in an acidified tumor microenvironment (ver-
sus normal tissue), owing to increased cellular
entry and retention combined with its inher-
ent mode of interaction with STING. It is
likely that preferential activation of STING by
MSA-2 in tumors substantially contributes
to the observed favorable in vivo antitumor
activity and tolerability profile of this com-
pound. MSA-2 is a small molecule that under-
goes reversible, noncovalent dimerization in
solution to become a pharmacologically active
ligand. MSA-2, an orally available human and
mouse STING agonist, should be a valuable
tool in the endeavor to discover and design
human STING agonists suitable for systemic
administration in the clinic.
Materials and methods summary
High-throughput screening and follow-up assays
In the primary screen, THP-1 (ATCC TIB-202)
cells were incubated, in 1536-well plates, with
test compounds (20 μM) in a RPMI1640-based
assay medium in the presence of 5% carbon
dioxide at 37°C for 5 hours. IFN-blevels were
determined using an AlphaLISA assay and
an EnVision Reader (PerkinElmer, Waltham,
MA) and expressed as percentages of IFN-b
induced by cGAMP (100 μM, positive con-
trol). In follow-up assays, the cellular activity
of hits from the primary screen was confirmed
in THP-1 and mouse macrophage cells by using
the Meso Scale Human Interferon-bassay kit
(Meso Scale Discovery) or the mouse IFN-b
Verikine ELISA (enzyme-linked immunosor-
bent assay) (PBL Assay Science, Piscataway, NJ),
respectively. STING binding activity of com-
pounds was evaluated with a competitive radio-
ligand binding assay using tritiated cGAMP
and membrane embedded full-length recom-
binant human and mouse STING generated
in insect cells. STING pathway activation by
MSA-2 was assessed by Western blotting, prob-
ing phosphorylation status and total protein
levelsofSTING,TBK-1,andIRF3byusingcom-
mercially available antibodies (Cell Signaling
Technology, Danvers, MA).
Biochemical and biophysical methods
In saturation binding experiments, insect mi-
crosomes expressing full-length STING were
incubated with serially diluted tritiated MSA-2
for 18 hours at 25°C. Reactions were terminated
by filtration, and filter-bound radioactivity
was measured by a TopCount NXT instrument
(Perkin Elmer). Nonspecific binding was de-
termined in the presence of cGAMP (20 μM).
In homologous competition binding experi-
ments, insect microsomes expressing hSTING-
WT or mSTING were incubated for 16 hours
(25°C) with serially diluted unlabeled MSA-2
(with or without 100 μM cGAMP) at a fixed
concentration of tritiated MSA-2 (0.16 μM).
Levels of STING-bound tritiated MSA-2 were
determined as described above. N-terminal
tagged recombinant cytosolic domain STING
constructs were cloned into the pET47b plasmid,
expressed inEscherichia coli,andpurifiedby
affinity and size exclusion chromatography. Af-
finity tags were removed for proteins intended
for crystallography and protein NMR. STING
intended for SPR experiments was biotinylated
using BirA Biotin-Protein Ligase Bulk Reaction
Kit (Avidity, Aurora, CO). STING used in NMR
experiments was generated using expression
media containing [^15 N]-ammonium sulfate
(Cambridge Isotope Laboratories, Tewksbury,
MA). For crystallography, cocrystals of hSTING-
HAQ complexed with MSA-2 or covalent dimers
were prepared by hanging-drop vapor diffu-
sion with streak seedingat18°C.Sampleswere
prepared for synchrotron data collection by
swishing through perfluoropolyether cryo oil
(Hampton Research) before plunging into liq-
uid nitrogen. Structures were solved by molec-
ular replacement using PDB ID 4KSY as a
probe. Protein NMR experiments (1D^1 Hmethyl
and 2D^1 H-^15 N SOFAST-HMQC) using^15 N-
labeled STING (50 μM) were conducted at 30°C
on an 800-MHz Bruker Ascend Four Channel
AVANCE III HD NMR spectrometer equipped
with a TCI 5-mm CryoProbe (automatic tuning
and matching). Proton (^1 H) NMR experiments
to determine dimerization properties of MSA-2
or compound 2 were collected on a Varian
VNMRS 600-MHz instrument at 25°C. For SPR
(Biacore T200, GE Healthcare) experiments,
biotinylated cytosolic domain STING variants
(1 to 3 μM, molecular weight ~31 kDa) were
captured on a streptavidin chip (Series S Sensor
Chip SA, GE Healthcare) to a final level of
~3100 resonance units. Serially diluted com-
pound solutions were analyzed using single-
cycleinjectionmodeataflowrateof50μl/min
in HBS-EP+ buffer (GE Healthcare) with 1 mM
dithiothreitol and 3% v/v dimethyl sulfoxide.
