S-phase kinase-associated protein 2 (Skp2) [1]
S-phase kinase-associated protein 2 (Skp2) [2]
S-phase kinase-associated protein 2 (Skp2) [3]
S-phase kinase-associated protein 2 (Skp2) (interacts with Skp2/Cks1 E3 ligase complex to block p27 binding,) [4]

Skp2 Inhibitor C1 (10–50 μM; 12 hours) reduces THP-1, U266, and RPMI 8226 cell viability.[1]. By blocking ubiquitination, Skp2 Inhibitor C1 (25 μM) raises the levels of p27 protein in U266 and RPMI 8226 cells[1]. Skp2 Inhibitor C1 (25 μM) inhibits U266 and RPMI 8226 cells' cell cycle.[1].

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1. SKPin C1 significantly inhibited the viability of multiple myeloma (MM) U266 and RPMI 8226 cells at 10 μM, with the inhibitory effect enhanced by increasing doses; 50 μM SKPin C1 only marginally decreased the viability of normal B lymphocytes within 12 h. Skp2 expression was higher and p27 expression was lower in U266 and RPMI 8226 cells compared with normal B lymphocytes. Treatment with SKPin C1 or Skp2 knockdown increased p27 protein levels in U266 and RPMI 8226 cells by preventing p27 ubiquitination, which slowed the cell cycle, inhibited cell proliferation, and triggered apoptosis (evaluated by MTT, EdU staining, cell cycle assays, western blot, and immunoprecipitation). [1]
2. C1 (Skp2 inhibitor) exhibited antidepressant-like effects in vitro screening (tail suspension test, forced swimming test, social interaction test) at doses of 5 and 10 mg/kg (but not 1 mg/kg) in male/female mice; 5 mg/kg C1 induced antidepressant-like effects in stress-naïve mice with three administrations within 24 h (24, 5, 1 h before test) but not with acute administration (1 h before test); co-administration of C1 and fluoxetine produced additive antidepressant-like effects, and C1 had no effect on locomotor activity. [2]
3. C1 selectively inhibited Skp2-mediated p27 ubiquitylation in vitro by binding to the Skp2-Cks1 interface (formed by Skp2-R294, Skp2-Y346, Cks1-R44, Cks1-Q52) and reducing p27 binding to Skp2; it increased p27 protein levels and half-life in metastatic melanoma cell lines (501 Mel, SK-MEL-147, SK-MEL-173) at 10 μM, induced cell cycle arrest (G1 phase in 501 Mel cells, G2/M phase in MCF-7 cells, G1 phase in T47D cells), and had no effect on E2-Ubc3/E2-Ubc5 ubiquitin transfer, CyclinE/CDK2 phosphorylation of p27, or off-target E3 ligases (MDM2, SCF-βTrCP). [3]
4. C1 increased p27 protein levels in endometrial carcinoma ECC-1 cells and primary endometrial carcinoma (ECA) cells; it increased both cytoplasmic and nuclear p27 levels in ECC-1 cells (unlike C2/C20 which specifically increased nuclear p27); it inhibited cell proliferation of primary ECA cells (grade III/III and I/III tumors). [4]

By using the tail suspension test (TST), forced swimming test (FST), and social interaction test (SIT), Skp2 Inhibitor C1 (5 mg/kg and 10 mg/kg; three times within 24 h: 24, 5, and 1 h before the test) shows the antidepressant-like effect in mouse models following chronic treatment.[2]

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1. Long-term treatment with 5 mg/kg C1 (Skp2 inhibitor) ameliorated depression-like behaviors in chronic social defeat stress-exposed mice, showing antidepressant-like actions under stress conditions. [2]

