Anaphase-promoting complex (APC); - Anaphase-Promoting Complex/Cyclosome (APC/C) bound to Cdc20 (APC/Cⁿᵈᶜ²⁰) - Apcin-A is a selective inhibitor of APC/Cⁿᵈᶜ²⁰, with an IC₅₀ of 1.2 μM for recombinant human APC/Cⁿᵈᶜ²⁰ in HTRF-based activity assays [2]
- It shows no significant inhibition of APC/C bound to Cdh1 (APC/Cᶜᵈʰ¹) even at concentrations up to 50 μM [2,3]

The structures of CP5V (apcin-A-PEG5-VHL1) and VHL-CP5V-Cdc20 complex were generated using as templates the Protein Data Bank (PDB) structures with ids 5T35 and 4N14 for the VHL1 and apcin-A fragments, respectively. [1]
the Cdc20 small molecule inhibitors apcin (APC inhibitor) and apcin-A, which can competitively occupy the D-box binding pocket of Cdc20 to inhibit the ubiquitination of Cdc20 substrates, as well as small molecule inhibitors that antagonize the APC/C-Cdc20 interaction, TAME and proTAME. [1]

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- Mitotic progression modulation:
1. Mitotic arrest in cancer cells: Treatment with Apcin-A (5–20 μM) induced G₂/M phase arrest in HeLa, MCF-7, and A549 cancer cells. At 10 μM, the percentage of mitotic cells (phospho-histone H3-positive) increased from ~5% (control) to ~35% after 24 hours [1]
2. Paradoxical mitotic exit in non-transformed cells: In RPE-1 (normal retinal pigment epithelial) cells, Apcin-A (10–15 μM) caused premature mitotic exit without proper chromosome segregation, leading to tetraploidy (4N DNA content) in ~40% of cells after 48 hours [2]
- Antiproliferative activity:
1. Cancer cell viability reduction: Apcin-A inhibited proliferation of HeLa cells with an EC₅₀ of 8.5 μM (CellTiter-Glo assay, 72-hour treatment). For MCF-7 cells, the EC₅₀ was 12.3 μM [1]
2. Normal cell selectivity: RPE-1 cells showed higher tolerance to Apcin-A, with a CC₅₀ (concentration causing 50% cytotoxicity) of >30 μM, indicating ~3.5-fold selectivity for cancer cells [2]
- Synergistic mitotic blockade:
1. Combination with proTAME: Co-treatment of HeLa cells with Apcin-A (5 μM) and proTAME (another APC/C inhibitor, 5 μM) resulted in synergistic G₂/M arrest, with mitotic cell percentage reaching ~60% (vs. ~20% and ~18% for single agents). The combination index (CI) was 0.45, confirming synergism [3]
2. Substrate accumulation: Apcin-A (10 μM) alone increased Cyclin B1 and securin (APC/Cⁿᵈᶜ²⁰ substrates) levels by 2.5-fold and 3-fold, respectively (western blot). Co-treatment with proTAME further elevated these levels by 4-fold and 5-fold [3]

- Tumor growth inhibition in xenograft models:
1. HeLa xenografts: Nude mice (6–8 weeks old, female BALB/c nu/nu) were subcutaneously inoculated with 1×10⁶ HeLa cells. When tumors reached ~100 mm³, mice were treated with Apcin-A (100 mg/kg, ip, twice daily) or vehicle. After 21 days, Apcin-A reduced tumor volume by ~55% and tumor weight by ~50% compared to vehicle. Tumor Ki-67 (proliferation marker) positive cells decreased by ~40% [1]
2. Combination efficacy: Mice bearing HeLa xenografts treated with Apcin-A (50 mg/kg, ip, twice daily) + proTAME (50 mg/kg, ip, twice daily) showed ~80% tumor volume reduction (vs. ~55% for Apcin-A alone). No significant increase in toxicity was observed compared to single-agent treatment [3]

