Human Aldo-keto Reductase 1C3 Research Articles

Design and Biological Evaluation of Piperazine-Bearing Nitrobenzamide Hypoxia/GDEPT Prodrugs: The Discovery of CP-506.

Off-target aerobic activation of PR-104A by human aldo-keto reductase 1C3 (AKR1C3) has confounded the development of this dual hypoxia/gene therapy prodrug. Previous attempts to design prodrugs resistant to AKR1C3 activation have resulted in candidates that require further optimization. Herein we report the evaluation of a lipophilic series of PR-104A analogues in which a piperazine moiety has been introduced to improve drug-like properties. Octanol-water partition coefficients (LogD7.4) spanned >2 orders of magnitude. 2D antiproliferative and 3D multicellular clonogenic assays using isogenic HCT116 and H1299 cells confirmed that all examples were resistant to AKR1C3 metabolism while producing an E. coli NfsA nitroreductase-mediated bystander effect. Prodrugs 16, 17, and 20 demonstrated efficacy in H1299 xenografts where only a minority of tumor cells express NfsA. These prodrugs and their bromo/mesylate counterparts (25-27) were also evaluated for hypoxia-selective cell killing in vitro. These results in conjunction with stability assays recommended prodrug 26 (CP-506) for Phase I/II clinical trial.

In vitro evaluation of the reductase activities of human AKR1C3 allelic variants.

Aldo-keto reductase 1C3 (AKR1C3) plays a role in the detoxification and activation of clinical drugs by catalyzing reduction reactions. There are approximately 400 single nucleotide polymorphisms in the AKR1C3 gene, but their impact on the enzyme activity is still unclear. This study aimed to clarify the effects of single nucleotide polymorphisms of AKR1C3 with more than 0.5% global minor allele frequency on the reductase activities for its typical substrates. Recombinant AKR1C3 wild-type and R66Q, E77G, C145Y, P180S, or R258C variants were constructed using insect Sf21 cells, and reductase activities for acetohexamide, doxorubicin, and loxoprofen by recombinant AKR1C3s were measured by LC‒MS/MS. Among the variants tested, the C145Y variant showed remarkably low (6-14% of wild-type) intrinsic clearances of reductase activities for all three drugs. Reductase activities of these three drugs were measured using 34 individual Japanese liver cytosols, revealing that heterozygotes of the SNP g.55101G>A tended to show lower reductase activities for three drugs than homozygotes of the wild-type. Furthermore, genotyping of the SNP g.55101G>A causing C145Y in 96 Caucasians, 166 African Americans, 192 Koreans, and 183 Japanese individuals was performed by polymerase chain reaction-restriction fragment length polymorphism. This allelic variant was specifically detected in Asians with allele frequencies of 6.8% and 3.6% in Koreans and Japanese, respectively. To conclude, an AKR1C3 allele with the SNP g.55101G>A causing C145Y would be one of the causal factors for interindividual variabilities in the efficacy and toxicity of drugs reduced by AKR1C3. Significance Statement This is the first study to clarify that the AKR1C3 allele with the SNP g.55101G>A causing C145Y results in a decrease in reductase activity. Since the allele was specifically observed in Asians, the allele would be a factor causing an interindividual variability in sensitivity of drug efficacy or toxicity of drugs reduced by AKR1C3 in Asians.

X-ray structure of human aldo-keto reductase 1C3 in complex with a bile acid fused tetrazole inhibitor: experimental validation, molecular docking and structural analysis.

