Science - USA (2020-08-21)

various substitutions—represents a general
pharmacophore for potent STING agonism.
We selected compound 3 as the most rea-
sonable surrogate for the noncovalent dimer
of MSA-2 (Fig. 6, B and C) and determined the
kinetic parameters by SPR. Compound 3
exhibited 1:1 binding with hSTING-WT and
a slow on-rate (ka= 1.1 × 10^4 M−^1 s−^1 ) that is
very similar to that calculated for the non-
covalent MSA-2 dimer based on Model 3 (ka2=
4.9 × 10^4 M−^1 s−^1 ; Fig. 5F). These observations
further support our interpretation that the
NMR-derived equilibrium constant for MSA-2
homodimerization (KD1) predominately re-
flects formation of a bioactive dimer. In sum-
mary, the relatively simple Model 3 can fully
account for all of the data. To our knowledge,
MSA-2 is currently the only small molecule
reported to undergo reversible, noncovalent
dimerization in solution to become a pharma-
cologically active ligand.

Enhanced cellular potency of MSA-2 predicted
in acidic tumor microenvironments

Considering physiologically relevant condi-
tions, the fraction of uncharged MSA-2 mole-
cules (pKa= 4.78 ± 0.05, whereKais the acid
dissociation constant) at pH 7.4 is ~0.2% and
increases with decreasing pH (fig. S9A). As-
suming that cellular influx and efflux of MSA-2

Panet al.,Science 369 , eaba6098 (2020) 21 August 2020 6of10

O S OMe

HO

O

O S

MeO O

OH

O

O

S

O OMe

HO

O

O S

MeO O

OH

O

O

O S O

HO

O

S

O O

OH

O

MeMe

O S OMe

HO

O

Me

Me

S

O

MeO O

OH

O

Me

Me

O

F F

3

5/5 S

5/5

5/5

5/5

5/6

6/6

4/6

5/5

O O

HO

O

O S O

OH

O

Me Me

4

O S O

HO

O

O S O

OH

O

O

Me Me

5

O S O

HO

O

O S O

OH

O

O O

Me Me

6

9

O S O

HO

O

O S O

OH

O

OO

Me Me

F

11

12

13

hSTING-WT + 3

11.1 33.3 100 300 900 nM

KD = 86 ± 2 nM

ka = 1.08 ± 0.03 × 10^4 M-1s-1

kd = 9.2 ± 0.2 × 10-4 s-1

0

5

10

15

RU

-500 500 1500 2500 sec

Structure cGAMP displacement assays

IC 50 (HAQ) IC 50 (WT)

THP-1 IFN-β

cellular EC 50

Compound

3

4

9

10

6

11

12

90°

ROCS Reference Tversky Combo Similarity

Freeform Configurational

ΔG

1.5 1.6 1.7 1.8 1.9 2

18

16

14

12

10

8

6

4

2

0

54

3

3

8±7 nM 23±7 nM 70±50 nM

16±10 nM 65±15 nM 400±250 nM

710±190 nM 1090±450 nM >30000 nMa

4±1 nM 11±7 nM 1330±300 nMa

1.5±0.6 nM 20±10 nM 470±25 nMa

17±9 nM 270±40 nM 4170±80 nM

10±3 nM 19±4 nM 450±210 nM

2±1 nMa 2±1 nMa 8±7 nMa

A

C

B

DE

Fig. 6. Identification of covalently linked MSA-2
analogs.(A) Enumeration of thousands of possible
linked benzothiophene core dimers identified linkage
arrangements predicted to adopt low-energy con-
formations that provide optimal overlay with the MSA-2
STING-bound structure. Teal circles, predicted optimal
conformations of 3 , 4 ,and 5 ;DG, Gibbs free energy.
(B) X-ray crystal structure of dimer 3 (purple)
confirms adoption of an MSA-2 (dark and light green
overlay)–like conformation in the STING binding site.
(C) Variation of linker composition and attachment
positions with assay data summary (mean ± SD,
n= 2 unless noted with superscripta,inwhichcase
n≥3). IC 50 , half-maximal inhibitory concentration;
EC 50 , half-maximal effective concentration.
(D) Superposition of crystal structures of STING in
complex with seven covalent dimeric MSA-2
analogs with various combinations of linker length
(three, four, or five atoms), C-linked or O-linked,
symmetric or asymmetric, and substitution positions
(5/5, 6/6, 5/6, 4/6). Each compound binds in the
same location and positions theg-ketoacid group for
identical interactions with Arg^238 (see Fig. 4A),
even if this requires the benzothiophene group to
be flipped relative to the parental MSA-2 orientation.
(E) SPR sensorgram for the interaction of compound 3
with hSTING-WT (purple line), successfully fitted
with a direct 1:1 binding model (black line). Resultant
kinetic parameters are summarized (n= 3), and
tested concentrations are noted in gray (top).
RU, resonance units.

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