Supplementary data/figures Khanim et al

Supplementary Figure 1 Figure 1A: Schematic of the two-step fluorescent NADPH assay used for identifying compounds with AKR1C inhibitory activity Figure 1B: Phenanthrenequinone (PQ) is a very stable non-limiting substrate for identification of inhibitors of AKR enzymes. 15 μg recombinant AKR1C3 protein was incubated in 50mM potassium phosphate buffer (pH 6.5) and 20μM PQ at 35°and OD340nm measured following the addition of β-NADPH. The product appears to spontaneously oxidise back to PQ because the initial rate of NADPH consumption was restored when fresh NADPH was added to the assay after 20mins (arrowheads). In these assays, NADPH was added in excess compared to PQ at each time-point. NADPH

Supplementary Figure 2; Structure of AKR1C3 selective NCI compounds NSC NSC NSC NSC

A1 (CRT ) 3-(3,4-Dihydro-1H-isoquinoline-2-sulfonyl)-benzoic acid A6 (CRT ) 1-[4-(2-Methyl-piperidine-1-sulfonyl)-phenyl]-pyrrolidin-2-one A9 (CRT ) [4-(4-Chloro-phenyl)-piperazin-1-yl]-morpholin-4-yl-methanone Supplementary Figure 3; Structure of AKR1C3 selective CRT compounds

Supplementary Table 1: Chemical Shift of A in DMSO

Supplementary Table 2: Chemical Shift of B in DMSO

To confirm that N(CH 3 ) 2 group is only present in A but not B, 15 N HSQC and long range 15 N HSQC were conducted. For A, we identified the NH 2 group in 15 N HSQC and both NH 2 and N(CH 3 ) 2 groups in the long range 15 N HSQC (Table 3) whereas for B, only the NH 2 group was identified regardless of whether we conduct a normal or long range 15 N HSQC. Furthermore, additional resonances in 13 C HSQC matched well the methyl group identified in the 13 C direct observed experiment of B. To confirm that bond shift rearrangement that altered A into B, 13 C HSQC was conducted. The CH group signatures at position 4 and 4a of A were completely absent in B. Table 3: 15 N Chemical Shift of A 15 N δ in ppmAssignment 105.1NH N Supplementary Table 3: 15 N Chemical Shift of A