Discovery of a Novel cGAMP Competitive Ligand of the Inactive Form of STING

Article abstract of DOI:10.1021/acsmedchemlett.8b00466

Drugging large protein pockets is a challenge due to the need for higher molecular weight ligands, which generally possess undesirable physicochemical properties. In this communication, we highlight a strategy leveraging small molecule active site dimers

Full text of DOI:10.1021/acsmedchemlett.8b00466

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Letter
Discovery of a Novel cGAMP Competitive
Ligand of the Inactive Form of STING
Tony Siu, Michael D Altman, Gretchen A Baltus, Matthew Childers, J Michael Ellis,
Hakan Gunaydin, Harold Hatch, Thu Ho, James Jewell, Brian M Lacey, Charles A
Lesburg, Bo-Sheng Pan, Berengere Sauvagnat, Gottfried K Schroeder, and Serena Xu
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00466 • Publication Date (Web): 06 Dec 2018
Downloaded from http://pubs.acs.org on December 10, 2018
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Discovery of a Novel cGAMP Competitive Ligand of the Inactive
Form of STING
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
,†
§
‡
†
†
Tony Siu, Michael D. Altman, Gretchen A. Baltus, Matthew Childers, J. Michael Ellis,
§

¥
†

Hakan Gunaydin, Harold Hatch, Thu Ho, James Jewell, Brian M. Lacey, Charles A.
∞




Lesburg, Bo-Sheng Pan, Berengere Sauvagnat, Gottfried K. Schroeder, and Serena Xu
†
‡
§

¥
Departments of Chemistry, Immunology, Chemistry Modeling and Informatics, In Vitro Pharmacology, Target
∞
Protein Design and Structural Chemistry, Merck & Co., Inc., 33 Avenue Louis Pasteur, Boston, MA 02115
KEYWORDS: STING, active site dimers, ligand-ligand interactions, permeability, inflammatory disease, druggability.
ABSTRACT: Drugging large protein pockets is a challenge due to the need for higher molecular weight ligands which
generally possess undesirable physicochemical properties. In this communication, we highlight a strategy leveraging small
molecule active site dimers to inhibit the STING protein, which contains a large symmetric binding pocket. By taking
advantage of the 2:1 binding stoichiometry, maximal buried interaction with STING protein can be achieved while
maintaining the ligand physicochemical properties necessary for oral exposure. This mode of binding requires unique
considerations for potency optimization including simultaneous optimization of protein-ligand as well as ligand-ligand
interactions. Successful implementation of this strategy led to the identification of 18, which exhibits good oral exposure,
slow binding kinetics, and functional inhibition of STING mediated cytokine release.
The activation of the innate immune system through
nucleic acid sensing is a key mechanism of host defense
from viruses and bacteria. Recent discoveries in the
cyclic GMP-AMP synthase–stimulator of interferon genes
Additionally, loss of function mutations in the DNA
exonuclease TREX1 lead to the excessive type 1 interferon
signature found in Aicardi–Goutières syndrome (AGS)
and SLE, implicating the role of self dsDNA in auto-
1
-2
1
1
(
cGAS-STING) pathway have captured the attention of
inflammation.
Furthermore, monogenic Mendelian
the pharmaceutical industry due to the pathway’s role in
diseases with STING gain of function mutations such as
familial chilblain lupus (FCL) support the role of STING
in autoimmune disease. These studies collectively
suggest that inhibition of STING might regulate DNA-
driven inflammatory diseases.
host
pathogen
defense,
immuno-oncology
and
autoimmune diseases.3 Upon activation by double
stranded DNA (dsDNA), cGAS synthesizes the cyclic
dinucleotide secondary messenger 2’,3’-cyclic GMP-AMP
12
(
(
cGAMP) which then activates the endoplasmic reticulum
4-5
ER)–membrane adaptor protein STING.
Activated
STING initiates a cascade that ultimately primes the
immune system to restrict viral spread through the
activation and production of type 1 interferons (IFN) and
pro-inflammatory cytokines such as tumor necrosis
factor- (TNF-).
