Table 5.

Mode of Inhibition of Cysteine-less Mouse MDR3 P-Glycoprotein by Group 1 and Group 2 Compound 29 Variants^a

compound	stimulated ATPase (% DMSO, significance)		effect on stimulated ATPase	basal ATPase (% DMSO, significance)		effect on basal ATPase	cellular accumulation: ratio of plus tariquidar over no tariquidar		transport substrate for P-gp	maximum ATP binding (mol of SL-ANP bound/mol of P-gp)	SL-ANP binding to P-gp, apparent Kd (μM)	effect on SL-ANP binding
DMSO	100 ± 8			100 ± 7						1.8 ± 0.1	36.5 ± 3.6
SMU29	49 ± 2	**	inhibitor	95 ± 11	NS	none	1.0	NS	no	1.9 ± 0.1b	71.0 ± 12.0	no
Group 1: ChemGen- and docking-selected
SMU29–216	108 ± 2	NS	none	105 ± 7	NS	none	1.1	NS	no	1.7 ± 0.1	23.1 ± 3.9	no
SMU29–227	50 ± 2	**	inhibitor	88 ± 4	NS	none	1.0	NS	no	1.8 ± 0.1	36.9 ± 4.1	no
SMU29–231	141 ± 18	*	stimulator	70 ± 0	*	inhibitor	0.9	NS	no	1.6 ± 0.1	22.2 ± 3.8	marginally
SMU29–541	68 ± 7	*	inhibitor	101 ± 12	NS	none	1.0	NS	no	1.8 ± 0.1	24.1 ± 4.0	no
SMU29–551	62 ± 5	**	inhibitor	150 ± 6	**	stimulator	1.1	*	no	1.7 ± 0.1	22.8 ± 3.6	no
Group 2: rationally designed/no docking selection
SMU29–238	194 ± 22	**	stimulator	1143 ± 46	**	stimulator	1.9	***	yes	1.2 ± 0.1	20.1 ± 4.0	yes
SMU29–255	123 ± 15	NS	none	355 ± 7	***	stimulator	1.2	*	yes	1.9 ± 0.1	25.6 ± 4.7	no
SMU29–278	41 ± 7	**	inhibitor	78 ± 4	*	inhibitor	1.0	NS	no	1.3 ± 0.1	22.9 ± 4.5	yes
SMU29–280	116 ± 4	*	stimulator	148 ± 4	*	stimulator	1.2	NS	yes	1.6 ± 0.1	21.6 ± 4.6	marginally
SMU29–286	98 ± 2	NS	none	143 ± 32	NS	none	1.3	*	yes	1.9 ± 0.1	25.7 ± 4.9	no

^aATP hydrolysis assays using purified P-gp were performed without added transport substrate (“basal ATPase”) or in the presence of verapamil (“stimulated ATPase”). Results are presented compared to DMSO control ± standard deviation (three independent experiments with duplicate samples). The basal activity of P-gp was 20 to 30 nmol min⁻¹ mg⁻¹; verapamil-stimulated rates were 200 to 400 nmol min⁻¹ mg⁻¹ of P-gp. Stimulation of basal ATPase by 29-variants was used as an indicator that a compound may be a P-gp transport substrate. Effects on stimulated P-gp ATPase activity indicated whether a compound directly interfered with ATP usage by the protein (***, p < 0.001; **, p < 0.01; *, p < 0.1; NS, not significant). Quantitative cellular accumulation of 29-variants was performed using LC-MS/MS (Experimental Section) and is presented as a ratio of the cellular amounts of 29-variants in the presence of the P-gp inhibitor tariquidar, divided by amounts accumulated in its absence. A ratio of >1 indicates that the compound likely is a transport substrate of P-gp (***, very significant; *, significant; NS, not significant). The binding of an ATP analog, SL-ATP, to P-gp was used to determine whether ATP binding to P-gp was affected by the 29-variants (see Experimental Section). Values ± standard deviations are shown for at least three different P-gp preparations and three independent SL-ATP titration experiments.

^bThe values for SL-ATP binding in the presence of 29 were taken directly from Brewer et al.²⁶