Amlexanox (AA673)

Kinase experiment:

The in vitro kinase assays is performed by incubating purified kinase (IKKε or TBK1) in kinase buffer containing 25 mM Tris (pH7.5), 10 mM MgCl2, 1 mM DTT, and 10 µM ATP for 30 minutes at 30°C in the presence of 0.5 µCi γ-[32P]-ATP and 1 µg MBP per sample as a substrate. The kinase reaction is stopped by adding 4x sodium dodecyl sulfate (SDS) sample buffer and boiling for 5 minutes at 95°C. Supernatants are resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and analyzed by autoradiography using a Typhoon 9410 phosphorimager.

Cell experiment:

To examine cell proliferation, a Cell Counting Kit-8 is used according to the manufacturer’s instructions. BMMs are seeded at a density of 5×103 cells/well in 96-well plates. After 24 hours, cells are treated with different concentrations of AmLexanox (0, 1.5, 3, 6, 12, 25 μM) every 2 days in the presence of M-CSF (30 ng/mL) for 7 days. After 1, 3, 5 and 7 days, the culture medium is replaced by the medium containing 10% CCK-8 and cells are incubated at 37°C for an additional 2 h. The absorbance is then measured at a wavelength of 450 nm on an ELX800 absorbance microplate reader.

Animal experiment:

Wildtype male C57BL/6 mice are fed with a HFD consisting of 45% of calories from fat starting at eight weeks of age for 12-24 weeks, while ND C57BL/6 controls are maintained on normal chow diet consisting of 4.5% fat. C57BL/6 diets are fed containing ω-3 fatty acids. Rosiglitazone treatment is administered for three weeks by addition of the compound to the diet in mice that have been on HFD for 16 weeks. Each mouse consumes on average 3.5 mg per kg rosiglitazone per day. AmLexanox is administered by daily oral gavage. For the prevention groups, amLexanox (25 mg per kg or 100 mg per kg) administration is begun concurrently with HFD feeding at eight weeks of age. For the treatment groups, 25 mg per kg amLexanox treatment is begun at 20 weeks of age after 12 weeks of HFD. To test the effect of amLexanox withdrawal, mice in the treatment group are switched from amLexanox gavage to vehicle control after eight weeks of amLexanox treatment. Control and ob/ob mice are fed with a normal chow diet and gavaged with 100 mg per kg amLexanox or vehicle control beginning at ten weeks of age. Animals are housed in a specific pathogen-free facility with a 12-hour light/12-hour dark cycle and given free access to food and water.

References:

[1]. Reilly SM, et al. An inhibitor of the protein kinases TBK1 and IKK-e improves obesity-related metabolic dysfunctions in mice. Nat Med. 2013 Mar;19(3):313-21.
[2]. Bell, J. AmLexanox for the treatment of recurrent aphthous ulcers. Clin Drug Investig, 2005. 25(9): p. 555-66.
[3]. Zhang Y, et al. AmLexanox Suppresses Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss. Sci Rep. 2015 Sep 4;5:13575.