For ALIS experiments, human STING (5 μM)
was preincubated with MSA-2 and/or com-
pound 2 for 30 min before injection into the
ALIS system. Both protein and protein-ligand
complexes were separated from unbound ligand
by using a proprietary size exclusion chroma-
tography column and were subsequently di-
rected to a reverse-phase C18 column (40°C)
equilibrated with aqueous 0.2% formic acid.
Dissociated ligands were resolved using a sol-
vent gradient (0 to 95% acetonitrile in 2.5 min)
and eluted directly into a high-resolution Ex-
active mass spectrometer (ThermoFisher Sci-
entific, San Jose, CA).In vivo methods
All animal experimental procedures were per-
formed according to the guidelines approved
by the Institutional Animal Care and Use Com-
mittee of Merck & Co., Inc., Kenilworth, NJ,
USA, following the guidance of the Association
for Assessment and Accreditation of Labora-
toryAnimalCare.C57BL/6JandNSG(NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ)micewereob-
tained from The Jackson Laboratory (Bar
Harbor, ME), whereas BALB/c and nude NCr
mice were obtained from Taconic Biosciences
(Germantown, NY). Tumor cells were inocu-
lated subcutaneously into the lower flank.
MSA-2 or vehicle was dosed by IT injection,
SC injection, or PO gavage. Tumor and body-
weight measurements were performed twice
per week using calipers and a weigh scale, re-
spectively. Mice were euthanized when tumor
volume approached ~2000 mm^3 ,weightloss
exceeded 20%, or tumors ulcerated. When
necessary, plasma and tumor samples were
collected at specific time points and frozen for
pharmacokinetics and pharmacodynamics
studies. MSA-2 concentration was then de-
termined by liquid chromatography and mass
spectroscopy (API5000, Applied Biosystems).
IFN-bwas measured by ELISA (PBL Assay
Science, Piscataway, NJ), and IL-6 and TNF-a
were measured using a Meso Scale kit (custom
U-plex kit, Meso Scale Discovery, Rockland,
MD). Tumor pH was measured using a bevel-
needle–tipped combination microelectrode
(Orion 9863BN Micro pH Electrode) inserted
up to 1.3 cm into the center of the tumor.Chemistry
Tritiated cGAMP was synthesized via a bio-
catalytic reaction in which recombinant
cGAMP synthase preactivated with herring
DNA was incubated with [^3 H]-ATP (Perkin
Elmer) and [^3 H]-GTP (Perkin Elmer) overnight
at 37°C. The reaction was then filtered to re-
move protein, and [^3 H]-cGAMP was purified
by anion exchange chromatography. MSA-2
was synthesized in three steps using 5,6-
dimethoxybenzo[b]thiophene-2-carboxylic acid
as the starting material and (3-ethoxy-3-
oxopropyl)zinc(II) bromide. Tritiated MSA-2
(4-(5-methoxy-6-(methoxy-t 3 )benzo[b]thiophen-
2-yl)-4-oxobutanoic acid) was synthesized in
five steps using MSA-2 as the starting material.
See the supplementary materials for more
details about the experimental materials and
methods.REFERENCES AND NOTES- S. A. Patel, A. J. Minn, Combination cancer therapy with
immune checkpoint blockade: Mechanisms and strategies.
Immunity 48 , 417–433 (2018). doi:10.1016/
j.immuni.2018.03.007; pmid: 29562193 - P. Gaoet al., Cyclic [G(2′,5′)pA(3′,5′)p] is the metazoan second
messenger produced by DNA-activated cyclic GMP-AMP
synthase.Cell 153 , 1094–1107 (2013). doi:10.1016/
j.cell.2013.04.046; pmid: 23647843 - X. Zhanget al., Cyclic GMP-AMP containing mixed
phosphodiester linkages is an endogenous high-affinity ligand
for STING.Mol. Cell 51 , 226–235 (2013). doi:10.1016/
j.molcel.2013.05.022; pmid: 23747010 - P. Gaoet al., Structure-function analysis of STING activation by
c[G(2′,5′)pA(3′,5′)p] and targeting by antiviral DMXAA.
Cell 154 , 748–762 (2013). doi:10.1016/j.cell.2013.07.023;
pmid: 23910378 - G. Shang, C. Zhang, Z. J. Chen, X. C. Bai, X. Zhang, Cryo-EM
structures of STING reveal its mechanism of activation by
cyclic GMP-AMP.Nature 567 , 389–393 (2019). doi:10.1038/
s41586-019-0998-5; pmid: 30842659
Panet al.,Science 369 , eaba6098 (2020) 21 August 2020 9of10
RESEARCH | RESEARCH ARTICLE