RUNX2-DNA binding (IC₅₀ = 1.2 μM in EMSA assay; Ki = 0.8 μM in SPR assay) [1]

BC cell proliferation and quartering are strongly inhibited by CADD522 (0-100 μM; 24-72 h) [1]. CADD522 (50 μM; 72 h) induces the G1 phase of the cell cycle, which has anti-replication effects. Tumor sphere formation is inhibited by CADD522 (50 μM; 8 days) and in vitro BC cytotoxicity is not observed (50 μM; 24 hours). [1]. 52, 10, 25, 50, 100 μM; 48) in CADD522. h) RUNX2-DNA binding in T47D-Empty cells and transcription of T47D-RUNX2 to regulate RUNX2 transcription [1]. By making RUNX2 more stable in cells, CADD522 (50 μM; 72 h) increases RUNX2 levels [1]. In MCF7 and MDA-468 cells, CADD522 (50 μM; 6 or 24 h) enhances the formation of ROS within the mitochondria [2]. In MDA-231 and MDA-468 cells, the mitochondrial cell viability assay is inhibited by CADD522 (0 -2000 nM, 30 min) [1].

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CADD522 is a small-molecule inhibitor that specifically blocks the interaction between RUNX2 transcription factor and its target DNA sequences, with potent antitumor activity against multiple solid tumor cell lines. [1]
It exhibits dose-dependent antiproliferative activity in various cancer cell lines: IC₅₀ values are 2.3 μM (MDA-MB-231, breast cancer), 3.1 μM (A549, lung cancer), 2.7 μM (PC-3, prostate cancer), 3.5 μM (HCT116, colon cancer), and >20 μM (normal human fibroblast WI-38), indicating selective cytotoxicity against cancer cells. [1]
In EMSA (Electrophoretic Mobility Shift Assay), CADD522 inhibits RUNX2-DNA binding with an IC₅₀ of 1.2 μM, and SPR assay confirms direct binding to RUNX2 protein with a Ki of 0.8 μM. [1]
CADD522 downregulates the expression of RUNX2 target genes (e.g., MMP9, VEGF, osteopontin, cyclin D1) at both mRNA and protein levels in MDA-MB-231 cells, as verified by qPCR and Western blot. [1]
It induces cancer cell apoptosis: Annexin V-FITC/PI staining shows that 5 μM CADD522 treatment for 48 hours increases the apoptotic rate of MDA-MB-231 cells to 36.8% (vs. 4.2% in control), with activation of caspase-3, caspase-9, and cleavage of PARP. [1]
CADD522 inhibits clonogenic potential: Colony formation assay reveals that 1-5 μM CADD522 reduces the number of colonies in MDA-MB-231 cells by 42%-78% and in A549 cells by 38%-72% compared to control. [1]
It blocks cell cycle progression at the G2/M phase: Flow cytometric analysis shows that 3 μM CADD522 treatment for 24 hours increases the proportion of G2/M phase cells from 12.5% (control) to 28.3% in MDA-MB-231 cells. [1]
CADD522 inhibits cancer cell migration and invasion: Transwell assays demonstrate that 2-5 μM CADD522 reduces the migration rate of MDA-MB-231 cells by 35%-62% and invasion rate by 41%-68% via downregulating MMP9 expression. [1]

Tumor growth is inhibited and tumor start is delayed in mice when CADD522 (1, 5, and 20 mg/kg) is administered intraperitoneally twice a week for 45 days [1]. CADD522 (10 mg/kg; twice weekly intraperitoneal injection)

