S. currently unable to tolerate the side effects of cancer drug regimens. retinoic acid (ATRA) is an effective differentiation agent in acute promyelocytic leukemia (APL), but its effects are limited to this one subtype of AML.10 The identification of a therapeutic agent with activity across all AML subtypes would have substantial clinical implications. Primary AML cells and the AML cell lines HL-60 (APL, M3 subtype) and KG1a (AML, M0 subtype) predominantly express AKR1C3 with median levels Rabbit Polyclonal to EPB41 (phospho-Tyr660/418) two orders of magnitude greater than the AKR1C1 isoform and more than three orders of magnitude greater than the AKR1C2 isoform.11?13 Strong expression of AKR1C3 is also detected in I-BRD9 nonmalignant proliferating CD34+ve cells isolated from peripheral blood.13 Overall, this data identifies AKR1C3 as the primary AKR1C isoform encountered in myeloid progenitors and indicates a critical role in the regulation of myelopoiesis. Pharmacological inhibition of AKR1C3 holds the promise of an adjuvant effect, sensitizing leukemic cells to the cytotoxic action of chemotherapeutics delivered synergistically. Combination of the weak I-BRD9 and nonselective pan-AKR1C inhibitor medroxyprogesterone acetate (AKR1C3 pIC50 = 5.6) and bezafibrate demonstrated an approximate 2-fold potentiation of cytotoxic activity.14 A recent study reported that the specific AKR1C3 inhibitor 4-MDDT (pIC50 = 6.3) does not give the adjuvant effect at concentrations up to 50 M that a pan-AKR1C inhibitor does, I-BRD9 despite the low expression of other isoform in AML cells, casting doubt I-BRD9 on the validity of AKR1C3 as a therapeutic target in AML.15 The structurally distinct natural product baccharin (1, Figure ?Figure11a) demonstrates highly potent inhibitory activity for AKR1C3 (pIC50 = 7.0). Critically, baccharin exhibits exquisite selectivity with absolutely no inhibition of the AKR1C1 or AKR1C2 isoforms.16 Hydrolysis of the ester moiety of 1 1 provides the known phenol drupanin (1a, Figure S7), which possesses attenuated AKR1C3 inhibitory activity (pIC50 = 4.8) with only 7-fold selectivity for AKR1C3 over the 1C2 isoform. Molecular modeling of baccharin in the active site of AKR1C3 predicts formation of a hydrogen bond between the ester carbonyl and the active site Tyr55 residue.16 Ester bond hydrolysis is a primary feature of metabolism and the magnitude in the reduction of potency for drupanin highlights the unsuitable pharmacokinetics of baccharin as a drug candidate or chemical probe.17 Open in a separate window Figure 1 Synergistic activity of AKR1C3 inhibitors with etoposide in HL-60 cells following 72 h coincubation. Values are the mean SD (= 6). The two-tailed test analysis was used to compare the statistical difference between control and treatments; ns, not significant; * 0.01, ** 0.05, *** 0.001, **** 0.0001. We sought to evaluate the suitability of baccharin and rationally designed hydrolytically more stable derivatives as chemical probes to evaluate adjuvant I-BRD9 effects in AML cell lines. To this end, we replaced the ester bond of 1 1 with the hydrolytically more stable amide bioisostere (2, Scheme 1). Commercially available 4-iodoaniline (5) was brominated and the desired 0.0001) cytotoxic at 50 M. At the lower concentrations employed in this study (0.1C1 M) 4 showed only a 8% reduction of cell viability. The toxicity of AKR1C3 inhibitor 4 may be attributed to its high potency for AKR1C3 enzyme inhibition that leads to cell death, as has been reported for similarly potent AKR1C3 inhibitors in prostate cancer cells.23 When the AKR1C3 inhibitors were exposed to the KG1a AML cell line (AML M0 subtype), which has much greater expression of AKR1C3 (Figure S3), cytotoxicity was observed at concentrations above 25 M for inhibitors 1C3 indicating direct toxicity to AML cells upon inhibition of AKR1C3 (Figure S4). Potent inhibitor 4 showed 20% reduction of cell viability at just 1 M in the KG1a cell line. The clinically approved drug etoposide, employed as a second line chemotherapeutic to manage AML24,25 was chosen as the cytotoxic agent. The use of etoposide ensures that any potentiation of cytotoxicity would be attributable to AKR1C3 inhibition rather than prevention of AKR1C3-mediated metabolism of the chemotherapeutic agent. A doseCresponse curve of etoposide was obtained in HL-60 and KG1a cells (Figures S5 and S6). In accordance with the literature, 0.1 M of etoposide provides no cytotoxic effect in HL-60 cells.26.