1. In vitro ubiquitylation assay: Recombinant proteins (wild-type/mutant Skp2, Cks1, p27) were generated by in vitro transcription-translation; the assay contained UBE1, His-UbcH3/His-UbcH5c, ubiquitin, ATP, and C1 (50 μM) or vehicle, incubated at 30°C for 60 min; reactions were stopped with non-denaturing sample buffer (with/without β-mercaptoethanol) and analyzed by western blotting to detect p27 polyubiquitylation. [3]
2. Ubiquitin charging assay: UBE1 (100 nM) and His-UbcH3 (10 ng/μl) were incubated with ubiquitin (2.5 μg/μl), ATP (2 mM), and 10 μM C1 or vehicle at 30°C for 60 min; reactions were analyzed by western blotting to assess E2 charging activity (no inhibition by C1). [3]
3. Differential scanning fluorimetry: Recombinant His-6Skp1-Skp2-Cks1 (1.5 μM) was preincubated with 75 μM C1, unmatched compound (UM-C1), or vehicle (0.5% DMSO); fluorescence (465 nm excitation/580 nm detection) was measured at a ramp rate of 0.06°C/s (20-85°C) to calculate melting temperature (Tm), showing C1 shifted Tm of Skp2-Cks1 complex (48.51°C vs vehicle 44.09°C, UM-C1 45.06°C). [3]
4. Surface plasmon resonance (SPR) assay: Purified Skp2-Cks1 complex was used to confirm C1 binding to the complex. [3]

Cell Line:THP-1, U266, RPMI 8226, and B lymphocytes
Concentration: 0, 5, 10, 25, and 50 μM
Incubation Time: 12 hr, 24 hr, 36 hr, and 48 hr
Result: significantly reduced U266 and RPMI 8226 cell viability at 10 μM for a 12-hour period.
had no discernible effect on the viability of B cells at 50 μM.
THP-1 cell viability was reduced for 12 hours at 50 μM.

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1. Multiple myeloma cell assay: Normal B lymphocytes, THP-1, U266, and RPMI 8226 cells were exposed to various doses of SKPin C1 for 48 h; cell viability was measured by MTT at different time points; cell cycle was evaluated by flow cytometry after 12 h treatment; western blot was used to assess Skp2, p27, cleaved caspase-3 protein levels; immunoprecipitation was performed to determine p27 ubiquitination levels; EdU staining was used to evaluate cell proliferation in U266/RPMI 8226 cells treated with 25 μM SKPin C1 or Skp2 siRNA. [1]
2. Melanoma cell assay: 501 Mel, SK-MEL-147, SK-MEL-173 cells were treated with 10 μM C1 or vehicle for 16 h; whole-cell extracts were prepared for western blotting to detect p27, p21, Skp2, Skp1, Cul1 levels; cycloheximide chase assay (20 ng/ml cycloheximide) was performed to assess p27 half-life (extended from 1 h to >5 h by C1); lentiviral tet-inducible shRNAs (shSkp2/shp27) were used to knockdown Skp2/p27, followed by C1 treatment (10 μM for 16 h) and flow cytometry for cell cycle analysis. [3]
3. Breast/prostate cancer cell assay: MCF-7, T47D (breast cancer) and LNCaP (prostate cancer) cells were treated with 5 μM (MCF-7/T47D) or 10 μM (LNCaP) C1 for 16 h; flow cytometry was used for cell cycle analysis (G2/M arrest in MCF-7, G1 arrest in T47D/LNCaP); western blotting was performed to detect p27, Skp2, Skp1 levels. [3]
4. Endometrial carcinoma cell assay: ECC-1 cells were treated with C1 (dose not specified) for 18 h; subcellular fractionation (nuclear/cytoplasmic) was performed, and western blotting was used to detect p27 levels (α-tubulin/Specificity protein 1 as fraction purity controls); MTS assay was used to evaluate cell proliferation inhibition by C1 ; super resolution fluorescence localization microscopy was used to quantify nuclear p27 clusters (no numerical change in density, 1.8-fold increase in cluster area). [4]
5. Primary ECA cell assay: Primary ECA cells (grade I/I, III/III, I/III, II/III tumors) were treated with C1 (dose not specified); western blotting was used to detect p27 levels in total cell lysates/nuclear-cytoplasmic fractions; MTS assay was used to assess proliferation inhibition. [4]

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1. Antidepressant effect assay in mice: Male/female mice (strain not specified) were administered C1 at doses of 1, 5, 10 mg/kg via unspecified route; acute administration (1 h before test) and chronic administration (8 days treatment) were tested; three administrations within 24 h (24, 5, 1 h before test) were used for stress-naïve mice; chronic social defeat stress-exposed mice were treated with 5 mg/kg C1 for long-term to evaluate depression-like behaviors (tail suspension test, forced swimming test, social interaction test); locomotor activity was measured to exclude non-specific effects. [2]
2. Endometrial epithelial cell proliferation assay in mice: Ovariectomized C57Bl/6 mice were primed with 100 ng 17-β-estradiol (E2) intramuscularly for 2 days, followed by 2 days rest; mice were injected intraperitoneally with C1 (dose not reported) or vehicle for 2 consecutive days, euthanized 24 h later; immunohistochemistry (IHC) was performed on endometrial tissue to detect p27 (nuclear) and PH3 (proliferation marker) levels (no data for C1 in this assay, only C5/C2 reported). [4]

