Deubiquitinase USP2a (IC50 = 9.7 μM)

With GI50 of 0.87±0.09 and 0.96±0.29μM, respectively, and LD50 of 27.8±3.9 and 26.5±0.1μM, respectively, LCAHA suppresses the viability of HCT116wt and HCT116 p53-/-colon cancer cells [1]. The most potent LCA derivative, LCA hydroxyamide (LCAHA), inhibits USP2a, leading to a significant Akt/GSK3β-independent destabilization of cyclin D1, but does not change the expression of p27. This leads to the defects in cell-cycle progression. As a result, LCAHA inhibits the growth of cyclin D1-expressing, but not cyclin D1-negative cells, independently of the p53 status. We show that LCA derivatives may be considered as future therapeutics for the treatment of cyclin D1-addicted p53-expressing and p53-defective cancer types.[1]

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For one LCA derivative, LCAHA, a biphasic response was observed for both cell types tested: a low-concentration GI effect and a high-concentration toxic effect (Figure 1A). LDH release was observed only at the concentrations corresponding to the high-concentration toxic effect (Figure 1B). The GI50 values calculated from MTT results were below 1 μM for both cell lines (Table 2) and the growth of cells was inhibited by about 60% in the concentration range 2–20 μM (Figure 1A). For the lethal effect, LD50 values were above 25 μM for both cell lines, giving selectivity of the GI effect over the lethal effect of 31.9-fold for p53wt and 27.6-fold for p53−/− cells (Table 2).[1]

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To evaluate whether the observed effects are accompanied by the induction of apoptosis, we examined the activation of caspases 3 and 7 in HCT116 p53wt cells. A weak and non-significant increase of caspase activity was detected in the cells treated with 1 or 5 μM LCAHA for 48 or 72 hr. Only at 10 μM LCAHA was the increase of caspase activity significant (Figure 1C). Thus, apoptosis is not efficiently induced in the GI concentration range of the compound. Treatment with staurosporine, a known inducer of apoptosis, led to a strong increase of the activity of caspases 3 and 7 (Figure 1C). [1]

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LCAHA Arrests HCT116 Cells in G0/G1 [1]
Low concentrations of LCAHA inhibit the growth of HCT116 cells without affecting their survival. To investigate this effect, we analyzed the cell-cycle distribution. Cells treated for 48 hr with LCAHA or DMSO were pulse-labeled with BrdU for 1 hr and stained with fluorescein isothiocyanate (FITC)-anti-bromodeoxyuridine (BrdU) antibody and propidium iodide (PI). Flow cytometry analysis revealed mild, but significant G0/G1 arrest of both HCT116 p53wt and HCT116 p53−/−cells treated with 5 or 10 μM LCAHA (Figure 1D). The analysis of BrdU incorporation revealed a dose-dependent decrease in the rate of DNA synthesis in LCAHA-treated cells (Figure 1E). This suggests that besides G0/G1 arrest, LCAHA also affects the progression through the S phase.

To verify this, we pulse-labeled HCT116 p53wt cells treated with LCAHA for 48 hr for 1 hr with BrdU and harvested or cultured them for an additional 3, 6, or 9 hr. To track the progression through the S phase and G0/G1 arrest precisely, we analyzed FITC-positive and -negative cells separately for cell-cycle distribution using ModFit LT software. FITC-positive cells represented the cells that incorporated BrdU (the population in the S phase during pulse-labeling that progressed toward G2/M in succeeding hours), and FITC-negative cells represented the cells that did not incorporate BrdU (the population that was either in G0/G1 or G2/M phase during pulse-labeling and entered S phase or divided in succeeding hours).

