DMXAA responses depend on species, model and co-stimulation. The sodium-salt preparation did not activate TBK1–IRF3 readouts in human THP-1 cells at 1 mM for 4 h. A later study found that 100 µg/ml DMXAA pretreatment reduced CDN-stimulated transcriptional/reporter responses in differentiated human THP1 cells, while early TBK1/IRF3 phosphorylation increased. The latter is a combination response and does not by itself establish direct human-STING binding or partial agonism; supplemental viability data were not independently retrieved. [1,3]
Direct binding to the mouse STING C-terminal domain
Biochemical assay · Purified mouse STING C-terminal domain, residues 139–378 [1]
ITC detected binding of the sodium-salt DMXAA preparation to mouse STING CTD. The authors report an approximate dissociation constant of 130 nM. This is a binding endpoint for isolated protein, not a cellular EC50 or treatment dose.
Literature-Reported Treatment Conditions
- Treatment concentration
- ITC methods: 400 μM ligand titrated into 40 μM STING protein.
- Method
- ITC at 25°C.
- Vehicle
- 20 mM Tris, 150 mM NaCl, pH 7.5.
- Reported limits
- The approximate Results Kd is retained rather than a more precise fit annotation. The figure caption does not specify an ITC replicate count; no n or error interpretation is assigned. No free-acid/sodium-salt equivalence or mass conversion is asserted.
Methods and source locations
Compound reported in the paper: DMXAA (5,6-dimethylxanthenone-4-acetic acid); Compound label in the paper: DMXAA
Mouse-protein binding of the reported sodium-salt preparation; no Adooq lot validation or free-acid potency assignment.
Final ACS PDF p. 1397, Figure 1C-D and mouse direct-binding Results; PMC Methods: Reagents and antibodies; Isothermal titration calorimetry assay
Binding to mouse STING CTD: Isothermal titration calorimetry; one-set-of-sites fit. c-di-GMP binding provided a positive ligand comparison. — Final ACS PDF p. 1397, Figure 1C-D and “DMXAA Binds to mSTING Directly”; PMC Methods: Isothermal titration calorimetry assay
No detectable TBK1–IRF3 activation in THP-1 cells under the tested conditions
Cell-based study · Human THP-1 monocytic leukemia cells and an IRF3-driven luciferase reporter [1]
At 1 mM, the sodium-salt DMXAA preparation did not activate the assayed TBK1/IRF3 phosphorylation and IRF3 reporter readouts after 4 h, whereas c-di-GMP produced a response. This condition-specific negative result does not establish absence of all human-cell effects.
Literature-Reported Treatment Conditions
- Treatment concentration
- DMXAA 1 mM.
- Exposure duration
- 4 h.
- Method
- Flavonoid treatment; phosphorylation and reporter assays.
- Co-treatment
- No DMXAA combination treatment; c-di-GMP was a separate positive comparator delivered by digitonin permeabilization.
- Reported limits
- Final DMXAA vehicle fraction and replicate/error semantics for Figure 3C are not specified in its caption; no numerical fold induction estimated from bars.
Methods and source locations
Compound reported in the paper: DMXAA (5,6-dimethylxanthenone-4-acetic acid); Compound label in the paper: DMXAA
Negative response in this human cell model and exposure only.
Figure 3C, caption and associated Results; Methods: Reagents and antibodies; cell culture context; SEAP and luciferase assay
TBK1/IRF3 phosphorylation and IRF3-driven luciferase: Immunoblotting and IRF3-driven luciferase reporter. — Figure 3C and caption; Results paragraph beginning “FAA and DMXAA lacked definitive anti-cancer efficacy”; Methods: SEAP and luciferase assay
DMXAA comparator responses in STING-reconstituted HepG2 cells
Cell-based study · HepG2/mSTING, parental HepG2, C-terminally truncated hSTING-expressing HepG2/STINGΔC, and full-length hSTING-expressing HepG2/STING cells [2]
DMXAA induced IFN-β mRNA in mouse-STING-expressing HepG2 cells, with little response in parental or truncated-hSTING cells. Immunofluorescence showed mouse-STING perinuclear redistribution and IRF3 nuclear redistribution with DMXAA; the full-length human-STING comparator did not show the corresponding response. BNBC was a separate human-STING-active comparator, not a DMXAA combination.
