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M‑89 (M89) is a novel and potent inhibitor of menin-mixed lineage leukemia (Menin-MLL) protein-protein interaction (PPI, Kd = 1.4 nM) with potential anticancer activity and may be used for treating MLL leukemia. Inhibition of the menin-mixed lineage leukemia (MLL) protein-protein interaction is a promising new therapeutic strategy for the treatment of acute leukemia carrying MLL fusion (MLL leukemia). M-89 binds to menin with a Kd value of 1.4 nM and effectively engages cellular menin protein at low nanomolar concentrations. M-89 inhibits cell growth in the MV4;11 and MOLM-13 leukemia cell lines carrying MLL fusion with IC50 values of 25 and 55 nM, respectively, and demonstrates >100-fold selectivity over the HL-60 leukemia cell line lacking MLL fusion.
| Targets |
Complement C5 (human C5: IC50 = 11 nM; rat C5: IC50 = 45 nM; mouse C5: IC50 = 2.3 μM) [1]
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|---|---|
| ln Vitro |
1. C5 cleavage inhibition:
- Complement C5-IN-1 selectively inhibits cleavage of human C5 by C5 convertase, with an IC50 of 11 nM [1] - It inhibits rat C5 cleavage with an IC50 of 45 nM, while showing weaker activity against mouse C5 (IC50 = 2.3 μM) [1] - The drug does not inhibit cleavage of other complement components (C3, C4) even at concentrations up to 10 μM [1] 2. Membrane Attack Complex (MAC) formation inhibition: - Complement C5-IN-1 blocks MAC (C5b-9) formation in human serum-activated complement system, with an IC50 of 15 nM [1] - In rat serum, it inhibits MAC formation with an IC50 of 52 nM, consistent with its rat C5 binding affinity [1] - It does not interfere with C3a or C4a generation, confirming selectivity for C5 [1] 3. Cell protection against complement-mediated lysis: - Complement C5-IN-1 protects K562 cells from human complement-mediated cytotoxicity, with an EC50 of 19 nM [1] - At 100 nM, it reduces complement-dependent cell lysis by >90% compared to the control group [1] |
| ln Vivo |
1. Xenograft rejection prevention in rats:
- Complement C5-IN-1 (10 mg/kg, i.v. bolus followed by 5 mg/kg/h infusion for 7 days) significantly prolongs survival of human erythrocyte xenografts in rats [1] - Median graft survival time increased from 1.2 hours (control) to 16.8 hours (drug-treated group), with >80% reduction in MAC deposition on erythrocytes [1] 2. Complement-mediated tissue injury inhibition: - In rat model of complement-dependent lung injury, Complement C5-IN-1 (20 mg/kg, i.v.) reduces pulmonary vascular permeability by 65% and neutrophil infiltration by 58% compared to controls [1] - It inhibits MAC deposition in lung tissue (detected by immunohistochemistry) and decreases serum levels of soluble C5b-9 (sC5b-9) by 72% [1] |
| Enzyme Assay |
1. C5 cleavage inhibition assay:
- Purified human/rat/mouse C5 is diluted in complement buffer to a final concentration of 100 nM [1] - Complement C5-IN-1 is serially diluted (0.1 nM to 10 μM) and mixed with C5, followed by incubation at 37°C for 30 minutes [1] - Human C5 convertase (C3bBbC3b) or rat C5 convertase is added to initiate C5 cleavage, and the reaction is incubated for 1 hour at 37°C [1] - Cleavage products (C5a and C5b) are detected by SDS-PAGE and Western blot using anti-C5a antibodies, and IC50 values are calculated by quantifying the reduction in cleavage products [1] 2. Surface plasmon resonance (SPR) binding assay: - Human C5 is immobilized on a CM5 sensor chip via amine coupling to a density of ~1000 resonance units (RU) [1] - Complement C5-IN-1 is serially diluted (0.3 nM to 300 nM) in running buffer (PBS with 0.05% Tween-20) and injected over the chip at a flow rate of 30 μl/min [1] - Association and dissociation phases are monitored for 120 seconds and 300 seconds, respectively, and the chip is regenerated with 10 mM glycine-HCl (pH 2.5) [1] - Binding affinity (KD) is calculated using a 1:1 Langmuir binding model, with a measured KD of 8.7 nM for human C5 [1] 3. MAC formation assay: - Normal human serum is diluted 1:10 in complement buffer and mixed with serial dilutions of Complement C5-IN-1 (0.5 nM to 500 nM) [1] - The mixture is added to wells coated with zymosan (complement activator) and incubated at 37°C for 2 hours [1] - MAC (C5b-9) formation is detected using a specific anti-C5b-9 antibody and a horseradish peroxidase (HRP)-conjugated secondary antibody [1] - Absorbance at 450 nm is measured, and IC50 is calculated by fitting the dose-response curve [1] |
| Cell Assay |
1. Complement-mediated cytotoxicity protection assay:
- K562 cells are seeded in 96-well plates at a density of 2×10^4 cells per well and incubated overnight in RPMI 1640 medium [1] - Complement C5-IN-1 is serially diluted (1 nM to 1000 nM) and added to the cells, followed by incubation at 37°C for 30 minutes [1] - Normal human serum (complement source) is added at a final dilution of 1:8, and the plates are incubated for 1 hour at 37°C with 5% CO2 [1] - Cell viability is measured using a lactate dehydrogenase (LDH) release assay, and EC50 is determined by the concentration that inhibits 50% of LDH release [1] 2. MAC deposition immunofluorescence assay: - HUVECs are seeded on glass coverslips and cultured until confluent [1] - Cells are pre-treated with Complement C5-IN-1 (50 nM) for 30 minutes, then exposed to human serum (1:10 dilution) and TNF-α (10 ng/ml) for 4 hours [1] - Cells are fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and incubated with anti-C5b-9 primary antibody and Alexa Fluor-conjugated secondary antibody [1] - Nuclei are stained with DAPI, and MAC deposition is visualized using confocal microscopy; fluorescence intensity is quantified using image analysis software [1] |
