NGF (IC50 ~1 µM); MSI RNA-binding (IC50 = 2.7 μM)
MUSASHI RNA-binding proteins (MSI2 and MSI1). MSI2

(IC50 for RNA-binding inhibition = 2.7 ± 0.4 µM in FP assay). MSI1.

By attaching to the NGF dimer, Ro 08-2750 most likely causes a change in conformation that prevents NGF from further attaching to p75NTR[2].
Ro 08-2750 (10 nM) completely prevents NGF-induced cell death in SK-N-MC 103 cells[2].
In myeloid leukemia cells, Ro 08-2750 (5-10 μM; 8 hours) increases differentiation and apoptosis[3].
Ro 08-2750 prevents human AML lines and patient cells from surviving[3].
Ro 08-2750 modifies the MSI2 gene signature and inhibits MSI2 RNA-binding[3].

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Anti-proliferative and Cytotoxic Effects: Ro 08-2750 inhibited the proliferation of murine MLL-AF9+ leukemic bone marrow cells with an EC50 of 2.6 ± 0.1 µM. In human leukemia cell lines, it demonstrated anti-proliferative effects with EC50 values of ~8 µM in both MOLM13 (AML) and K562 (CML-BC) cells. The compound was less potent against normal human CD34+ cord blood cells (EC50 ~22 µM), suggesting a therapeutic window. [3]
Induction of Differentiation: Treatment with Ro 08-2750 (5 µM for 48h) increased differentiation in murine MLL-AF9+ cells, as shown by flow cytometry (increased Mac1 and Gr1 markers) and morphological analysis. In human K562 and MOLM13 cells, Ro (20 µM for 48h) induced differentiation markers (CD14 in MOLM13, Glycophorin A in K562) and morphological changes indicative of differentiation. No effect on differentiation was observed in normal human CD34+ cord blood cells. [3]
Induction of Apoptosis: Ro 08-2750 treatment led to a significant increase in apoptosis (Annexin V+ population) in murine MLL-AF9+ cells, observed as early as 8h and more pronounced at 48h with 10 µM treatment. Similarly, in human MOLM13 and K562 cells, Ro (20 µM) increased the percentage of Annexin V+ cells over 48-96 hours. [3]
Inhibition of Colony Formation: Ro 08-2750 reduced colony formation in a dose-dependent manner. In murine MLL-AF9+ cells, colony formation was inhibited by >50% at 1 µM and ~75% at 5 µM. In human AML cell lines (MOLM13, K562), plating activity was inhibited by >80% at 20 µM. The compound also showed differential sensitivity in three AML patient samples, with >50% inhibition of colony formation at 5 µM, compared to a modest reduction in normal human CD34+ cord blood cells even at 20 µM. [3]
Inhibition of MSI2-RNA Binding and Target Expression: Ro 08-2750 (10 µM for 1h) significantly decreased the binding of MSI2 to its target mRNAs (TGFBR1, c-MYC, SMAD3, CDKN1A) in K562 cells, as shown by RNA immunoprecipitation. Western blot analysis confirmed a dose-dependent (1-20 µM for 4h) and time-dependent (1-24h with 20 µM) reduction in MSI2 target proteins (TGFβR1, c-MYC, SMAD3, HOXA9) and an increase in P21 in MOLM13 and K562 cells, while non-target β-ACTIN remained unchanged. This effect was post-transcriptional, as mRNA levels of these targets were only modestly affected. [3]
On-target Specificity: Overexpression of MSI2 in murine MLL-AF9+ cells reduced the anti-proliferative and target inhibition effects of Ro 08-2750, and overexpression of Ro-binding deficient MSI2 mutants (K22A, F66A, F97A, R100A) further rescued colony-forming ability. The analogs Ro-OH and Ro-NGF, which showed reduced or no binding to MSI2, had diminished or no effect on cell proliferation and RNA-binding inhibition, suggesting that Ro's activity is primarily through MSI2 inhibition. [3]
Gene Expression Signature: RNA-seq analysis after 4h treatment with Ro 08-2750 (20 µM) in MOLM13 and K562 cells showed a gene expression signature that strongly overlapped with the signature obtained from shRNA-mediated depletion of MSI2 in various leukemia cell lines. This included shared pathways related to c-MYC, mRNA processing, and leukemia-associated gene sets. [3]

In a myeloid leukemia model, Ro 08-2750 (13.75 mg/kg; i.p.) inhibits leukemogenesis[3].