While stimulation of STING and the production of type 1
interferons is an important mechanism for pathogen
defense and tumor control, failure to regulate chronic
6
inflammatory signaling can lead to autoimmunity. The
mis-localization of nuclear and mitochondrial dsDNA in
the cytoplasm combined with the inability of cGAS to
differentiate foreign from self dsDNA is a potential trigger
Figure 1: Conformational comparison of STING
apoprotein (“open” conformation, green, PDB 6MX0)
with a STING:cGAMP complex (“closed”, white, from PDB
4KSY). Residues which become ordered upon cGAMP
binding are shown as dark gray β-strands in the closed
conformation and green dashes in the open
7-9
for type 1 interferon production and auto-inflammation.
Type 1 interferon and mis-localized dsDNA are hallmarks
and key drivers for the pathogenesis of autoimmune
1
0
diseases such as systemic lupus erythematosus (SLE).
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are bound to a single STING homodimer (Figure 2).15
conformation. cGAMP is shown as sticks in the center of
the STING homodimer. Arrows indicate the direction of
movement of the central α-2 helix upon cGAMP binding
and the ordering of the central β-sheet.
This 2:1 binding ratio is possible due to the C2 symmetry
of the STING protein which allows each DMXAA
molecule to interact with a single STING monomer. Our
strategy to maximize binding efficiency was to exploit the
intrinsic symmetry of the STING protein by identifying
small molecules that can bind to the “open” conformation
in a 2:1 ratio to the STING homodimer. Such a binding
stoichiometry could afford fuller occupation of the large
binding site, and thus effectively compete with cGAMP,
while still maintaining physiochemical properties
compatible with oral drugs.
Although the biological rationale linking STING to
inflammatory diseases supports the development of a
STING antagonist, the general lack of biochemical
mechanistic understanding of STING activation
combined with the absence of known small molecule
tools suggested that identification of binders to the
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13-14
STING protein might pose a significant challenge.
Based on biophysical and X-ray crystallographic data, the
STING C-terminal domain (referred to as STING
henceforth) exists as a symmetrical dimer with the ligand
binding site located at the interface between the two
monomers, which has been shown to be the binding site
of the natural agonist cGAMP and the mouse-specific
1
5
agonist DMXAA. Comparison of the structures of STING
apoprotein with cGAMP-bound protein highlights two
distinct conformations (Figure 1). The STING apoprotein
adopts an “open” conformation, with residues 226 to 241
in each monomer. In contrast, STING complexed with an
agonist adopts a “closed” conformation wherein, the
central α-2 helix from each monomer (approximately
residues 171 to 185) tilt toward each other by roughly 15°
and residues 226 to 241 in each monomer, disordered in
the “open” conformation, adopt a four-stranded β-sheet.
These distinct conformational states suggest that the
stabilization of the “open” conformation might lead to
inactivation whereas stabilization of the “closed”
Figure 2: X-ray structure of DMXAA molecules bound to
1
5
mouse STING in the “closed” conformation (PDB 4LOL)
.
STING protein is colored green, and DMXAA is shown in
blue sticks. Intermolecular interactions between DMXAA
and Thr-262 and Thr-266 are indicated by black dashes.
1
5-17
conformation might lead to activation of the protein.
There is also
a salt bridge between the DMXAA
With the knowledge of the ligand binding site, a
druggability assessment was performed on the apoprotein
to probe the feasibility of discovering a suitable ligand
which might preferentially bind to the “open”
carboxylate group and Arg-237 (not shown; corresponds
to Arg-238 in human STING).
1
8
conformation. The total protein solvent accessible
Table 1: Profile of screening hit compound 1.
2
surface area (SASA) is calculated to be 390 Å , which
O
OH
O
1
9
would require a ligand of ~700 Da to occupy. Of the
total SASA, 60% is polar SASA which limits maximal
binding affinity driven by hydrophobic interactions.
N
O
Finally, the calculated volume of the ligand binding site is
3
9
52 Å , suggesting the possibility for binding a large
complex
ligand.
Unfortunately,
physicochemical
1 (rac)
properties of ligands of this nature are not generally
consistent with orally bioavailable drug-like small
HAQ STING IC50 (nM)a
7,300
430
20
molecules. Another complexity in targeting the STING
MW
protein is the requirement for
a small molecule
cLogP
6.21
antagonist to be competitive with the high molecular
weight, high affinity endogenous ligand, cGAMP.
Together, these considerations highlight the challenges
for identifying small molecule STING antagonists with
the appropriate properties necessary for oral exposure.
