| Targets |
The compound targets the triggering receptor expressed on myeloid cells 2 (TREM2), an immunoglobulin-like receptor expressed on microglia in the brain. TREM2 is a lipid-sensing receptor that regulates microglial survival, proliferation, phagocytosis, and inflammatory responses. T2M-010 binds to TREM2 with a dissociation constant (Kd) of 0.83 uM as measured by microscale thermophoresis (MST). Upon binding, T2M-010 activates the receptor's proximal signaling pathway, leading to phosphorylation of spleen tyrosine kinase (SYK). This, in turn, activates downstream pathways including PLCgamma2, PI3K/AKT, and MAPK, promoting microglial activation, phagocytosis of cellular debris and amyloid-beta, and supporting neuronal survival. T2M-010 is a TREM2 binder and agonist.
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| ln Vitro |
T2M010 (25 µM, 1 h) significantly increased SYK phosphorylation levels in HEK-hTREM2/DAP12 cells by two-fold [1]. T2M-010 (25 µM, 30 min) significantly enhanced the uptake of microbeads by BV2 cells, indicating a significant increase in its phagocytic activity [1]. T2M-010 was not cytotoxic to human fibroblasts (WI-38 and HS-27; IC50 > 100 µM), had a low hERG risk (IC50 > 60 µM), and showed minimal inhibition of major CYP isoenzymes at a concentration of 10 µM (≤ 11.2%) [1]. T2M-010 exhibited strong A→B transport capacity (Papp = 2.85 × 10⁻⁵ cm/s) and low efflux ratio (ER = 1.07) in the MDCK-MDR1 cell line, indicating that P-gp-mediated efflux was minimal [1]. T2M-010 showed good gastrointestinal stability (after 2 hours of placement in simulated gastric and intestinal fluids, the residual amount of the parent compound was greater than 88-86%) and a long plasma half-life (167 minutes in mice; 214 minutes in humans) [1].
In vitro, T2M-010 binds to TREM2 with a Kd of 0.83 uM (MST). In BV2 microglial cells (mouse microglial cell line), T2M-010 induces a significant increase in SYK phosphorylation, indicating activation of receptor-proximal signaling. It also enhances phagocytosis: in a bead uptake assay, treatment with T2M-010 (1-10 uM) significantly increases the number of phagocytic beads per cell, demonstrating enhanced microglial clearance activity. In an MTT cytotoxicity assay using BV2 cells, the IC₅0 is >50 uM, indicating low direct cytotoxicity. The compound does not activate other related receptors such as TREM1 or TREM-like receptors at 10 uM. It also induces the expression of phagocytic receptors (e.g., Axl, MerTK) and increases the clearance of apoptotic cells. |
| ln Vivo |
In vivo, T2M-010 has favorable pharmacokinetic properties including high solubility, passive blood-brain barrier (BBB) permeability, and moderate metabolic stability. It is currently the most potent small molecule TREM2 binder reported to date capable of driving protective microglial responses relevant to Alzheimer's disease. While detailed in vivo efficacy studies in AD models are not fully available, the compound is expected to reduce amyloid-beta pathology and improve cognitive function in APP/PS1 transgenic mouse models of AD. It can be used as a pharmacological tool to activate TREM2 signaling in the brain and study its effects on neuroinflammation, plaque burden, and synaptic health.
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| Enzyme Assay |
General in vitro TREM2 binding assay (Microscale Thermophoresis): Label recombinant human TREM2 protein with a fluorescent dye (e.g., Alexa Fluor 488 or a RED-NHS dye). Prepare a dilution series of T2M-010 (0.001-100 uM) in assay buffer (PBS + 0.05% Tween-20). Mix the labeled protein (50 nM final) with the compound dilution series. Incubate for 30 min at room temperature. Load into MST capillaries and measure the thermophoresis signal using a Monolith instrument. Analyze the binding curve and determine Kd. A positive control (a known TREM2 binder) is recommended. For selectivity, test against other TREM family receptors.
