interferon-b(IFN-b) secretion, a known effect
of STING agonism. Using human monocytic
THP-1 cells that express the naturally occurring
human HAQ STING isoform (hSTING-HAQ)
( 19 ), we screened a diverse library of ~2.4 mil-
lion compounds and identified a small number
of molecules, including MSA-2 (benzothiophene
oxobutanoic acid) (Fig. 2A), that induced IFN-b
production in THP-1 cells (Fig. 2B). MSA-2 did
not exhibit such activity in STING−/−THP-1
cells (fig. S1A). Moreover, treatment of THP-1
cells with MSA-2 induced phosphorylation of
both TBK1 and IRF-3, consistent with STING
pathway activation (fig. S1B). MSA-2 also in-
duced IFN-bin mouse macrophages (Fig. 2C).
In biochemical assays, MSA-2 inhibited binding
of radiolabeled cGAMP to full-length, membrane-
anchored wild-type human STING (hSTING-WT)
and hSTING-HAQ (Fig. 2D). Additionally, MSA-2
appeared to be selective, exhibiting no signif-
icant effect in binding assays against a panel of
108 receptor, transporter, ion channel, and en-
zyme assays when tested at 10mM(tableS1).
Consistent with its small size, MSA-2 also ex-
hibited higher permeability than CDNs such as
the phosphorothioate analog MSA-1 (Fig. 1A)
in an in vitro permeability assay (apparent
permeability = 23.7 × 10−^6 cm/s versus un-
detected in LLC-PK1 cells).Orally dosed MSA-2 exhibits durable
STING-dependent antitumor activity in vivo
To evaluate the in vivo pharmacokinetic and
pharmacodynamic properties and antitumor
activity of MSA-2, it was administered by in-
tratumoral (IT), subcutaneous (SC), or oral
(PO) routes in the MC38 (colon carcinoma)
syngeneic mouse tumor model (Fig. 3A). Phar-
macokinetic studies (Fig. 3, B and C) demon-
strated that MSA-2 dosed via either PO or SC
regimens achieved comparable exposure in
both tumor and plasma (table S2). MSA-2 also
exhibited dose-dependent antitumor activity
when administered by IT, SC, or PO routes,
and dosing regimens were identified that in-
duced complete tumor regressions in 80 to
100% of treated animals (Fig. 3, D to F). Well-
tolerated (assessed by body weight loss and
recovery; Fig. 3G and fig. S2, A to C) PO or SC
doses of MSA-2 that effectively inhibited tu-
mor growth induced substantial elevations of
IFN-b, interleukin-6 (IL-6), and tumor necrosis
factor–a(TNF-a) in tumor and plasma (Fig. 3, H
to J, and fig. S2, D and E), with peak levels at
2 to 4 hours and a return to baseline within
~24 hours (Fig. 3, I to J, and fig. S2, D and E).
All mice that experienced complete tumor
regression were subsequently rechallenged with
MC38 cells. Tumors did not grow in 95% of
rechallenged animals (Fig. 3K), which suggests
that MSA-2 induced long-term antitumor im-
munity. Furthermore, inMC38tumor-bearing
STINGgt/gt(Goldenticket) mice, which lack de-
tectable STING protein ( 20 ), MSA-2 exhibited
no antitumor activity, weight loss, or cytokine
induction (fig. S2, F to H), demonstrating that
the observed MSA-2 activity is STING dependent.
Moreover, as evaluated by in vivo antitumor
activity and tolerability, MSA-2 administered
orally in mice was equal to or better than MSA-1
(cGAMP analog; Fig. 1A) dosed by IT or SC routes
(fig. S3). Orally dosed MSA-1 [10 mg per kilogram
of body weight (mg/kg)] exhibited poor exposure
and was undetectable in plasma (<0.01mM).MSA-2 predimerizes in solution before
binding to STING
The x-ray crystal structure of MSA-2 bound to
human STING (Fig. 4A) shows a closed con-
formation of STING, with the“lid”residues(disordered in the ligand-free protein) form-
ing a four-strandedbsheet atop two MSA-2
molecules and the twoa 2 helices forming a
smaller angle (Fig. 5A) than in the open con-
formation (Fig. 1B), similar to the cGAMP
complex (Fig. 1C). Binding in the same site
as cGAMP, the two MSA-2 molecules make
substantial contact with each other via their
aromatic cores [316 Å^2 of total buried solvent-
accessible surface area (SASA); table S3] and
stack against Tyr^167 from each STING subunit
(Fig. 4A). Bound MSA-2 also forms polar in-
teractions with a network of water molecules
and several surrounding side chains such as
Ser^162 (Fig. 4A; pink dashed lines to methoxy
oxygen atoms). Each MSA-2 ketone forms a
hydrogen bond (green dashed lines) with the
Arg^238 guanidinium group of the proximal
STING monomer (e.g., chain A), and the car-
boxylate forms a hydrogen bond with the
proximal Thr^263 (e.g., chain A). These same
carboxylate groups additionally form hydrogen
bonds (yellow dashed lines) with the Arg^238
side chain across the STING homodimer (e.g.,
chain B), noncovalently cross-linking the pro-
tein homodimer and stabilizing the“closed-
lid”conformation. This binding mode is a
distinctive characteristic of MSA-2 and is not
observed in structures with other STING ago-
nists ( 4 , 18 ). Thus, the MSA-2 dimer fills the
CDN binding pocket as effectively as cGAMP
(MSA-2 dimer: 1047 Å^2 , cGAMP: 1145 Å^2 of
total buried SASA; table S3) and functions as
aSTINGagonistdespitehavingamarkedly
lower molecular weight than that of cGAMP
(294 versus 674 Da).
The solution^1 H nuclear magnetic resonance
(NMR) spectrum of^15 N-labeled hSTING-HAQ
(Fig. 4B, magenta) exhibits several well-separated
resonances below 0 parts per million (ppm).
When incubated with either cGAMP (black) or
MSA-2 (blue), these resonances undergo anal-
ogous upfield shifts relative to ligand-free pro-
tein(Fig.4B,dashedlines),suggestingasimilar
environment for these unassigned protein pro-
tons. Moreover, two-dimensional (2D)^1 H-^15 N
heteronuclear correlation NMR spectra of
hSTING (Fig. 4C) exhibited several distinct
diagnostic peak shifts (or“fingerprints”) cor-
responding to the open conformation (ligand-
free, magenta) or closed conformation (with
bound cGAMP or MSA-2, black or blue, respec-
tively). Substantial chemical shift perturba-
tions were observed for lid residues such as
Gly^230 and Gly^234 , consistent with the pro-
nounced changes in local environment pre-
dicted for the“closed-lid”conformation versus
the largely unstructured lid of the ligand-free
state (Fig. 1B).
Conceptually, the STING–MSA-2 complex
maybeformedbyoneofthethreemechanisms
depicted in Fig. 5C. In Model 1, monomeric
MSA-2 molecules bind independently to two
identical, noninteracting ligand-binding sitesPanet al.,Science 369 , eaba6098 (2020) 21 August 2020 2of10
Fig. 2. Confirmation of MSA-2 as a STING ligand.
(A) Chemical structures of MSA-2 and compound 2.
Me, methyl group. (B) In vitro evidence of STING
agonism included induction of IFN-bin human
THP-1 monocytes and (C) mouse macrophages.
(D) MSA-2 displaces [^3 H]-cGAMP from hSTING-WT
and hSTING-HAQ membranes in a filtration-based
competition assay. Error bars indicate SD.
RESEARCH | RESEARCH ARTICLE