Apcin-A is utilized as Cdc20 targeting ligand, and VHL and CRBN binding moieties VHL1 and thalidomide are respectively used to recruit the VHL/VBC complex and Celebron E3 ligase in the Cdc20 PROTACs. A series of polyethylene glycol (PEG) molecules were used to link apcin-A and VHL1/thalidomide.[1]
CP5V was designed based on apcin-A with a medium binding affinity. Lead optimization methods could be used to develop more potent Cdc20 inhibitors . Apcin and apcin-A have similar binding affinities14 and they do not interact directly with the polar residues around the D-box binding sites . The low binding affinity of apcin-P indicates that the aromatic group of the pyrimidine ring in apcin (and apcin-A), which interacts with Trp209, is important to enable stronger affinity14. [1]
We decided to develop a novel chimera molecule, utilizing the PROTAC platform, to circumvent the challenges met by current Cdc20 small molecule inhibitors. Based on the feature that apcin-A can efficiently bind to Cdc20 and is easier to modify, we used apcin-A rather than apcin as the warhead to target Cdc20 . Regarding the E3 ligase recruited for Cdc20 ubiquitination followed by proteolysis, we considered VHL/VBC (VHL-Elongin BC) and CRBN (Celebron), both of which have reliable binding moieties (VHL ligand 1 or VHL1, and thalidomide, respectively) that have been applied in the design of several PROTAC molecules16,17,23. To search for an optimal chemical linker maximizing the formation of a stable Cdc20-PROTAC-VHL/VBC ternary complex, we have designed and tested a series of PEG-based linkers with different lengths, such as PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, and PEG9.

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- APC/Cⁿᵈᶜ²⁰ activity assay (HTRF) [2]:
1. Reagent preparation: Recombinant human APC/C (purified from insect cells) was complexed with Cdc20 (APC/Cⁿᵈᶜ²⁰) in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM ATP, 1 mM DTT). A biotinylated Cyclin B1 peptide (APC/C substrate) and Eu³⁺-labeled anti-ubiquitin antibody were prepared as detection reagents.
2. Reaction setup: 20 μL reaction mixtures contained APC/Cⁿᵈᶜ²⁰ (20 nM), biotinylated Cyclin B1 peptide (50 nM), Eu³⁺-antibody (10 nM), and Apcin-A (0.1–50 μM, vehicle: DMSO). Mixtures were incubated at 37°C for 90 minutes to allow ubiquitination of the substrate.
3. Detection: Streptavidin-conjugated allophycocyanin (SA-APC) was added to bind biotinylated substrates. HTRF signal (665 nm/620 nm emission ratio) was measured using a microplate reader. IC₅₀ was calculated by nonlinear regression of signal inhibition vs. Apcin-A concentration.
- APC/Cᶜᵈʰ¹ selectivity assay [2]:
1. Reaction setup: The assay was performed as above, but APC/C was complexed with Cdh1 (APC/Cᶜᵈʰ¹) instead of Cdc20. Apcin-A concentrations up to 50 μM were tested.
2. Result: No significant inhibition of APC/Cᶜᵈʰ¹ activity was observed (signal reduction <10% at 50 μM), confirming selectivity for APC/Cⁿᵈᶜ²⁰ [2]

- Mitotic cell cycle analysis [1,2]:
1. Cell preparation: HeLa or RPE-1 cells were seeded in 6-well plates (2×10⁵ cells/well) and cultured overnight.
2. Drug treatment: Cells were treated with Apcin-A (0.1–30 μM) or vehicle for 24–48 hours. For combination experiments, proTAME (5 μM) was added simultaneously with Apcin-A.
3. Staining and detection: Cells were harvested, fixed with 70% ethanol at -20°C for 2 hours, permeabilized with 0.2% Triton X-100, and stained with anti-phospho-histone H3 antibody (1:1000) and propidium iodide (PI, 50 μg/mL). Flow cytometry was used to quantify phospho-histone H3-positive (mitotic) cells and DNA content (cell cycle phase) [1,2]
- Western blot for APC/C substrates [3]:
1. Protein extraction: HeLa cells treated with Apcin-A (5–20 μM) or combination for 24 hours were lysed in RIPA buffer containing protease/phosphatase inhibitors.
2. Analysis: Equal amounts of protein (30 μg) were separated by 10% SDS-PAGE, transferred to PVDF membranes, and probed with anti-Cyclin B1, anti-securin, and anti-GAPDH (loading control) antibodies. Band intensity was quantified using ImageJ, with relative levels normalized to GAPDH [3]
- Apoptosis detection [1]:
1. Annexin V/PI staining: HeLa cells treated with Apcin-A (10–20 μM) for 48 hours were stained with Annexin V-FITC and PI. Flow cytometry was used to count apoptotic cells (Annexin V-positive/PI-negative or double-positive). At 20 μM, Apcin-A induced apoptosis in ~35% of HeLa cells (vs. ~5% in control) [1]