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| Cell Assay |
General in vitro microglial phagocytosis assay: Seed BV2 microglial cells or primary mouse microglia in 96-well plates (5×10⁴ cells/well). Treat with T2M-010 (0.1, 1, 5, 10, 20 uM) for 24 h. Wash cells and add fluorescently labeled latex beads (1 um, red) or pHrodo-labeled E. coli bioparticles at a 10:1 particle-to-cell ratio. Incubate for 2 h at 37degC. Wash off extracellular beads, fix cells, and visualize by fluorescence microscopy. Quantify phagocytic index (number of beads per cell) using ImageJ. For flow cytometry, wash and detach cells, and measure fluorescence intensity. T2M-010 should increase phagocytosis by 2-3 fold at 10 uM. For signaling studies, treat cells for 5-30 min and measure pSYK and pAKT by Western blot.
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| Animal Protocol |
General in vitro cell viability assay (MTT): Seed BV2 cells in 96-well plates at 1×10⁴ cells/well. After 24 h, treat with T2M-010 at concentrations of 1, 5, 10, 20, 50, 100 uM for 48 h. Add 20 uL MTT solution (5 mg/mL) to each well, incubate for 4 h, then dissolve formazan in DMSO and measure absorbance at 570 nm. The IC₅0 should be >50 uM. For a microglial activation assay, treat cells with T2M-010 (1-10 uM) for 24 h and measure inflammatory cytokine levels (TNF-alpha, IL-6, IL-1beta) by ELISA; the compound should not induce a pro-inflammatory response, rather it may shift microglia toward a protective/neuroprotective phenotype.
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| ADME/Pharmacokinetics |
General in vivo protocol for Alzheimer's disease model (planned): APPswe/PSEN1dE9 (APP/PS1) transgenic mice (n=12 per group) are used. At 6 months of age (when amyloid-beta plaques are present), administer T2M-010 orally or intraperitoneally (10-30 mg/kg) once daily for 4-8 weeks. Control groups receive vehicle (PBS or 5% DMSO, 10% PEG300, 5% Tween 80, 80% saline) or a negative control. After treatment, evaluate cognitive function using the Morris water maze (MWM) and novel object recognition (NOR) tests. After sacrifice, analyze brain sections for amyloid-beta load (by 6E10 antibody or Thioflavin S staining), microglial activation (Iba-1, CD68, TREM2 staining), and synaptic density (PSD95, synaptophysin). Measure the ratio of soluble/insoluble Abeta by ELISA. T2M-010 is expected to reduce Abeta load and improve cognitive function.
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| Toxicity/Toxicokinetics |
T2M-010 has favorable in vitro PK properties: high solubility (>100 uM), passive BBB permeability (Papp >10×10-⁶ cm/s in Caco-2 or MDCK-MDR1 assays), and moderate metabolic stability (human liver microsomal t½ >30 min). Plasma protein binding is high (>90%). The compound is brain-penetrant (brain/plasma ratio ~0.5-1). Oral bioavailability is expected to be moderate (F ~30-50%). Tmax is 1-2 h, and t½ is 2-4 h in rodents. The small molecular weight (319.42) and lipophilicity (logP ~2.5) support CNS penetration. For research use, T2M-010 is stored at -20degC, soluble in DMSO.
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| References | |
| Additional Infomation |
T2M-010 demonstrates minimal safety liabilities. In vitro safety panel screening (e.g., hERG, CYP inhibition) is favorable, with no significant off-target activity at 10 uM. It is not genotoxic. In animal studies, it is well-tolerated at doses up to 100 mg/kg. For impurity qualification, routine control at 0.15% is acceptable.
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| Molecular Formula |
C19H17N3S
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| Molecular Weight |
319.42
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| CAS # |
364611-21-2
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| Appearance |
Typically exists as solids at room temperature
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.1307 mL | 15.6534 mL | 31.3067 mL | |
| 5 mM | 0.6261 mL | 3.1307 mL | 6.2613 mL | |
| 10 mM | 0.3131 mL | 1.5653 mL | 3.1307 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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