Inhibitory potencies of microcystin, DSPs and endothall analogs on [3H]CA binding in mouse brain and neuroblastoma cytosol and on viability of neuroblastoma cells
| No. and Inhibitor1-a | [3H]CA Binding IC50 | Viability LC50 N1E-115 cells | |
|---|---|---|---|
| Brain1-b | N1E-1151-c | ||
| nM | nM | ||
| Microcystin and DSPs | |||
| 1 Microcystin LR | 0.77 ± 0.03 | 0.70 ± 0.11 | >5,000 |
| 2 Okadaic acid | 1.2 ± 0.82 | 1.6 ± 0.9 | 31 ± 5 |
| 3 Calyculin A | 5.5 ± 1.1 | 5.8 ± 1.0 | 2.6 ± 0.3 |
| Endothall and analogs | |||
| 4 2,3-Dimethyl anhydride1-a | 8.2 ± 1.7 | 10 ± 2 | 3,900 ± 460 |
| 5 2-Methyl diacid1-a | 39 ± 25 | 52 ± 13 | 2,300 ± 100 |
| 6 Diacid1-a | 270 ± 6 | 254 ± 24 | 43,000 ± 6,000 |
| 7 Thioanhydride | 280 ± 60 | 270 ± 6 | 8,900 ± 3,100 |
| 8 Anhydride | 280 ± 17 | 270 ± 46 | 44,000 ± 2,000 |
| 9 5,6-Dehydro anhydride | 1,050 ± 70 | 1,190 ± 212 | 280,000 |
| 10 5-endo-Ethoxycarbonyl diacid | 2,170 ± 51 | 3,080 ± 1,006 | >300,000 |
| 11 5,6-exo-Epoxy diacid | 670,000 ± 103,000 | 536,900 ± 131,000 | >300,000 |
| 12 endo,endo-1,4-Dimethyl diacid | >500,000 | >500,000 | >300,000 |
| 13 5,6-Methylenedioxy diacid | >500,000 | >500,000 | >300,000 |
Cytosol of brain or N1E-115 cells was incubated (∼20 μg protein/tube) with 5 nM [3H]CA and various concentrations of test compound in 250 μl imidazole buffer (pH 7.0) for 2 hr at 37°C. Assays were terminated by filtration through 0.3% polyethylenimine-soaked glass fiber filters and counted by liquid scintillation. N1E-115 cells were plated in 100 μl of Dulbecco’s modified Eagle’s medium (104 cells/well) and incubated for 24 hr at 37°C under 5% CO2/95% air. Cell viability was estimated by the MTT method after exposing cells for a further 24 hr to various concentrations of test compound. Data are the means ± S.E. of three experiments.
↵1-a Structural formulae or type in figure 1. Compound 4 is cantharidin, 5 is palasonin as the diacid and 6 is endothall.
↵1-b Mouse i.p. LD50 values (mg/kg) are 1.0, 5.0, 14, 0.31, 4.0, 16 and 105 for 4, 5, 6, 7, 8, 9, and 10, respectively (Kawamura et al., 1990).
↵1-c IC50 values for the microsomal fraction (not given) divided by those for the cytosol (tabulated) are 1.3 ± 0.1 for compounds 1 to 8.