As expected, the majority of BrdU+ cells were in the S phase at time point t0, and the majority of BrdU− cells were in G0/G1 or G2/M phases (Figure 1F). Nine hours later only 23% of BrdU+ cells treated with DMSO were still in the S phase and more than 50% of cells had finished the cell cycle and were located in G0/G1 phase (Figure 1F, top panel). The treatment with LCAHA dose-dependently inhibited the transition of BrdU+ cells through the S phase, as evidenced by the significantly higher proportion of cells remaining in the S phase (up to 56% at 5 μM LCAHA) and significantly lower proportion of cells in G0/G1 phase (down to 24%). At the same time, around 44% of BrdU− cells treated with DMSO left G0/G1 phase and entered the S phase, while treatment with LCAHA significantly and dose-dependently inhibited this transition down to 20% at 5 μM concentration of the compound (Figure 1F, bottom three panels). These results reveal a much stronger G0/G1 arrest than suggested by a simple analysis of the whole population of the cells (Figure 1C). The complete outcome of the test, presenting the progress of the cell cycle with 3-h intervals, is presented in Figure S2.

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LCAHA-Induced G0/G1 Arrest Is Accompanied by Decreased Expression of Cyclin D1 [1]
The progression from G1 to S phase relies largely on the expression and activity of D cyclins. To verify whether the observed impairment in cell-cycle progression following LCAHA treatment is associated with D cyclins, we investigated the expression of cyclins D1 and D3 in HCT116 p53wt and HCT116 p53−/− cells. A significant, dose-dependent decrease of cyclin D1 expression was observed in both cell lines treated with 5 or 20 μM LCAHA; however, this effect was more evident in HCT116 p53wt cells (Figure 2A). LCA did not affect the expression of cyclin D1, and LCAE, which did not present a biphasic effect on the survival of HCT116 cells, decreased the expression of the protein in HCT116 p53wt cells, but this effect was much weaker than for LCAHA (Figure 2A). A 24-hr treatment with any of the compounds did not affect the expression of cyclin D1 in HCT116 cell lines (Figure S3B).

Thermal Shift Assay (TSA) [1]
TSA analysis was carried out by monitoring the fluorescence of SYPRO Orange Dye in the presence of USP2a and tested compounds at increasing temperatures (form 22 to 98oC). Experiments were performed in PBS pH=7.4 buffer. USP2a (1 μM) was incubated alone and with LCAE or LCAHA compounds (both at 50 μM). Constant temperature gradient was applied (0.2°C/min) and fluorescence changes were monitored using real time thermocycler. Melting temperature (Tm) was estimated from the first derivative of fluorescence intensity as a function of temperature.

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MALDI TOF Based High Throughput DUB Activity Assay [1]
The assay was performed as described before (Ritorto et al., 2014). Thirty one human DUBs were freshly diluted in reaction buffer (40mM Tris–HCl, pH 7.6, 5mM DTT, 0.005% BSA) to the proper concentrations. Enzymes were incubated with the LCAHA and LCAE at 100 μM final concentration for 30 min at room temperature. Diubiquitin topoisomers (K63, K48, K11 and M1) were diluted to 0.2 μl/μg and added to the reaction mixture using a Nanoliter pipetting system to the final concentration of 1.5 μM. The plate was sealed and incubated for 30 min at room temperature and stopped by adding TFA to a final concentration of 2% (v/v). The terminated reaction was then transferred (1.050 μl) to a 384 plate, spiked with 15N-ubiquitin internal standard (0.15 μl, 16 μM) and mixed 1:1 with 2.5 DHAP matrix freshly prepared (7.6 mg of 2,5 DHAP in 375 ml ethanol and 125 ml of an aqueous 12 mg/ml diammonium hydrogen citrate). The resulting matrix/reaction mixture was spotted in 200 μl aliquots onto an MTP AnchorChip 1,536 TF (600 mm anchor). Mass spectrometry data was acquired on an UltrafleXtreme MALDI-TOF mass spectrometer with Compass 1.3 control and processing software. The sample carrier was taught before each analysis to optimize and centre laser shooting. Internal calibration was performed before each analysis using the 15N-Ub peak [M+H]+ average = 8,569.3). Samples were analyzed in automatic mode as previously reported (Ritorto et al., 2014). For area calculation, the complete isotopic distribution was taken into account. An in-house made script was used to report - 15N and mono-ubiquitin areas; plotting of graphs, calculation of standard deviation and coefficient of variation (%) were processed in Microsoft Excel.

