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MYCi361 (NUCC0196361) is a novel and potent MYC inhibitor (Kd = 3.2 μM) with potential anticancer activity, it demonstrated a narrow therapeutic index. MYCi361 phosphorylated MYC within cells, shattered MYC/MAX dimers, and reduced MYC-stimulated transcription. Additionally, it increased the phosphorylation of MYC on threonine-58, which increased the degradation of MYC by proteasomes. MYCi361 has the ability to improve anti-PD1 immunotherapy and inhibit tumor growth.
| Targets |
MYC (Kd = 3.2 μM)
MYCi361 (NUCC-0196361) targets MYC (Kd = 3.2 μM for MYC binding as determined by fluorescence polarization competition assay) and disrupts MYC/MAX interaction. It does not inhibit 468 kinases (including GSK3β) in a kinome screen, nor does it inhibit phosphatases in a 10-dose screening panel up to 100 μM. [1] |
|---|---|
| ln Vitro |
MYCi361 inhibits MYC-driven gene expression, breaks down MYC/MAX dimers, and activates MYC inside of cells. MYCi361 increases MYC phosphorylation on threonine-58, which in turn increases MYC degradation by the proteasome.[1]
MYCi361 (NUCC-0196361) exhibits low micromolar IC50s against MYC-dependent cancer cells including prostate cancer (MycCaP, LNCaP, PC3), leukemia (MV4-11), lymphoma (HL-60, P493-6) and neuroblastoma (SK-N-B2), while showing little effect on MYC/MAX-independent PC12 cells. [1] It reduces MYC and MYCN but not MAX protein levels without altering MYC mRNA. The reduction is rescued by proteasome inhibitor MG132, and cycloheximide chase assay shows MYC half-life decreased from 66 min to 28 min in PC3 cells. [1] It increases MYC T58 phosphorylation selectively, not S62, and this precedes MYC protein reduction. MYC T58A mutant is resistant to degradation, and in vitro kinase assay confirms that MYCi361 directly enhances GSK3β-mediated MYC pT58. [1] It disrupts MYC/MAX interaction as shown by co-immunoprecipitation and proximity ligation assay (PLA) at 6 μM for 1 hr. [1] It suppresses MYC target gene expression (e.g., CDC25A, MYB) and impairs cell cycle progression with downregulation of CDC25, Cyclins, E2F, CDKs, Skp2 and upregulation of p21, p15, p16 without inducing γ-H2AX. [1] It induces immunogenic cell death in MycCaP cells at 4 μM for 72 hr, evidenced by increased cell surface calreticulin, HMGB1 release, and ATP release. [1] It increases cleaved caspase-3 expression at 6 μM for 48 hr. [1] |
| ln Vivo |
MYCi361 upregulates PD-L1 on tumors, enhances tumor immune cell infiltration, inhibits in vivo tumor growth in mice, and makes tumors more susceptible to anti-PD1 immunotherapy.[1]
MYCi361 (NUCC-0196361) administered at 100 mg/kg/day i.p. induces tumor regression in FVB mice bearing established MycCaP allografts, but with an average 10% body weight loss. Re-starting at 70 mg/kg/day controls tumor growth without further weight loss. [1] It shows anti-tumor efficacy in a prostate PDX model (TM00298) with modest MYC expression. Ki67 proliferation marker decreases and MYC pT58 increases in tumor tissues. [1] It exhibits stronger tumor inhibition in immunocompetent FVB mice compared to immunodeficient NSG mice (50 mg/kg/day for 4 days), indicating dependence on an intact immune system. [1] It enhances tumor infiltration of CD3+ T cells, upregulates PD-L1 on tumor cells, increases CD4+ and CD8+ T cells, IFNγ-expressing T cells, dendritic cells and NK cells, with a trend for decreased Tregs. [1] It synergizes with anti-PD1 immunotherapy: alternating doses of MYCi361 50 mg/kg/day for 2 days and anti-PD1 100 μg/day for 2 days (4 cycles) results in synergistic suppression of tumor growth in MycCaP allografts. [1] |
| Enzyme Assay |
