HDAC1 (IC50 = 0.31 nM); HDAC3 (IC50 = 0.83 nM); HDAC6 (IC50 = 71.8 nM); HDAC8 (IC50 = 877 nM)
Resminostat hydrochloride (Resminostat [HCl], 5 μM) causes histone acetylation in myeloma cells. With a mean K_i value of 27 nM, resminostat hydrochloride exhibits a substrate competitive binding mode. In myeloma cells, resminostat hydrochloride (5 μM) causes histone hyperacetylation. Resminostat suppresses MM cell proliferation, triggers apoptosis, and prevents cell growth. Additionally, resminostat (5 μM) inhibits signalling downstream of Akt and modifies the expression of proteins in the bcl-2 family. When combined with both established and novel anti-myeloma agents, resminostat exhibits synergistic activity against myeloma cells[1]. Resminostat shows IC50s ranging from 0.775 μM to 1.572 μM (IC50 for SCC25: 0.775 μM, CAL27: 1.572 μM, and FaDu: 0.899 μM) in head and neck squamous cell carcinoma cell lines, indicating that it inhibits cell growth. HNSCC cell lines respond well to both irradiation and resminostat (1.25 and 2.5 μM). One can downregulate survivin by combining resminostat with cisplatin. On Mcl-1 and p-AKT expression, however, Resminostat has no effect[2]. With IC50s ranging from 0.89 ± 0.12 μM to 0.07 ± 0.01 μM, respinostat decreases the viability of HCC cells when used in conjunction with AZD-2014[3].

Resminostat hydrochloride (Resminostat [HCl], 5 μM) causes histone acetylation in myeloma cells. With a mean K_i value of 27 nM, resminostat hydrochloride exhibits a substrate competitive binding mode. In myeloma cells, resminostat hydrochloride (5 μM) causes histone hyperacetylation. Resminostat suppresses MM cell proliferation, triggers apoptosis, and prevents cell growth. Additionally, resminostat (5 μM) inhibits signalling downstream of Akt and modifies the expression of proteins in the bcl-2 family. When combined with both established and novel anti-myeloma agents, resminostat exhibits synergistic activity against myeloma cells[1]. Resminostat shows IC50s ranging from 0.775 μM to 1.572 μM (IC50 for SCC25: 0.775 μM, CAL27: 1.572 μM, and FaDu: 0.899 μM) in head and neck squamous cell carcinoma cell lines, indicating that it inhibits cell growth. HNSCC cell lines respond well to both irradiation and resminostat (1.25 and 2.5 μM). One can downregulate survivin by combining resminostat with cisplatin. On Mcl-1 and p-AKT expression, however, Resminostat has no effect[2]. With IC50s ranging from 0.89 ± 0.12 μM to 0.07 ± 0.01 μM, respinostat decreases the viability of HCC cells when used in conjunction with AZD-2014[3].

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Resminostat HCl inhibited the growth of multiple myeloma (MM) cell lines (OPM-2, NCI-H929, RPMI-8226, U266) in a concentration-dependent manner. The IC50 values for growth inhibition after 48-96 hours ranged from approximately 2.5 to 3 μmol/L in three out of four cell lines. [1]
Resminostat HCl (0.5 - 10 μmol/L, 48h) strongly induced apoptosis in MM cell lines and primary MM cells from patients, as assessed by annexin-V/PI staining. At 10 μmol/L, apoptosis rates reached up to 93% in some cell lines and up to 90% in primary cells. [1]
Resminostat HCl (5 or 10 μmol/L, 48h) induced cleavage/activation of caspases 3, 8, and 9 in MM cell lines (OPM-2, U266). [1]
Resminostat HCl (up to 10 μmol/L, 72h) strongly inhibited MM cell proliferation, with up to 92% inhibition in a BrdU incorporation assay. [1]
Resminostat HCl (1 μmol/L) induced G0/G1 cell cycle arrest in three out of four MM cell lines (OPM-2, NCI-H929, U266). At 10 μmol/L, it led to accumulation of cells in the subG1 phase. [1]
Western blot analysis showed that treatment with Resminostat HCl (5 μmol/L) in U266 cells led to downregulation of cyclin D1, Cdk4, phosphorylated Rb (p-Rb), cdc25a, and p53, and upregulation of p21. [1]
Resminostat HCl (5 μmol/L) treatment of U266 cells decreased phosphorylation of Akt pathway downstream effectors 4E-BP1 and p70S6k. [1]
Resminostat HCl (5 μmol/L) treatment modulated expression of Bcl-2 family proteins in U266 cells, increasing pro-apoptotic Bim and Bax levels while decreasing anti-apoptotic Bcl-xL levels. [1]
Resminostat HCl (5 μmol/L) induced hyperacetylation of histone H4 in U266 myeloma cells, confirming its HDAC inhibitory activity in cells. [1]
Combination studies showed synergistic effects (Combination Index, CI < 0.9) when Resminostat HCl was combined with melphalan, bortezomib, or the proteasome inhibitor S-2209 in U266 cells, as analyzed by isobologram method. [1]

