of the classical NF- B pathway is initiated through tran-
sient assembly of a large multiprotein signalsome that contains the
key components of NF- B signaling, such as the I B (inhibitor of
NF-
B) kinase (IKK) complex.8–10 Despite the involvement of dis-
tinct receptor proteins, NF- B stimuli induce signaling within the
IKK-NF- B module as well as parallel activation of other signaling
porter system and LPS as stimulus resulted in the identification of
both napthyl- (NP1) and bisphenyl-based (BP1 and BP2) scaffolds
j
as potential modulators of NF-
data, Fig. 1). To investigate whether these compounds indeed affect
NF- B signaling, the effects of BP1, BP2 and NP1 on the kinetics of
LPS- or TNF-induced I phosphorylation, degradation and resyn-
jB activity (see Supplementary
j
j
j
j
j
jBa
j
thesis in macrophages were studied. As specificity control, the
phosphorylation of p38 kinase (p-p38), a marker of a MAPK cas-
cade,15 was also assayed. Similar levels of p-p38 were observed
in response to LPS alone or in combination with the inhibitors.
pathways, including the p38 mitogen- (or stress-) activated protein
kinase (MAPK/SAPK) cascade.11
The temporal patterns of NF-
the presence of BP1 or BP2; in contrast, addition of NP1 resulted
in substantial inhibition of LPS-induced I phosphorylation
and degradation shown in Figure 1b (and Supplementary data,
jB signaling were also unaffected in
⇑
Corresponding authors. Tel.: +1 858 784 2516; fax: +1 858 784 2595 (K.D.J.).
E-mail addresses: kaufmann@scripps.edu (G.F. Kaufmann), kdjanda@scripps.edu
jBa
(K.D. Janda).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.024

2044
V. V. Kravchenko et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2043–2045
adaptors are utilized by TLR4 (Fig. 2a). To better understand the
signaling mechanisms targeted by NP1, we examined the effect
of NP1 on activation of the NF- B and p38 pathways in response
to IL-1R-, TLR2-, TLR3- or TLR9-specific ligands. As seen in Figure 2a
and b, NP1 substantially blocked I phosphorylation and degra-
j
jBa
dation induced by IL-1R and TLR2 ligands, suggesting a role of NP1
in targeting MyD88-dependent processes. However, a complemen-
tation assay demonstrated that NP1 had no effect on the direct
interaction between the cytoplasmic domains of TLR4 and
MyD88 (Supplementary data, Fig. 4). Moreover, although NP1
blocked MyD88-dependent TLR9-mediated activation of the NF-
jB pathway, it also disrupted the MyD88-independent signaling
mediated through TLR3-TRIF interactions shown in Figure 2c and
d. Interestingly, in contrast to cell surface-expressed IL-1R, TLR2
and TLR4, both TLR3 and TLR9 are intracellular proteins.12,16 Thus,
these data indicate that NP1 acts in the cytoplasm to affect IL-1R/
TLR-mediated activation of NF-
jB signaling. Although further
investigations are required to identify the exact mechanisms
Figure 1. NP1 acts as a specific modulator of NF-jB signaling. (a) Generic structures
of bisphenyl (BP)- and naphthyl (NP)-based members of the ‘credit card’ library. (b)
Western blot analysis of I , p38 and their phosphorylated forms (p-I and p-
jB
a
jBa
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 lM of each). (c) Western blot analysis of IKKb, IjBa, RelA and their phosphor-
ylated 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 IjBa 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 IjBa and MCP-1 genes induced by LPS or TNF.
Fig. 2). Notably, neither BP1/2 nor NP1 had any effect on TNF-in-
duced p-p38 and activation of NF-
data, Fig. 3).
jB signaling (Supplementary
The phosphorylation-dependent activation of IKKb (also known
as IKK-2), a subunit of the IKK complex, is essential for induction of
NF-
B proteins and RelA subunit of NF-
quence of these processes is transcriptional induction of I
and other NF-
B target genes.10 Therefore, to better define the
specificity of NP1, its effects on activation of the IKKb–I –RelA
signaling module in response to TNF or LPS was investigated.
