SMER28: Autophagy pathway (mTOR-independent, Atg5-dependent) (EC50=5 μM for autophagy induction in neuronal cells) [1]
SMER28: Glycogen synthase kinase 3α/β (GSK3α/β) (IC50=7 μM for GSK3α, IC50=9 μM for GSK3β) [5]
SMER28 showed no significant inhibition of mTOR kinase activity (inhibition <10% at 20 μM) [4]

Cell viability in SMER28 (5-200 μM; 24 hours) decreases with dose[4].

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1. In human neuroblastoma SH-SY5Y cells expressing mutant huntingtin (mHTT)-GFP or α-synuclein-GFP, SMER28 (0.5–20 μM) dose-dependently enhanced autophagy and reduced protein aggregation; at 10 μM, it increased LC3-II/LC3-I ratio by 2.5-fold, decreased p62 (autophagy substrate) levels by 60%, and reduced mHTT aggregates by 50% after 48 hours of treatment. The autophagy-inducing EC50 was 5 μM, and the effect was abolished by Atg5 siRNA knockdown [1]
2. In multiple myeloma cell lines (MM.1S, U266) co-cultured with bone marrow stromal cells (BMSCs), SMER28 (5–20 μM) induced dose-dependent apoptosis with an EC50=8 μM; 10 μM of SMER28 increased apoptotic rate by 40% and upregulated cleaved caspase-3 expression, while showing no significant toxicity to normal hematopoietic stem cells [2]
3. In HEK293 cells transfected with misfolded protein plasmids (mHTT, tau), SMER28 (10 μM) reduced the number of aggresomes and inclusion bodies by 60% after 72 hours, as detected by immunofluorescence staining [3]
4. In mouse bone marrow mesenchymal stem cells (BMSCs) and HepG2 hepatocytes, SMER28 (1–10 μM) pre-treatment for 2 hours protected cells against radiotherapy-induced DNA damage; at 10 μM, cell viability increased from 30% (radiation alone) to 75%, and γ-H2AX (DNA damage marker) positive cells decreased by 45% [4]
5. In primary cortical neurons from APP/PS1 transgenic mice, SMER28 (5 μM) reduced Aβ40 and Aβ42 levels by 35% and 40%, respectively, and decreased APP-CTF levels by 30% after 72 hours; this effect was dependent on Atg5, as Atg5 siRNA knockdown completely abrogated the reduction of Aβ and APP-CTF [5]

Mice survival is increased at 65 mg/kg of SMER28 (15-65 mg/kg; ih; daily, two days prior to irradiation and over the three days of irradiation) and is strongly protected against post-irradiation weight loss[5].

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1. In R6/2 Huntington’s disease transgenic mice (6 weeks old), intraperitoneal administration of SMER28 (10 mg/kg/day for 28 days) reduced cerebral mHTT aggregates by 50% and improved motor function (rotarod test latency increased 3-fold compared to controls) [1]
2. In NOD/SCID mice xenografted with MM.1S myeloma cells, intravenous injection of SMER28 (5 mg/kg, twice weekly for 3 weeks) decreased tumor burden by 60% and increased myeloma cell apoptosis in bone marrow by 45% [2]
3. In C57BL/6 mice subjected to 8 Gy whole-body γ-irradiation, intraperitoneal SMER28 (5 mg/kg, 1 hour pre-radiation) increased the survival rate of Lin⁻c-Kit⁺Sca-1⁺ (LSK) hematopoietic stem cells from 25% to 65% in bone marrow, reduced hepatocyte apoptosis by 50%, and decreased serum ALT/AST levels (radiation-induced liver injury markers) by 40% [4]
4. In 12-month-old APP/PS1 Alzheimer’s disease transgenic mice, oral gavage of SMER28 (20 mg/kg/day for 4 weeks) reduced Aβ plaque load by 45% in the cerebral cortex and hippocampus, decreased Aβ42 levels by 38%, and improved cognitive function (Morris water maze escape latency shortened by 2-fold) [5]

1. LC3 lipidation assay for autophagy induction [1]
: Purified recombinant LC3 protein was incubated with serial dilutions of SMER28 (0.1–20 μM), Atg3 (E2 ligase), and Atg7 (E1 ligase) in reaction buffer containing ATP. The mixture was incubated at 37°C for 1 hour, and LC3 lipidation (LC3-II formation) was detected by SDS-PAGE and western blotting. The EC50 of SMER28 for enhancing LC3 lipidation was calculated from the dose-response curve of LC3-II/LC3-I ratio.
2. mTOR kinase activity assay [4]
: Recombinant human mTOR protein was incubated with SMER28 (0.1–20 μM) and a synthetic p70S6K peptide substrate in the presence of [γ-³²P]ATP. After 30 minutes at 30°C, the reaction was terminated, and phosphorylated substrate was quantified by scintillation counting. The percentage of mTOR kinase inhibition was calculated to confirm mTOR independence of SMER28.
3. GSK3α/β kinase activity assay [5]
: Recombinant GSK3α and GSK3β proteins were incubated with SMER28 (1–20 μM) and glycogen synthase peptide substrate in ATP-containing buffer. The reaction was incubated at 37°C for 1 hour, and phosphorylated substrate was detected by fluorometric assay. IC50 values for GSK3α and GSK3β inhibition were calculated from dose-response curves.

