Inosine monophosphate dehydrogenase (IMPDH) – noncompetitive inhibitor against both human IMPDH isoforms I and II (Ki = 6-10 nM) [2]

In a dose-dependent manner, AVN-944 (0-1 μM, 48 hours) suppresses the growth of human multiple myeloma (MM) cell lines [1]. AVN-944 (800 nM, 0-72 hours) induces apoptosis in MM cell lines through the caspase-independent Bax/AIF/Endo G pathway [1]. Melphalan and doxorubicin are more cytotoxic when AVN-944 (0-200 nM) is added [1]. AVN-944 inhibits the proliferation of human MV-4-11 and murine Ba/F3-Flt3-ITD-dependent cell lines with IC50 values of 26 and 30 nM respectively [2]. AVN-944 (0-32 μM, 48 h) exhibits good activity against arenavirus infection at low doses (7.5 μM) with less cytotoxicity [3]. AVN-944 (0-6.4 μM, 48 hours) does not reduce peripheral blood mononuclear cell (PBNC) viability [1]. Cell proliferation experiment [1]

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AVN-944 inhibited growth of K562, Raji, and CCRF-CEM cells in a dose-dependent manner with IC50 values ranging from 0.13 to 0.16 μM; growth inhibition was completely reversible by guanosine [2]

AVN-944 induced apoptosis in Raji (Burkitt lymphoma) and CCRF-CEM (T cell leukemia) cell lines, while K562 cells underwent erythroid differentiation [2]

In U2OS osteosarcoma cells, AVN-944 (1 μM) induced translocation of nucleolar proteins (nucleolin, nucleophosmin, and nucleostemin) from the nucleolus to the nucleoplasm after 4 hours of treatment [2]

In U2OS and MCF-7 cells expressing wild-type p53, AVN-944 (1 μM) induced marked p53 expression starting at 4 hours, sustained up to 24 hours [2]

In 293T cells, AVN-944 (1 μM) induced ARF translocation at 4 hours, with nearly complete translocation occurring at 8 hours [2]

AVN-944 (1 μM) inhibited total RNA synthesis as measured by [³H]-uridine incorporation in K562 and CCRF-CEM cells, with >80% inhibition at 4 hours and sustained inhibition lasting for 24 hours [2]

AVN-944 markedly inhibited 45S precursor rRNA synthesis in CCRF-CEM cells (at 2 hours) and K562 cells (at 4 hours), with inhibition sustained up to 8 hours as determined by semi-quantitative RT-PCR and autoradiography of [³H]-uridine-labeled RNA [2]

AVN-944 induced co-translocation of EGFP-tagged PAF53 (Pol I subunit) and myc-tagged TIF-IA (Pol I-associated initiation factor) to the nucleolar cap region in transfected U2OS cells [2]

AVN-944 (0-150 mg/kg, orally, twice daily) considerably prolongs the median life time of leukemia model mice [2].

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Cell proliferation experiment [1]
Cell Types: RPMI8226, MM.1S, U266 Cell
Tested Concentrations: 0, 100, 200, 300, 400, 600, 1000 nM
Incubation Duration: 48 h
Experimental Results: Dramatically inhibited the growth of RPMI8226 and MM. Acts in a dose-dependent manner on 1S and U266 cells with 50% inhibition (IC50) values of 450, 450 and 600 nM at 48 hrs (hours), respectively. Inhibits the growth of drug-resistant cell lines, including doxorubicin (Dox)-resistant RPMI8226-Dox40, melphalan (Mel)-resistant RPMI8226-LR5, and Dex (dexamethasone)-resistant MM.1R cells, with IC50 values similar to Parental drug-sensitive cell-like cell lines.

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Apoptosis analysis [1]
Cell Types: MM.1S and RPMI8226 Cell
Tested Concentrations: 800 nM
Incubation Duration: 48 and 72 hrs (hours)
Experimental Results: Induction of apoptosis in MM cell lines.

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Western Blot Analysis[1]
Cell Types: MM.1S and RPMI8226 Cell
Tested Concentrations: 800 nM
Incubation Duration: 12, 24, 48 hrs (hours)
Experimental Results: Moderate cleavage of caspases 3, 8 and 9 was induced in MM.1S cells and RPMI8226 cells. Bax and Ba

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Cell proliferation was measured by modified MTT assay. Exponentially growing CCRF-CEM, K562, and Raji cells were plated in 48-well plates at 2×10⁴ cells per well, incubated with various concentrations of AVN-944 or vehicle alone for 96 hours, then cytotoxicity was determined by MTT method. All samples were tested in triplicate [2]

To measure RNA synthesis, exponentially growing U2OS and CCRF-CEM cells were plated at 1×10⁵ cells/mL, incubated with or without AVN-944 (1 μM), then labeled with [³H]-uridine for 30 minutes. Total RNA was isolated, quantitated, and radioactivity determined by liquid scintillation counting. Triplicate samples were analyzed for each treatment [2]

For analysis of 45S pre-rRNA synthesis, K562 cells were treated with AVN-944 (1 μM) for 1.5, 3.5, or 7.5 hours, then metabolically labeled with ²H-uridine for 30 minutes. RNA was extracted, separated on formaldehyde-agarose gel, transferred to membrane, and subjected to autoradiography [2]