LBE/ LLEb
0.22/1.4
aValues in this table are determined by the HAQ STING cGAMP
displacement assay and are the means from at least n = 2 experiments.
b
Values are calculated from pIC50 − ALogP98
The large ligand binding surfaces and volumes calculated
Encouraged by the possibility of utilizing 2:1 binding
stochiometry to offset the challenge of a large binding
pocket, we turned to our Automated Ligand
for STING necessitated
a
unique approach. Our
investigation was inspired by the recognition that two
molecules of the mouse-specific STING agonist DMXAA
2
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Identification System (ALIS), which has emerged as a
robust platform for hit identification. Compound 1 was
initially identified as a low activity hit (IC50 = 7,300 nM).
in the ligand binding site. The aromatic ring of the
isoquinolone not only engages in face-to-face π-stacking
with the aromatic ring of the other copy of 1, but also
21
engages
in
edge-to-face
π-stacking
with
the
Crystallographic studies confirmed 2:1 binding. Not only
do two molecules of 1 complement each other’s shape and
bind to one dimer of STING, but they also bind to the
22
methoxyphenyl group. This network of interlocking π-
stacking interactions between the two copies of the
molecules reinforces binding to the STING protein and is
likely the source of observed cooperative binding (vida
infra). The two monomers form a substantial dimer
“open” conformation (Figure 3) suggesting these
molecules might function as an antagonist. Two
interlocking copies of the compound are bound in the
central cleft of the STING homodimer, and the absolute
configuration is unambiguously assigned as (S, S).
1
1
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interface within the active site, burying approximately 274
2
Å
of water accessible surface area, emphasizing the
importance of ligand-ligand interactions in the observed
23
binding mode.
Scheme 1: Synthesis of tetrahydroisoquinolone
analogs
O
O
R2
a
O
4
O
OH
N
X
Y
Route 1
H2N
X
Y
O
R2
R1
O
R1
2
3
6
Route 2
O
b
OH
OH
R2
O
5
3
a) InCl , Cs
2 3
CO , MeCN, microwave 100 °C, 2-38% ; b)
toluene, reflux, 31-81%.
An advantage of the tetrahydroisoquinolone series is the
flexibility of the synthesis which allows for rapid diversity
generation in a one-pot synthesis. From the initial hit,
SAR was generated using the procedures highlighted in
Figure 3: X-ray structure of 1 in STING protein showing
2:1 binding and ligand interactions (PDB 6MX3). Top:
STING is shown as a white surface, with 1 colored yellow.
The inset shows a close-up view of the two copies of 1
from the direction indicated by the arrow. Bottom: close-
up of polar interactions between 1 and side chains of Thr-
Scheme 1.
In this initial approach, aniline (2),
homopthalic anhydride (4), and benzaldehyde (3) were
heated in a microwave reactor with cesium carbonate
(
2 3 3
Cs CO ) and indium chloride (InCl ) as a catalyst.
263 and Thr-267 of both STING chains. Sidechain atoms
However, the yields were poor and the purification was
complicated by mixtures of side products. By switching to
the diacid (5) and removing InCl , reactions proceeded
3
of Tyr-167 are also shown as sticks. Dashed lines indicate
selected van der Waals interactions and hydrogen bonds.
more cleanly to desired products.
Compound
1
makes predominantly hydrophobic
Despite
a
clear view of the compound-protein
interactions, punctuated by several polar contacts. The
carboxylate group forms a hydrogen bond with the side
chain of Thr-263 and the exocyclic oxygen forms a
hydrogen bond with the side chain of Thr-267. The
methoxyphenyl group maintains van der Waals contact
with the protein, and the para-tert-butyl group projects
towards open solvent. There are a number of disordered
residues which have been omitted from the
crystallographic model, including many which become
ordered upon agonist binding. In addition, there are
various interactions between the two molecules of 1 found
interactions, early SAR highlighted the challenge of
designing molecules with improved affinity. Table 2 lists
a few examples to illustrate initial struggles to improve
potency. While computational studies supported the
replacement of the methoxyphenyl with various
substitutions, these modifications did not lead to
improvements in activity with the exception of the
benzodioxane compound 7. Because these compounds are
bound to STING as dimers, special consideration is
required to rationalize the observed SAR. In addition to
stabilizing the protein-ligand complex relative to the
3
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unbound state, the interaction between the bound
enhance both ligand-ligand and protein-ligand
interactions. Toward that end, various heteroatoms were
incorporated to increase the ligand π interactions. In
order to compare the analogs as matched pairs, the
compounds were synthesized as single enantiomers with
the more potent chloro t-butyl phenyl solvent piece
(Table 3). Although none of these analogs were
dramatically differentiated from compound 13, the
experimental results were consistent with our hypothesis
that activity was dependent on both ligand and protein
interactions when compared to Table 2.