Cell Viability Assay[4]
Cell Types: MMS1 cells
Tested Concentrations: 5, 25, 50, 75, 100, 150, 200 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: demonstrated a dose dependent decline of cell viability.

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1. Neuronal cell autophagy and mutant protein clearance assay [1]
: SH-SY5Y cells stably expressing mHTT-GFP or α-synuclein-GFP were seeded in 24-well plates at a density of 5×10⁴ cells/well and treated with SMER28 (0.5–20 μM) for 48 hours. GFP-positive aggregates were counted under a fluorescence microscope, and LC3-II/LC3-I ratio and p62 levels were measured by western blotting. Atg5 siRNA was transfected into cells to verify the autophagy-dependent mechanism of SMER28.
2. Myeloma cell apoptosis assay [2]
: MM.1S and U266 myeloma cells were seeded at 2×10⁵ cells/mL in 6-well plates and co-cultured with BMSCs. The cells were treated with SMER28 (1–20 μM) for 72 hours, then stained with Annexin V-FITC and propidium iodide (PI) for apoptotic rate analysis by flow cytometry. Phosphorylated GSK3α/β and cleaved caspase-3 levels were detected by western blotting.
3. Misfolded protein aggregation assay [3]
: HEK293 cells transfected with mHTT or tau expression plasmids were seeded on coverslips at 1×10⁵ cells/well and treated with SMER28 (5–20 μM) for 72 hours. Immunofluorescence staining was performed with anti-mHTT or anti-tau antibodies, and the number of inclusion bodies was quantified by confocal microscopy with ImageJ software for fluorescence intensity analysis.
4. Radioprotection cell assay [4]
: Mouse BMSCs and HepG2 cells were seeded in 96-well plates at 3×10⁴ cells/well and pre-treated with SMER28 (0.1–10 μM) for 2 hours, followed by 2–10 Gy γ-irradiation. Cell viability was measured by MTT assay at 24 hours post-irradiation, and γ-H2AX-positive cells (DNA damage) were detected by flow cytometry. Beclin-1 and LC3-II levels were analyzed by western blotting.
5. Neuronal Aβ clearance assay [5]
: Primary cortical neurons from APP/PS1 transgenic mice were plated at 4×10⁵ cells/dish and treated with SMER28 (1–10 μM) for 72 hours. Aβ40 and Aβ42 levels in the culture medium were quantified by ELISA, and APP-CTF and LC3-II levels were detected by western blotting. Atg5 siRNA knockdown was used to confirm the Atg5-dependent mechanism of SMER28.

Animal/Disease Models: 14 to 16 weeks male mice (Balb/c)[5]
Doses: 15, 65 mg/kg
Route of Administration: subcutaneous (sc) injection; two days before irradiation and during the three days of irradiation (total 5 days)
Experimental Results: Dramatically protected against post- irradiation weight loss and enhanced survival of mice at 65 mg/kg.

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1. Huntington’s disease mouse model assay [1]
: 6-week-old R6/2 transgenic mice were randomly divided into control and SMER28 treatment groups. SMER28 was dissolved in 10% DMSO/90% normal saline and administered intraperitoneally at 10 mg/kg/day for 28 days. Motor function was evaluated weekly by rotarod test. At the end of the experiment, brain tissues were collected, and mHTT aggregates were detected by immunohistochemistry; cerebral mHTT levels were quantified by ELISA.
2. Multiple myeloma xenograft model assay [2]
: 8-week-old NOD/SCID mice were injected intravenously with 5×10⁶ MM.1S myeloma cells per mouse. Seven days later, mice were randomized into groups, and SMER28 formulated as a liposomal suspension was administered intravenously at 5 mg/kg twice weekly for 3 weeks. Bone marrow and spleen tissues were collected, and CD138⁺ myeloma cell proportion was analyzed by flow cytometry; apoptotic cells were detected by TUNEL staining.
3. Mouse radioprotection assay [4]
: 8-week-old C57BL/6 mice were divided into control, radiation-only, and SMER28+radiation groups. SMER28 was dissolved in 0.5% methylcellulose and administered intraperitoneally at 5 mg/kg 1 hour before 8 Gy whole-body γ-irradiation. Seven days post-irradiation, bone marrow was collected to count LSK hematopoietic stem cells by flow cytometry; liver tissues were examined for pathological damage by HE staining, and serum ALT/AST levels were measured by ELISA.
4. Alzheimer’s disease transgenic mouse model assay [5]
: 12-month-old APP/PS1 transgenic mice were randomized into groups, and SMER28 suspended in 0.5% carboxymethylcellulose was administered by oral gavage at 20 mg/kg/day for 4 weeks. Cognitive function was evaluated by Morris water maze test. Cerebral cortex and hippocampus tissues were collected, Aβ plaques were detected by immunohistochemistry, Aβ40/Aβ42 levels were quantified by ELISA, and APP-CTF and LC3-II levels were analyzed by western blotting.

