DYRK1A; 5-HT_2A Receptor (Ki = 397 nM)
- DYRK1A (dual-specificity tyrosine-phosphorylation regulated kinase 1A) – IC50 = 0.3 μM (in cellulo tau phosphorylation inhibition assay) [2]
- 5-HT2A serotonin receptor – Ki = 397 ± 13 nM (agonist-labeled) [1]
- MAO-A (monoamine oxidase A) – mentioned as a target but no IC50/Ki provided [3]
- Imidazoline receptors – mentioned but no affinity data provided [3]
- Homologous recombination (HR) repair pathway – inhibition via disruption of Rad51 recruitment, no IC50 provided [3]

Harmine has an IC50 of 190 nM and inhibits tau phosphorylation of DYRK1A through specific DANDY [2]. Harmine causes severe cytotoxicity in liver cancer cells by obstructing Rad51 recruitment, which negatively regulates homologous recombination (HR). Furthermore, Hep3B cells were considerably more sensitive to the anti-proliferative effects of Harmine when Nu7441, an NHEJ inhibitor, was used [3].

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- Harmine hydrochloride inhibited DYRK1A-mediated tau phosphorylation in HEK293 cells transiently co-transfected with DYRK1A and tau, with an IC50 of approximately 0.3 μM. [2]
- In HEK293 cells, Harmine hydrochloride showed an in cellulo/in vitro inhibitory potency ratio of 48 compared to its in vitro DYRK1A inhibition. [2]
- Harmine hydrochloride inhibited KB cell proliferation with 49% growth inhibition at 10^-5 M and 0% at 10^-6 M. [2]
- In Hep3B and HuH7 hepatoma cells, Harmine hydrochloride dose-dependently decreased homologous recombination (HR) efficiency but did not affect nonhomologous end joining (NHEJ). [3]
- Harmine hydrochloride treatment (12, 24, 48 h) reduced proliferation of Hep3B cells with IC50 values of 12.80 μM (12 h), 6.20 μM (24 h), and 0.70 μM (48 h) as measured by MTT assay. [3]
- In HuH7 cells, IC50 values were 11.90 μM (12 h), 6.90 μM (24 h), and 0.70 μM (48 h). [3]
- In normal liver cell lines Chang liver and QSG-7701, Harmine hydrochloride showed relatively mild cytotoxicity. [3]
- Harmine hydrochloride (40 μM, 48 h) did not induce significant apoptosis or necrosis in Hep3B cells as measured by Annexin V-FITC/PI staining. [3]
- Harmine hydrochloride (40 μM, 12 h and 48 h) induced significant S and G2/M phase arrest in Hep3B cells. [3]
- ATM inhibitor Ku55933 and ATR inhibitor VE-821 rescued the S phase arrest induced by Harmine hydrochloride in Hep3B cells. [3]
- Harmine hydrochloride (20 μM) combined with Nu7441 (4 μM) increased S and G2/M phase arrest to 90% in Hep3B cells. [3]
- Western blot analysis showed that Harmine hydrochloride increased γ-H2Ax and phospho-Chk1 levels in a dose-dependent manner in Hep3B cells. [3]
- Harmine hydrochloride reduced the number of Rad51 foci in Hep3B cells following ionizing radiation. [3]

The TBI group's brain water content increased dramatically, according to the results. When compared to the TBI group, the hormone treatment considerably decreased the amount of water in the tissue on days 1, 3, and 5. When compared to the TBI group, the escape delay on days 3 and 5 was dramatically reduced by dehydrokine therapy. Rats given harmine 1, 3, and 5 days post-trauma showed a markedly better recovery in motor function than the group of rats given no treatment. When comparing the TBI group to the Harmine-treated group, there was a significant increase in the rates of neural survival. GLT-1 expression was much higher after receiving harmine than it was in the TBI group. When Harmine was administered, the expression of caspase 3 was considerably lower than in the TBI group [4].

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- In Ts65Dn Down syndrome mouse model, Harmine hydrochloride was not used; instead, a fluoro-DANDY derivative (compound 5a) was used. No in vivo data for Harmine hydrochloride were reported in this study. [2]