Resminostat taken orally at 600 mg QD every day for 1–5 days in a 14-day cycle is well tolerated. With low inter-pt variability and high bioavailability, resminostat exhibits a favorable pharmacokinetic profile. Oral resminostat's apparent t 1/2 varied from 2.7 to 4.4 hours. Additional evidence of medication activity is the modulation of plasma biomarkers.

A 96-well microtitre plate is filled with 40 microliters of enzyme buffer (15 mM Tris HCl pH 8.1, 0.25 mM EDTA, 250 mM NaCl, 10% v:v glycerol) containing HDAC1, 3, 6, or 8 activity, 29 microliters of enzyme buffer, and 1 microliter of resminostat [HCl] at varying concentrations. The reaction is initiated by adding 30 microliters of substrate peptide Ac-NH-GGK(Ac)-AMC (HDAC1, 3 and 6 assays, final concentrations of 6 μM for HDAC1, 10 μM for HDAC6, and 25 μM for HDAC3/DAD) or Ac-RHK(Ac)K(Ac)-AMC (HDAC8 assay, final concentration 50 μM). The reaction is stopped by adding 25 μL of stop solution (50 mM Tris HCl pH 8, 100 mM NaCl, 0.5 mg/mL trypsin, and 2 μM trichostatin A [TSA]) after the enzymes have been incubated for 180 min (HDAC1, HDAC6, HDAC8) or 120 min (HDAC3) at 30°C. The quantification of AMC produced by tryptic cleavage of the deacetylated peptide is done using a Wallac Victor2 1420 multilabel counter (extinction 355 nm, emission 460 nm) following an additional 40 minutes of room temperature incubation. The fluorescence in wells without test compound (1% DMSO, negative control) is set to 100% enzymatic activity for the calculation of the 50% inhibitory concentration (IC50) values, while the fluorescence in wells with 2 μM TSA (positive control) is set to 0% enzymatic activity (background fluorescence substracted)[1].

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For HDAC inhibition assays, recombinant HDAC enzymes (HDAC1, 3, 6, 8) were used. Enzyme buffer containing the enzyme and varying concentrations of Resminostat HCl was added to a microtiter plate. The reaction was started by adding a fluorogenic peptide substrate (Ac-NH-GGK(Ac)-AMC for HDAC1, 3, 6; Ac-RHK(Ac)K(Ac)-AMC for HDAC8). After incubation at 30°C (120-180 min), the reaction was terminated by adding a stop solution containing trypsin and trichostatin A. Fluorescence resulting from tryptic cleavage of the deacetylated peptide was measured (Ex 355 nm, Em 460 nm). IC50 values were calculated relative to controls (no inhibitor = 100% activity, trichostatin A = 0% activity). [1]
For HDAC1 enzyme kinetics and Ki determination, enzyme buffer containing HDAC1 and varying concentrations of Resminostat HCl was mixed in a plate. The reaction was started by adding substrate peptide at different final concentrations (10-100 μmol/L). After 60 min incubation at 30°C, the reaction was stopped, and fluorescence was measured. Data were analyzed using Lineweaver-Burk plots and linear regression to determine the inhibition constant (Ki). Resminostat HCl displayed a substrate-competitive binding mode for HDAC1. [1]

The antiproliferative action of resminostat on HNSCC cells is examined using a CCK-8 cell proliferation assay. 3 × 10⁵ cells per well are seeded into 96-well plates. The cells are treated with resminostat and cisplatin, either separately or together, after growing for 24 hours, and they are then incubated for 72 hours. As a control, untreated cells were kept in RPMI with equal amounts of dimethyl sulfoxide. CCK-8 is used to measure cell proliferation after 72 hours. Three times, each experiment is run in triplicate[2].

