p53 Y220C mutant (mutant p53 core domain) - binds to the mutation-induced surface cavity; Kd = 167 ± 12 μM (by ¹H-¹⁵N HSQC NMR at 20°C); Kd = 125 ± 10 μM (by isothermal titration calorimetry at 20°C); Kd ≈ 140 ± 73 μM (from thermal denaturation data at 316–318 K); Kd = 170 μM (by analytical ultracentrifugation at 10°C). [1]

PhiKan 083, a derivative of carbazole, has a Kd of 167 μM[1] and a relative binding affinity (Kd) of 150 μM for p53Y220C in Ln229 cells[3]. It binds to surface cavities and stabilizes Y220C (p53 mutant). Its thermal denaturation rate is slowed down by PhiKan 083 [2]. Engineered Ln229 cell variations' cell viability is decreased by PhiKan 083 (125 μM, 48 hours) [3]. In Ln229 cells, combining PhiKan 083 (100 μM) with NSC 123127 (1 μM) increases the pro-apoptotic activity of all variants (p53wt, p53Y220C, p53G245S, and p53R282W) [3].

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PhiKan-083 binds to T-p53C-Y220C with a dissociation constant of 167 ± 12 μM as measured by ¹H-¹⁵N HSQC NMR spectroscopy at 20°C. [1]
Isothermal titration calorimetry (ITC) gave a Kd of 125 ± 10 μM with 1:1 stoichiometry at 20°C. [1]
Analytical ultracentrifugation gave a Kd of 170 μM at 10°C. [1]
PhiKan-083 increased the apparent melting temperature (Tm) of T-p53C-Y220C in a concentration-dependent manner; at 2.5 mM ligand, Tm was raised by nearly 2°C from 316 K (apparent Tm in the absence of ligand), and the data fitted to a simple binding model yielded an approximate Kd of 140 ± 73 μM at 316–318 K. [1]
At 37°C (310 K), PhiKan-083 slowed the thermal denaturation kinetics; the half-life of T-p53C-Y220C increased from 3.8 min in the absence of ligand to 15.7 min at saturating concentrations of the compound. [1]
The crystal structure of the T-p53C-Y220C:PhiKan-083 complex was solved at 1.5 Å resolution (PDB 2VUK), showing unambiguous electron density for PhiKan-083 bound to the mutation-induced cleft. The central carbazole moiety is largely buried, with the 9-ethyl group occupying the deepest part of the hydrophobic pocket, and the N-methylmethanamine moiety forms a hydrogen bond with the main-chain carbonyl of Asp-228. Only small structural shifts occur upon binding, with the most significant shift observed for Thr-150 side chain (displaced by up to 1.4 Å). [1]

¹H-¹⁵N HSQC NMR spectroscopy for binding: ¹⁵N-labeled T-p53C-Y220C protein at 70 μM in 25 mM sodium phosphate (pH 7.2), 150 mM NaCl, 5 mM DTT, and 4.5% d6-DMSO was titrated with PhiKan-083 at varying concentrations. Spectra were acquired at 20°C on 700 or 800 MHz spectrometers with a cryogenic probe. Chemical shift changes for 15 characteristic resonances were used to derive an average Kd by fitting to a single-site binding model. [1]
Isothermal titration calorimetry (ITC): Binding was measured using a VP-ITC calorimeter at 20°C in 25 mM sodium phosphate (pH 7.2), 150 mM NaCl, 1 mM DTT, with 5% d6-DMSO. PhiKan-083 (5 mM) was titrated into the cell containing 100 μM protein. Injection steps of 10 μL (first 3 μL) with 600 s spacing were used; data were evaluated with the MicroCal Origin program. [1]
Analytical ultracentrifugation: Sedimentation equilibrium experiments were performed to measure binding, giving Kd values at 10°C. [1]
Thermal stability assay (differential scanning calorimetry or SYPRO Orange fluorescence): Thermal unfolding was monitored by DSC at a scan rate of 250 K/h or by SYPRO Orange fluorescence at 270 K/h in 25 mM sodium phosphate (pH 7.2), 150 mM NaCl, 5 mM DTT, with 10 μM protein. The apparent Tm was determined in the presence of varying concentrations of PhiKan-083, and data were fitted to a binding model to extract Kd. [1]
Kinetics of thermal denaturation: Unfolding kinetics were followed at 37°C by monitoring tryptophan emission at 340 nm (excitation 280 nm) in 50 mM Hepes (pH 7.2), 1 mM Tris(2-carboxyethyl)phosphine. Reactions were followed for 10,000 s; data were fitted to a single exponential plus a linear drift term. [1]
X-ray crystallography: Crystals of T-p53C-Y220C in space group P2₁2₁2₁ were grown by sitting-drop vapor diffusion. PhiKan-083 was soaked into crystals by stepwise addition of cryo buffer with increasing ligand concentration up to 10 mM over 2.5 h. Data to 1.5 Å were collected at 100 K; structure was solved and refined with CNS, and the ligand was built into chain B with clear electron density. [1]

Cell Viability Assay [1]
Cell Types: Ln229, Ln229-p53-wt, Ln229-p53-Y220C, Ln229-p53-G245S, Ln229-p53-R282W Cell
Tested Concentrations: 125 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Caused ∼70 ± The Ln229 cell variant had a 5% reduction in cell viability.

