RESEARCH ARTICLE
◥DRUG DEVELOPMENT
An orally available non-nucleotide STING agonist
with antitumor activity
Bo-Sheng Pan^1 , Samanthi A. Perera^1 †, Jennifer A. Piesvaux^1 , Jeremy P. Presland^1 ,
Gottfried K. Schroeder^1 †, Jared N. Cumming^2 ‡, B. Wesley Trotter^2 *†§, Michael D. Altman^2 ,
Alexei V. Buevich^2 , Brandon Cash^2 , Saso Cemerski^3 ¶, Wonsuk Chang^2 , Yiping Chen^1 ,
Peter J. Dandliker^1 #, Guo Feng^1 , Andrew Haidle^2 , Timothy Henderson^2 , James Jewell^2 , Ilona Kariv^1 ,
Ian Knemeyer^4 , Johnny Kopinja^1 , Brian M. Lacey^1 , Jason Laskey^1 , Charles A. Lesburg^2 , Rui Liang^2 **,
Brian J. Long^1 , Min Lu^2 , Yanhong Ma^1 , Ellen C. Minnihan^5 ††, Greg O’Donnell^1 , Ryan Otte^2 ,
Laura Price^1 , Larissa Rakhilina^1 , Berengere Sauvagnat^1 , Sharad Sharma^3 ‡‡, Sriram Tyagarajan^2 ,
Hyun Woo^4 , Daniel F. Wyss^2 , Serena Xu^1 , David Jonathan Bennett^2 †, George H. Addona^1 †
Pharmacological activation of the STING (stimulator of interferon genes)–controlled innate immune
pathway is a promising therapeutic strategy for cancer. Here we report the identification of MSA-2, an
orally available non-nucleotide human STING agonist. In syngeneic mouse tumor models, subcutaneous
and oral MSA-2 regimens were well tolerated and stimulated interferon-bsecretionintumors,inducedtumor
regression with durable antitumor immunity, and synergized with anti–PD-1 therapy. Experimental
and theoretical analyses showed that MSA-2 exists as interconverting monomers and dimers in solution,
but only dimers bind and activate STING. This model was validated by using synthetic covalent
MSA-2 dimers, which were potent agonists. Cellular potency of MSA-2 increased upon extracellular
acidification, which mimics the tumor microenvironment. These properties appear to underpin the
favorable activity and tolerability profiles of effective systemic administration of MSA-2.
R
ecent clinical experience with new cancer
therapies that block immune checkpoint
pathways, such as antibodies to PD-1
(anti–PD-1), has led to intense efforts
focused on other immune pathways
that may be pharmacologically modulated to
enhance the therapeutic benefits of check-
point inhibitors ( 1 ). STING (stimulator of
interferon genes) is an endoplasmic reticulum–
associated homodimeric protein and the receptor
for 2′,3′-cyclic guanosine monophosphate–
adenosine monophosphate (cGAMP) (Fig. 1A),
which is a second messenger produced by
cGAMP synthase, a cytosolic double-stranded
DNA sensor ( 2 , 3 ). Crystallographic ( 4 )and
cryo–electron microscopy ( 5 , 6 ) studies have
revealed that cGAMP binding to STING induces
a pronounced conformational change from
the open form of the ligand-free structure
(Fig. 1B) to a closed-form complex that com-
pletely sequesters the bound ligand from
solution(Fig.1C).ActivationofSTINGby
cGAMP triggers downstream signaling events
initially via interactions of the closed-form
complex with TBK1 kinase ( 5 , 6 )andlater
by the transcription factors IRF3 and NFkB,
culminating in increased synthesis and secre-
tion of type I interferons and proinflammatory
cytokines ( 3 ). Type I interferons are essential to
the development of robust adaptive antitumor
immunity owing to their ability to stimulate
T cell cross-priming ( 7 ), potentially rendering
tumors more susceptible to checkpoint block-
ade ( 8 ). The therapeutic potential of STING
agonism has been demonstrated in preclinical
studies with syngeneic mouse tumor models
in which a cyclic dinucleotide (CDN) STING
agonist exhibited marked antitumor activity
when administered intratumorally either alone
or in combination with an inhibitor of PD-1 or
PD-L1 ( 7 , 9 – 12 ). However, owing to ubiquitous
STING expression, systemically administered
CDN-based STING agonists induced inflam-
matory cytokines in both tumor and normal
tissues. Thus, the dosing routes of CDN-based
STING agonists are largely limited to direct
intratumor injection ( 7 ), which restricts their
application to a subset of tumors.
To address a broad range of malignancies,
STING agonists that are suitable for systemic
administration are required. Feasibility oftreating tumors with a systemically dosed
STING agonist has been demonstrated in vivo
with the xanthone DMXAA, a mouse-specific
STING agonist ( 13 – 15 ) that binds mouse STING
in the same closed form as that induced by
cGAMP( 4 ) but does not activate human STING
( 16 ). Unfortunately, efforts to use DMXAA as a
starting point for medicinal chemistry have
thus far not succeeded ( 17 ). Recently, a non-
CDN–based human STING agonist intended
for intravenous administration was reported
( 18 ). Herein, we describe a previously unknown
non-nucleotide STING agonist (MSA-2) that
preferentially targets tumor tissue, owing to
its distinctive mechanism of action. Moreover,
MSA-2 can be dosed by oral administration,
which is a convenient, low-cost delivery route.Discovery of the non-CDN STING agonist MSA-2
To identify cell-permeable STING agonists,
we developed a high-throughput, cell-based
phenotypic screen to detect stimulation ofRESEARCH
Panet al.,Science 369 , eaba6098 (2020) 21 August 2020 1of10
(^1) Department of Quantitative Biosciences, Merck & Co., Inc.,
Kenilworth, NJ, USA.^2 Department of Discovery Chemistry,
Merck & Co., Inc., Kenilworth, NJ, USA.^3 Department of
Discovery Oncology, Merck & Co., Inc., Kenilworth, NJ, USA.
(^4) Department of Pharmacokinetics, Merck & Co., Inc., Kenilworth,
NJ, USA.^5 Department of Discovery Pharmaceutical Sciences,
Merck & Co., Inc., Kenilworth, NJ, USA.
*These authors contributed equally to this work.
†Corresponding author. Email: [email protected]
(S.A.P.); [email protected] (G.K.S.); wes.trotter@
kronosbio.com (B.W.T.); [email protected] (D.J.B.);
[email protected] (G.H.A.)
‡Present address: LifeMine Therapeutics, Cambridge, MA, USA.
§Present address: Kronos Bio, Inc., Cambridge, MA, USA.
¶Present address: Cue Biopharma, Cambridge, MA, USA.
#Present address: Dewpoint Therapeutics, Inc., Boston, MA, USA.
**Present address: Tri-Institutional Therapeutics Discovery Institute,
New York, NY, USA.
††Present address: Bill & Melinda Gates Medical Research Institute,
Cambridge, MA, USA.
‡‡Present address: Sanofi US, Cambridge, MA, USA.
Fig. 1. Structures of CDN ligands and human
STING.(A) Chemical structures of human endoge-
nous STING agonist cGAMP and synthetic analog
MSA-1. (BandC) The ligand-free open-form
structure (magenta) (B) of the STING homodimer
(PDB ID 4EF5) undergoes conformational change
to a closed form (gray) (C) upon binding cGAMP
(PDB ID 4KSY). In the closed form, the angle
between the central helices (a 2 ) becomes more
acute, and the lid residues form a four-stranded
bsheet atop the binding site.