Science - USA (2020-08-21)

(Antfer) #1

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


  1. 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

  2. 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

  3. 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

  4. 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

  5. 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


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