Ribonucleotide Reductase (RR) small subunit M2 (RRM2) [1]
IC50 for recombinant RR enzyme activity = 8.23 μM [1]
IC50 for cellular RR activity in HepG2.2.15 cell lysates = 760.8 μM [1]

It was determined that osalmid might be a ribonucleotide reductase small subunit M2 (RRM2) molecule. Osalmid strongly reduces HBV DNA and cccDNA synthesis in HepG2.2.15 cells in a time- and dose-dependent manner. It also inhibits ribonucleotide reductase (RR) activity ten times more than hydroxyurea. Following an 8-day Osalmid treatment, 16.5 μM was the EC50 for HBV DNA inhibition in cells and 11.1 μM in the culture supernatant. With a concentration-dependent effect and an IC50 of 8.23 μM, osalmid suppresses RR activity. Osalmid's strong action against HBV strains resistant to 3TC has been demonstrated [1], indicating that it may be used to treat drug-resistant HBV infections.

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Inhibition of Recombinant RR Activity: Using a [³H]CDP reduction assay with recombinant RRM1 and RRM2 proteins, osalmid suppressed RR activity in a concentration-dependent manner. The IC50 was 8.23 μM, compared to 95.7 μM for hydroxyurea (HU), indicating approximately 10-fold greater potency than HU. [1]
Inhibition of Cellular RR Activity: In HepG2.2.15 cells, osalmid decreased cellular RR activity in a time- and dose-dependent manner. After 36 hours of treatment, the IC50 in cell lysates was 760.8 μM. In contrast, HU showed no significant inhibition of cellular RR activity even at doses as high as 10 mM. [1]
Inhibition of HBV DNA Replication: Osalmid significantly inhibited HBV DNA replication in HepG2.2.15 cells in a time- and dose-dependent manner. After 8 days of treatment, the EC50 for HBV DNA inhibition was 11.1 μM in culture supernatants and 16.5 μM in cells. The selectivity index (SI = CC50/EC50) was >10, indicating efficient HBV inhibition with limited cytotoxicity. [1]
Inhibition of HBV cccDNA Synthesis: Osalmid promoted a dose-dependent inhibition of HBV cccDNA levels in HepG2.2.15 cells after 8 days of treatment, with an EC50 of 45.8 μM. This suggests it can inhibit both HBV genomic DNA and viral cccDNA synthesis. [1]
Inhibition of HBsAg and HBeAg Secretion: Osalmid effectively inhibited the secretion of HBsAg (EC50 = 55.2 μM) and HBeAg (EC50 = 61.7 μM) from 8-day treated HepG2.2.15 cells. [1]
Activity Against 3TC-Resistant HBV Mutant: Osalmid showed similar effectiveness against both wild-type and 3TC-resistant (rtL180M/M204V double mutation) HBV strains, with EC50 values of 15.8 μM and 19.8 μM, respectively. [1]
Synergistic Effect with Lamivudine (3TC): Combination of osalmid and 3TC at a 40:1 molar ratio showed dramatically greater HBV inhibitory activity compared to either drug alone. The combination index (CI) was <1.0, indicating synergistic effects. The combinational EC50 values were only about 20% of the EC50s for each drug alone. Cytotoxicity of the combination was similar to either drug alone. [1]
Cytotoxicity (CC50): The CC50 (concentration reducing cell viability to 50%) for osalmid in HepG2.2.15 cells was 122.5 μM after 8 days of treatment. [1]

In HBV transgenic mice, osalmid can lower RR activity and HBV replication. It also exhibits a synergistic impact with 3TC without being visibly harmful. Time-dependent suppression of HBV DNA replication is observed upon oral treatment of osalmid 400 mg/kg/d. Osalmid reduced HBV DNA replication in mouse serum and liver tissue by about 40–45% after 4 weeks of treatment as compared to the control group [1].

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Anti-HBV Efficacy in Transgenic Mice: In HBV-transgenic mice, oral administration of osalmid at 400 mg/kg/day for 28 days resulted in time-dependent inhibition of HBV DNA replication. After 4 weeks, osalmid suppressed HBV DNA replication by approximately 40-45% compared to control in both sera and liver tissues. [1]
Synergistic Efficacy with 3TC in Vivo: Combination of osalmid (400 mg/kg) and 3TC (100 mg/kg) showed synergistic anti-HBV effects, with significantly greater HBV DNA reduction than either drug alone in both sera and liver tissues. [1]
RR Activity Inhibition in Vivo: Osalmid treatment significantly inhibited RR activity in mouse liver tissues after 28 days of treatment, verifying its in vivo mechanism of action. [1]

