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, 23 (17), 5999-6013

Vinyl Sulfone Analogs of Lysophosphatidylcholine Irreversibly Inhibit Autotaxin and Prevent Angiogenesis in Melanoma

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Vinyl Sulfone Analogs of Lysophosphatidylcholine Irreversibly Inhibit Autotaxin and Prevent Angiogenesis in Melanoma

Mandi M Murph et al. Bioorg Med Chem.

Abstract

Autotaxin (ATX) is an enzyme discovered in the conditioned medium of cultured melanoma cells and identified as a protein that strongly stimulates motility. This unique ectonucleotide pyrophosphatase and phosphodiesterase facilitates the removal of a choline headgroup from lysophosphatidylcholine (LPC) to yield lysophosphatidic acid (LPA), which is a potent lipid stimulator of tumorigenesis. Thus, ATX has received renewed attention because it has a prominent role in malignant progression with significant translational potential. Specifically, we sought to develop active site-targeted irreversible inhibitors as anti-cancer agents. Herein we describe the synthesis and biological activity of an LPC-mimetic electrophilic affinity label that targets the active site of ATX, which has a critical threonine residue that acts as a nucleophile in the lysophospholipase D reaction to liberate choline. We synthesized a set of quaternary ammonium derivative-containing vinyl sulfone analogs of LPC that function as irreversible inhibitors of ATX and inactivate the enzyme. The analogs were tested in cell viability assays using multiple cancer cell lines. The IC50 values ranged from 6.74 to 0.39 μM, consistent with a Ki of 3.50 μM for inhibition of ATX by the C16H33 vinyl sulfone analog CVS-16 (10b). A phenyl vinyl sulfone control compound, PVS-16, lacking the choline-like quaternary ammonium mimicking head group moiety, had little effect on cell viability and did not inhibit ATX. Most importantly, CVS-16 (10b) significantly inhibited melanoma progression in an in vivo tumor model by preventing angiogenesis. Taken together, this suggests that CVS-16 (10b) is a potent and irreversible ATX inhibitor with significant biological activity both in vitro and in vivo.

Keywords: Angiogenesis; Anticancer therapy; Autotaxin; Lysophosphatidylcholine; Melanoma; Vinyl sulfone.

Figures

Figure 1
Figure 1
Synthesis of Quaternary Ammonium Vinyl Sulfone Analogs 10 (CVS-18, CVS-16). Reagents and conditions: (a) K2CO3, acetone, reflux, 2 h, 98%; (b)TrCl, triethylamine, dichloromethane, 94%;(c) mCPBA, dichloromethane, 0 °C to room temperature, 8 h, 95%; (d) NaH, RI, DMF, 72-88%; (e) LiAlH4, diethyl ether, at 0 °C, 10 min, 34-51%; (f) TFA, dichloromethane, 2 h, 66-81%; (g) MsCl, DIPEA, dichloromethane; (h) NaI, acetone; (i) DIPEA, EtOAc, 45-81% for three steps; (j) trimethylamine in EtOH, dichloromethane, 4 days, 71-85%.
Figure 2
Figure 2
Synthesis of Quaternary Ammonium Vinyl Sulfone Analogs 17 (CVS-12, CVS-6). Reagents and conditions: (a) K2CO3, acetone, reflux, 2 h, 89-93%; (b) LiAlH4, THF, at 60 °C, overnight, 88-90%; (c) m-CPBA, dichloromethane, 0 °C to room temperature, 8 h, 77-81%; (d) MsCl, DIPEA, dichloromethane; (e) NaI, acetone; (f) DIPEA, EtOAc, 69-82% for three steps; (g) trimethylamine in EtOH, dichloromethane, 4 days, 58-72%.
Figure 3
Figure 3
Synthesis of Non-quaternary ammonium Vinyl Sulfone 21 (PVS-16). Reagents and conditions: (a) NaH, C16H33I, DMF, 95%;; (b) LiAlH4, Diethyl ether, -78 °C, 15 min, 33%; (c) MsCl, DIPEA, dichloromethane; (d) NaI, acetone; (e) NaOtBu, EtOAc/DCM, 77% for three steps.
Figure 4
Figure 4
CVS-16 (10b) reduces cell viability in multiple cancer cell lines. Cell viability was measured using CellTiter® Blue after treating cells with the indicated concentration of CVS-16 (10b) for 48 h. The data is presented as a viability curve (A) and bar graph (B). *p<0.05 comparing 0 μM to CVS-16-treated condition for each cell line using the student’s t-test. The negative control, PVS-16 (21), a phenyl vinyl sulfone compound was used against multiple cancer cell lines. The data is presented as a viability curve (C) and bar graph (D).
Figure 5
Figure 5
ATX inhibitors reduce the cell viability of MeWo melanoma cells, in contrast with chemotherapy. Cell viability was measured using CellTiter® Blue after treating cells with the indicated concentration of: CVS-16 (10b), HA-130, PF-8380, dacarbazine (DTIC), paclitaxel (PTX/TAX), vincristine (VCR) and cisplatin (CDDP) for 48 h. *p<0.05 comparing CVS-16 (10b) to PF-8380-treated cells or **p<0.01 comparing CVS-16 (10b) to chemotherapy (DTIC, PTX/TAX, VCR and CDDP) using the student’s t-test.
Figure 6
Figure 6
CVS-16 (10b) blunts tumor progression by inhibiting angiogenesis in a melanoma xenograft model. (A) Animals with melanoma tumors were treated with the indicated concentrations of CVS-16 (10b) and established tumors were measured with calipers every other day. The arrow indicates the reduction of dosage from 50 mg/kg to 40 mg/kg on day 47 (see text for details). *p<0.05 and ***p<0.001, control (n=10) vs. 50-40 mg/kg (n=5). There was no significant difference among the control group (n=10) vs. 20 mg/kg (n=5). (B) Images of tumors on day 57 from control and treated animals. (C) Serum was collected and analyzed for KC/CXCL1. *p<0.05, control vs. 20 mg/kg and ***p<0.001, control vs. 50-40 mg/kg. (D) Quantification of specimens in the pathology report indicates the increase in tumor necrosis among animals treated with 50-40 mg/kg of CVS-16 (10b). **p<0.01, control vs. 40-50 mg/kg and *p<0.05, 20 mg/kg vs. 40-50 mg/kg. (E) Analysis of tumor specimens for regions with endothelial cells present indicates differences among the groups. (F) ATX was measured from the serum of treated and control animals. *p<0.05, comparing drug treated animals to controls using ANOVA followed by the Bonferroni multiple comparison test.
Figure 7
Figure 7
The autotaxin inhibitors HA-130 and PF-8380 did not impact tumor progression in a xenograft model of melanoma. Animals with melanoma tumors were treated with the indicated concentrations of either: HA-130 (A), PF-8380 (B) or dimethylformamide (DMF) solvent control. The established pigmented tumors were measured with calipers every other day. (C) ATX was measured from the serum of treated and control animals.

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