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For cell growth inhibition assays (WST-1 assay), MM cell lines were seeded and incubated with increasing concentrations of Resminostat HCl for 48 or 96 hours. The tetrazolium salt WST-1 was added, and after further incubation, the formazan dye produced by metabolically active cells was quantified by measuring absorbance at 450 nm (reference 690 nm). [1]
For apoptosis analysis, cells treated with Resminostat HCl were stained with annexin-V-FITC and propidium iodide (PI) and analyzed by flow cytometry. [1]
For cell cycle analysis, treated cells were fixed in ethanol, stained with PI and RNase A, and analyzed by flow cytometry to determine DNA content. [1]
For cell proliferation assay (BrdU incorporation), cells were seeded in 96-well plates and treated with Resminostat HCl for approximately 57 hours. BrdU labeling solution was then added, and cells were cultured for another 15 hours. Cells were fixed, DNA was denatured, and incorporated BrdU was detected using an anti-BrdU-POD antibody and a colorimetric TMB substrate. Absorbance was measured at 450 nm. [1]
For Western blot analysis, cells were lysed, and protein concentrations were determined. Proteins were separated by SDS-PAGE, transferred to membranes, blocked, and incubated with primary antibodies overnight. After washing, membranes were incubated with peroxidase-conjugated secondary antibodies, and signals were detected using chemiluminescence. [1]

MM Xenograft Mouse Model Protocol: Female SCID mice (6–8 weeks old) were injected subcutaneously with 5×10⁶ U266 MM cells into the right flank. When tumors reached 100 mm³, mice were randomly divided into 2 groups (n=6/group): control group (intraperitoneal injection of 0.9% saline, once daily) and Resminostat (RAS2410) group (intraperitoneal injection of 50 mg/kg Resminostat (RAS2410) dissolved in 0.9% saline, once daily). Treatments continued for 21 days. Measure tumor volume (using calipers, formula: volume = length × width² / 2) and mouse body weight every 3 days. At the end of treatment, sacrifice mice, excise tumors, and weigh them. Collect tumor tissues for immunohistochemistry and Western blot analysis. Monitor mouse survival for 60 days to calculate median survival time [1]

[1]. The novel inhibitor of histone deacetylase resminostat (RAS2410) inhibits proliferation and induces apoptosis in multiple myeloma (MM) cells. Br J Haematol. 2010 May;149(4):518-28.

[2]. Effect of the histone deacetylase inhibitor resminostat on head and neck squamous cell carcinoma cell lines. Head Neck. 2017 May;39(5):900-907.

p>[3]. mTOR inhibition sensitizes human hepatocellular carcinoma cells to resminostat. Biochem Biophys Res Commun. 2016 Sep 2;477(4):556-562.

Resminostat (RAS2410) is a novel hydroxamic acid-based histone deacetylase (HDAC) inhibitor. Its chemical structure differs from vorinostat in the linker region and hydrophobic cap region, while the metal-bound hydroxamic acid moiety is the same. [1]
Resminostat hydrochloride (Resminostat HCl) exhibits potent in vitro anti-myeloma activity by inhibiting cell proliferation, inducing cell cycle arrest and apoptosis, regulating cell cycle and apoptosis-related proteins, and interfering with the Akt/mTOR signaling pathway downstream of Akt. [1]
In vitro experiments have shown that resminostat hydrochloride has a synergistic effect when used in combination with conventional anti-myeloma drugs (melphalan) and novel anti-myeloma drugs (bortezomib, S-2209), which supports its potential application in combination therapy. [1]

C16H20CLN3O4S

385.86

385.086

C, 49.80; H, 5.22; Cl, 9.19; N, 10.89; O, 16.59; S, 8.31

1187075-34-8

Resminostat;864814-88-0

16662815

White to off-white solid powder

3.579

3

5

6

25

548

0

Cl[H].S(C1C([H])=C([H])C(=C([H])C=1[H])C([H])([H])N(C([H])([H])[H])C([H])([H])[H])(N1C([H])=C([H])C(/C(/[H])=C(\[H])/C(N([H])O[H])=O)=C1[H])(=O)=O

BVXPKDRKHXARHY-HAAWTFQLSA-N

InChI=1S/C16H19N3O4S.ClH/c1-18(2)11-13-3-6-15(7-4-13)24(22,23)19-10-9-14(12-19)5-8-16(20)17-21;/h3-10,12,21H,11H2,1-2H3,(H,17,20);1H/b8-5+;

(E)-3-[1-[4-[(dimethylamino)methyl]phenyl]sulfonylpyrrol-3-yl]-N-hydroxyprop-2-enamide;hydrochloride

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)

Solubility in Formulation 1: 2.08 mg/mL (5.39 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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: 2.08 mg/mL (5.39 mM) 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; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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: ≥ 2.08 mg/mL (5.39 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 20.8 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.)