Recombinant RR Activity Assay ([³H]CDP Reduction Assay): His-tagged RRM1 and RRM2 recombinant proteins were expressed in E. coli and purified using nickel-nitrilotriacetic acid-agarose resin affinity chromatography. RR activity was measured using a [³H]CDP reduction assay. The 100 μL reaction mixture contained 0.125 μmol/L [³H]CDP, 50 mM Hepes (pH 7.2), 100 mM KCl, 6 mM DTT, 4 mM magnesium acetate, 2 mM ATP, 0.05 mM CDP, and 0.25 μM RR holoenzyme (5 μg RRM1 and 2.5 μg RRM2). After incubation at 37°C for 15-30 minutes and dephosphorylation, samples were analyzed by HPLC and liquid scintillation counting. RR activity was calculated as: RR activity = dCDP/(CDP+dCDP) × 100%. For inhibition analysis, serially diluted osalmid was incubated with recombinant RR proteins at room temperature for 30 minutes before assaying enzyme activity. [1]
Cellular RR Activity Assay: Cultured HepG2.2.15 cells were lysed in low salt homogenization buffer (10 mM Hepes, pH 7.2, and 2 mM DTT) by passing through a 27-gauge needle on ice. An equal volume of high salt buffer (1 M Hepes, pH 7.2, 2 mM DTT) with protease inhibitors was added. Supernatants were collected after centrifugation and used for RR activity measurement as described above. [1]
Tissue RR Activity Assay: Mouse liver tissues were triturated with liquid nitrogen and homogenized in 50 mM Tris-HCl, pH 7.6, 2 mM DTT, and protease inhibitors. The mixture was centrifuged at 14,000 g at 4°C for 30 minutes, and supernatants were collected for RR activity measurement as described above. [1]

HBV DNA Replication Assay: HepG2.2.15 cells (1×10⁵ cells/well in 24-well plates) were treated with various concentrations of osalmid for 6 or 8 days, with media changed every 2 days. Culture supernatants and cells were collected separately by centrifugation. HBV DNA was extracted from supernatants using a Viral DNA kit, and from cells using phenol/chloroform extraction and ethanol precipitation. HBV DNA levels were measured by quantitative PCR (Q-PCR) using SYBR Green mix and specific primers (sense: 5'-CTGGAAAGTAAAGCGCTACTAA-3'; antisense: 5'-CCTCGGCAGTCAAAAGGAG-3'). EC50 values were calculated as the concentration inhibiting HBV DNA synthesis to 50% of control. [1]
HBV cccDNA Assay: HBV cccDNA was extracted from 3×10⁵ HepG2.2.15 cells using a QIAamp Mini DNA kit. Extracts were further purified with mung bean nuclease to remove HBV relaxed circular DNA. The purification reaction contained 44 μL extracted DNA, 1 μL mung bean nuclease (10,000 U/mL), and 5 μL of 10× mung bean nuclease buffer, incubated at 37°C for 30 minutes, then stopped with 2 μL of 100 mM EGTA (pH 7.4). cccDNA was quantified by Q-PCR using primers: forward 5'-TGAATCCTGCGGACGACC-3' and reverse 5'-ACAGCTTGGAGGCTTGAACAG-3'. [1]
Cell Viability Assay (CCK-8): HepG2.2.15 cell viability was measured using CCK-8 assay after treatment with compounds. CC50 values (concentration reducing cell viability to 50%) were calculated. [1]
Drug Combination Effect Analysis: Based on EC50 values, osalmid and 3TC were mixed at different molar ratios (20:1 to 80:1) and two-fold serial dilutions covering EC90, EC75, EC50, and EC25 were added to cells. After 8 days, HBV DNA was measured by Q-PCR. Combination effects were determined using the median effect method of Chou and Talalay with the CalcuSyn program. Combination index (CI) < 1.0 indicates synergism. [1]
Activity Against 3TC-Resistant HBV Mutant: HepG2 cells were transfected with plasmids encoding wild-type or 3TC-resistant mutant (rtL180M/rtM204V) HBV 1.3-copy genome. After 24 hours, cells were treated with serial dilutions of osalmid for 8 days. HBV DNA levels in supernatants were assessed by Q-PCR. [1]
HBsAg and HBeAg Assays: Secretion of HBsAg and HBeAg from treated HepG2.2.15 cells was measured by ELISA. [1]

HBV-Transgenic Mouse Study:** HBV-transgenic mice (BALB/c background, 8 weeks old) containing 1.3 copies of the HBV ayw complete genome were used. Osalmid was suspended in 0.05% CMC-Na and administered once daily by gavage at 400 mg/kg for 28 days. For combination studies, mice received a mixture of osalmid (400 mg/kg) and 3TC (100 mg/kg). Control mice received 0.05% CMC-Na solution. Blood samples were collected at various time points for serum HBV DNA and ALT/AST activity measurements. After 28 days, mice were sacrificed, and liver tissues were excised. The left lobe was removed for histological analysis, and the remaining liver tissues were frozen at -80°C for HBV DNA and RR activity measurements. [1]
* **Serum ALT and AST Activity Measurement:** Serum samples were collected after 28 days of treatment. ALT and AST activities were measured using commercial detection kits according to manufacturer's instructions. [1]
* **Histopathological Examination:** Mouse liver tissues were fixed in formalin, embedded in paraffin, sectioned at 5 μm, stained with hematoxylin and eosin (HE), and examined by light microscopy. [1]