monomers must also be considered. To this end, we
redesigned our computational models to include an
evaluation of dimer association energy using density
functional theory (DFT) to estimate active site ligand-
ligand complementarity in addition to docking studies to
24
model protein-ligand interactions.
Table
2:
Initial
representative
SAR
on
0
1
2
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7
8
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tetrahydroisoquinoline screening hit
Compound
7 (rac)
Structure
IC50 (nM)a
Table 3: Benzodioxane variants
O
OH
O
O
O
OH
N
2700
R
O
N
O
OH
N
O
Cl
O
8
9
(rac)
(rac)
>20000
>20000
>20000
IC50 (nM)a
84
Compound
R
O
1
3
O
OH
N
O
O
S
14
15
16
384
41
S
O
O
O
OH
CN
S
S
10 (rac)
N
670
O
O
O
17
106
O
OH
N
N
N
aValues in this table are determined by the HAQ STING cGAMP
displacement assay and are the means from at least n = 2 experiments.
1
1 (rac)
>20000
O
O
OH
O
F
F
Although 13 exhibited potent activity in the cell-free
ligand displacement assay, it displayed low oral
bioavailability (%F = 5) which may be a consequence of
O
1
2 (rac)
N
>20000
O
-6
26
the modest permeability (MDCK Papp = 9 x 10 cm/s).
We postulated that an increased ionization of 13
negatively impacted the passive permeability. The pK is
calculated to be 3.4, lower than the typical value (pK = 4-
) for an aliphatic carboxylic acid, likely due to an
aValues in this table are determined by the HAQ STING cGAMP
displacement assay and are the means from at least n = 2 experiments.
a
a
Consistent with previous observations regarding
substituent effects on edge-to-face aromatic interactions,
the predicted dimer association energies track with the
electron richness of the accepting ring as well as steric
interaction/repulsion across the dimer interface (see
5
inductive effect from the aromatic isoquinolone core.
Accordingly, extension of the carboxylic acid to the
homologated acid (compound 18) preserved activity and
a
increased the calculated pK to 4.3. To rule out any
25
SI). For example, the electron-rich benzodioxyl 7 has a
unexpected conformational change induced by 18,
crystallographic studies were conducted (Figure 4). As
with 1, 2 molecules of extended acid 18 bind a single
STING homodimer in the “open” conformation. The
extended acid group and the lone pair of the
isoquinonlone amide maintain the same interactions with
Thr-263 and Thr-267, which are found in the identical
conformation as in the complex with 1. Unique to this
structure is the benzodioxane stacking with Tyr-167. The
aromatic fluorine fills a pocket created by Gly-158 and
Leu-159 at the bottom of the central binding groove, while
more favorable predicted dimer association energy which
may, in part, explain its improved binding activity, while
naphthyl 8 and p-cyanophenyl 10, being more electron
poor, are expected to exhibit less self-interaction which
may
contribute
to
their
weaker
STING
binding. Difluorobenzodioxole 12 is predicted to have a
steric clash across the dimer interface where fluorine on
one monomer is in close proximity to the carboxylate
group of the other monomer.