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Efficacy in a Murine AML Model: In an aggressive MLL-AF9 murine leukemia model, administration of Ro 08-2750 (13.75 mg/kg, i.p., every 3 days for a total of 5 doses) reduced disease burden. At the time of sacrifice (day 19 post-transplantation), treated mice showed a significant reduction in spleen weights and white blood cell counts compared to the control group. [3]
Pharmacodynamic Effects in Vivo: Acute treatment with Ro 08-2750 (13.75 mg/kg, i.p.) in mice bearing MLL-AF9 leukemia resulted in reduced c-KIT protein abundance (at 4h and 12h) and decreased intracellular c-MYC levels (at 4h and 12h) in spleen cells, as measured by flow cytometry. [3]

Fluorescence Polarization (FP) Assay: To validate the inhibition of MSI-RNA binding by Ro 08-2750, a fluorescence polarization-based assay was used in a 384-well format for dose-response curve studies. An RNA oligo (Cy3-C9[spacer]-rGUAGUAGU) containing two MSI motifs (GUAGU) was used. Manual pipetting was employed to plate the reagents, and the FP reading was performed on a plate reader. The IC50 for MSI2 RNA-binding inhibition was determined to be 2.7 ± 0.4 µM. [3]
MicroScale Thermophoresis (MST) Assay: For binding affinity studies, purified recombinant GST-MSI2 (WT and mutants), GST-RBP controls (SYNCRIP, SRSF2, HUR, RBMX, TIA-1), and bovine serum albumin were fluorescently labeled using an amine-coupling kit. Proteins were diluted in MST buffer (50 mM HEPES, 100 mM NaCl, 0.05% Tween-20, pH 7.4). Labeled proteins or protein/RNA complexes (pre-incubated for 15 min) were mixed with increasing concentrations of Ro 08-2750 (0.015 to 500 μM) or a control RNA oligo (0.0015 to 50 μM, rGUAGUAGUAGUAGUA) and loaded into capillaries. MST measurements were taken at room temperature with a fixed IR-laser power. The KD of Ro for MSI2 was determined to be 12.3 ± 0.5 µM. For the MSI2/RNA complex, the KD was 27.5 ± 2.6 µM. Binding to BSA was negligible (KD >500 µM). [3]
Isothermal Titration Calorimetry (ITC) Assay: To confirm Ro's interaction with MSI2 and rule out direct RNA binding, ITC was performed. Recombinant GST-MSI2 (30 μM) in ITC buffer (10 mM HEPES + 10% 10 mM Citrate Phosphate and 0.05% Tween-20, pH 7.0) was titrated against 100 μM and 300 μM Ro 08-2750 in the same buffer. To test for RNA binding, a 15-nt RNA probe (rGUAGUAGUAGUAGUA) or poly(A) RNA (10 μM) was titrated against 100 μM Ro or palmitate (positive control). The thermodynamic parameters were analyzed using dedicated software. Ro bound to MSI2 with a KD of 13.3 ± 0.27 µM, but showed no binding to the MSI-target RNA or poly(A) RNA. [3]
Chemiluminescent Electrophoresis Mobility Shift Assay (EMSA): To assess MSI2-RNA complex inhibition by Ro 08-2750, an EMSA approach was used. GST-MSI2 (125-250 ng) was pre-incubated with DMSO or the compound (typically 20 µM) for 1 hour at room temperature in 1X RNA EMSA binding buffer (10 mM HEPES, 20 mM KCl, 1 mM MgCl2, 1 mM DTT) supplemented with 5% glycerol, 100 µg/mL tRNA, and 10 mM KCl. Then, 40 nM of biotinylated-RNA (biotin-rGUAGUAGUAGUAGUA) was added and incubated for another hour. Samples were run on a 4-20% TBE polyacrylamide gel, transferred to a membrane, UV-crosslinked, and developed using a chemiluminescence kit. Ro competed with RNA for MSI2 binding in a concentration-dependent manner (5 to 40 µM). [3]

