COMMUNICATION
pubs.acs.org/JACS
Small-Molecule Inhibitors of the TLR3/dsRNA Complex
Kui Cheng, Xiaohui Wang, and Hang Yin*
Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, Colorado 80309, United States
S Supporting Information
b
Despite the significant potential, the discovery of small-
ABSTRACT: The protein-RNA interface has been re-
garded as “undruggable” despite its importance in many
biological processes. The toll-like receptor 3 (TLR3)/
double-stranded RNA (dsRNA) complex provides an excit-
ing target for a number of infectious diseases and cancers.
We describe the development of a series of small-molecule
probes that were shown to be competitive inhibitors of
dsRNA binding to TLR3 with high affinity and specificity. In
a multitude of assays, compound 4a was profiled as a potent
antagonist to TLR3 signaling and also repressed the expres-
sion of downstream signaling pathways mediated by the
TLR3/dsRNA complex, including TNF-R and IL-1β.
molecule inhibitors of TLR3 has been slow due to the complexity
associated with disrupting the protein-RNA contact: immense
effort is required to design individual compounds that target
specific RNA-binding domains with high binding affinity and
selectivity.1 Herein, we describe the successful identification and
characterization of small-molecule probes for the TLR3/dsRNA
complex.
In search of small-molecule probes, the 1.2 million-compound
Enamine database was screened against the dsRNA-binding
domain of TLR3 (crystal structure PDB: 3CIY12) using the
Glide 5.6 program.13 Initially, nine hits (Figure 1) were selected
for cell assay screening. Interestingly, almost all of the hits
identified, with the exception of T5528092, from the in silico
screening generally share the common motif of a D-amino acid
conjugated with an aromatic substituent, implying a novel
pharmacophore to target the RNA-binding site of TLR3.
These initial hits were first evaluated using our previously
nterfering with protein-protein interactions or protein-
nucleic acid interactions has been regarded as a daunting goal
established high-throughput cell assay of TLR3 activation.14
A
I
in drug discovery.1 Major strides have been made during the past
decade in developing small-molecule agents to target protein-
protein interactions. However, regulation of protein-RNA
interactions lags behind, arguably due to the fact that RNA
molecules pose a particular challenge with their high flexibility.2
RNA-binding proteins play key roles in post-transcriptional
modifications, which, along with transcriptional regulation, is a
main method of controlling gene expression during develop-
ment. In the present study, we report novel molecular probes that
disrupt double-stranded (ds) RNA binding to toll-like receptor 3
(TLR3) as a demonstration of the use of specific small-molecule
agents to target the protein-RNA interface.
dsRNA, polyriboinosinic:polyribocytidylic acid (Poly(I:C)), was
employed to selectively activate TLR3 signaling, resulting in the
activation of nitric oxide (NO) synthase and the production of
NO in RAW 264.7 macrophage cells.15 We monitored the NO
level as an indicator of Poly(I:C)-induced TLR3 activation to
evaluate the drug’s inhibitory activity.
Two compounds, T5626448 and T5260630 (shown in boxes
in Figure 1), demonstrated mild inhibitory activities in whole
cells, with IC50 values of 154 ( 6 and 145 ( 4 μM, respectively.
Both of these two compounds are derivatives of D-phenylalanine,
suggesting the D-phenylalanine backbone as the scaffold to
develop small-molecule inhibitors of TLR3. Computational
docking results also implied that T5626448 and T5260630
could be further optimized by varying the substituents on the
benzene or thiophene rings (Supporting Information (SI),
Figure S1).
With the hit compounds selected, we developed concise
synthetic routes for both T5626448 and T5260630 (SI, Scheme
S1), which allows an extensive structure-activity relationship
(SAR) analysis. Various substitutions with different size and
electron-withdrawing/donating capability were examined on
the aromatic systems. To inspect the impact on the activities
imposed by the stereogenic center, both R- and S-isomers were
prepared.
An improvement of 2 orders of magnitude in inhibitory
potency of T5626448 was achieved, with compound 4a
(Figure 2A) showing a low micromolar (3.44 ( 0.41 μM)
IC50 value. By contrast, no significant activity improvement for
Toll-like receptors (TLRs) are highly conserved transmem-
brane proteins that detect pathogen-associated molecular pat-
terns and elicit pathogen-specific immune responses.3 TLR3
signaling is activated by dsRNA released from necrotic cells
during inflammation or viral infection.4 TLR3 activation induces
secretion of type I interferons and pro-inflammatory cytokines,
such as TNF-R, IL-1, and IL-6, triggering immune cell activation
and recruitment that are protective during certain microbial
infections.5 A dominant-negative TLR3 allele has been asso-
ciated with increased susceptibility to herpes simplex encepha-
litis, a serious illness with significant risks of morbidity and death,
upon primary infection with HSV-1 in childhood.6 In mice,
TLR3 deficiency is associated with decreased survival upon
coxsackie virus challenge.7 In addition, uncontrolled or sustained
innate immune response via TLR3 has been shown to contribute
to morbidity and mortality in certain viral infection models,
including the West Nile disease, phlebovirus, vaccinia, and
influenza A.8-11 Therefore, modulation of TLR3 pathways offers
an attractive strategy to fight a variety of diseases.
Received: December 15, 2010
Published: February 28, 2011
r
2011 American Chemical Society
3764
dx.doi.org/10.1021/ja111312h J. Am. Chem. Soc. 2011, 133, 3764–3767
|