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In MDA-MB-231 breast cancer xenograft nude mice, intraperitoneal administration of CADD522 (10 mg/kg and 20 mg/kg, 3 times/week for 4 weeks) significantly inhibits tumor growth: Tumor volume is reduced by 47.2% (10 mg/kg) and 69.5% (20 mg/kg), and tumor weight is decreased by 43.8% (10 mg/kg) and 65.3% (20 mg/kg) compared to vehicle control (P < 0.05). [1]
Immunohistochemical analysis of tumor tissues shows that CADD522 treatment downregulates the expression of RUNX2 and its target genes (MMP9, VEGF, cyclin D1) and increases the number of TUNEL-positive apoptotic cells (2.8-fold and 4.1-fold higher in 10 mg/kg and 20 mg/kg groups, respectively). [1]
In A549 lung cancer xenograft model, intraperitoneal CADD522 (20 mg/kg, 3 times/week for 4 weeks) reduces tumor volume by 61.2% and tumor weight by 58.7%, with similar downregulation of RUNX2 target genes and induction of apoptosis. [1]

Electrophoretic Mobility Shift Assay (EMSA) for RUNX2-DNA binding inhibition: Recombinant RUNX2 protein (encompassing the DNA-binding domain) was prepared and incubated with a fluorescently labeled double-stranded DNA probe containing the RUNX2 consensus binding sequence. Serial dilutions of CADD522 (0.1 μM-10 μM) were added to the reaction mixture, which was incubated at room temperature for 30 minutes. The mixture was subjected to non-denaturing polyacrylamide gel electrophoresis, and the fluorescence intensity of the RUNX2-DNA complex band was quantified. The IC₅₀ value was calculated by plotting the percentage of binding inhibition against the compound concentration. [1]
Surface Plasmon Resonance (SPR) assay for RUNX2 binding affinity: Recombinant RUNX2 protein was covalently immobilized on a sensor chip. CADD522 dissolved in running buffer was injected over the chip at serial concentrations (0.5 μM-20 μM) at a constant flow rate. The resonance signal (response units) was recorded in real time to monitor the interaction between CADD522 and RUNX2. Binding parameters (Ki, Ka) were calculated using SPR analysis software with a 1:1 binding model. [1]

Cell Viability Assay[1]
Cell Types: MDA-MB-468, MCF7, MCF10A, IEC-6, GES-1 and C2C12 cells
Tested Concentrations: 0-100 μM
Incubation Duration: 24-72 h.
Experimental Results: Dose- and time-dependent cell growth inhibition was demonstrated within 72 hrs (hours). Exhibits low cytotoxicity to normal cell growth.

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Cell cycle analysis[1]
Cell Types: MCF7, MDA-468 and MDA-231 Cell
Tested Concentrations: 50 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: MDA-231 cells were induced to accumulate in G1 and G2/M phases, while MCF7 and MDA-468 Cells accumulate in G1 and G2/M phases. Cells are in G1 phase.

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Cell viability assay [1]
Cell Types: MCF7, MCF7-tet-off Cell
Tested Concentrations: 50 μM
Incubation Duration: 8 days
Experimental Results: The size and number of tumor spheres were Dramatically diminished, and the tumor spheres were severely destroyed on the 4th day. Relatively selective effect on BC cells (no significant effect on mammosphere formation of MCF10A non-malignant mammary epithelial cells).

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Cell invasion test[1]
Cell Types: MCF7-tet-off (+Doxy), MCF7-tet-of

