Panet al.,Science 369 , eaba6098 (2020) 21 August 2020 3of10
Fig. 3. MSA-2 is an orally bioavailable STING agonist with in vivo antitumor activity.(A) Illustration
of administration routes. (BandC) Pharmacokinetics of indicated MSA-2 doses in tumors (B) and
plasma (C) after single intratumoral (IT; blue circles), subcutaneous (SC; red squares), and oral (PO; yellow
diamonds) administration in MC38 tumor-bearing C57BL6 mice (n= 3 to 6). (DtoF)Effectofindicated
IT (D), SC (E), and PO (F) regimens of MSA-2 (triangles below thexaxis indicate dosing days) on
MC38 tumor growth (initial volume ~100 mm^3 ,n= 10 mice per group). Dashed lines indicate tumor start size
of 81 mm^3 (D) or 112 mm^3 (E and F). (G) Tolerability, illustrated by percentage of body weight change of
mice in (D) to (F), on days 1 and 2 for the indicated MSA-2 doses. (HtoJ)IFN-b(H) and IL-6 (I to J) levels
in MC38 tumors and plasma from C57BL6 mice after 4 hours or over 24 hours after the indicated single doses
of MSA-2 (n= 3 to 5). (K) Tumor-growth kinetics and tumor-take frequency after reinoculation of MC38
tumors in mice from (D) to (F) that had previously experienced complete regression in response to indicated
IT, SC, or PO regimens of MSA-2. Treatment-naïve mice (n= 10) were used as positive controls (triangles).
Data points in (B), (C), and (G) to (J) represent mean ± SD and in (D) to (F) and (K) represent mean ± SEM.
Statistical significance was determined by one-way analysis of variance (ANOVA). ns, not significant;
P< 0.05; P< 0.01; P< 0.001; ****P< 0.0001.
Fig. 4. MSA-2 is bound to a closed form of
hSTING.(A) The 1.7-Å x-ray structure of an MSA-2:
hSTING-HAQ complex shows two copies of MSA-2
(dark and light green) in the CDN binding site of
human hSTING-HAQ. cGAMP (from PDB ID 4KSY) is
depicted as faint transparent sticks to illustrate the
overlapping binding site. Interactions between MSA-2
and the surrounding protein are shown as dashed
lines. Lid interactions are shown in green (proximal)
and yellow (cross-linking). Interactions at the base
of the binding site are shown in pink. Specifically,
MSA-2 forms both proximal and cross-linking
interactions with lid residue Arg^238 .ResidueTyr^167 (A)
has been removed for clarity. R, Arg; S, Ser; T, Thr;
Y, Tyr. (B)Solution^1 H NMR spectra of hSTING-HAQ
shows that multiple well-resolved chemical shift
perturbations in the presence of MSA-2 (blue, top
spectrum) are more closely aligned with those in
the presence of cGAMP (black, middle) than ligand-free
protein (magenta, bottom). (C) Solution^1 H-^15 N
heteronuclear correlation NMR spectra of hSTING-
WT and hSTING-HAQ (magenta, ligand-free; black,
cGAMP; blue, MSA-2), highlighting resonances of
lid residues Gly^230 and Gly^234 for hSTING-WT (top) and
unassigned resonances for hSTING-HAQ (bottom),
which undergo marked chemical shift perturbations
when human STING adopts the closed form.RESEARCH | RESEARCH ARTICLE