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Transient transfection and stable cell line generation: HEK293T, Ln229, and U87MG cells were used. Lentiviral vectors expressing split-Renilla luciferase (RLUC) complementation biosensors (NRLUC-p53-CRLUC) with different p53 variants (wt, Y220C, G245S, R282W) were constructed. Stable Ln229 cells were generated by lentiviral transduction and FACS sorting for equal dTomato expression. [3]
Drug treatment and bioluminescence imaging: Cells were treated with PhiKan-083 at various concentrations (15.6, 31.25, 62.5, 125, 250 μM) for durations from 1 to 24 hours. After treatment, cells were washed and coelenterazine (2 μg in 50 μL PBS) was added, and RLUC complementation signal was measured using IVIS optical imaging system with 1-minute integration times over 15 minutes. Signal was quantified by drawing regions of interest. [3]
MTT cell viability assay: Cells plated in 96-well plates (5,000 cells/well) were treated with drugs individually or in combinations for 24-72 hours. After treatment, MTT reagent (12.5 μM) was added in phenol red-free DMEM for 2 hours, formazan crystals dissolved in DMSO, and absorbance measured at 540 nm. [3]
Apoptosis and cell cycle analysis by FACS: Cells in 12-well plates (75,000 cells/well) were treated, harvested by trypsinization (including dead cells), fixed in ice-cold 70% ethanol for 2 hours at -20°C, stained with propidium iodide (10 μg/mL) with RNase A (100 μg/mL) and 0.05% Triton X-100 for 15 minutes, and analyzed using a Guava FACS analyzer with InCyte and FlowJo software. [3]
Immunoblot analysis: Cells were lysed in RIPA buffer with protease inhibitors, sonicated, centrifuged, and protein quantified. Samples (200 μg total protein) were resolved on 4-12% SDS-PAGE, transferred to PVDF membranes, blocked, and probed with primary antibodies against p53, p21, Bcl2, SOD2, and loading controls (GAPDH, tubulin, β-actin), followed by HRP-conjugated secondary antibodies and chemiluminescent detection using IVIS-Lumina imaging system. [3]

PhiKan-083 at high concentrations (125 μM and above) is highly toxic to cells, as observed by significant cell death even without chemotherapy. However, at lower concentrations (e.g., 3.75 μM), it shows limited toxicity but significant synergy with chemotherapeutics. The relative binding affinity of PhiKan-083 (150 μM) is considerably toxic, limiting its in vivo use due to unintended toxicity to normal cells. [3]

[1]. Targeted rescue of a destabilized mutant of p53 by an in silico screened drug. Proc Natl Acad Sci U S A. 2008 Jul 29;105(30):10360-5.

[2]. Effect of Y220C mutation on p53 and its rescue mechanism: a computer chemistry approach. Protein J. 2013 Jan;32(1):68-74.

[3]. A protein folding molecular imaging biosensor monitors the effects of drugs that restore mutant p53 structure and its downstream function in glioblastoma cells. Oncotarget. 2018 Apr 20;9(30):21495-21511.

The Y220C mutation in p53 occurs in approximately 75,000 new cancer cases per year worldwide. The mutation creates a surface cavity that is distant from the DNA-binding and protein-protein interaction interfaces, making it an attractive target for small-molecule stabilization therapy. PhiKan-083 binds to this cavity and stabilizes the mutant protein by increasing its melting temperature and slowing denaturation, representing a lead compound for developing anticancer drugs based on protein stabilization. The degree of stabilization is related to the binding affinity and the entropy of denaturation; for a given ratio of ligand concentration to Kd, the increase in Tm can be calculated. The crystal structure reveals that the carbazole scaffold could be further modified to introduce hydrogen-bond donors (e.g., to the carbonyl of Leu-145) or to extend into the pre-existing part of the crevice not addressed by the current ligand, offering multiple opportunities for affinity improvement. [1]

C16H19CLN2

274.79

274.123

1050480-30-2

PhiKan 083;880813-36-5

16255105

White to off-white solid powder

2

1

3

19

278

0

Cl.N1(CC)C2C=CC=CC=2C2C=C(CNC)C=CC1=2

IXWVUPURFWFRNE-UHFFFAOYSA-N

InChI=1S/C16H18N2.ClH/c1-3-18-15-7-5-4-6-13(15)14-10-12(11-17-2)8-9-16(14)18;/h4-10,17H,3,11H2,1-2H3;1H

1-(9-ethylcarbazol-3-yl)-N-methylmethanamine;hydrochloride

PhiKan 083; PhiKan083; PhiKan-083;

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, 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 : ~62.5 mg/mL (~227.45 mM)
H2O : ~2 mg/mL (~7.28 mM)

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.)