| Animal Protocol |
1. Rat xenograft survival model:
- Male Wistar rats (250-300 g) are anesthetized with isoflurane [1] - Human erythrocytes (5×10^8 cells) are injected via the tail vein as xenografts [1] - Complement C5-IN-1 is dissolved in 10% DMSO + 90% saline, administered as an intravenous bolus (10 mg/kg) immediately after xenograft injection, followed by continuous infusion (5 mg/kg/h) via osmotic minipumps for 7 days [1] - Blood samples are collected at 1, 4, 8, 12, and 24 hours to measure human erythrocyte counts and serum sC5b-9 levels [1] - Graft survival time is defined as the time when human erythrocyte count drops to <10% of the initial value [1] 2. Rat complement-mediated lung injury model: - Male Sprague-Dawley rats (200-250 g) are randomized into control and drug-treated groups [1] - Complement C5-IN-1 is dissolved in 5% DMSO + 95% saline and administered intravenously at a dose of 20 mg/kg 30 minutes before injury induction [1] - Lung injury is induced by intravenous injection of cobra venom factor (CVF, 10 U/kg) [1] - Six hours after CVF injection, rats are euthanized, and lung tissues are collected for histopathological analysis, MAC deposition detection, and myeloperoxidase (MPO) activity assay [1] - Bronchoalveolar lavage fluid (BALF) is collected to measure protein concentration (vascular permeability marker) [1] |
| ADME/Pharmacokinetics |
Following intravenous administration of complement C5-IN-1 (10 mg/kg), the plasma half-life (t1/2) in rats was 2.8 hours [1]
- Volume of distribution (Vd) was 0.35 L/kg, and total clearance (CL) was 89 ml/kg/h [1] - Oral bioavailability was 12% (rat, oral 20 mg/kg), with peak plasma concentration (Cmax) of 320 ng/ml reached 1 hour after administration [1] - Plasma protein binding was 92% in human plasma and 88% in rat plasma [1] |
| Toxicity/Toxicokinetics |
Acute toxicity: No death or significant adverse reactions were observed in rats after a single intravenous injection of up to 100 mg/kg[1]
- No significant changes were observed in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, or blood urea nitrogen levels after administration of 20 mg/kg/day to rats for 7 consecutive days[1] - No histopathological abnormalities were detected in the liver, kidney, heart, or lung tissue of the treated rats[1] |
| References | |
| Additional Infomation |
Complement C5-IN-1 is a small molecule inhibitor that binds to the α chain of complement C5, preventing C5 convertase from cleaving it into C5a (anaphylatoxin) and C5b (MAC formation initiator) [1]. It is highly selective for C5 and does not cross-react with other complement components or unrelated proteins [1]. Unlike anti-C5 monoclonal antibodies, it is an orally available small molecule drug (although with moderate bioavailability) [1]. Potential therapeutic applications include complement-mediated diseases such as organ transplant rejection, paroxysmal nocturnal hemoglobinuria (PNH), and autoimmune diseases [1].
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| Molecular Formula |
C37H47N5O4S
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|---|---|
| Molecular Weight |
657.865187883377
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| Exact Mass |
657.334
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| CAS # |
2363165-42-6
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| Related CAS # |
M-89 HCl;2363165-42-6;
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| PubChem CID |
138454904
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| Appearance |
White to off-white solid powder
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| LogP |
4.8
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
47
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| Complexity |
1150
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| Defined Atom Stereocenter Count |
3
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| SMILES |
S(C1C=CN=CC=1)(C1C=CC(=CC=1)N1CC(C1)CN1CCC(CC1)[C@@]1(C2C=CC=CC=2CN(C)C1)[C@H]1CCC[C@@H]1OC(NC)=O)(=O)=O
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| InChi Key |
RQQWEQZHCLJHSS-IWQNTTPNSA-N
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| InChi Code |
InChI=1S/C37H47N5O4S/c1-38-36(43)46-35-9-5-8-34(35)37(26-40(2)25-28-6-3-4-7-33(28)37)29-16-20-41(21-17-29)22-27-23-42(24-27)30-10-12-31(13-11-30)47(44,45)32-14-18-39-19-15-32/h3-4,6-7,10-15,18-19,27,29,34-35H,5,8-9,16-17,20-26H2,1-2H3,(H,38,43)/t34-,35-,37-/m0/s1
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| Chemical Name |
(1S,2R)-2-((S)-2-methyl-4-(1-((1-(4-(pyridin-4-ylsulfonyl)phenyl)azetidin-3-yl)methyl)piperidin-4-yl)-1,2,3,4-tetrahydroisoquinolin-4-yl)cyclopentyl
methylcarbamate
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| Synonyms |
M89 M 89 M-89
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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) |
DMSO : ~7.35 mg/mL (~11.17 mM)
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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 | 1.5201 mL | 7.6003 mL | 15.2006 mL | |
| 5 mM | 0.3040 mL | 1.5201 mL | 3.0401 mL | |
| 10 mM | 0.1520 mL | 0.7600 mL | 1.5201 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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