A high-throughput screen for alpha particle radiation protectants

Abstract

Alpha-particle-emitting elements are of increasing importance as environmental and occupational carcinogens, toxic components of radiation dispersal devices and accidents, and potent therapeutics in oncology. Alpha particle radiation differs from radiations of lower linear energy transfer in that it predominantly damages DNA via direct action. Because of this, radical scavengers effective for other radiations have had only limited effect in mitigating alpha particle toxicity. We describe here a simple assay and a pilot screen of 3,119 compounds in a high-throughput screen (HTS), using the alpha-particle-emitting isotope, ²²⁵Ac, for the discovery of compounds that might protect mammalian cells from alpha particles through novel mechanisms. The assay, which monitored the viability of a myeloid leukemic cell line upon alpha particle exposure, was robust and reproducible, yielding a Z' factor of 0.66 and a signal-to-noise ratio of nearly 10 to 1. Surprisingly, 1 compound emerged from this screen, epoxy-4,5-α-dihydroxysantonin (EDHS), that showed considerable protective activity. While the value of EDHS remains to be determined, its discovery is a proof of concept and validation of the utility of this HTS methodology. Further application of the described assay could yield compounds useful in minimizing the toxicity and carcinogenesis associated with alpha particle exposure.

Fig. 1.

Assay optimization and prevalidation. (A) HL60 cell density was titrated from 19,200 cells/well (circle) (EC₅₀ = 2201 nCi/mL, 95% CI 1,114–4,347 nCi/mL); 9600 cells/well (diamond) (EC₅₀ = 1,130 nCi/mL, 95% CI 903.5–1,413 nCi/mL); 4,800 cells/well (inverted triangle) (EC₅₀ = 573.5 nCi/mL, 95% CI 469.1–701.2 nCi/mL); 2400 cells/well (triangle) (EC₅₀ = 392.7 nCi/mL, 95% CI 331.6–465.1 nCi/mL); and 1,200 cells/well (square) (EC₅₀ = 252.2 nCi/mL, 95% CI 214.3–371.5 nCi/mL) in the presence of increasing amounts of ²²⁵Ac to determine optimal Alamar Blue conditions at 48 h. (B) Dimethylsulfoxide (DMSO) at 1% final concentration (v/v) (diamond) (EC₅₀ = 166.8 nCi/mL, 95% CI 82.2–338.5 nCi/mL) had no discernable effect on HL60 ²²⁵Ac toxicity compared to the normal culture medium (square) (EC₅₀ = 275.8 nCi/mL, 95% CI 165.9–458.4 nCi/mL) at 48 h. (C) Prevalidation results of HL60 representing triplicate 384-well plates. Untreated cells containing only 1% DMSO (v/v) served as positive controls (pc), and compared to high controls (hc) containing EC₅₀ ²²⁵Ac (150 nCi/mL), and low controls (lc) containing 25 μM staurosporine in 0.1% sodium dodecyl sulfate. In all panels, data presented are the mean value (n ≥ 3), and error bars indicate ± standard error of the mean (SEM). EC₅₀ values were determined by nonlinear regression curve fitting, sigmoidal dose–response model, using GraphPad Prism software.

Fig. 2.

High-throughput assay results. Collated internal high (hc, open circle) and low (lc, triangle) control data from successive (A and B) validation runs with LEADseeker™ Multimodality Imaging System. (C) Duplicate results from same assays for 3,119 validation compounds were plotted on opposite axes as indicated, expressed as percent inhibition of growth. Protective compounds are in the lower left quadrant, toxic compounds are in the upper right quadrant, and compounds with no effect are at the center. Protective compounds were selected from the indicated circular region, and epoxy-4,5-α-dihydroxysantonin was found to be the most protective compound (arrow).

Fig. 3.

Titration of select compounds with Alamar Blue (AB) reagent. Thirteen selected compounds were titrated in the presence of ²²⁵Ac and assayed by AB reagent to confirm screen results. Epoxy-4,5-α-dihydroxysantonin was found to have a clear dose response (olive square) that was over 2 standard deviations (dashed lines) above the mean (solid line), whereas others, lanatoside C (orange circle) and rotenone (magenta square), were found to be toxic. Remaining compounds are 5α-cholestan-3β-ol-6-on (green diamond), quercetin (light green diamond), nerol (magenta diamond), enoxolone (magenta cross), gandleoidin acetate (green circle), flumethazone pivalate (orange square), formononetin (violet inverted triangle), naphazoline hydrochloride (blue circle), aconitine (blue square), and cuneatin methyl ether (olive diagonal cross). All values are means (n = 5), and error bars indicate ± SEM.

Fig. 4.

Epoxy-4,5-α-dihydroxysantonin (EDHS) protective and toxic effects. (A) Forty-eight-hour Alamar Blue (AB) assay with HL60 compared to (B) a 24-h ATP-Lite assay, (C) RV+ in AB at 48 h, and (D) RV+ in a 24-h ATP-Lite assay, with 150 nCi/mL ²²⁵Ac (triangle) or no radioactivity (circle), and normalized to vehicle control with no ²²⁵Ac. *P value < 0.05 for ²²⁵Ac-treated samples. ⁺P value of <0.05 for unirradiated samples, as compared to vehicle control, by one-way ANOVA with Bonferroni's multiple comparison posttest. For all panels, values are means (n ≥ 3), and error bars indicate ± SEM.

Fig. 5.

Comparison of epoxy-4,5-α-dihydroxysantonin (EDHS) (A) to its parent compound, santonin (B). The structure of the sesquiterpene lactone parent compound santonin compared to the epoxidated derivative, EDHS. (C) In an ATP-Lite assay with the HL60 variant, RV+ cells, no protective or mitogenic activity was detected in the absence of ²²⁵Ac (circle) and presence (triangle) of isotope. Means were not significantly different among all groups. (D) Treatment of HL60 (open) or RV+ cells (closed) with EDHS has no effect on nuclide accumulation. EDHS (square) or 1% dimethylsulfoxide (v/v) (circle) was incubated along with ²²⁵Ac for indicated time points before pellets were washed and counted for gamma activity. For each panel, values are means (n ≥ 3), and error bars indicate ± SEM.