DRUG DEVELOPMENT
Antitumor activity of a systemic STING-activating
non-nucleotide cGAMP mimetic
Emily N. Chin^1 , Chenguang Yu1,2, Vincent F. Vartabedian^3 , Ying Jia1,3*, Manoj Kumar^2 , Ana M. Gamo^2 ,
William Vernier^2 , Sabrina H. Ali^1 , Mildred Kissai^1 , Daniel C. Lazar^3 , Nhan Nguyen^3 , Laura E. Pereira^1 ,
Brent Benish^2 , Ashley K. Woods^2 , Sean B. Joseph^2 , Alan Chu^2 , Kristen A. Johnson^2 ,
Philipp N. Sander^1 , Francisco Martínez-Peña^1 , Eric N. Hampton^2 , Travis S. Young^2 , Dennis W. Wolan^4 ,
Arnab K. Chatterjee^2 , Peter G. Schultz1,2, H. Michael Petrassi^2 †, John R. Teijaro^3 †, Luke L. Lairson^1 †
Stimulator of interferon genes (STING) links innate immunity to biological processes ranging
from antitumor immunity to microbiome homeostasis. Mechanistic understanding of the
anticancer potential for STING receptor activation is currently limited by metabolic instability of
the natural cyclic dinucleotide (CDN) ligands. From a pathway-targeted cell-based screen, we
identified a non-nucleotide, small-molecule STING agonist, termed SR-717, that demonstrates
broad interspecies and interallelic specificity. A 1.8-angstrom cocrystal structure revealed
that SR-717 functions as a directcyclic guanosine monophosphate–adenosine monophosphate
(cGAMP) mimetic that induces the same“closed”conformation of STING. SR-717 displayed
antitumor activity; promoted the activation of CD8+T, natural killer, and dendritic cells in
relevant tissues; and facilitated antigen cross-priming. SR-717 also induced the expression
of clinically relevant targets, including programmed cell death 1 ligand 1 (PD-L1), in a STING-
dependent manner.
T
he cyclic guanosine monophosphate
(GMP)–adenosine monophosphate (AMP)
synthase (cGAS)–stimulator of inter-
feron genes (STING) (cGAS-STING) sig-
naling pathway plays a critical role in
the innate response to infection ( 1 , 2 ). It also
serves as a direct link between inflammation
and diverse physiological processes, includ-
ing micronuclei surveillance in the context
of DNA damage ( 3 , 4 ), age-associated in-
flammation ( 5 ), mitochondrial DNA–related
inflammatory phenotypes ( 6 ), and microbiome-
dependent intestinal homeostasis ( 7 ). STING
is an endoplasmic reticulum signaling protein,
partially localized to mitochondria-associated
membranes, that is broadly expressed in both
immune and nonimmune cell types. STING
binds cyclic dinucleotides (CDNs)—including
2 ′,3′–cyclic GMP-AMP (2′,3′-cGAMP) produced
by cGAS in response to cytosolic DNA ( 8 )—
and the scaffolding function rapidly induces
type I interferon (IFN) and proinflamma-
tory cytokines in a TBK1–IRF3-dependent
fashion ( 9 , 10 ). STING has been demonstrated
to play essential roles in antitumor immu-
nity. For example, efficient tumor-initiated
T cell activation requires STING pathway–
dependent IFN-bexpression, as well as ex-
pression of STING in dendritic cells (DCs)
( 11 , 12 ).
Initial STING agonist small molecules were
synthesized as derivatives of the CDN natural
ligand; however, because of poor stability prop-
erties, CDN-based agonist administration is
limited to intratumoral delivery. Although
intratumoral delivery of CDN agonists has
consistently shown regression of established
tumors in syngeneic models ( 13 , 14 ), intra-
tumor CDN administration in humans has
been met with mixed success. Activation of
the STING pathway has also been demon-
strated to contribute notably to the antitumor
effect of radiation andchemotherapeutics
( 4 , 15 , 16 ). As such, a systemic STING-activating
agent has considerable potential utility, not
only as a therapeutic for cancer and infec-
tious disease but also as a pharmacological
probe to enable mechanistic discovery in the
context of STING-dependent antitumor im-
munity and diverse STING-dependent bio-
logical processes.