LPS-induced phosphorylation of IKKb, I and RelA was substan-
tially disrupted in the presence of NP1. However, functional integ-
rity of the IKKb–I –RelA signaling module remained intact in
cells stimulated with TNF in the presence of NP1 shown in Figure
1c and d. Thus, the core components of NF- B signaling are not af-
fected by NP1. Northern blot analysis independently confirmed
that NP1 selectively blocked the induction of NF- B target genes
jB activity by TNF or LPS; once activated IKKb phosphorylates
Ij
jB. The functional conse-
jBa
j
jBa
jBa
jBa
j
j
in LPS- but not TNF-treated macrophages (Fig. 1e). These findings
are consistent with the fact that TNFR and TLR4 use distinct adap-
ter protein components to assemble signaling complexes essential
for the activation of receptor-proximal IKK cascades.12,13
Figure 2. NP1 selectively impairs IL-1R/TLR-mediated activation of the NF-jB
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 oligonucleo-
tide containing unmethylated CpG motifs). (b and c) Western blot analysis of total
TLR4 engagement initiates assembly of multicomponent recep-
tor-associated signaling complexes that activate NF-jB and p38
pathways.12 Several of these components are also recruited by
the interleukin-1 receptor (IL-1R) and other TLRs; namely, TLR3
signals through TRIF adaptor protein, while the remaining known
TLRs as well as IL-1R use MyD88 as adaptor protein; notably both
protein extracts prepared from BMDMs monitors the effect of NP1 on
IjBa
degradation and resynthesis as well as I phosphorylation induced by IL-1 and
jBa
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.

V. V. Kravchenko et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2043–2045
2045
tially improves the efficacy of their planar-aromatic motif as inhib-
itor of IL-1R/TLR-mediated activation of the IKK signaling cascade.
Our current understanding of signal transduction through
growth factor, cytokine and innate immune receptors suggests that
both assembly and dissociation of multiprotein signaling com-
plexes at/from intracellular receptor domains are required for the
regulation of receptor-proximal MAPK and IKK cascades.11–13,23,24
For example, it has been reported that CD40, a TNFR superfamily
member, activates IKK through a receptor-associated complex
while the MAPK cascade is triggered after translocation of the mul-
ticomponent signaling complex from the intracellular CD40 do-
main into the cytosol.24 Our findings strongly support this model
of spatial and temporal separation of MAPK and IKK signaling cas-
cades, and show how the principles of chemical biology can be ap-
plied to begin to dissect receptor-proximal signaling events.
Acknowledgments
This work was supported by the National Institutes of Health
(AI088229 to Y.J.K., AI080715 to G.F.K., and AI077644 to K.D.J.),
the Worm Institute for Research and Medicine (K.D.J.) and the
Skaggs Institute for Chemical Biology (K.D.J.). This is manuscript
# 21546 from The Scripps Research Institute.
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
degradation and resynthesis as well as I phosphorylation induced by LPS (left
panel) and TNF (right panel) as indicated.
jBa
jBa
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2012.01.024.
involved, it is unlikely that NP1 interferes with primary assembly
of IL-1R/TLR-associated signaling complexes, because receptor-
proximal activation of the p38 pathway still occurred in the pres-
ence of NP1 shown in Figure 2d (and Supplementary data, Fig. 5).
Several thiol-reactive molecules have been shown to inhibit
References and notes
1. Keskin, O.; Gursoy, A.; Ma, B.; Nussinov, R. Chem. Rev. 2008, 108, 1225.
2. McNeill, H.; Woodgett, J. R. Nat. Rev. Mol. Cell Biol. 2010, 11, 404.
3. Wilson, A. J. Chem. Soc. Rev. 2009, 38, 3289.
4. Bogan, A. A.; Thorn, K. S. J. Mol. Biol. 1998, 280, 1.
5. Clackson, T.; Wells, J. A. Science (New York, NY) 1995, 267, 383.
6. Workman, P.; Collins, I. Chem. Biol. 2010, 17, 561.
7. Baltimore, D. Nat. Immunol. 2011, 12, 683.
8. DiDonato, J. A.; Hayakawa, M.; Rothwarf, D. M.; Zandi, E.; Karin, M. Nature
1997, 388, 548.
9. Mercurio, F.; Zhu, H.; Murray, B. W.; Shevchenko, A.; Bennett, B. L.; Li, J.; Young,
D. B.; Barbosa, M.; Mann, M.; Manning, A.; Rao, A. Science (New York, NY) 1997,
278, 860.
