responsible for the clinical failure of 5,6-
dimethylxanthone-4-acetic acid (DMXAA),
a vascular disrupting agent that can function
as a murine STING agonist but fails to bind
to the human form of the protein ( 17 , 18 ).
Characterization of SR-717 in a homogeneous
time-resolved fluorescence-based cGAMP
competition assay, involving wild-type (WT)
human STING protein, revealed that SR-717
binds to STING with an apparent affinity
[mean inhibitory concentration (IC 50 )=7.8mM;
fig. S3B] that is comparable to its observed
cell-based potency. Compound washout ex-
periments revealed that a robust induction of
reporter signal is achieved within 1 hour of
exposure (fig. S3C). Further, SR-717 was found
to possess mouse pharmacokinetic properties
that would enable its in vivo characterization
in the context of systemic administration (fig.
S4A). Cell-free profiling of SR-717 at a concen-
tration of 5mM against a panel of 468 kinases
revealed the compound to be exceptionally
selective, with only a single kinase (serine/
threonine kinase PIM3) being identified as
an interacting partner (fig. S5; percent of
control < 35). Kinetic characterization of the
cell-based activity of SR-717, at the calculated
EC 80 concentration (3.6mM), revealed that
interferon-stimulated response element (ISRE)–
luciferase reporter activity translated to ac-
tivation of target gene expression. Specifically,
SR-717 stimulatesIFNB1expression within
1 hour of incubation and achieves maximal
activation by 2 hours of treatment, whereas
activation ofCXCL10, a transcript activated
by both IRF3 and IFN-bsignaling, peaks at
6hours(Fig.1E).Importantly,theseki-
netics of activation are maintained in primary
human peripheral blood mononuclear cells
(PBMCs) (Fig. 1F). On-target pathway acti-vation by SR-717 was further validated by
analyzing the phosphorylation of TBK1,
IRF3, and p65 signal transduction proteins
(Fig. 1G), which are downstream of STING,
as well as the secondary activation of STAT1
and STAT3 (Fig. 1G), which are downstream
of IFN-band IL-6 signaling, respectively.
Further, inhibition of the downstream effector
TBK1, using small molecule inhibitor BX795,
was observed to block the induction of reporter
signal (fig. S6, A and B) and downstream IRF-3
and secondary STAT1 phosphorylation events
(fig. S6C) by SR-717.
The soluble cytosolic C-terminal region of
hSTING exists in solution as a dimer and
binds to naturally occurring and synthetic
CDNs with varying degrees of affinity. X-ray
crystallographic analysis of this CDN-binding
domain has revealed that STING exists as a
closely associated dimer that takes the shapeChinet al.,Science 369 , 993–999 (2020) 21 August 2020 2of7
N
HN
NN NOMeO ON
HN
NN NOHO ON
HNNN NOHO OF
A F
WT
(EC 50 = 2.1 μM)
STING KO
(EC 50 >50 μM)
cGAS KO
(EC 50 = 2.2 μM)8006004002000
0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
log [SR-717], nMRLU0min10 min30 min1h2h3h4h6h
p-TBK1 (S172)p-STING (S366)p-STAT1 (Y701)γ-tubulinp-IRF3 (S386)p-p65 (S536)p-STAT3 (Y705)G + SR-717BCDEF2460500100015002000300040005000Time (h)hPBMCRelative expressionIFNB1
CXCL10
IL6IFNB1
CXCL10
IL6246
Time (h)Relative expressionTHP105010020030035040025 35 45 55 65 75^150
Temperature (°C)25 35 45 55 65 75
Temperature (°C)d(Fluor)/dT4
2
06d(Fluor)/dT2
1
03Δ Tm (°C)
hSTING
REFmSTING
LigandcGAMPSR-717WT AQ Q WT3.2011.210.38.1911.99.3213.58.1922.210.0Δ Tm (°C)SR-001 SR-012 SR-717DMSO
cGAMP
SR-717hSTINGREFDMSO
cGAMP
SR-717mSTINGFig. 1. Discovery and profile of small-molecule STING agonist SR-717.
(A) Structure of prodrug screening hit SR-001 and elucidated STING agonist
SR-012. Me, methyl. (B) Structure of optimized STING agonist SR-717.
(C) Cell-based activity of SR-717 in ISG-THP1 (WT), ISG-THP1 cGAS KO (cGAS
KO), and ISG-THP1 STING KO (STING KO) cell lines was quantified by normalizing
SR-717–induced reporter activity to DMSO-treated reporter activity for each
cell and reported as relative light units (RLU). (D) Impact of SR-717 (100mM) or
cGAMP (100mM) on the melting temperature (DTm) of recombinant common
human alleles of STING protein (hSTING), as well as recombinant mSTING.
REF, R232H; AQ, G230A, R293Q; Q, R293Q; d(Fluor)/dT, first derivative of
fluorescence melt curve. (E) Quantitative reverse transcription polymerase
chain reaction (qRT-PCR) analysis of time-dependent target gene expression in
THP1 cells treated with SR-717 (EC 80 =3.6mM) normalized to DMSO-treated
control. (F) qRT-PCR analysis of time-dependent target gene expression in freshly
isolated human PBMCs treated with SR-717 (10mM) normalized to DMSO-treated
control. (G) Western blot analysis of the kinetics of activation of the cGAS-STING
and FN-a/breceptor (IFNAR) signaling pathways in THP1 cells stimulated with
SR-717 (EC 80 =3.6mM).g-Tubulin was used as a loading control. Data are
representative of three independent experiments [(C) to (G)], and values are
the mean of three replicates ± SD [(C) to (F)].RESEARCH | REPORT