MYCi361 (NUCC-0196361) was assessed in an electrophoretic mobility shift assay (EMSA) to disrupt MYC/MAX/DNA complex formation. The binding reaction buffer contained 0.005% IGEPAL CA-630, 5% glycerol, 1 mM EDTA in 1x PBS. MYC (residues 353-439, 60 nM) was incubated with compound for 1 hr at room temperature, then mixed with MAX(S) (60 nM) and E-box oligonucleotide (20 nM) for 15 min before native gel electrophoresis. [1]
Fluorescence polarization competition assay was performed to determine binding affinity. Human MYC bHLHZip domain (residues 353-439) was purified. Competition experiments were performed over 3-25 μM of nonfluorescent MYCi361 against 10 μM 10074-G5 in the presence of 10 μM MYC. Measurements were at excitation 470 nm and emission 560 nm. Data were analyzed using "One site - Fit Ki" analysis, yielding a Kd of 3.2 μM. [1] In vitro kinase assay: recombinant MYC was first phosphorylated on S62 by activated recombinant ERK2, then incubated with GSK3β kinase and 6 μM of MYCi361 or inactive analog. The reaction was performed in 1x Kinase Buffer (25 mM Tris-HCl pH 7.5, 5 mM beta-glycerophosphate, 2 mM DTT, 0.1 mM sodium vanadate, 10 mM MgCl2) for 2 hr at room temperature, terminated by adding Laemmli buffer and boiling. Western blot assessed pT58 levels. [1] |
| Cell Assay |
The supernatants are obtained after treating MycCaP cells with 4 μM MYCi361 for a duration of 72 hours. To quantify high mobility group protein B1 (HMGB1) and secreted ATP, cell counts are carried out. After incubating cells with rabbit anti-Calreticulin for 60 minutes, they are subjected to an analysis by flow cytometry using Alexa Flour 488 anti-rabbit secondary antibody to detect the presence of surface Calreticulin.
MYCi361 (NUCC-0196361) was tested in cell viability assays using MTS kit or viable cell counting. Cells (1000-5000/well in 96-well plates) were treated for 2-7 days, then MTS reagent added and absorbance at 490 nm measured. [1] Cellular thermal shift assay (CETSA): PC3 cells treated with MYCi361 (4-10 μM) or DMSO for 30 min were harvested, suspended in PBS with protease/phosphatase inhibitors, distributed into PCR tubes, heated at designated temperatures for 2 min, cooled, freeze-thawed, centrifuged, and lysates subjected to Western blot. [1] Biotin pull-down: Biotin-361 (5-10 μM) was incubated with nuclear extracts or recombinant MYC, captured on streptavidin beads, washed, eluted, and analyzed by Western blot. Competition assays used excess Phosphate-361, G5, or JKY-2-169. [1] Co-immunoprecipitation: Cells treated with MYCi361 (6 μM for 1 hr) were lysed, nuclear proteins released by enzymatic shearing, and 1 mg protein incubated with MYC antibody pre-coated magnetic beads overnight at 4°C, then washed and eluted. [1] Proximity ligation assay (PLA): PC3 cells grown in chamber slides treated with MYCi361 (6 μM for 1-2 hr), fixed, permeabilized, incubated with anti-MYC and anti-MAX antibodies, then Duolink kit was used. PLA signals per cell quantified. [1] RNA-seq: PC3 cells treated with 6 μM MYCi361 for 24 hr in triplicates, RNA extracted, libraries prepared using TruSeq RNA Access Library Prep Kit, sequenced on HiSeq4000. GSEA and GO analysis performed. [1] Immunogenic cell death assays: MycCaP cells treated with 4 μM MYCi361 for 72 hr, supernatants collected for ATP (bioluminescence) and HMGB1 (ELISA) measurement; cell surface calreticulin detected by flow cytometry. [1] |
| Animal Protocol |
6-8 week old FVB mice, prostate PDX model, NSG mice, C57BL/6 mice, CB17/Icr-Prkdcscid/IcrIcoCrl mice, CD-1 mice