10. Oeckinghaus, A.; Hayden, M. S.; Ghosh, S. Nat. Immunol. 2011, 12, 695.
11. Baud, V.; Karin, M. Trends Cell Biol. 2001, 11, 372.
12. Beutler, B. Nature 2004, 430, 257.
13. Chen, G.; Goeddel, D. V. Science (New York NY) 2002, 296, 1634.
14. Xu, Y.; Shi, J.; Yamamoto, N.; Moss, J. A.; Vogt, P. K.; Janda, K. D. Bioorg. Med.
Chem. 2006, 14, 2660.
15. Zarubin, T.; Han, J. Cell Res. 2005, 15, 11.
16. Nishiya, T.; Kajita, E.; Miwa, S.; Defranco, A. L. J. Biol. Chem. 2005, 280,
37107.
17. Hehner, S. P.; Heinrich, M.; Bork, P. M.; Vogt, M.; Ratter, F.; Lehmann, V.;
Schulze-Osthoff, K.; Droge, W.; Schmitz, M. L. J. Biol. Chem. 1998, 273,
1288.
18. Hehner, S. P.; Hofmann, T. G.; Droge, W.; Schmitz, M. L. J. Immunol. 1999, 163,
5617.
19. Rossi, A.; Kapahi, P.; Natoli, G.; Takahashi, T.; Chen, Y.; Karin, M.; Santoro, M. G.
Nature 2000, 403, 103.
20. Toledano, M. B.; Leonard, W. J. Proc. Nat. Acad. Sci. U.S.A. 1991, 88, 4328.
21. Garcia-Pineres, A. J.; Castro, V.; Mora, G.; Schmidt, T. J.; Strunck, E.; Pahl, H. L.;
Merfort, I. J. Biol. Chem. 2001, 276, 39713.
22. Ha, K. H.; Byun, M. S.; Choi, J.; Jeong, J.; Lee, K. J.; Jue, D. M. Biochemistry 2009,
48, 7271.
23. Schlessinger, J. Cell 2000, 103, 211.
24. Matsuzawa, A.; Tseng, P. H.; Vallabhapurapu, S.; Luo, J. L.; Zhang, W.; Wang, H.;
Vignali, D. A.; Gallagher, E.; Karin, M. Science (New York NY) 2008, 321, 663.
phosphorylation and degradation of IjBa in cells stimulated with
TNF and LPS by blocking IKKb kinase activity via a Michael addition
reaction with a specific cysteine residue within the activation loop
of IKKb.17–19 Similar to N-ethyl maleimide,20 these inhibitors also
target a free cysteine residue in the NF-
j
B subunit RelA, and in
B/RelA-medi-
turn such modification results in reduction of NF-
j
ated transcription.21,22 NP1 contains a maleimide moiety; how-
ever, our data clearly demonstrate that NP1 did not alter the
signal processing within the IKKb–IjBa–RelA signaling module.
Nevertheless, the inhibitory effect of NP1 on LPS-induced NF-
jB
signaling required the maleimide group (Supplementary data,
Fig. 6), suggesting that covalent target modification contributes
to the observed signaling inhibition. To ascertain whether chemical
reactivity and inhibitory functions of NP1 are linked, we synthe-
sized compounds NP2 and NP3 depicted in Figure 3a, representing
a chemically-inert as well as a reversibly reactive derivative of NP1,
respectively (see Supplementary data). Comparison of NP1, NP2 or
NP3 effects on LPS-induced NF-jB signaling shown in Figure 3b re-
vealed that NP2 was a very week inhibitor, whereas NP3-mediated
inhibition was comparable with the effect seen for NP1. Impor-
tantly, both NP1 derivatives retain selectivity for TLR-dependent
induction of the NF-
jB pathway, as NP2 or NP3 had no effect on
TNFR-mediated activation of the IKKb–IjBa–RelA signaling mod-
ule. However, the induction of the p38 cascade through cytokine
and immune receptors still occurred in the presence of the com-
pounds as evident in Figure 3c (and Supplementary data, Fig. 7).
Thus, potential covalent protein reactivity of NP1 and NP3 substan-