Encouraged by these calculations, efforts were made to
simultaneously design subtle variations of the aryl ring to
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the aromatic chlorine further fills the pocket created by
Ile-165 and Ala-270.
functioning as STING antagonists. Considering that the
assay is stimulated with nonphysiological levels of
cGAMP, the cellular IC50 may not be an accurate
Consistent with our hypothesis, the increase in calculated
27
representation of antagonist potency.
pK
a
translated to a combination of improved permeability
-6
(
Papp = 18 x 10 cm/s) as well as improved oral
In conclusion, we have identified weak antagonists of
STING-mediated signaling that binds to the cGAMP
binding site in the inactive “open” conformation. By
exploiting the natural symmetry of the STING protein and
utilizing 2:1 binding stoichiometry, these compounds are
able to fully occupy the binding pocket while mitigating
the undesirable physicochemical properties associated
with larger ligands. As a consequence of the 2:1 binding
ratio, two dimensional optimization of the protein-ligand
and ligand-ligand interactions was necessary to improve
potency. This approach led to the discovery of compound
18 with slow dissociation kinetics, good oral
bioavailability, and ability to inhibit cGAMP dependent
signaling in vitro.
bioavailability (%F = 60; solubility and Clint remains
constant allowing for the interpretation that permeability
is the main driver for improvement, Table 4). Overall, the
pharmacokinetics properties for compound 18 are modest
with high intrinsic clearance. Kinetic binding assessment
of 13 and 18 by surface plasmon resonance (Biacore)
revealed a slow koff (13, t1/2 = 35 min; 18, t1/2 = 62 min).
Moreover, consistent with cooperative binding, data
fitting required a two-step model (Figure S1A-C and
supporting information). These observations may reflect
an overall increase in stability when a second molecule
binds to STING.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4: In vitro profile of lead STING ligands
O
OH
O
O
O
O
HO
O
N
N
O
F
O
Cl
Cl
13
18
HAQ STING IC50 (nM)a
LBE/LLEb
84
68
0.28/2.87
0.27/2.58
4.3
pKac
3.4
9
MDCK Papp (x 10-6 cm/s)26
18
%F
5
60
FASSIF (uM) pH(6.5)
151
187
Hep Clint(r/h)(mL/min/kg)d
158/28
38
240/31
45
(mL/min/kg)e
Figure 4: Co-crystal structure of 18 bound to STING protein
(PDB 6MXE) showing interactions with Thr-263 and Thr-267,
with sidechain atoms depicted as sticks. As observed with 1, the
compound binds in a 2:1 ratio to the STING homodimer and
makes the same interactions, with additional van der Waals
contact with the sidechain phenol of Tyr-167. The homologated
carboxylate group maintains a hydrogen bond interaction with
Thr-263.
Rat Cl
p
SPR t 1/2 (min)d
35
62
THP1 Cell IC50 (nM)f
THP1 Cell EC50 (nM)d
11500
>30000
11000
>30000
aValues in this table are determined by the HAQ STING cGAMP
displacement assay and are the means from at least n = 2 experiments.
b
c
Values are calculated from pIC50 − ALogP98. Values calculated from
d
e
ACD LABS 11.0. See supporting information. Dose; rat iv 0.5 mpk as a
solution in PEG400:H O (60:40 (v/v)), po: 1 mpk as a solution in
PEG400:H O (60:40 (v/v)). Values in this table are determined by
2
f
2
Supporting Information. Synthetic procedures and
analytical data of selected compounds, conditions for all
biological assays, and X-ray crystallographic methods and
statistics, DFT methods, Surface Plasmon Resonance and
protein preparation, off-target profile. This material is
available free of charge on the internet at
http://pubs.acs.org.
inhibiting IFNβ production in cGAMP stimulated THP1 cells and are the
means from at least n = 2 experiments.
Although we have identified molecules that are able to
displace cGAMP and stabilize the “open” conformation of
STING, a key assumption is that stabilizing the “open”
conformation will prevent all STING signaling. To test
this hypothesis, THP1 cells were incubated with 13 and 18
with and without cGAMP stimulation. The compounds
did not stimulate IFN production (13 EC50= >30,000 nM;
18 EC50=>30000 nM; Table 4), but modestly inhibited
cGAMP-induced IFN production (13 IC50 = 11,500 nM; 18
IC50 = 11,000 nM; Table 4), with a >100-fold shift in
potency from binding to the functional cell assay. Our
observations are consistent with these compounds
Acknowledgements. We thank J. Presland for helpful
discussions and P. Schwartz for assistance with
developing the kinetic model for Biacore fitting.
Accession Codes : STING apoprotein: 6MX0; STING
complex with 1: 6MX3; STING complex with 18: 6MXE.
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c[G(2',5')pA(3',5')p] and targeting by antiviral DMXAA. Cell
013, 154 (4), 748-62.