55 mg/kg, 50 mg/kg IP, Oral gavage For in vivo efficacy, FVB or NSG male mice (6-8 weeks old, ~25 g) were subcutaneously injected with 1×10^6 MycCaP cells in Matrigel. When tumors reached 150-200 mm³, mice were grouped. MYCi361 was dissolved in PBS with 10% DMSO and 20% TWEEN80. For rapid screening, compound was given i.p. for 3 days at low dose (30-50 mg/kg/day) then 3 days at high dose (100-200 mg/kg/day) as tolerated. [1] For efficacy study, MYCi361 was dosed at 100 mg/kg/day for 2 days (50 mg/kg twice daily), then paused until tumor regained original size, then 70 mg/kg/day for 9 days. [1] For short-term treatment (4 days), 50 mg/kg/day i.p. was given, tumor volume measured before and after. [1] For combination with anti-PD1, MYCi361 50 mg/kg/day for 2 days alternating with anti-PD1 antibody (100 μg/day) or IgG2a isotype control for 2 days, for 4 cycles (16 days). [1] For tumor immunophenotyping, mice bearing MycCaP allografts were treated with MYCi361 50 mg/kg/day 2 days on/2 days off for 2 rounds, then tumors and lymph nodes analyzed by flow cytometry. [1] |
| ADME/Pharmacokinetics |
Pharmacokinetic analysis in C57BL/6 mice after intraperitoneal (i.p.) or oral (p.o.) dosing of MYCi361 at 50 mg/kg showed plasma half-lives of 44 hr (i.p.) and 20 hr (p.o.). Maximum plasma concentrations (Cmax) were 27200 ng/ml (46 μM) i.p. and 13867 ng/ml (23 μM) p.o. At 24 hr post-exposure, plasma concentrations were 12733 ng/ml (21 μM) i.p. and 5283 ng/ml (9 μM) p.o. [1]
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| Toxicity/Toxicokinetics |
MYCi361 (NUCC-0196361) has a narrow therapeutic index. Acute toxicity studies indicated a maximum tolerated dose (MTD) of 240 mg/kg/day p.o. Histopathological analysis of major organs showed suppression of splenic white pulp and hepatocyte hypertrophy. In efficacy studies at 100 mg/kg/day, mice lost an average 10% of body weight; weight was regained after treatment cessation. [1]
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| References | |
| Additional Infomation |
MYCi361 (NUCC-0196361) is a small molecule that directly binds MYC (amino acids 366-378 region, same as G5 and JKY-2-169) and disrupts MYC/MAX heterodimerization. It promotes MYC degradation by enhancing phosphorylation on threonine 58 (T58), leading to proteasome-mediated degradation. The compound shows dual mechanism: impairing MYC/MAX complex formation independent of its effects on MYC stability. Due to its narrow therapeutic window, an improved analog MYCi975 (NUCC-0200975) was developed with better tolerability. [1]
|
| Molecular Formula |
C26H16CLF9N2O2
|
|---|---|
| Molecular Weight |
594.8561
|
| Exact Mass |
594.075
|
| Elemental Analysis |
C, 52.50; H, 2.71; Cl, 5.96; F, 28.74; N, 4.71; O, 5.38
|
| CAS # |
2289690-31-7
|
| Related CAS # |
2289690-31-7;
|
| PubChem CID |
139600319
|
| Appearance |
White to off-white solid powder
|
| LogP |
7.9
|
| Hydrogen Bond Donor Count |
1
|
| Hydrogen Bond Acceptor Count |
12
|
| Rotatable Bond Count |
5
|
| Heavy Atom Count |
40
|
| Complexity |
813
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
CN1C(=CC(=N1)C(F)(F)F)C2=C(C(=C(C=C2)OCC3=CC=C(C=C3)Cl)C4=CC(=CC(=C4)C(F)(F)F)C(F)(F)F)O
|
| InChi Key |
CKLCWLSEYDDTCN-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C26H16ClF9N2O2/c1-38-19(11-21(37-38)26(34,35)36)18-6-7-20(40-12-13-2-4-17(27)5-3-13)22(23(18)39)14-8-15(24(28,29)30)10-16(9-14)25(31,32)33/h2-11,39H,12H2,1H3
|
| Chemical Name |
2-[3,5-bis(trifluoromethyl)phenyl]-3-[(4-chlorophenyl)methoxy]-6-[2-methyl-5-(trifluoromethyl)pyrazol-3-yl]phenol
|
| Synonyms |
NUCC-0196361; NUCC0196361; NUCC 0196361; MYC i361; MYCi361; MYC-i361
|
| HS Tariff Code |
2934.99.9001
|
| 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)
|
| Solubility (In Vitro) |
DMSO: ~100 mg/mL (~168.1 mM)
Ethanol: ~14 mg/mL (~23.5 mM) |
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (3.50 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6811 mL | 8.4053 mL | 16.8107 mL | |
| 5 mM | 0.3362 mL | 1.6811 mL | 3.3621 mL | |
| 10 mM | 0.1681 mL | 0.8405 mL | 1.6811 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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