Cell viability assay[1]
Cell Types: HCT116wt and HCT116 p53-/- colon cancer cells
Tested Concentrations: 0.01, 0.1, 1, 10 and 100 μM
Incubation Duration: 6 days
Experimental Results: GI50 were 0.87±0.09 and 0.96±0.29 μM, respectively For HCT116wt and HCT116 p53-/- colon cancer cells.

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Cell Cycle Analysis [1]
The cells were treated with DMSO or LCAHA for 48 hours and pulse-labelled with 10 μM bromodeoxyuridine (BrdU) for the last hour of the treatment. After that, the cells were either harvested by trypsinization and fixed with 96% ethanol, or cultured for additional 3-9 hours in the absence of BrdU before harvesting and fixation. The cells were stained with PI and FITC-conjugated anti-BrdU antibody and analysed with Fortessa flow cytometer. Cell cycle distribution was analysed using ModFit LT Software.

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Caspase 3/7 Activity [1]
The cells were plated on the 96-well white, flat bottom plates and treated with DMSO, LCAHA or Staurosporine. For the detection of caspase activity Caspase-Glo 3/7 Assay System was used according to the manufacturer’s instructions. Due to the observed growth-inhibitory properties of LCAHA the results were normalized to cell numbers, calculated from the pictures of Hoechst-stained cells seeded on transparent culture plates and treated identically as the cells seeded for caspase activity assay (mean nuclei number per picture was 6452 for 3 days treatment with DMSO, and 3957, 4229 and 2783 for the cells treated with increasing concentrations of LCAHA).

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Colony Formation Assay [1]
For the colony formation assay MCF-7 or SAOS-2 cells were treated with DMSO or 5 μM LCAHA for 5 days. One thousand of the cells was then seeded on 6-well plates and cultured for 2-4 weeks until the generation of well-visible clones. The plates were stained with crystal violet, imaged with the ChemiDoc MP system and analyzed using the ImageJ software (Schneider et al., 2012) and ‘Analyze particles’ tool. The surviving fraction (SF) was calculated using the equation: SF = (PE of treated sample / PE of control) * 100%, where PE (plating efficiency) = Ncolonies / Ncells plated.

[1]. Lithocholic Acid Hydroxyamide Destabilizes Cyclin D1 and Induces G 0/G1 Arrest by Inhibiting Deubiquitinase USP2a. Cell Chem Biol. 2017 Apr 20;24(4):458-470.e18.

In summary, we show that cell-growth inhibition induced by LCAHA is accompanied by a significant decrease in cyclin D1 level, which is a consequence of decreased stability of the protein. We also show that LCAHA antagonizes proliferative signals delivered by the presence of serum in the culture medium and describe the mechanism of engagement of USP2a protein in the observed destabilization of cyclin D1 by LCAHA. Our work constitutes a starting point for the design of more potent USP inhibitors based on an LCA scaffold for improved treatment of cancer.[1]

C24H41NO3

391.587247610092

391.308

C, 73.61; H, 10.55; N, 3.58; O, 12.26

117094-40-3

126961696

White to off-white solid powder

5.5

3

3

4

28

594

9

O[C@@H]1CC[C@@]2(C)C(C1)CC[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)CCC(NO)=O)CC[C@H]21

WZXAGWREMCSWMF-HVATVPOCSA-N

InChI=1S/C24H41NO3/c1-15(4-9-22(27)25-28)19-7-8-20-18-6-5-16-14-17(26)10-12-23(16,2)21(18)11-13-24(19,20)3/h15-21,26,28H,4-14H2,1-3H3,(H,25,27)/t15-,16-,17-,18+,19-,20+,21+,23+,24-/m1/s1

(4R)-N-hydroxy-4-[(3R,5R,8R,9S,10S,13R,14S,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanamide

LCAHA; 117094-40-3; (4R)-N-hydroxy-4-[(3R,5R,8R,9S,10S,13R,14S,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanamide; Lithocholic acid hydroxyamide;

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 : ~45 mg/mL (~114.92 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.31 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: ≥ 2.08 mg/mL (5.31 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 20.8 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.)