Immunocytochemistry: Cells grown on coverslips were treated with AVN-944 (1 μM) for indicated times, fixed in paraformaldehyde, permeabilized with Triton X-100, blocked with BSA, then incubated with primary antibodies against nucleolin, nucleophosmin, nucleostemin, ARF, or p53, followed by fluorophore-conjugated secondary antibodies. Nuclei were stained with DAPI and cells examined by fluorescent microscopy [2]

Immunoblotting: Cells treated with AVN-944 were lysed, protein quantified, separated by SDS-PAGE, transferred to PVDF membrane, probed with anti-p53 antibody, and visualized by enhanced chemiluminescence [2]

Semi-quantitative RT-PCR for 45S precursor rRNA: Total RNA was extracted, first-strand cDNA synthesized using random primers, then PCR amplified using primers spanning from the 3’ end of 18S rRNA to the internal transcribed sequence between 18S and 5.8S rRNA. GAPDH was used as control [2]

Animal/Disease Models: Balb/c mouse (leukemia model using Ba/F3 cells transduced with activated human Flt-3 mutation injected into mice) [2]
Doses: 75 or 150 mg/kg
Route of Administration: Oral , twice (two times) daily
Experimental Results: provided a significant increase in median survival time. When the study was terminated, 3 of 12 mice treated with 150 mg/kg AVN-944 were still alive at day 35.

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[1]. Inosine-5'-monophosphate dehydrogenase: regulation of expression and role in cellular proliferation and T lymphocyte activation. Prog Nucleic Acid Res Mol Biol. 1998;61:181-209.

[2]. Guanine nucleotide depletion inhibits pre-ribosomal RNA synthesis and causes nucleolar disruption. Leuk Res. 2008 Jan;32(1):131-41.

[3]. Antiproliferative effects of AVN944, a novel inosine 5-monophosphate dehydrogenase inhibitor, in prostate cancer cells. Int J Cancer. 2008 Nov 15;123(10):2294-302.

AVN944 is a biotech drug that has been shown to have statistically significant effects on IMPDH and other proteins crucial to cancer cell activity, including nucleotide biosynthesis, energy metabolism, DNA replication, apoptosis, and cell cycle regulation. Clinical trials have demonstrated that AVN944 is associated with cancer cell death. Its efficacy in treating patients with advanced hematologic malignancies is currently under investigation.
AVN944, an inosine monophosphate dehydrogenase inhibitor, is an orally synthesized small molecule drug with potential antitumor activity. AVN944 inhibits inosine monophosphate dehydrogenase (IMPDH), an enzyme involved in the de novo synthesis of guanosine triphosphate (GTP), a purine molecule essential for DNA and RNA synthesis. Inhibition of IMPDH leads to a lack of GTP in cancer cells, resulting in impaired DNA and RNA synthesis, inhibited cell proliferation, and induced apoptosis. AVN944 appears to have a selective effect on cancer cells; GTP deficiency in normal cells only leads to a temporary slowdown in cell growth. IMPDH is overexpressed in certain cancer cells, particularly in hematologic malignancies. Drug Indications It has been investigated for the treatment of cancer/tumor (unspecified), leukemia (unspecified), lymphoma (unspecified), multiple myeloma, and pancreatic cancer. Mechanism of Action AVN944 inhibits IMPDH and appears to induce apoptosis. Specifically, studies have found that even at the lowest doses in trials, the stress response marker HspA1A gene (which is associated with GTP pool depletion in cancer cell lines) is induced within hours of the first dose. Following continued administration of AVN944, this disease-related cellular stress marker remains elevated even with zero circulating drug levels between doses. Pharmacodynamics IMPDH is highly upregulated in most hematologic malignancies and many solid tumors. AVN944 is a biotech drug that has been shown to have statistically significant effects in inhibiting IMPDH and other proteins crucial to cancer cell activity, including nucleotide biosynthesis, energy metabolism, DNA replication, apoptosis, and cell cycle regulation. Clinical trials have demonstrated that AVN944 is associated with cancer cell death. Its efficacy in treating patients with advanced hematologic malignancies is currently under investigation.

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AVN-944 is a selective, noncompetitive inhibitor of IMPDH developed based on structural analysis of the enzyme’s active site. It is currently in Phase I clinical trials for the treatment of refractory hematologic malignancies [2]

The ability of AVN-944 to induce apoptosis in a number of leukemic cell lines supports its potential utility in the treatment of hematologic malignancies [2]

C25H27N5O5

477.512

477.201

297730-17-7

9918559

White to off-white solid powder

5.764

3

7

10

35

743

2

O=C(O[C@@H](CC#N)CC)N[C@H](C1=CC=CC(NC(NC2=CC=C(C3=CN=CO3)C(OC)=C2)=O)=C1)C

GYCPCOJTCINIFZ-OXJNMPFZSA-N

InChI=1S/C25H27N5O5/c1-4-20(10-11-26)35-25(32)28-16(2)17-6-5-7-18(12-17)29-24(31)30-19-8-9-21(22(13-19)33-3)23-14-27-15-34-23/h5-9,12-16,20H,4,10H2,1-3H3,(H,28,32)(H2,29,30,31)/t16-,20+/m0/s1

[(2R)-1-cyanobutan-2-yl] N-[(1S)-1-[3-[[3-methoxy-4-(1,3-oxazol-5-yl)phenyl]carbamoylamino]phenyl]ethyl]carbamate

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 (~209.42 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.24 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 (5.24 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.)