Literature-Reported Treatment Conditions
- Treatment concentration
- Figure 2B: DMXAA 0, 6.25, 25 and 100 μM. Figure 2C: DMXAA 50 μM.
- Exposure duration
- qRT-PCR: 6 h; immunofluorescence: 2 h.
- Method
- Separate DMXAA and BNBC treatment arms compared with control/mock conditions.
- Co-treatment
- None; BNBC was tested separately.
- Reported limits
- DMXAA stock solvent, final vehicle fraction, catalog number, lot, purity and salt form are not supplied in the retrieved Chemicals paragraph. The 50 mM DMSO stock described there belongs to BNBC/analogs, not automatically to DMXAA. Figure 2C replication is not specified in its caption.
Methods and source locations
Compound reported in the paper: DMXAA (5,6-dimethylxanthenone-4-acetic acid); Compound label in the paper: DMXAA
Non-infectious STING-species comparator assays only; no antiviral efficacy or clinical implication.
Figure 2B-C and caption; Results paragraph introducing Figure 2; Methods: Chemicals; Analysis of cytokine expression; Immunofluorescent assay
IFN-β mRNA after DMXAA treatment: qRT-PCR normalized to β-actin; Figure 2B shows mean ± standard deviation, n=4; the caption does not define n as biological versus technical replicates. — Figure 2B, caption; Results paragraph introducing Figure 2; Methods: Analysis of cytokine expression
STING and IRF3 subcellular localization: Immunofluorescence staining for STING and IRF3, with DAPI nuclear counterstain. — Figure 2C and caption; Methods: Immunofluorescent assay
Reduced agonist-stimulated transcriptional responses in differentiated THP1 cells
Cell-based study · PMA-differentiated human THP1 dual reporter cells [3]
DMXAA pretreatment reduced CDN-stimulated IFNB/IL6 mRNA and IRF/NF-κB reporter outputs in the reported human-cell assays. In contrast, early TBK1 and IRF3 phosphorylation was enhanced rather than suppressed. These results describe responses to DMXAA plus a second agonist; they do not demonstrate direct human-STING binding or establish the proposed partial-agonist mechanism.
Literature-Reported Treatment Conditions
- Treatment concentration
- DMXAA 100 µg/ml; indicated CDNs 10 µg/ml.
- Exposure duration
- DMXAA pretreatment 90 min; CDN stimulation 12 or 24 h for Figure 1B-C, 3 h for Figure 1D.
- Method
- PMA-differentiated THP1 dual reporter cells; paired CDN-stimulated control and DMXAA-pretreated groups.
- Vehicle
- DMXAA dissolved in DMSO; final fraction and explicit vehicle matching not established in the selected caption/methods.
- Co-treatment
- DMXAA plus separately indicated CDN (3′3′-cGAMP, 2′3′-cGAMP, c-di-GMP or c-di-AMP); no single-agent inhibition potency assigned.
- Reported limits
- Supplementary Figure 1A viability data were cited in the main text but not retrieved. No cytotoxicity-independent mechanism is claimed here. Authors discuss partial agonism and a downstream mechanism as alternatives.
Methods and source locations
Compound reported in the paper: DMXAA (5,6-dimethylxanthenone-4-acetic acid); Compound label in the paper: DMXAA
Condition-specific human-cell combination response; not proof of direct receptor competition, clinical benefit or absence of cytotoxicity.
Figure 1B-D and caption; first Results subsection; Methods: Cell lines and reagents; reporter and qPCR assays; Discussion
CDN-stimulated IFNB/IL6 mRNA and IRF/NF-κB reporter outputs: qRT-PCR normalized to 18S rRNA; IRF secreted luciferase and NF-κB SEAP. Caption: representative data from three independent experiments, mean ± SD of triplicates. No numerical inhibition percentage extracted. — Figure 1B-C and caption; first Results subsection; Methods: Measurement of NF-κB and IRF activity; Real time-PCR analysis
Early TBK1 and IRF3 phosphorylation after CDN stimulation: Western blot with total protein and β-actin controls; caption describes mean ± SD, n=3 biological replicates for densitometry. — Figure 1D and caption; first Results subsection; Discussion paragraph addressing alternative downstream mechanisms