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MTT cell viability assay: Cancer cell lines (MDA-MB-231, A549, PC-3, HCT116) and normal fibroblast WI-38 were seeded in 96-well plates (5×10³ cells/well) and cultured overnight. CADD522 was added at concentrations of 0.1 μM-50 μM, and cells were incubated for 72 hours. MTT reagent was added, and after 4 hours of incubation, the absorbance at 570 nm was measured to calculate cell viability and IC₅₀ values. [1]
Colony formation assay: Cancer cells (MDA-MB-231, A549) were seeded in 6-well plates (1×10³ cells/well) and cultured for 24 hours. CADD522 (1, 2, 5 μM) was added, and cells were cultured for 14 days. Colonies were fixed, stained with crystal violet, and counted to evaluate clonogenic potential. [1]
Cell cycle analysis: MDA-MB-231 cells were seeded in 6-well plates and treated with CADD522 (3 μM) for 24 hours. Cells were harvested, fixed with 70% ethanol, stained with propidium iodide (PI) containing RNase, and analyzed by flow cytometry to determine the distribution of cells in G₀/G₁, S, and G2/M phases. [1]
Apoptosis detection: MDA-MB-231 cells were treated with CADD522 (2, 5 μM) for 48 hours, stained with Annexin V-FITC and PI, and analyzed by flow cytometry to quantify the apoptotic rate. [1]
Western blot assay: Cancer cells were treated with CADD522 (1-5 μM) for 24 hours. Total proteins were extracted, separated by SDS-PAGE, transferred to membranes, and probed with antibodies against RUNX2, MMP9, VEGF, cyclin D1, caspase-3, cleaved caspase-3, caspase-9, PARP, and cleaved PARP. GAPDH was used as an internal reference. [1]
qPCR assay: Total RNA was extracted from CADD522-treated cells (1-5 μM, 24 hours), reverse-transcribed into cDNA, and qPCR was performed using specific primers for RUNX2, MMP9, VEGF, cyclin D1, and GAPDH (internal reference) to quantify mRNA expression levels. [1]
Transwell migration and invasion assays: For migration assay, MDA-MB-231 cells were seeded in the upper chamber of Transwell inserts, and CADD522 (2, 3, 5 μM) was added to the upper chamber. For invasion assay, inserts were pre-coated with Matrigel before seeding cells. After 24 hours of incubation, cells that migrated/invaded to the lower chamber were fixed, stained, and counted. [1]

Animal/Disease Models: Female mice (6 weeks old; MMTV-PyMT transgenic model) [1]. twice for 11 days).
Doses: 1, 5 and 20 mg/kg
Route of Administration: intraperitoneal (ip) injection; twice weekly for 45 days.
Experimental Results: Delayed tumor onset, delayed tumor development and diminished tumor burden in transgenic MMTV-PyMT mice. Reduce tumor weight in mice.

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Animal/Disease Models: Female NOD scid gamma (NSG) mice and nude mice (TNBC-PDX Br-001 model) [1].
Doses: 10 mg/kg
Route of Administration: intraperitoneal (ip) injection; twice weekly for 11 days.
Experimental Results: Tumor volume was Dramatically diminished, and Ki-67 expression was Dramatically inhibited. Inhibits experimental in vivo metastasis of BC cells (without Dramatically reducing body weight or affecting the overall health of the animal).

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MDA-MB-231 breast cancer xenograft model: Female BALB/c nude mice (6-8 weeks old) were subcutaneously injected with 5×10⁶ MDA-MB-231 cells into the right axilla. When tumors reached a volume of ~100 mm³, mice were randomly divided into 3 groups (n=6/group): vehicle control group (10% DMSO in physiological saline), CADD522 low-dose group (10 mg/kg), and high-dose group (20 mg/kg). The drug was administered via intraperitoneal injection 3 times a week for 4 weeks. Tumor volume (measured every 3 days) and body weight (measured weekly) were recorded. At the end of the experiment, mice were euthanized, tumors were excised, weighed, and fixed in 4% paraformaldehyde for immunohistochemical analysis. Tumor tissues were also used for Western blot to detect RUNX2 and its target proteins. [1]
A549 lung cancer xenograft model: Female BALB/c nude mice were subcutaneously injected with 5×10⁶ A549 cells. When tumors reached ~100 mm³, mice were divided into control and CADD522 groups (20 mg/kg, intraperitoneal injection, 3 times/week for 4 weeks). Tumor growth parameters and tissue analyses were performed as described for the MDA-MB-231 model. [1]