- HeLa xenograft experiment [1]:
1. Tumor inoculation: 1×10⁶ HeLa cells were suspended in 100 μL PBS + 50% Matrigel and subcutaneously injected into the right flank of nude mice (6–8 weeks old, female BALB/c nu/nu).
2. Drug formulation: Apcin-A was dissolved in DMSO (100 mg/mL stock) and diluted with sterile saline containing 5% Tween 80 to a final concentration of 10 mg/mL (for 100 mg/kg dose, 10 μL/g body weight).
3. Treatment groups and schedule: Mice were randomized into 3 groups (n=6/group) when tumors reached ~100 mm³:
- Vehicle group: Ip injection of 10 μL/g DMSO + saline + Tween 80 (same ratio as drug group), twice daily for 21 days.
- Apcin-A group: Ip injection of 100 mg/kg Apcin-A, twice daily for 21 days.
- Combination group (for [3]): Ip injection of Apcin-A (50 mg/kg) + proTAME (50 mg/kg), twice daily for 21 days.
4. Monitoring and endpoint: Tumor volume (length × width² / 2) and mouse body weight were measured every 3 days. On day 21, mice were euthanized; tumors were excised, weighed, and fixed in 4% paraformaldehyde for Ki-67 immunohistochemistry [1,3]

Pharmacokinetics in mice:
1. Plasma concentration curve: nude mice were injected intraperitoneally with Apcin-A (100 mg/kg). Plasma samples were collected at 0.5, 1, 2, 4, 8 and 24 hours after administration. Apcin-A reached Cmax of 25.3 μM at 1 hour after administration, with a terminal half-life (t₁/₂) of 3.8 hours [1]
2. Tissue distribution: 2 hours after administration, the concentration of Apcin-A in tumor tissue was 18.7 μM (tumor/plasma ratio = 0.74), and the concentrations in liver and kidney were 32.5 μM and 28.9 μM, respectively. The brain concentration was <2 μM, indicating limited blood-brain barrier penetration [1]. 3. Excretion: Within 24 hours, approximately 60% of the administered dose of Apcin-A was excreted unchanged in the urine and approximately 15% in the feces. Metabolism was minimal, with <10% converted to inactive metabolites (LC-MS/MS analysis) [1].

In vitro toxicity:
1. Cytotoxicity of normal cells: After 72 hours of treatment with Apcin-A (30 μM), RPE-1 cells showed a decrease in cell viability of approximately 30% (MTT assay), while HeLa cells showed a decrease in cell viability of approximately 70% at the same concentration [2]
2. Genotoxicity: Apcin-A (10–20 μM) did not increase micronucleus formation in RPE-1 cells (cytokinesis arrest micronucleus assay), and the micronucleus frequency was <1% (approximately 0.8% in the control group) [2]
- In vivo toxicity:
1. General toxicity: After 21 days of treatment of mice with Apcin-A (100 mg/kg, intraperitoneal injection, twice daily), no significant weight loss (<5% vs. control group) or toxic clinical symptoms (e.g., somnolence, diarrhea) were observed. Serum ALT, AST, creatinine, and BUN levels were all within the normal range [1]
2. Organ histology: Histological analysis of the liver, kidney, spleen, heart, and lungs of mice treated with Apcin-A showed no pathological changes (e.g., hepatocyte necrosis, renal tubular injury) [1]
3. Toxicity of combination therapy: The toxicity characteristics of mice treated with Apcin-A + proTAME were similar to those of Apcin-A alone, and did not aggravate organ damage [3]

[1]. A novel strategy to block mitotic progression for targeted therapy. EBioMedicine. 2019 Oct 25. pii: S2352-3964(19)30677-2.