Cytotoxicity/Proliferation Assay (Cell-Titer Glo): Cells (murine MLL-AF9+ BM, human MOLM13, or K562) were plated in 96-well plates and treated with increasing concentrations of Ro 08-2750 (up to 100 µM, 1:2 serial dilutions) for 72 hours at 37°C. After cooling to room temperature, cells were mixed with Cell-Titer Glo reagent, incubated for 15 minutes, and luminescence was read. Data was normalized to determine percentage viability and EC50 values. [3]
Flow Cytometry for Differentiation and Apoptosis: For differentiation, cells treated with DMSO or Ro 08-2750 were stained with fluorochrome-conjugated antibodies against specific surface markers (e.g., Mac1-PE, Gr1-APC for murine cells; CD14-PE, CD235a-PE for human cells) for 20 minutes in the dark, washed, and analyzed. For apoptosis, cells were stained using an Annexin V and Dead Cell Assay Kit and analyzed on a cell analyzer. For intracellular c-MYC detection, cells were fixed in 2% paraformaldehyde, permeabilized with cold methanol, stained with a primary antibody against c-MYC and then a fluorophore-conjugated secondary antibody before analysis. [3]
Colony Forming Unit (CFU) Assay: Leukemic cells (e.g., 10,000 murine MLL-AF9+ BM cells) or normal progenitor cells (e.g., Lin-Sca+Kit+ cells, human CD34+ cord blood cells) were plated in methylcellulose-based culture media containing various concentrations of Ro 08-2750. Colonies were scored after 5-14 days of incubation, depending on the cell type. [3]
RNA Immunoprecipitation (RIP): K562 cells (empty vector or FLAG-MSI2 overexpressing) were treated with Ro 08-2750 (10 µM) or DMSO for 1 hour. Cells were then lysed, and FLAG-MSI2 was immunoprecipitated using an anti-FLAG antibody. RNA bound to the immunoprecipitated complex was isolated, and the enrichment of specific MSI2 target mRNAs (TGFBR1, c-MYC, SMAD3, CDKN1A) was quantified by real-time quantitative PCR. [3]
Western Blot Analysis: Cells (MOLM13 or K562) treated with various concentrations of Ro 08-2750 (1-20 µM for 4h) or for different time points (1-24h with 20 µM) were lysed in RIPA buffer. Total protein was quantified, separated by SDS-PAGE, and transferred to a nitrocellulose membrane. Membranes were blotted with antibodies against MSI2 targets (e.g., TGFBR1, SMAD3, HOXA9, c-MYC, P21) and β-ACTIN as a loading control, then developed using chemiluminescence. [3]

Pharmacodynamic Study in MLL-AF9 Model:** 10,000 MLL-AF9 GFP+ secondary murine leukemia cells were transplanted retro-orbitally into sublethally irradiated (475 cGy) female C57BL/6 mice. Three weeks post-transplantation, when signs of disease appeared, mice were injected intraperitoneally with a single dose of Ro 08-2750 (13.75 mg/kg in DMSO) or DMSO vehicle. Mice were sacrificed for analysis at 4 and 12 hours post-injection. Spleen cells were collected for flow cytometry analysis of c-Kit receptor and intracellular c-MYC levels. [3]
* **Efficacy Study in MLL-AF9 Model:** 10,000 MLL-AF9 GFP+ secondary murine leukemia cells were transplanted retro-orbitally into sublethally irradiated (475 cGy) female C57BL/6 mice. Starting three days post-transplantation, mice received intraperitoneal injections of Ro 08-2750 (13.75 mg/kg in DMSO) or DMSO vehicle on days 1, 4, 7, 10, and 13 (a schedule of one day on, two days off). On day 19 post-transplantation, mice were sacrificed. Disease burden was assessed by measuring spleen weight, white blood cell counts, and intracellular c-MYC levels in spleen cells via flow cytometry. [3]

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Pharmacodynamic Study in MLL-AF9 Model: 10,000 MLL-AF9 GFP+ secondary murine leukemia cells were transplanted retro-orbitally into sublethally irradiated (475 cGy) female C57BL/6 mice. Three weeks post-transplantation, when signs of disease appeared, mice were injected intraperitoneally with a single dose of Ro 08-2750 (13.75 mg/kg in DMSO) or DMSO vehicle. Mice were sacrificed for analysis at 4 and 12 hours post-injection. Spleen cells were collected for flow cytometry analysis of c-Kit receptor and intracellular c-MYC levels. [3]
Efficacy Study in MLL-AF9 Model: 10,000 MLL-AF9 GFP+ secondary murine leukemia cells were transplanted retro-orbitally into sublethally irradiated (475 cGy) female C57BL/6 mice. Starting three days post-transplantation, mice received intraperitoneal injections of Ro 08-2750 (13.75 mg/kg in DMSO) or DMSO vehicle on days 1, 4, 7, 10, and 13 (a schedule of one day on, two days off). On day 19 post-transplantation, mice were sacrificed. Disease burden was assessed by measuring spleen weight, white blood cell counts, and intracellular c-MYC levels in spleen cells via flow cytometry. [3]

