Target: G‑quadruplex (G4) DNA (stabilizer) – no IC₅₀/Kd reported; MYC mRNA/protein downregulation (IC₅₀ for anti‑proliferation in AML cells ranges from 57 nM to 1.75 μM) [1].
Target: Metal‑regulatory transcription factor‑1 (MTF1) – inhibitor; in rheumatoid arthritis synovial fibroblasts, APTO-253 (0.5–2 μM) reduces IL‑6 and CCL5 expression; in CIA model, 15 mg/kg twice daily shows therapeutic effect [3].

In Vitro: In AML cell lines (MV4‑11, EOL‑1, KG‑1, etc.), APTO-253 produced concentration‑dependent and time‑dependent reduction in MYC mRNA and protein levels. IC₅₀ values for anti‑proliferation (5‑day MTS assay) ranged from 57 nM to 1.75 μM. In MV4‑11 cells, IC₅₀ at 48, 72 and 120 h were 0.47, 0.40 and 0.24 μM respectively [1].
APTO-253 (0.5–1 μM, 24 h) induced G₀/G₁ cell cycle arrest (increase from ~50% to >80% in MV4‑11), decreased CDK4 and CCND3 protein levels, and increased CDKN1A (p21) mRNA and protein [1].
APTO-253 induced apoptosis: annexin V/PI staining showed concentration‑dependent increase in apoptotic cells; PARP1 cleavage (c‑PARP) was detected by Western blot. Time course showed apoptosis at 3–6 h [1].
APTO-253 (500 nM, 1–24 h) induced DNA damage: increased TP53 (total and phospho‑Ser15, acetyl‑K382), γH2AX (H2AX phospho‑S139), and phospho‑CHEK1. RNA‑seq and RPPA analyses confirmed upregulation of DNA damage response and ER stress pathways [1].
APTO-253 and its intracellular iron complex Fe(253)₃ stabilized G‑quadruplex DNA motifs (MYC, KIT promoters, telomeres, rRNA) in FRET melting assays, with selectivity over double‑stranded DNA. ΔT₁/₂ values increased with concentration [1].
In rheumatoid arthritis synovial fibroblasts (RASFs), APTO-253 (0.5–2 μM, 24 h) suppressed IL‑6 and CCL5 mRNA and protein expression in a concentration‑dependent manner (qRT‑PCR and ELISA) [3].
Knockdown of MTF1 (a transcription factor whose binding motif is enriched in super‑enhancers induced by 8‑cytokine mix) reduced expression of 8‑mix SE‑contacted genes; APTO-253 similarly inhibited those genes, and its effect was more pronounced on SE‑contacted genes than TE‑contacted genes [3].

In Vivo: In a collagen‑induced arthritis (CIA) mouse model, APTO-253 (15 mg/kg, intravenous injection twice per day for two consecutive days per week, starting after arthritis onset) significantly reduced clinical arthritis scores and histopathological scores compared to control (Mann‑Whitney U test, p<0.05 and p<0.001 respectively) [3].
In a previous study (referenced but not detailed in this paper), APTO-253 showed anti‑tumor activity in murine xenograft models of solid tumors and was advanced to Phase 1 clinical trials (no specific in vivo data provided in this manuscript) [1].

Cell Assay (AML cells): AML cell lines (MV4‑11, EOL‑1, KG‑1, etc.) were cultured in complete media. Cytotoxicity measured by MTS assay (CellTiter 96 AQueous One Solution) after 5‑day treatment with 10 concentrations of APTO-253; IC₅₀ calculated using GraphPad Prism 7 [1].
Cell cycle analysis: Treated cells stained with EdU (5‑ethynyl‑2´‑deoxyuridine) Alexa Fluor 488 and propidium iodide (PI) with Live/Dead fixable far red dead cell stain; analysis on BD Accuri C6 flow cytometer [1].
Apoptosis assay: Cells stained with FITC‑annexin V and PI, analyzed by flow cytometry. Western blot for cleaved PARP1 (c‑PARP) and other proteins [1].
Western blot: Whole cell lysates separated by SDS‑PAGE, transferred to nitrocellulose, probed with antibodies against MYC, CDK4, CCND3, p21, TP53, phospho‑TP53, γH2AX, phospho‑CHEK1, PARP1, etc.; densitometry normalized to GAPDH [1].
RNA‑seq: MV4‑11 cells treated with 500 nM APTO-253 for 6 h, total RNA extracted, sequenced on Illumina HiSeq4000; differential expression analysis (≥2‑fold change, p<0.05) identified 1,643 genes. RPPA: protein lysates analyzed on reverse phase protein array (>300 proteins) [1].
ChIP‑qPCR: Cells treated with 500 nM APTO-253 for 2–24 h, crosslinked, chromatin sonicated, immunoprecipitated with anti‑H3K27ac, qPCR for MYC promoter [1].
RNA decay assay: Cells pre‑treated with 500 nM APTO-253 for 3 h, then 1 μM actinomycin D added; MYC mRNA levels measured by RT‑qPCR at various times [1].
FRET assay: Dual‑labeled oligos (5′FAM‑3′BHQ1) containing G4 sequences (MYC, KIT, telomere, rRNA) or dsDNA hairpin were incubated with APTO-253 or Fe(253)₃ at increasing concentrations (1 nM–10 μM). Melting curves obtained on LightCycler 96; ΔT₁/₂ calculated [1].
Uptake/efflux assay: KG‑1 cells exposed to APTO-253, lysed in acetonitrile with deuterated internal standard, analyzed by LC‑MS (Agilent 1260 LC with Thermo LCQdeca MS, positive ESI) [1].
Cell Assay (synovial fibroblasts): RASFs from RA patients (n=30) and OASFs from OA patients (n=30) were stimulated with cytokines (IFN‑α, IFN‑γ, TNF‑α, IL‑1β, IL‑6/sIL‑6R, IL‑17, TGF‑β1, IL‑18, or 8‑mix). For APTO-253 treatment, cells (n=13) were treated with 0.5, 1, 2 μM for 24 h; mRNA quantified by qRT‑PCR (IL‑6, CCL5); protein by ELISA. Knockdown experiments used siRNA against MTF1, RUNX1, TCF4, SNAI1 [3].