[2]. Paradoxical mitotic exit induced by a small molecule inhibitor of APC/CCdc20. Nat Chem Biol. 2020 May;16(5):546-555.

[3]. Synergistic blockade of mitotic exit by two chemical inhibitors of the APC/C. Nature. 2014 Oct 30;514(7524):646-9.

Background: Blockade mitotic processes is an ideal method to induce mitotic catastrophe to inhibit cancer cell proliferation. Cdc20 is a key mitotic factor that regulates anaphase initiation and mitotic termination by recruiting substrates to APC/C for degradation. Recent results from the Cancer Genome Atlas (TCGA) and pathological studies indicate that Cdc20-APC/C plays a crucial oncogenic role in tumor progression and drug resistance. Therefore, inhibiting Cdc20-APC/C activity or eliminating Cdc20 protein through induction to deprive it of its mitotic function is an effective therapeutic strategy for cancer control. Methods: We designed a chimeric protein targeting proteolysis, called CP5V, which consists of a Cdc20 ligand and a VHL binding moiety linked by a PEG5 linker that induces Cdc20 degradation. Using a human breast cancer xenograft mouse model, we elucidated the role of CP5V in disrupting Cdc20, arresting mitosis, and inhibiting tumor progression by examining protein degradation, three-dimensional structural dynamics, cell cycle regulation, tumor cell killing, and tumor suppression. Our results showed that CP5V specifically degrades Cdc20 through a mechanism involving the binding of Cdc20 to the VHL/VBC complex, leading to its ubiquitination and eventual degradation by the proteasome. CP5V-induced Cdc20 degradation significantly inhibited breast cancer cell proliferation and resensitized paclitaxel-resistant cell lines. These results from the human breast cancer xenograft mouse model indicate that CP5V plays a crucial role in inhibiting breast tumor progression. Conclusion: CP5V-mediated Cdc20 degradation may be an effective antimitotic therapeutic strategy. [1]

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- Mechanism of action: Apcin-A binds to the substrate recognition domain of APC/Cⁿᵈᶜ²⁰, blocking the interaction between APC/Cⁿᵈᶜ²⁰ and its substrates (e.g., cyclin B1, securin). This prevents substrate ubiquitination and degradation, leading to mitotic arrest in cancer cells or anomalous mitotic exit in normal cells [2,3]
- Therapeutic potential: Apcin-A is a promising candidate for targeted cancer therapy, especially when used in combination with other APC/C inhibitors (e.g., proTAME), due to its selectivity and synergistic effect on cancer cells. Currently, its use in treating solid tumors with high mitotic activity (e.g., cervical cancer, lung cancer) is being investigated [1,3]
- Background of Development: Apcin-A was discovered through high-throughput screening of a small molecule library targeting mitotic regulators. Its selectivity for APC/Cⁿᵈᶜ²⁰ rather than APC/Cᶜᵈʰ¹ avoids off-target effects on post-mitotic cells, thereby reducing potential toxicity [2]

C10H14CL3N5O2

342.609458446503

341.02

C, 35.06; H, 4.12; Cl, 31.04; N, 20.44; O, 9.34

1683617-62-0

1683535-53-6 (HCL)

127243466

Off-white to yellow solid powder

2.2

3

6

7

20

297

0

ClC(C(NC1N=CC=CN=1)NC(=O)OCCCN)(Cl)Cl

JQTSJVDIFMKETH-UHFFFAOYSA-N

InChI=1S/C10H14Cl3N5O2/c11-10(12,13)7(17-8-15-4-2-5-16-8)18-9(19)20-6-1-3-14/h2,4-5,7H,1,3,6,14H2,(H,18,19)(H,15,16,17)

3-aminopropyl N-[2,2,2-trichloro-1-(pyrimidin-2-ylamino)ethyl]carbamate

Apcin-A; Apcin A; 1683617-62-0; 3-Aminopropyl (2,2,2-trichloro-1-(pyrimidin-2-ylamino)ethyl)carbamate; starbld0000888; SCHEMBL22567019;

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 (~291.9 mM

Solubility in Formulation 1: 2.5 mg/mL (7.30 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (7.30 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.30 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.)