Table 1. In Vitro Inhibitory Activity of Tested Compounds against BoNT/A LC.

	BoNT/A LC
compd	% inhibitiona	IC50 (μM)	Ki (μM)e
NSC 240898b	73.31c	2.62
12	74.00	3.80	6.99 ± 0.46
13	81.43	2.28	5.67 ± 0.37
14	69.04
15	83.35
16	69.44	4.68
17	69.88	5.18
18	80.83
19	62.88
22	67.09	5.70
23	72.86	2.32
24	81.45
25	77.23
20	33.28		34.51 ± 4.72
21	61.07	4.48	7.69 ± 0.39
36	73.95	2.46
37	77.30	4.92
38	74.38	5.09
39	72.92	3.25
40	67.37	4.90
41	74.91	3.24
42	33.75	24.97
43	55.64	8.21
44	29.97	6.78
45	59.63	6.62
50	93.51	1.02
51	92.59	1.34
52	89.34	0.81	3.22 ± 0.32
53	84.80	1.04	3.45 ± 0.35
54	89.48	1.14
55	81.38	1.13
56	85.54	2.27
57	86.86	1.29
60	82.22	3.05
59	6.42
61	92.81	0.63
62	96.47		0.341 ± 0.042
63	97.24		0.171 ± 0.013
64	96.68		0.300 ± 0.065
65	89.57		0.389 ± 0.059
66	95.46		0.285 ± 0.056
67	95.43		0.103 ± 0.024
68	93.70		0.300 ± 0.059
9d	6.51

^aPercent inhibition calculated at 20 μM. Percent inhibition measurements were performed in duplicate, and standard deviations were less than (25%) for all. IC₅₀ calculations were determined by measuring enzyme activity at nine different SMNPI concentrations and in the absence of the small molecule. The small molecule concentrations used in the measurements were determined by estimating the IC₅₀ value and moving in 1 log increments in either direction of the estimated value.

^bNSC240898, used as the control for comparison, displayed dose dependent inhibition of BoNT/A induced SNAP-25 cleavage in neurons with no toxicity at concentrations as high as 40 μM (ref (57)).

^cAverage value from more than 20 measurements.

^dTested as diphosphate salt.

^eFor K_i determination, reaction velocity versus substrate concentration was plotted for multiple small molecule concentrations. These plots were analyzed using global kinetic analysis. Subsequently, the data were fit to a model of competitive inhibition and analyzed by nonlinear regression analysis.