- DYRK1A kinase activity was measured using a UFLC-based approach with a fluorescent peptide substrate derived from the human transcription factor FKHR (sequence: KISGRLSPIMTEQ). Purified recombinant rat DYRK1A catalytic domain (DYRK1A-ΔC) was used. The reaction was performed in 96-well plates in 50 μL kinase buffer (50 mM Tris-HCl pH 7.5, 10 mM DTT, 5 mM MgCl₂) containing 20 ng DYRK1A-ΔC and peptide substrate (5–60 μM). The reaction was initiated by ATP (50–800 μM), incubated for 30 min at 37°C, stopped with 15% HClO₄, and analyzed by UFLC on a C8 reverse-phase column. Phosphorylated and unphosphorylated peptides were separated by isocratic flow (85% solvent A/15% solvent B) and monitored by fluorimetry (excitation 485 nm, emission 530 nm). Inhibition studies were performed by adding Harmine hydrochloride at various concentrations prior to ATP addition. [2]
- In cellulo DYRK1A inhibition assay: HEK293 cells were transiently co-transfected with full-length human DYRK1A and Tau. Cells were incubated with Harmine hydrochloride for 2 hours at various concentrations. Cells were then lysed, and an ELISA was performed to capture phosphorylated Tau and detect phosphorylation levels using an antibody to phospho-Tau T212. Kinase-deficient DYRK1A[K188R] was used as a negative control. [2]

To avoid cell cycle arrest or apoptosis, rapidly proliferating cancer cells have to promote DNA double strand break (DSB) repair to fix replication stress induced DSBs. Therefore, developing drugs blocking homologous recombination (HR) and nonhomologous end joining (NHEJ) - 2 major DSB repair pathways - holds great potential for cancer therapy. Over the last few decades, much attention has been paid to explore drugs targeting DSB repair pathways for cancer therapy. Here, using 2 well-established reporters for analyzing HR and NHEJ efficiency, we found that both HR and NHEJ are elevated in hepatoma cell lines Hep3B and HuH7 compared with normal liver cell lines Chang liver and QSG-7701. Our further study found that Harmine, a natural compound, negatively regulates HR but not NHEJ by interfering Rad51 recruitment, resulting in severe cytotoxicity in hepatoma cells. Furthermore, NHEJ inhibitor Nu7441 markedly sensitizes Hep3B cells to the anti-proliferative effects of Harmine. Taken together, our study suggested that Harmine holds great promise as an oncologic drug and combination of Harmine with a NHEJ inhibitor might be an effective strategy for anti-cancer treatment.[3]

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- KB cell proliferation assay: Human KB epidermal carcinoma cells were grown in DMEM with 10% FBS, 1% glutamine, penicillin, streptomycin, and fungizone. Cells were seeded in 96-well plates (5×10³ cells/well) and after 24 h treated with Harmine hydrochloride (10^-5 M or 10^-6 M) for 72 h. Cell viability was assessed using resazurin (20 μL, 2 h incubation), and fluorescence was recorded at λex=560 nm, λem=590 nm. [2]
- HR and NHEJ reporter assays in hepatoma cells: Hep3B and HuH7 cells were pretreated with Harmine hydrochloride (various concentrations) for 6 h, then co-transfected with linearized HR or NHEJ reporter plasmids and a DsRed expression plasmid. 24 h later, cells were harvested and analyzed by flow cytometry. The ratio of GFP⁺ to DsRed⁺ cells was used as a measure of repair efficiency. [3]
- MTT cytotoxicity assay: Hep3B, HuH7, Chang liver, and QSG-7701 cells were cultured in 96-well plates and treated with Harmine hydrochloride for 12, 24, or 48 h. 5 mg/mL MTT solution was added for 4 h at 37°C, formazan crystals dissolved in DMSO, and absorbance read at 570 nm. IC50 values were calculated using Dose-Effect Analysis software. [3]
- Apoptosis assay: Hep3B cells (1–5×10⁵ per sample) were treated with Harmine hydrochloride (40 μM, 48 h), collected, washed, resuspended in binding buffer, stained with Annexin V-FITC and PI for 10 min in the dark, and analyzed by flow cytometry. [3]
- Cell cycle analysis: Hep3B cells were treated with Harmine hydrochloride (40 μM for 12 or 48 h; or 20 μM for 24 h), fixed in 75% ethanol, stained with PI after RNase treatment, and DNA content analyzed by flow cytometry. [3]
- Immunofluorescence: Hep3B cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with goat serum, and incubated overnight at 4°C with anti-γ-H2Ax or anti-Rad51 antibody (1:50). After washing, cells were incubated with FITC-conjugated secondary antibody (1:100) for 1 h at room temperature, stained with DAPI, and observed under confocal microscopy. [3]
- Western blot: Hep3B cells were lysed in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1, protease inhibitors). Protein concentration was determined, and equal amounts were separated on 8–15% SDS-PAGE, transferred to PVDF membranes, blocked, and incubated with primary antibodies (γ-H2Ax, Rad51, NBS1, p-NBS1, RPA, Rad50, BRCA1, Chk1, p-Chk1, Actin, Tubulin), followed by HRP-conjugated secondary antibodies and enhanced chemiluminescence detection. [3]

Rats: The study uses 150 male Sprague-Dawley rats, weighing between 280 and 320 g and aged between 10 and 12 weeks. Three groups of rats are randomly assigned: the TBI group (n=35); the TBI + Harmine-treated group (n=35); and the Sham-operated group (n= 15). Immediately after traumatic brain injury, heroine (i.p., 30 mg/kg daily) is given for a maximum of five days. Equal volumes of 0.9% saline solution are given to the TBI and sham groups (i.p.). For the purpose of examining behavioral recovery, the rats are divided into three groups: Sham (n = 3), TBI (n = 7), and Harmine (n = 7). The NSS is assessed 1, 3, and 5 days after a traumatic brain injury. An observer who is blind to the animal treatment evaluates each rat individually[4].

