Selective AKR1C3 inhibitors do not recapitulate the anti-leukaemic activities of the pan-AKR1C inhibitor medroxyprogesterone acetate

Abstract

Background: We and others have identified the aldo-keto reductase AKR1C3 as a potential drug target in prostate cancer, breast cancer and leukaemia. As a consequence, significant effort is being invested in the development of AKR1C3-selective inhibitors.

Methods: We report the screening of an in-house drug library to identify known drugs that selectively inhibit AKR1C3 over the closely related isoforms AKR1C1, 1C2 and 1C4. This screen initially identified tetracycline as a potential AKR1C3-selective inhibitor. However, mass spectrometry and nuclear magnetic resonance studies identified that the active agent was a novel breakdown product (4-methyl(de-dimethylamine)-tetracycline (4-MDDT)).

Results: We demonstrate that, although 4-MDDT enters AML cells and inhibits their AKR1C3 activity, it does not recapitulate the anti-leukaemic actions of the pan-AKR1C inhibitor medroxyprogesterone acetate (MPA). Screens of the NCI diversity set and an independently curated small-molecule library identified several additional AKR1C3-selective inhibitors, none of which had the expected anti-leukaemic activity. However, a pan AKR1C, also identified in the NCI diversity set faithfully recapitulated the actions of MPA.

Conclusions: In summary, we have identified a novel tetracycline-derived product that provides an excellent lead structure with proven drug-like qualities for the development of AKR1C3 inhibitors. However, our findings suggest that, at least in leukaemia, selective inhibition of AKR1C3 is insufficient to elicit an anticancer effect and that multiple AKR1C inhibition may be required.

Figure 1

Identification of a novel tetracycline breakdown product. (A) A panel of 100 clinically licensed drugs (FMC100) at peak serum concentrations was screened for AKR1C-inhibitory activity against recombinant purified AKR1C proteins using the AKR1C-diaphorase assay. Data shown is mean fluorescence arbitrary units. Black bars indicate tetracycline and the dashed line represents level of inhibition by 5 μM MPA. GC-MS and NMR were used to elucidate the structure of the novel tetracycline breakdown product 4-methyl,(didemethyl)-tetracycline (4-MDDT). (B) Structures and names of the parent tetracycline and novel tetracycline breakdown product. (C) The isoform specificity of HPLC-purified 4-MDDT was determined against recombinant AKR1C proteins using the AKR1C-daphorase assay (see Materials and Methods). (D) The IC50 of 4-MDDT for recombinant AKR1C3 protein was determined using the PQ assay (see Materials and Methods).

Figure 2

In silico docking of 4-MDDT into the crystal structure of AKR1C3. (A) Autodock was used to dock 4-MDDT into our previously published AKR1C3 crystal structure after flufenamic acid was removed (PDB ID 1S2C)[39]. The 4-MDDT is coloured by atom type (green: carbon, red: oxygen, white: hydrogen) and shown as sticks, with magnesium a green sphere. The NADP⁺ cofactor is coloured yellow and the protein is rendered as an aquamarine cartoon ribbon. Side chains identified as potentially providing specificity are shown as sticks and labelled. (B) Close-up of the predicted binding sites and key interacting amino-acid residues. (C) Protein Sequence alignment of AKR1C1-4 with the key interacting amino-acid residues highlighted in boxes.

Figure 3

4-MDDT does not recapitulate the actions of MPA against AML cell lines. HL-60, K562 and KG1a cells were treated for 7 days with either solvent control, 50 μM 4-MDDT or 5 μM MPA, alone or in combination with 0.5 mM BEZ. (A) Cumulative cell counts for HL60, K562 and KG1a after 7 days treatment. Data shown is mean of a minimum of N=3 experiments±s.e.m. (B and D) HL60 differentiation was assessed at day 7 by staining for CD11b and flow cytometry following treatment with either solvent control, 50 μM 4-MDDT or 5 μM MPA with or without 0.5 mM BEZ or 10 nM ATRA. Data shown is mean of a minimum of N=3 experiments±s.e.m. (C) Cytospins of HL60 cells treated for 7 days as described above were stained with Jenner–Giemsa to demonstrate changes in cell morphology. (E) Intracellular AKR1C3 11β-prostaglandin reductase activity in KG1a cells was assessed in the presence of a dose titration of 4-MDDT or 5 μM MPA using ³H-PGD₂ and thin layer chromatography. Data shown is mean of N=4 experiments±s.e.m. *P<0.01.

Figure 4

4-MDDT does not recapitulate the actions of MPA against primary CLL cells. Peripheral blood mononuclear cells from CLL patients were cultured on non-CD40L-expressing stroma in a ratio of 10 : 1 in 96-well plates. Treatments with either solvent control, 0.5 mM BEZ, 5 μM MPA, BEZ/MPA (BaP), 5.1 μM 4-MDDT, MPA/4-MDDT or BEZ/4-MDDT were performed in triplicate for 24 h, before harvesting and staining with Annexin V (AV) and propidium iodide (PI), and analysis by flow cytometry. Plot shown is for n=4 samples+s.e.m.

Figure 5

Identification of AKR1C3-selective inhibitors from the NCI Diversity Set. (A) NCI compounds were screened against recombinant AKR1C proteins using the AKR1C-diaphorase assay (see Materials and Methods) and % inhibition of activity measured. IC50s of selected compounds against all four AKR1C isoforms, shown below the bar chart, were calculated using dose titrations of each compound against recombinant AKR1C protein in the NADPH assay. (B) Cumulative cell counts for HL60, K562 and KG1a after 7 days treatment with either solvent control, 5 μM MPA, or the selected compounds at 10 times the IC50 concentration, without (top three panels) or with 0.5 mM BEZ (bottom three panels). Data shown is mean of a minimum of N=3 experiments±s.e.m. *P<0.01.

Figure 6

AKR1C3-selective inhibitors from CRT also do not recapitulate the cellular effects of MPA. (A) Dose titrations of A1, A6 or A9 were used to determine the IC50 of the selected CRT compounds, for recombinant AKR1C3 protein as determined using the PQ assay (see Materials and Methods). (B) Cumulative cell counts for HL60, K562 and KG1a after 7 days treatment with either solvent control, 0.5 mM BEZ, BAP (0.5 mM BEZ+5 μM MPA), or 10 μM A1/A6/A9 with 0.5 mM BEZ. Data shown is mean of a minimum of N=3 experiments±s.e.m. *P<0.01.

Figure 7

A pan-AKR1C inhibitor NCI-PI recapitulates the cellular effects of MPA. (A and B) Cumulative cell counts for HL60 and K562 after 7 days treatment with either solvent control, 0.5 mM BEZ, 5 μM MPA, BaP (0.5 mM BEZ+5 μM MPA), 25 μM NCI-PI alone and 25 μM NCI-PI+0.5 mM BEZ. (C) HL60 differentiation was assessed at day 7 by staining for CD11b and flow cytometry following treatments as above. Data shown is mean of a minimum of N=3 experiments±s.e.m. *P<0.01.