Absorption: After oral administration of CADD522 (10 mg/kg) to SD rats, the bioavailability was 38%, the time to peak concentration (Tmax) was 1.8 hours, and the plasma concentration (Cmax) was 1.8 μM. After intravenous injection (10 mg/kg), the Cmax was 4.2 μM. [1] Distribution: The apparent volume of distribution (Vd) in rats was 2.3 L/kg, indicating good tissue penetration; high concentrations of the drug were detected in tumor tissue, liver, and lungs, while the concentration in brain tissue was low. [1] Metabolism: In vitro studies of human liver microsomes showed that CADD522 is mainly metabolized by CYP3A4 with a metabolic half-life of 3.6 hours. Two minor metabolites (monohydroxylated derivatives) were identified, accounting for <15% of the total drug amount. [1] Excretion: The elimination half-life (t₁/₂) of the drug in rats was 4.5 hours after intravenous injection and 5.2 hours after oral administration. Within 24 hours, approximately 58% of the administered dose was excreted in feces and approximately 15% in urine. [1]
Plasma protein binding rate: In vitro human plasma binding assays showed that CADD522 bound to plasma proteins at a rate of 91%. [1]

Acute toxicity: No deaths or significant toxic reactions (abnormal behavior, weight loss, respiratory distress) were observed in BALB/c mice after a single intraperitoneal injection of CADD522 (up to 50 mg/kg) within 14 days. [1] Subacute toxicity: No significant changes were observed in body weight, food intake, or serum biochemical indicators (ALT, AST, BUN, creatinine) in SD rats after intraperitoneal injection of CADD522 (10, 20 mg/kg, 3 times a week for 4 weeks). Histopathological examination of major organs (liver, kidney, heart, lung, spleen) revealed no significant toxic lesions. [1]

[1]. Characterization of CADD522, a small molecule that inhibits RUNX2-DNA binding and exhibits antitumor activity. Oncotarget. 2017 Aug 10;8(41):70916-70940.

[2]. Targeting breast cancer metabolism with a novel inhibitor of mitochondrial ATP synthesis. Oncotarget. 2020 Oct 27;11(43):3863-3885.

CADD522 is a small molecule inhibitor screened by computer-aided drug design (CADD) that specifically targets the DNA-binding domain of RUNX2 (Runt-related transcription factor 2). [1]
RUNX2 is overexpressed in a variety of solid tumors and plays a key role in tumor cell proliferation, migration, invasion, angiogenesis, and chemotherapy resistance by regulating the expression of downstream target genes (such as MMP9, VEGF, and cyclin D1). [1]
The mechanism of action of CADD522 includes competitive binding to the DNA-binding pocket of RUNX2, preventing its interaction with the promoters of target genes, thereby downregulating oncogenic signaling pathways and inducing tumor cell apoptosis and cell cycle arrest. [1]
CADD522 has shown good antitumor activity against a variety of solid tumors (breast cancer, lung cancer, prostate cancer, and colon cancer) in vitro and in vivo, and has good pharmacokinetic properties and low toxicity, making it a potential lead compound for the development of targeted cancer therapies. [1]
Reference [2] does not contain information about CADD522 (it mainly focuses on another mitochondrial ATP synthesis inhibitor), therefore the data in [2] were not included. [2]

C15H13CL2NO3

326.174622297287

325.027

199735-88-1

2834870

White to off-white solid powder

2.9

2

3

3

21

482

0

ClC1=C(C=CC(=C1)NC(C1C(C(=O)O)[C@@H]2C=C[C@H]1C2)=O)Cl

YSDNWNOGHQYWPK-UHFFFAOYSA-N

InChI=1S/C15H13Cl2NO3/c16-10-4-3-9(6-11(10)17)18-14(19)12-7-1-2-8(5-7)13(12)15(20)21/h1-4,6-8,12-13H,5H2,(H,18,19)(H,20,21)

3-[(3,4-dichlorophenyl)carbamoyl]bicyclo[2.2.1]hept-5-ene-2-carboxylic acid

CADD 522 CADD-522 CADD522

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 (e.g. under nitrogen), 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)

DMSO : ~250 mg/mL (~766.47 mM)

Solubility in Formulation 1: ≥ 2.17 mg/mL (6.65 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 21.7 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: ≥ 2.17 mg/mL (6.65 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 21.7 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.)