6. Zhang, X.; Shi, H.; Wu, J.; Zhang, X.; Sun, L.; Chen, C.;
Chen, Z. J., Cyclic GMP-AMP containing mixed
phosphodiester linkages is an endogenous high-affinity
ligand for STING. Mol Cell 2013, 51 (2), 226-35.
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Corresponding Author
Phone: 617-992-2386. E-mail: tony_siu@merck.com
References
1
. Ahlers, L. R.; Goodman, A. G., Nucleic acid sensing and
1
7. Ramanjulu, J. M.; Pesiridis, G. S.; Yang, J.; Concha, N.;
innate immunity: signaling pathways controlling viral
pathogenesis and autoimmunity. Curr Clin Microbiol Rep
2016, 3 (3), 132-141.
Singhaus, R.; Zhang, S. Y.; Tran, J. L.; Moore, P.; Lehmann, S.;
Eberl, H. C.; Muelbaier, M.; Schneck, J. L.; Clemens, J.; Adam,
M.; Mehlmann, J.; Romano, J.; Morales, A.; Kang, J.; Leister,
L.; Graybill, T. L.; Charnley, A. K.; Ye, G.; Nevins, N.; Behnia,
K.; Wolf, A. I.; Kasparcova, V.; Nurse, K.; Wang, L.; Li, Y.;
Klein, M.; Hopson, C. B.; Guss, J.; Bantscheff, M.; Bergamini,
G.; Reilly, M. A.; Lian, Y.; Duffy, K. J.; Adams, J.; Foley, K. P.;
Gough, P. J.; Marquis, R. W.; Smothers, J.; Hoos, A.; Bertin, J.,
Design of amidobenzimidazole STING receptor agonists with
systemic activity. Nature 2018.
2
. Wu, J.; Chen, Z. J., Innate immune sensing and signaling of
cytosolic nucleic acids. Annu Rev Immunol 2014, 32, 461-88.
. Junt, T.; Barchet, W., Translating nucleic acid-sensing
pathways into therapies. Nat Rev Immunol 2015, 15 (9), 529-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
4
4
4.
. Sun, L.; Wu, J.; Du, F.; Chen, X.; Chen, Z. J., Cyclic GMP-
AMP synthase is a cytosolic DNA sensor that activates the
type I interferon pathway. Science 2013, 339 (6121), 786-91.
1
8. Le Guilloux, V.; Schmidtke, P.; Tuffery, P., Fpocket: an
5
. Chen, Q.; Sun, L.; Chen, Z. J., Regulation and function of
open source platform for ligand pocket detection. BMC
Bioinformatics 2009, 10, 168.
the cGAS-STING pathway of cytosolic DNA sensing. Nat
Immunol 2016, 17 (10), 1142-9.
1
9. Cheng, A. C.; Coleman, R. G.; Smyth, K. T.; Cao, Q.;
6. Ahn, J.; Barber, G. N., Self-DNA, STING-dependent
signaling and the origins of autoinflammatory disease. Curr
Opin Immunol 2014, 31, 121-6.
Soulard, P.; Caffrey, D. R.; Salzberg, A. C.; Huang, E. S.,
Structure-based maximal affinity model predicts small-
molecule druggability. Nat Biotechnol 2007, 25 (1), 71-5.
7. Ahn, J.; Gutman, D.; Saijo, S.; Barber, G. N., STING
2
0. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P.
manifests self DNA-dependent inflammatory disease. Proc
Natl Acad Sci U S A 2012, 109 (47), 19386-91.
J., Experimental and computational approaches to estimate
solubility and permeability in drug discovery and
development settings. Adv Drug Deliv Rev 2001, 46 (1-3), 3-
26.
8
. Ahn, J.; Ruiz, P.; Barber, G. N., Intrinsic self-DNA triggers
inflammatory disease dependent on STING. J Immunol 2014,
93 (9), 4634-42.
. Gao, D.; Li, T.; Li, X. D.; Chen, X.; Li, Q. Z.; Wight-Carter,
1
9
2
1. Annis, D. A.; Nickbarg, E.; Yang, X.; Ziebell, M. R.;
Whitehurst, C. E., Affinity selection-mass spectrometry
screening techniques for small molecule drug discovery. Curr
Opin Chem Biol 2007, 11 (5), 518-26.
