Assay Report Card

Basic Information

ID: ALA3888180

Type: Binding

Description: Homogenous Time-Resolved Fluorescence Assay (HTRF2 Assay): The standard assay conditions for the in vitro HTRF assay consisted of a 50 ul total reaction volume in black 384-well Costar polypropylene plates in 1xPBS buffer pH 7.4, 1 mM DTT, 0.1% BSA, 0.2 nM GST-hMDM2 (aa 1-188), 0.5 nM biotinylated-p53 (aa 1-83), 0.18 nM SA-XLent (Cisbio; Bedford, Mass.), 0.6 nM anti-GST cryptate monoclonal antibody (Cisbio; Bedford, Mass.) and 100 mM KF. Amino acid residues 1-188 of human MDM2 were expressed as an amino-terminal glutathione-5-transferase (GST) fusion protein (GST-hMDM2) in Escherichia coli. Residues 1-83 of human p53 were expressed as an amino-terminal AviTag-TrxA-6×His fusion protein (biotinylated p53) in E. coli. Each protein was purified from cell paste by affinity chromatography. Specifically, 10 uL of GST-hMDM2 was incubated with 10 ul of diluted compound (various concentrations, serially diluted) in 10% DMSO for 20 minutes at room temperature. 20 uL of biotinylated-p53 was added to the GST-hMDM2+compound mixture, and then incubated at room temperature for 60 min. 10 uL of detection buffer consisting of SA-XLent, anti-GST cryptate antibody and KF was added to GST-hMDM2, biotinylated-p53 and compound reaction and left at room temperature to reach equilibrium for >4 hrs. The final concentration of DMSO in the reaction was 2%. Time-resolved fluorescence readings were measured on a microplate multilabel reader. Percentage of inhibition was calculated relative to nutlin-3.

Activity Charts

Compound Summary

Parent Molecular WeightALogPPolar Surface Area
478.425.695.69
558.515.935.93
464.395.305.30
583.586.036.03
597.616.376.37
593.575.735.73
595.595.985.98
567.545.205.20
579.555.355.35
593.575.745.74
526.485.355.35
540.515.745.74
552.525.895.89
554.546.136.13
568.566.526.52
566.556.146.14
566.556.286.28
580.576.676.67
568.525.125.12
582.596.776.77
580.576.526.52
582.596.776.77
566.556.136.13
540.515.605.60
538.495.355.35
568.566.386.38
492.446.086.08
520.506.866.86
541.504.864.86
553.514.864.86
581.565.645.64
607.606.136.13
609.574.974.97
511.457.087.08
511.457.087.08
525.487.327.32
545.897.737.73
434.365.945.94
460.406.336.33
617.606.606.60
567.545.395.39
649.047.837.83
546.416.626.62
581.565.735.73
596.575.905.90
533.505.645.64
564.535.895.89
512.446.476.47
664.706.206.20
600.595.865.86
581.565.785.78
600.545.985.98
651.705.335.33
558.505.885.88
653.007.587.58
606.496.296.29
628.586.166.16
578.566.146.14
531.485.935.93
518.446.026.02
638.016.946.94
534.527.257.25
586.556.666.66
610.604.894.89
587.476.726.72
643.637.257.25
580.576.676.67
654.664.124.12
572.536.276.27
628.586.166.16
580.576.536.53
596.575.765.76
552.525.745.74
568.524.794.79
580.576.536.53
633.596.306.30
638.016.946.94
632.587.317.31
554.546.146.14
567.545.345.34
616.616.216.21
538.546.476.47
518.486.886.88
594.606.916.91
506.435.875.87
545.515.785.78
604.565.685.68
541.486.176.17
460.406.476.47
636.547.067.06
620.647.457.45
518.486.746.74
541.505.145.14
630.646.526.52
553.515.285.28
560.447.017.01
579.458.108.10
583.534.634.63
544.475.495.49
595.596.126.12
614.597.097.09
595.596.126.12
569.555.925.92
494.464.964.96
573.515.395.39
624.634.764.76
561.556.426.42
608.637.167.16
568.566.386.38
594.606.926.92
568.566.526.52
581.565.595.59
595.596.126.12
582.555.135.13
595.475.755.75
616.616.216.21
597.616.376.37
597.565.115.11
562.415.945.94
597.564.884.88
610.605.335.33
618.556.926.92
539.456.266.26
609.477.197.19
632.587.317.31
582.596.916.91
492.446.086.08
578.566.286.28
555.525.205.20
490.436.296.29
569.555.645.64
580.576.526.52
608.516.536.53
568.566.526.52
554.546.136.13
584.565.505.50
434.365.725.72
490.435.555.55
614.597.177.17
592.596.676.67
630.646.526.52
609.626.516.51
478.425.695.69
617.606.606.60
566.556.286.28
581.565.785.78
623.526.136.13
592.526.656.65
581.565.735.73
547.526.036.03
581.565.045.04
629.556.336.33
573.525.095.09
534.486.656.65
587.475.925.92
617.546.186.18
587.545.735.73
533.455.765.76
574.555.475.47
668.684.514.51
494.464.964.96
638.665.155.15
605.595.255.25
635.017.447.44
606.617.067.06
554.546.146.14
578.566.286.28
520.506.726.72
600.566.786.78
602.585.825.82
586.525.645.64
570.516.036.03
636.645.425.42
654.664.124.12
603.576.296.29
601.556.186.18
490.435.695.69
615.586.516.51
616.616.136.13
574.477.407.40
594.606.916.91
533.505.645.64
574.477.407.40
611.595.275.27
596.585.165.16
561.556.426.42
478.425.695.69
604.565.685.68
595.595.985.98
635.017.447.44
492.445.945.94
568.566.526.52
680.705.445.44
580.576.536.53
608.585.765.76
632.587.317.31
552.525.745.74
567.545.255.25
654.627.337.33
607.606.126.12
602.585.825.82
518.486.936.93
630.595.825.82
581.565.645.64
595.545.265.26
533.506.226.22
547.526.476.47
654.627.337.33
518.445.875.87
476.405.305.30
581.565.645.64
605.585.785.78
592.466.246.24
554.545.995.99
658.674.734.73
515.487.257.25
492.445.945.94
523.467.087.08
569.555.505.50
621.636.566.56
727.784.244.24
596.585.055.05
629.616.786.78
463.415.795.79
486.456.006.00
670.087.357.35
554.545.995.99
553.514.864.86
630.596.176.17
486.446.726.72
504.456.086.08
434.365.725.72
616.616.136.13
473.406.226.22
554.546.146.14
494.424.664.66
476.405.905.90
583.586.036.03
566.555.225.22
569.537.317.31
630.596.176.17
566.556.146.14
661.627.317.31
452.356.086.08
490.435.695.69
520.456.266.26
462.426.726.72
656.057.007.00
643.637.177.17
593.575.785.78
621.616.356.35
673.666.786.78
535.476.056.05
607.606.176.17
649.666.836.83
655.647.317.31
434.387.187.18
581.565.645.64
661.627.317.31
478.425.695.69
595.596.036.03
540.515.745.74
647.606.926.92
594.606.916.91
554.546.136.13
581.565.785.78
463.406.126.12
645.606.966.96
432.355.555.55
492.405.485.48
581.565.645.64
595.596.036.03
532.466.266.26
582.554.714.71
579.555.345.34
593.575.735.73
630.595.825.82
504.456.496.49
568.566.386.38
635.646.696.69
567.545.355.35
629.616.436.43