Table 2.

Comparison of High-Throughput Screening (293 Cells) vs Competition Assay (U2OS Cells)

Compound	Concentration Used, μM		Percent Change in Amax			Absolute Change in EC50			Mechanism
	HT	Competition	HT	Competition	Compare	HT	Competition	Compare
AC93253	0.37–9.2	0.3–10	−95	Down	Same	0.1	RS	Same	C
Baclofen	0.37–9.2	0.5–12.5	≈0a	≈0		1.1	RS	Same	C
Camptothecin	0.37–9.2	0.5–3	−50	Down	Same	0.7	RS	Same	C
Dihydroouabain	0.37–9.2	1–6	107	Up	Same	0.5	RS	Same	A
Emetine	0.37–9.2	0.01–0.16	BP	BP	Same	0.6	≈RS	Same	C
Nocodazole	0.37–9.2	3.4–34	69	Up	Same	−0.2	LS	Same	A
NU6027	0.37–9.2	2.5–25	110	Up	Same	0.1	≈RS	Same	A
Phenanthroline	0.37–9.2	2–10	BP	Up	—b	0.7	≈0		A
Sanguinarine	0.37–9.2	0.1–2	BP	≈0	—c	1.1	RS	Same	C
Stattic	0.37–9.2	1–10	≈0	Down		1.0	RS	Same	C
Agreement between assays					≥60%			90%

A, accelerator; BP, biphasic (increase above no compound and then decrease with higher concentrations of compound); C, competitive decelerator; HT, high-throughput; LS, left shift to lower concentrations of compound; RS, right shift to higher concentrations of compound; ≈RS, right shift observed most of the time. Results from the high-throughput screen (n = 1) and competition assays (n = 3–9) are given for the concentration range of compound in each assay, percent change in A_max from that with 100 nM Dex and no added compound, and absolute change in EC₅₀ of dose-response curve for either no added compound or lowest concentration of compound used (a positive number means a shift to higher concentrations of compound). Precise numbers are given for the high-throughput results, whereas qualitative changes are listed for the competition assay because of the varying concentration ranges used, which are usually different from those for the high-throughput screen. Whether the 2 assays give qualitatively the same results is listed under “Compare.” The kinetically determined mechanism (from Table 3) is given under “Mechanism.”

^a Activity with no Dex = 70% of maximal, with 100 nM Dex = 0% to 15% below maximal or 100% to 85% maximal.

^b Response in the high-throughput screen is small and biphasic: up 20% at 0.37 and 1.84 μM and then down 30% at 9.2 μM to 10% less than control.

^c Response in the high-throughput screen is biphasic: goes up from 100% at 0.37 μM to 325% at 1.84 μM, with 1.1 increase in EC₅₀, and then down to 0 at 9.2 μM.