Animal Protocol (CIA model): Male DBA/1J mice (age not specified) were immunized with bovine type II collagen in complete Freund’s adjuvant. After arthritis onset (day 21–28), mice received intravenous injection of APTO-253 at 15 mg/kg (or vehicle control) twice per day for two consecutive days per week for 2 weeks. Clinical scores (0‑4 per paw) were recorded. At endpoint, hind paws were collected for histopathological scoring (inflammation, pannus, cartilage damage, bone erosion) [3].
Preventive study: In separate experiment, APTO-253 was administered from day 0 after immunization (15 mg/kg i.v. twice daily, two consecutive days per week) and showed significant reduction in arthritis incidence and severity (online supplemental figure 9) [3].
No animal experiments for AML were described in the provided manuscript; only reference to prior xenograft studies is mentioned without details [1].

Toxicity/Toxicokinetics: In a Phase 1 clinical trial in patients with advanced solid tumors (referenced but not detailed in this paper), APTO-253 was well tolerated and did not produce myelosuppression even at the maximum tested dose. Most common treatment‑emergent adverse events (any grade) were rash, peripheral neuropathy, hypersensitivity (<10%), and fatigue (>10%) [1].
No preclinical toxicity data (e.g., LD₅₀, organ toxicity) are provided in the two references [1][3].

[1].APTO-253 Stabilizes G-quadruplex DNA, Inhibits MYC Expression, and Induces DNA Damage in Acute Myeloid Leukemia Cells. Mol Cancer Ther. 2018 Jun;17(6):1177-1186.

[2].Inhibition of c-Myc By Apto-253 As an Innovative Therapeutic Approach to Induce Cell Cycle Arrest and Apoptosis in Acute Myeloid Leukemia. Blood 2016 128:1716.

[3].Parsing multiomics landscape of activated synovial fibroblasts highlights drug targets linked to genetic risk of rheumatoid arthritis. Ann Rheum Dis. 2020 Nov2;annrheumdis-2020-218189

APTO-253 HCl, an MTF-1 inhibitor, is the hydrochloride salt of a small molecule inhibitor of human metal-regulated transcription factor 1 (MTF-1) and possesses potential antitumor activity. APTO-253 inhibits MTF-1 activity, thereby inducing the expression of the MTF-1-dependent tumor suppressor Kruppel-like factor 4 (KLF4). This subsequently leads to the downregulation of cyclin D1, blocking cell cycle progression and proliferation. The drug also reduces the expression of genes involved in tumor hypoxia and angiogenesis.

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Additional Info: APTO-253 is converted intracellularly from a monomer to a ferrous complex [Fe(253)₃] which is the principal intracellular form. Both monomer and complex stabilize G‑quadruplex DNA, leading to MYC downregulation, DNA damage, and apoptosis. In AML cells, MYC basal expression is higher than in healthy PBMCs, correlating with selective cytotoxicity. In rheumatoid arthritis, APTO-253 acts as an MTF1 inhibitor, suppressing super‑enhancer‑driven inflammatory genes (IL‑6, CCL5) and showing therapeutic efficacy in CIA model. The drug is being evaluated in Phase 1 trials for relapsed/refractory hematologic malignancies (NCT02267863) and previously for solid tumors (NCT123226) [1][3].

C22H15CLFN5

403.839406251907

403.1

1691221-67-6

1691221-67-6 (HCl);916151-99-0 (Free base);1422731-37-0 (APTO-253 isomer HCl);1422826-80-9 (APTO-253 isomer free base);

91668263

Typically exists as solid at room temperature

3

4

1

29

589

0

XGEPFSYBBQZKKW-UHFFFAOYSA-N

InChI=1S/C22H14FN5.ClH/c1-11-17(15-10-12(23)6-7-16(15)26-11)22-27-20-13-4-2-8-24-18(13)19-14(21(20)28-22)5-3-9-25-19;/h2-10,26H,1H3,(H,27,28);1H

2-(5-fluoro-2-methyl-1H-indol-3-yl)-1H-imidazo[4,5-f][1,10]phenanthroline;hydrochloride

APTO 253 HCl APTO253 HCl LOR 253 LOR253APTO-253 hydrochloride APTO-253 HCl LOR-253 LT-253

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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