In Vivo Tolerability: In the murine efficacy study, administration of Ro 08-2750 (13.75 mg/kg i.p. every 3 days) was well tolerated. Treated mice exhibited little to no weight loss. Blood analysis showed equivalent red blood cell, platelet, mean corpuscular volume, hematocrit, and hemoglobin counts compared to the non-treated group. In healthy mice, no changes in liver enzymes were observed 24 hours after a single Ro treatment. [3]
Therapeutic Window in Vitro: Ro 08-2750 showed a degree of selectivity for malignant cells. The EC50 for anti-proliferative effects in human leukemia cell lines (MOLM13 and K562) was ~8 µM, while in normal human CD34+ cord blood cells, the EC50 was ~22 µM, indicating a ~2.6-fold higher concentration needed for toxicity in normal cells. Furthermore, Ro inhibited colony formation in AML patient samples at 5 µM but only showed a modest reduction in normal CD34+ cell colonies at 20 µM. [3]

[1]. Small-molecule inhibitors of protein-protein interactions: progressing towards the dream. Nat Rev Drug Discov. 2004 Apr;3(4):301-17.

[2]. NGF ligand alters NGF signaling via p75(NTR) and trkA. J Neurosci Res. 2000 Aug 1;61(3):263-72.

[3]. Small-molecule targeting of MUSASHI RNA-binding activity in acute myeloid leukemia. Nat Commun. 2019 Jun 19;10(1):2691.

7,10-Dimethyl-2,4-dioxo-8-benzo[g]pterocarboxaldehyde is a flavin.

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Background and Mechanism: Ro 08-2750 was identified as a hit from a fluorescence polarization-based screen of 6208 compounds aimed at identifying inhibitors of MUSASHI (MSI) RNA-binding proteins. This study characterizes it as a selective inhibitor of MSI2. It directly binds to the RNA-recognition motif 1 (RRM1) of MSI2, competing with RNA for the binding site. This interaction inhibits MSI2's function as a post-transcriptional regulator, leading to the downregulation of its oncogenic targets (e.g., c-MYC, HOXA9, SMAD3) and upregulation of targets like P21. The compound's activity is linked to its ability to inhibit MSI2, as its effects can be rescued by MSI2 overexpression or by mutations in MSI2 that prevent Ro binding. [3]
Chemical Analogs: Two analogs of Ro 08-2750 were studied: Ro-OH (a single alteration reducing an aldehyde to an alcohol) and Ro-NGF (a compound with high affinity for Nerve Growth Factor). Both analogs showed significantly reduced or complete loss of binding to MSI2 and were correspondingly less potent or inactive in inhibiting leukemia cell growth and MSI2-RNA binding, supporting the on-target mechanism of the parent compound. [3]
Significance: The study provides a framework for targeting RNA-binding proteins like MSI in cancer. It demonstrates that inhibiting MSI2's RNA-binding activity with a small molecule is a viable therapeutic strategy in acute myeloid leukemia (AML), reducing disease burden in a preclinical model. The compound serves as a proof-of-concept for developing more potent and clinically useful MSI family inhibitors. [3]

C13H10N4O3

270.2435

270.075

C, 57.78; H, 3.73; N, 20.73; O, 17.76

37854-59-4

37854-59-4

17756791

Orange to red solid powder

1.6±0.1 g/cm3

1.754

-0.81

1

4

1

20

554

0

O=CC1C(C)=CC2=C(N(C3=NC(=O)NC(=O)C3=N2)C)C=1

JDEMVNYMYPJJIM-UHFFFAOYSA-N

InChI=1S/C13H10N4O3/c1-6-3-8-9(4-7(6)5-18)17(2)11-10(14-8)12(19)16-13(20)15-11/h3-5H,1-2H3,(H,16,19,20)

7,10-dimethyl-2,4-dioxobenzo[g]pteridine-8-carbaldehyde

Ro-08-2750; Ro 082750; Ro -082750; Ro 08-2750; Ro08-2750; Ro082750

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: ~4 mg/mL (~14.8 mM)

Solubility in Formulation 1: 5 mg/mL (18.50 mM) in 50% PEG300 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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: 5 mg/mL (18.50 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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