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Supplementary data/figures Khanim et al

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. NADPH. NADPH. NADPH.

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Supplementary data/figures Khanim et al

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  1. Supplementary data/figures Khanim et al

  2. Supplementary Figure 1 Figure 1A: Schematic of the two-step fluorescent NADPH assay used for identifying compounds with AKR1C inhibitory activity NADPH NADPH NADPH 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.

  3. Supplementary Figure 2; Structure of AKR1C3 selective NCI compounds NSC 73100 NSC 75600 NSC 82147 NSC 211094

  4. Supplementary Figure 3; Structure of AKR1C3 selective CRT compounds A1 (CRT0036521) 3-(3,4-Dihydro-1H-isoquinoline-2-sulfonyl)-benzoic acid A6 (CRT0083914) 1-[4-(2-Methyl-piperidine-1-sulfonyl)-phenyl]-pyrrolidin-2-one A9 (CRT0093964) [4-(4-Chloro-phenyl)-piperazin-1-yl]-morpholin-4-yl-methanone

  5. Supplementary Table 1: Chemical Shift of A in DMSO

  6. Supplementary Table 2: Chemical Shift of B in DMSO

  7. Supplementary Table 3: 15N Chemical Shift of A To confirm that N(CH3)2 group is only present in A but not B,15N HSQC and long range 15N HSQC were conducted. For A, we identified the NH2 group in 15N HSQC and both NH2 and N(CH3)2 groups in the long range 15N HSQC (Table 3) whereas for B, only the NH2 group was identified regardless of whether we conduct a normal or long range 15N HSQC. Furthermore, additional resonances in 13C HSQC matched well the methyl group identified in the 13C direct observed experiment of B . To confirm that bond shift rearrangement that altered A into B, 13C HSQC was conducted. The CH group signatures at position 4 and 4a of A were completely absent in B.

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