We used a cGAS-STING pathway–targeted
cell-based phenotypic screening approach
to identify functional non-nucleotide small-
molecule STING agonists. Human monocytic
THP-1 cells, harboring an interferon regulatory
factor (IRF)–inducible luciferase reporter con-
struct (ISG-THP1), were used to screen a col-
lection of ~100,000 commercially available
structurally diverse druglike small molecules
in 1536-well plate format [5mM, 0.1% dimethyl
sulfoxide (DMSO)]. Confirmed primary hits
(plate-basedzscore >3) were evaluated in
secondary assays involving STING knockout
(ISG-THP1 STING-KO) or cGAS knockout(ISG-THP1 cGAS KO) cell lines (fig. S1) to
determine pathway specificity and potential
target identity, respectively. Protein thermal
shift assays, involving recombinant human
and mouse STING protein (hSTING and
mSTING), were used to profile compounds
for direct on-target binding activity, cross-
species activity, and human allele specificity.
This resulted in the identification of SR-001
(Fig. 1A), which was found to robustly induce
reporter signal in ISG-THP1 cells [fig. S2A;
mean effective concentration (EC 50 )~1.1mM]
and ISG-THP1 cGAS KO cells (fig. S2B) but was
completely inactive in ISG-THP1 STING KO
cells (fig. S2, A to C), suggesting that the com-
pound acts downstream of cGAS with a de-
pendence on STING expression. Commercial
SR-001 was also found to increase the ther-
mal stability of the soluble C-terminal CDN-
binding domain of recombinant human STING
protein (hSTINGREF) (fig. S2E), which is re-
sponsible for recruiting downstream signaling
proteins. Chemical resynthesis of SR-001 af-
forded a compound that was consistently
active in cell-based assays (fig. S2D) but was
now devoid of activity in the STING thermal
shift binding assay (fig. S2E). Analytical char-
acterization of the commercial material re-
vealed the presence of a minor but notable
amount of the de-esterified derivative SR-
012 (Fig. 1A). This suggested that SR-001
was acting as a prodrug, with ester substi-
tution being a requisite for cell permeabil-
ity and the active STING-binding species
being the carboxylic acid.
Consistent with this hypothesis, synthetic
SR-012 was found to bind both recombinant
hSTING and mSTING protein (fig. S2E) but
was inactive in cell-based assays (fig. S2D).
SR-001 was also observed to be rapidly con-
verted to SR-012 in cells (fig. S2F). To address
the cell permeability of SR-012 and intrac-
table rodent exposure properties of the SR-001
prodrug, we completed a medicinal chem-
istry effort focused on improved prodrug
stability or bioisosteric replacement of the
carboxylic acid or improved cell permeability
for the carboxylic acid. This resulted in the
identification of a carboxylic acid–containing
analog, containing difluoro-substitution of
the aniline ring system (SR-717; Fig. 1B),
which was found to possess equivalent cell-
basedactivitywhencomparedwithSR-001
prodrug (Fig. 1C; ISG-THP1, EC 50 =2.1mM;
ISG-THP1 cGAS KO, EC 50 =2.2mM; ISG-
THP1 STING KO, no activity up to the limit
of solubility). Critically, SR-717 increased the
thermal stability of the common human al-
leles of hSTING (Fig. 1D and fig. S3A), as well
as that of recombinant soluble mSTING pro-
tein (Fig. 1D), suggesting that the binding
mode of SR-717 is not affected by interallelic
or interspecies differences in amino acid
sequence. This latter issue was ultimatelyRESEARCH
Chinet al.,Science 369 , 993–999 (2020) 21 August 2020 1of7
(^1) Department of Chemistry, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
(^2) California Institute for Biomedical Research, 11119 North
Torrey Pines Road, La Jolla, CA 92037, USA.^3 Department
of Immunology and Microbiology, The Scripps Research
Institute, 10550 North Torrey Pines Road, La Jolla, CA
92037, USA.^4 Department of Molecular Medicine, The
Scripps Research Institute, 10550 North Torrey Pines Road,
La Jolla, CA 92037, USA.
*These authors contributed equally to this work.
†Corresponding author. Email: [email protected] (H.M.P.);
[email protected] (J.R.T.); [email protected] (L.L.L.)