M.; Chen, Z. J., Activation of cyclic GMP-AMP synthase by
self-DNA causes autoimmune diseases. Proc Natl Acad Sci U
S A 2015, 112 (42), E5699-705.
2
2. Meyer, E. A.; Castellano, R. K.; Diederich, F., Interactions
1
0. Rumore, P. M.; Steinman, C. R., Endogenous circulating
with aromatic rings in chemical and biological recognition.
DNA in systemic lupus erythematosus. Occurrence as
Angew Chem Int Ed Engl 2003, 42 (11), 1210-50.
multimeric complexes bound to histone. J Clin Invest 1990,
2
3. Buried accessible surface area was calculated using MOE
8
6 (1), 69-74.
by taking the difference in accessible surface area between
the dimer and two monomers. Molecular Operating
Environment (MOE), 2013.08; Chemical Computing Group
ULC, 1010 Sherbooke St. West, Suite #910, Montreal, QC,
Canada, H3A 2R7, 2018.
1
1. Rice, G. I.; Rodero, M. P.; Crow, Y. J., Human disease
phenotypes associated with mutations in TREX1. J Clin
Immunol 2015, 35 (3), 235-43.
1
2. Konig, N.; Fiehn, C.; Wolf, C.; Schuster, M.; Cura Costa,
E.; Tungler, V.; Alvarez, H. A.; Chara, O.; Engel, K.;
Goldbach-Mansky, R.; Gunther, C.; Lee-Kirsch, M. A.,
Familial chilblain lupus due to a gain-of-function mutation
in STING. Ann Rheum Dis 2017, 76 (2), 468-472.
2
4. See supplementary section for methods and free energy
calculations of analogs tested
5. Sinnokrot, M. O.; Sherrill, C. D., Substituent effects in pi-
2
pi interactions: sandwich and T-shaped configurations. J Am
Chem Soc 2004, 126 (24), 7690-7.
1
3. Koch, P. D.; Miller, H. R.; Yu, G.; Tallarico, J. A.; Sorger, P.
K.; Wang, Y.; Feng, Y.; Thomas, J. R.; Ross, N. T.; Mitchison,
T., A High Content Screen in Macrophages Identifies Small
Molecule Modulators of STING-IRF3 and NFkB Signaling.
ACS Chem Biol 2018, 13 (4), 1066-1081.
2
6. Irvine, J. D.; Takahashi, L.; Lockhart, K.; Cheong, J.;
Tolan, J. W.; Selick, H. E.; Grove, J. R., MDCK (Madin-Darby
canine kidney) cells: A tool for membrane permeability
screening. J Pharm Sci 1999, 88 (1), 28-33.
1
4. Haag, S. M.; Gulen, M. F.; Reymond, L.; Gibelin, A.;
27. Cell viability has been accounted for in the THP1 assay;
for list of off target hits for 13 see supporting information
Abrami, L.; Decout, A.; Heymann, M.; van der Goot, F. G.;
Turcatti, G.; Behrendt, R.; Ablasser, A., Targeting STING with
covalent small-molecule inhibitors. Nature 2018, 559 (7713),
269-273.
15. Gao, P.; Ascano, M.; Zillinger, T.; Wang, W.; Dai, P.;
Serganov, A. A.; Gaffney, B. L.; Shuman, S.; Jones, R. A.;
Deng, L.; Hartmann, G.; Barchet, W.; Tuschl, T.; Patel, D. J.,
Structure-function analysis of STING activation by
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Discovery of a Novel cGAMP Competitive Ligand of the
Inactive Form of STING

,†
§
‡
†
†
Tony Siu, Michael D. Altman, Gretchen A. Baltus, Matthew Childers, J. Michael Ellis,
§
⊥
¥
†
⊥
Hakan Gunaydin, Harold Hatch, Thu Ho, James Jewell, Brian M. Lacey, Charles A.
Lesburg, Bo-Sheng Pan, Berengere Sauvagnat, Gottfried K. Schroeder, and Serena Xu
∞
⊥
⊥
⊥
⊥
†
‡
§
⊥
Departments of Chemistry, Immunology, Chemistry Modeling and Informatics, In Vitro
¥
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Pharmacology, Target Protein Design and Structural Chemistry, Merck & Co., Inc., 33
Avenue Louis Pasteur, Boston, MA 02115
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