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Metabolism / Metabolites
Harmine 's known human metabolites include 6-hydroxyHarmine and harmoll.

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- Harmine hydrochloride showed mild cytotoxicity in normal liver cell lines Chang liver and QSG-7701. [3]
- In KB cells, Harmine hydrochloride showed 49% growth inhibition at 10^-5 M and 0% at 10^-6 M. [2]

[1]. Binding of beta-carbolines and related agents at serotonin (5-HT(2) and 5-HT(1A)), dopamine (D(2)) and benzodiazepine receptors. Drug Alcohol Depend. 2000 Aug 1;60(2):121-32.

[2]. DYRK1A inhibition and cognitive rescue in a Down syndrome mouse model are induced by new fluoro-DANDY derivatives. Sci Rep. 2018 Feb 12;8(1):2859.

[3]. Harmine suppresses homologous recombination repair and inhibits proliferation of hepatoma cells. Cancer Biol Ther. 2015;16(11):1585-92.

[4]. Treatment with harmine ameliorates functional impairment and neuronal death following traumatic brain injury. Mol Med Rep. 2015 Dec;12(6):7985-91.

Harmine is a Harmine alkaloid, with its Harmine skeleton substituted with a methoxy group at the C-7 position. It is a metabolite, an anti-HIV drug, and an EC 1.4.3.4 (monoamine oxidase) inhibitor. It is derived from the hydride of Harmine . Harmine has been reported in passionflower (Passiflora phoenicia), Sichuan aster (Symplocos setchuensis), and several other organisms with relevant data. Harmine is an alkaloid isolated from the seeds of Peganum harmala, a plant in the Zygophyllaceae family. It is identical to banisterine or telepathine found in Banisteria caapi, and is one of the active ingredients in hallucinogenic drinks made from related plants in the western Amazon. It has no therapeutic use, but (like banisterine) it was touted as a treatment for post-encephalitis Parkinson's disease in the 1920s.

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- Harmine hydrochloride is a naturally occurring β-carboline alkaloid found in Peganum harmala and other sources. [3]
- It has been reported to possess anxiolytic and behavioral effects both in vitro and in vivo. [3]
- Harmine hydrochloride inhibits breast cancer resistance protein (BCRP) and reverses drug resistance in BCRP-overexpressing breast cancer cells. [3]
- Harmine hydrochloride reduces proliferation of HL60 cells in a dose- and time-dependent manner, alone or in combination with ATRA and G-CSF. [3]
- In Hep3B cells (p53-deficient), Harmine hydrochloride induces S and G2/M phase arrest rather than apoptosis, likely due to low p53 expression. [3]
- In DOM-trained animal models, harmaline (a related β-carboline) substitutes for DOM, but Harmine hydrochloride was not tested in those behavioral assays. [1]
- Harmine hydrochloride is not a major drug abuse problem in the US but has been used in plant-derived forms by South American natives for centuries. [1]

C13H12N2O

212.2472

248.071

C, 62.78; H, 5.27; Cl, 14.25; N, 11.26; O, 6.43

343-27-1

Harmine; 442-51-3

5280953

Off-white to light yellow solid

421.4ºC at 760mmHg

265-270°C

139.8ºC

3.835

1

2

1

16

258

0

O(C([H])([H])[H])C1C([H])=C([H])C2=C(C=1[H])N([H])C1C(C([H])([H])[H])=NC([H])=C([H])C2=1

VNPLYCKZIUTKJM-UHFFFAOYSA-N

InChI=1S/C13H12N2O.ClH/c1-8-13-11(5-6-14-8)10-4-3-9(16-2)7-12(10)15-13;/h3-7,15H,1-2H3;1H

7-methoxy-1-methyl-9H-pyrido[3,4-b]indole;hydrochloride

Harmine Hydrochloride; 343-27-1; 7-Methoxy-1-methyl-9H-pyrido[3,4-b]indole hydrochloride; HARMINE HCl; Harmine monohydrochloride; Harmine (hydrochloride); Telepathine (hydrochloride); T89I34ODAA;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

H2O : ~10 mg/mL (~40.21 mM)
DMSO : ~5 mg/mL (~20.10 mM)

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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