Table 1 Comparison of IC₅₀ values determined for various small molecule and stapled peptide inhibitors of Mdm2/Mdm4 in the following assays: (1) Mdm2/Mdm4:p53 bimodal FLUOPPI assay, (2) the Mdm2/4 and p53 NanoBIT PPI assays, (3) intracellular ATP determination assay (CellTiterGlow, PROMEGA) and (4) the LDH release assay (CytoToxOne, PROMEGA).

Compound	FLUOPPI Mdm2:p53 (IC50, µM)	FLUOPPI Mdm4:p53 (IC50, µM)	NanoBIT Mdm2:p53 (IC50, µM)	NanoBIT Mdm4:p53 (IC50, µM)	Viability (IC50, µM)	LDH Release (EC50, µM)
Small Molecules
Nutlin 3A	5.6 ± 0.4/4.6 ± 0.6	NA/NA	1.6 ± 0.1/2.7 ± 0.4	40.5 ± 6.0/40.8 ± 7.2	34.0 ± 7.0/28.5 ± 4.7	35.7 ± 6.2/ND
MI-773	1.1 ± 0.1/0.9 ± 0.1	NA/NA	0.27 ± 0.01/0.25 ± 0.01	10.2 ± 1.6/2.7 ± 0.4	NA/23.2 ± 0.02	30.0 ± 6.2/ND
RO-5963	NA/30.0 ± 1.3	NA/38.7 ± 2.1	87.3 ± 18.4/41.4 ± 5.9	54.7 ± 9.3/12.5 ± 3.8	NA/NA	NA/NA
Staple Peptides
VIP-82	38.0 ± 18.0/12.3 ± 1.9	20.6 ± 4.6/2.9 ± 0.2	3.0 ± 1.7/0.8 ± 0.1	1.2 ± 0.1/0.9 ± 0.1	NA/8.5 ± 1.1	NA/10.0 ± 1,0
VIP-82^SCRAM	NA/49.9 ± 16.6	NA/NA	17.7 ± 8.9/12.7 ± 2.0	6.0 ± 0.9/4.5 ± 1.2	NA/12.3 ± 1.0	21.3 ± 2.7/10.1 ± 2.2

The first value quoted in each table cell refers to a compound treatment period of either 1 hours in the case of small molecules and 4 hours in the case of the stapled peptides. The second value in each cells refers to a compound treatment period of 24 hours. NA = No activity. ND = Not determined. IC₅₀ values were not determined for titrations that sampled insufficient points for the derivation of accurate values. Table does not include IC₅₀ values derived for normalised data. IC₅₀s were also determined for Nutlin-3A against FLUOPPI Mdm2:p53 and Mdm4:p53 systems at 1 hour, independently of each system. These were 4.9 ± 0.6 µM and N/A, respectively. IC₅₀s were also determined for RO-5963 against FLUOPPI Mdm2:p53 and Mdm4:p53 at 24 hour, seperately as well. These were 37.7 ± 1.9 µM and 37.1 ± 2.0 µM, respectively.

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