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HBV-Transgenic Mouse Study: HBV-transgenic mice (BALB/c background, 8 weeks old) containing 1.3 copies of the HBV ayw complete genome were used. Osalmid was suspended in 0.05% CMC-Na and administered once daily by gavage at 400 mg/kg for 28 days. For combination studies, mice received a mixture of osalmid (400 mg/kg) and 3TC (100 mg/kg). Control mice received 0.05% CMC-Na solution. Blood samples were collected at various time points for serum HBV DNA and ALT/AST activity measurements. After 28 days, mice were sacrificed, and liver tissues were excised. The left lobe was removed for histological analysis, and the remaining liver tissues were frozen at -80°C for HBV DNA and RR activity measurements. [1]
Serum ALT and AST Activity Measurement: Serum samples were collected after 28 days of treatment. ALT and AST activities were measured using commercial detection kits according to manufacturer's instructions. [1]
Histopathological Examination: Mouse liver tissues were fixed in formalin, embedded in paraffin, sectioned at 5 μm, stained with hematoxylin and eosin (HE), and examined by light microscopy. [1]

In Vitro Cytotoxicity: In HepG2.2.15 cells, osalmid showed acceptable selectivity index (SI > 10) with CC50 of 122.5 μM and EC50 of 11.1 μM for HBV DNA inhibition after 8 days. [1]
In Vivo Toxicity in Mice: Osalmid treatment at 400 mg/kg/day for 28 days in HBV-transgenic mice did not cause apparent toxicity as shown by: [1]
Body weight measurements: no significant changes during treatment [1]
Liver function tests: no significant elevation of ALT and AST activities in serum [1]
Histopathological examination: no significant pathological changes in liver tissue sections [1]
Clinical Safety: The study notes that no severe toxicity has been reported in clinical use of osalmid for hepatobiliary diseases, suggesting its safety in vivo. [1]

[1]. Inhibition of hepatitis B virus replication by targeting ribonucleotide reductase M2 protein. Biochem Pharmacol. 2016 Mar 1;103:118-28.

Osalmid is an organic molecular entity. Osalmid is currently being investigated in the Khellinical trial NCT03670173 (Evaluation of the safety and efficacy of Osalmid in multiple myeloma).

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Background and Identification: Osalmid is an approved choleretic drug whose molecular target and mechanism of action were previously unknown. It was identified as a potential RRM2-targeting compound through computer-assisted virtual screening against the crystal structure of RRM2 (PDB: 3OLJ) using the Comprehensive Medicinal Chemistry (CMC) database. Molecular docking showed interactions between osalmid and key residues of RRM2 including hydrogen bonding. [1]
Mechanism of Action: Osalmid inhibits HBV replication by targeting ribonucleotide reductase small subunit M2 (RRM2), thereby suppressing RR activity and reducing the dNTP pools required for HBV DNA and cccDNA synthesis. This represents a novel mechanism of action different from current anti-HBV drugs like nucleoside analogs. [1]
Key Advantages: Compared to existing RR inhibitors: [1]
Approximately 10-fold more potent than hydroxyurea (HU) in inhibiting RR activity [1]
Shows selective anti-HBV activity with acceptable selectivity index, unlike HU and gemcitabine which showed little selective activity [1]
Effective against 3TC-resistant HBV strains [1]
Synergistic with lamivudine (3TC) without increased cytotoxicity [1]
Inhibits HBV cccDNA synthesis, addressing a key limitation of current therapies [1]
Potential Applications: Osalmid could serve as a novel anti-HBV candidate for treatment of chronic HBV infection and potentially HBV-related hepatocellular carcinoma (HCC), as RRM2 is also overexpressed in HCC and essential for cancer cell proliferation. [1]

229.073

526-18-1

4602

White to off-white solid powder

1.4±0.1 g/cm3

350.8±27.0 °C at 760 mmHg

179ºC

165.9±23.7 °C

0.0±0.8 mmHg at 25°C

1.711

2.53

3

3

2

17

261

0

O=C(NC1=CC=C(O)C=C1)C2=CC=CC=C2O

LGCMKPRGGJRYGM-UHFFFAOYSA-N

InChI=1S/C13H11NO3/c15-10-7-5-9(6-8-10)14-13(17)11-3-1-2-4-12(11)16/h1-8,15-16H,(H,14,17)

2-hydroxy-N-(4-hydroxyphenyl)benzamide

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)

DMSO : ≥ 100 mg/mL (~436.24 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.91 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 (10.91 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 (10.91 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.)