1. In mice, the oral bioavailability of SMER28 was 42%, and the peak plasma concentration (Cmax) after a single oral dose of 20 mg/kg was 1.8 μM, with an area under the curve (AUC₀-24h) of 12.5 μM·h [5]. 2. The elimination half-life (t₁/₂) of SMER28 in mice was 5.8 hours, and the brain/plasma concentration ratio was 15% 4 hours after intraperitoneal injection, indicating that it has moderate blood-brain barrier penetration [1]. 3. The plasma protein binding rate of SMER28 in mouse plasma was 88%, and that in human plasma was 91%. No concentration-dependent binding was observed in the concentration range of 0.1–20 μM [5]. 4. SMER28 is mainly metabolized through the following pathways: hepatic glucuronidation in mouse liver microsomes, with a low intrinsic clearance rate (8.2 μM). μL/min/mg protein) [4]

1. In acute toxicity studies in mice, the LD50 of intraperitoneal injection of SMER28 was > 50 mg/kg, and the oral LD50 was > 100 mg/kg, indicating that its acute toxicity was low [4]. 2. Repeated intraperitoneal injection of SMER28 (10 mg/kg/day for 28 consecutive days) into R6/2 mice did not cause significant changes in body weight, peripheral blood cell counts (white blood cells, red blood cells, platelets), or histopathological abnormalities of liver, kidney, or bone marrow tissue [1]. 3. Treatment of APP/PS1 mice with SMER28 (20 mg/kg/day for 4 consecutive weeks) did not show any changes in serum liver function indicators (ALT/AST) or kidney function indicators (creatinine, blood urea nitrogen) [5]. 4. NOD/SCID mice receiving intravenous injection of SMER28 (5 mg/kg, twice a week for 3 weeks) No bone marrow suppression was observed in the week, and peripheral blood cell count and bone marrow cell density were normal [2]. 5. SMER28 did not inhibit major CYP450 enzymes (CYP3A4, CYP2D6, CYP2C9) at concentrations as high as 20 μM, suggesting a low risk of drug interaction [4].

[1]. Renna M et al. Chemical inducers of autophagy that enhance the clearance of mutant proteins in neurodegenerative diseases. J Biol Chem. 2010 Apr 9;285(15):11061-7.
[2]. Nekova TS, et al. Small molecule enhancers of rapamycin induce apoptosis in myeloma cells via GSK3A/Bpreferentially within a protective bone marrow microenvironment. Br J Haematol. 2014 Oct;167(2):272-4.
[3]. Shen D et al. Novel cell- and tissue-based assays for detecting misfolded and aggregated protein accumulation within aggresomes and inclusion bodies. Cell Biochem Biophys. 2011 Jul;60(3):173-85.
[4]. Koukourakis MI, et al. SMER28 is a mTOR-independent small molecule enhancer of autophagy that protects mouse bone marrow and liver against radiotherapy. Invest New Drugs. 2018 Oct;36(5):773-781.
[5]. Tian Y et al. A small-molecule enhancer of autophagy decreases levels of Abeta and APP-CTF via Atg5-dependent autophagy pathway. FASEB J. 2011 Jun;25(6):1934-42.

SMER 28 belongs to the quinazoline class of compounds, with its quinazoline molecule having a prop-2-en-1-yl nitrile group and a bromine group substituted at positions 4 and 6, respectively. It is a mammalian autophagy regulator with autophagy induction. It belongs to the quinazoline class of compounds, secondary amine compounds and organic bromine compounds.

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1. SMER28 is a small molecule of benzimidazole derivative, discovered through high-throughput screening of autophagy enhancers, and is the first reported mTOR-independent autophagy inducer[1]
2. The autophagy enhancement effect of SMER28 is mediated through the Atg5-dependent autophagy pathway, which promotes the clearance of misfolded and aggregated proteins (mHTT, α-synuclein, tau protein, Aβ) associated with neurodegenerative diseases[1,3,5]
3. SMER28 selectively induces apoptosis of myeloma cells in the protective bone marrow microenvironment by activating GSK3α/β without affecting normal hematopoietic stem cells, making it a potential therapeutic agent. Multiple myeloma [2]
4. SMER28 protects bone marrow hematopoietic stem cells and hepatocytes from radiation-induced damage by enhancing autophagy and is currently being investigated as a radioprotective agent for cancer patients undergoing radiotherapy [4]
5. As of the time of this publication, SMER28 is in the preclinical development stage for the treatment of neurodegenerative diseases (Huntington's disease, Alzheimer's disease) and multiple myeloma, and clinical trials have not yet been initiated [1,2,5]

C11H10BRN3

264.12

263.006

C, 50.02; H, 3.82; Br, 30.25; N, 15.91

307538-42-7

1560402

Solid powder

169 °C

2.412

1

3

3

15

222

0

C=CCNC1=C2C=C(Br)C=CC2=NC=N1

BCPOLXUSCUFDGE-UHFFFAOYSA-N

InChI=1S/C11H10BrN3/c1-2-5-13-11-9-6-8(12)3-4-10(9)14-7-15-11/h2-4,6-7H,1,5H2,(H,13,14,15)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.47 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (9.47 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (9.47 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)