1. 50 μM SKPin C1 only slightly reduced the viability of normal B lymphocytes within 12 hours, indicating that it has low in vitro toxicity to normal cells. [1]
2. C1 (5, 10 mg/kg) had no effect on the kinetic activity of mice, indicating that it has no acute toxicity related to kinetic function; (LD50, hepatotoxicity, nephrotoxicity, plasma protein binding) have been reported. [2]
3. C1 (10 μM) was not cytotoxic to 501 Mel, MCF-7, T47D and LNCaP cells. [3]
4. C1 was not cytotoxic to ECC-1 cells (no decrease in cell viability was observed, and no apoptosis was detected in PARP1/caspase-3 lysis). [4]

[1]. Braz J Med Biol Res. 2019;52(5):e8412.

[2]. Behav Pharmacol. 2021 Feb 1;32(1):62-72.

[3]. Chem Biol. 2012 Dec 21; 19(12)

[4]. 1515–1524.;Endocrinology.2013 Nov;154(11):4030-45.

1. SKPin C1 is a Skp2 inhibitor that has been shown to inhibit metastatic melanoma cells; it effectively inhibits the abnormal proliferation and immortalization of multiple myeloma by stabilizing p27 (preventing ubiquitination), slowing the cell cycle, and inducing apoptosis. [1]
2. C1 is a specific Skp2 inhibitor; inhibiting Skp2 may be a potential strategy for treating depression, and Skp2 may also be a target for the development of novel antidepressants; C1 combined with fluoxetine can enhance its effect in alleviating depressive-like behaviors. [2]
3. C1 is a small molecule inhibitor that, identified by computer virtual ligand screening (VLS), targets the Skp2-Cks1 interface (a pocket formed by Skp2-R294, Skp2-Y346, Cks1-R44, and Cks1-Q52). It blocks Skp2-mediated p27 degradation by competitively inhibiting Skp2-p27 interaction, and its cell cycle arrest effect depends on the cell type (G1 phase for melanoma/prostate cancer/endometrial cancer cells, and G2/M phase for breast cancer cells). [3]
4. C1 is a Skp2/Cks1 E3 ligase inhibitor (Skp2E3LI) that can stabilize p27 (nuclear and cytoplasmic) in endometrial cancer cells and inhibit E2-induced p27 degradation and cell proliferation; it has potential therapeutic value for endometrial cancer characterized by SCF-Skp2/Cks1-mediated destruction of nuclear p27 and is superior to general proteasome inhibitors. [4]

C18H13BRN2O4S2

465.34

463.95

C, 46.46; H, 2.82; Br, 17.17; N, 6.02; O, 13.75; S, 13.78

432001-69-9

5733396

White to yellow solid powder

3.646

1

7

6

27

631

0

S=C(S/1)N(CC2=CN=CC=C2)C(C1=C\C(C=C(Br)C=C3)=C3OCC(O)=O)=O

O=C(O)COC1=CC=C(Br)C=C1/C=C(SC(N2CC3=CC=CN=C3)=S)/C2=O

InChI=1S/C18H13BrN2O4S2/c19-13-3-4-14(25-10-16(22)23)12(6-13)7-15-17(24)21(18(26)27-15)9-11-2-1-5-20-8-11/h1-8H,9-10H2,(H,22,23)/b15-7-

2-[4-Bromo-2-[[4-oxo-3-(3-pyridinylmethyl)-2-thioxo-5-thiazolidinylidene]methyl]phenoxy]acetic acid

MDK-1699; MDK 1699; MDK1699; Skp2 inhibitor C1 and SKPin C1.

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 :20.83~ 93 mg/mL ( 44.76~199.85 mM )

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.47 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 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 2: 5% DMSO + Corn oil: 1.2mg/ml

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