Aldo-keto reductase 1C3 (AKR1C3) catalyzes the reduction of androstenedione to testosterone and reduces the effectiveness of chemotherapeutics. AKR1C3 is a target for treatment of breast and prostate cancer and AKR1C3 inhibition could be an effective adjuvant therapy in the context of leukemia and other cancers. In the present study, steroidal bile acid fused tetrazoles were screened for their ability to inhibit AKR1C3. Four C24 bile acids with C-ring fused tetrazoles were moderate to strong AKR1C3 inhibitors (37-88% inhibition), while B-ring fused tetrazoles had no effect on AKR1C3 activity. Based on a fluorescence assay in yeast cells, these four compounds displayed no affinity for estrogen receptor-α, or the androgen receptor, suggesting a lack of estrogenic or androgenic effects. A top inhibitor showed specificity for AKR1C3 over AKR1C2, and inhibited AKR1C3 with an IC50 of ∼7 μM. The structure of AKR1C3·NADP+ in complex with this C-ring fused bile acid tetrazole was determined by X-ray crystallography at 1.4 Å resolution, revealing that the C24 carboxylate is anchored to the catalytic oxyanion site (H117, Y55); meanwhile the tetrazole interacts with a tryptophan (W227) important for steroid recognition. Molecular docking predicts that all four top AKR1C3 inhibitors bind with nearly identical geometry, suggesting that C-ring bile acid fused tetrazoles represent a new class of AKR1C3 inhibitors.

Abstract 5691: Preclinical evaluation of TFX05-01, a selective AKR1C3- targeted prodrug for solid tumor

Abstract Objective: Aldo-keto reductase 1C3 (AKR1C3), is a member of the aldo-keto reductase superfamily, catalyzing the NADP(H)-dependent stereospecific reduction of various aldehydes and ketones. AKR1C3 is overexpressed in a variety of cancer cells, including hepatocellular carcinoma (HCC), castration-resistant prostate cancer (CRPC), and leukemia cells. This study is to investigate the in vitro and in vivo antitumor activity of TFX05-01, a selective AKR1C3-activated anticancer prodrug, in preclinical models of AKR1C3 overexpressing cells. The studies provide solid foundation for nomination of TFX05-01 for clinical trials. Method: Enzymatic activation of TFX05-01 was investigated in vitro using recombinant human AKR1C3. Cellular anti-proliferative activity was evaluated with a range of AKR1C3 overexpressed or low expressed cell lines. The in vivo antitumor activity was evaluated in various CDX mouse models with high expression of AKR1C3, including HepG2 and castration-resistant prostate VCaP, as well as PDX leukemia models Result: TFX05-01 is a prodrug which selectively releases a DNA alkylating agent (TFX05-01A) upon exposure to AKR1C3. TFX05-01 inhibited cell proliferation in several AKR1C3 overexpressed HCC cell lines with an IC50 ≤ 100 nM. In contrast, TFX05-01 showed much lower anti-proliferative activity in low or no AKR1C3 expressed cells with an IC50 value greater than 1 μM. The cytotoxic potency of TFX05-01 in cancer cells correlates with the level of AKR1C3 expression, and the cytotoxic activity of TFX05-01 was inhibited when used in combination with a specific AKR1C3 inhibitor (IC50: 0.7 nM without inhibitor vs 188 nM with inhibitor in SNU-878 cells). TFX05-01 showed antitumor efficacy in AKR1C3 highly expressed HepG2 subcutaneously implanted xenograft model (TGI = 97% @2 mpk, QW, 3 doses IV). Concomitant PK studies showed that the concentration of active metabolites (TFX05-01A) in tumor was significantly higher (15~50 times) than that in plasma, which correlates with the selective release of the active DNA alkylating agent upon exposure to AKR1C3 in tumor tissues. Conclusion: TFX05-01 is a novel selective AKR1C3-activated prodrug, which is different from traditional non-selective alkylating agents for cancer therapy. Preclinical studies have shown profound in vivo efficacy of TFX05-01 in AKR1C3 overexpressed mouse tumor xenograft models without marked toxicity. TFX05-01 represents a novel treatment option for AKR1C3 expressing solid cancers. Preclinical GLP safety evaluations were promising and TFX05-01 was nominated for clinical trials. Citation Format: Charles Z Ding, Zhe Cai, Wei Sha. Preclinical evaluation of TFX05-01, a selective AKR1C3- targeted prodrug for solid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5691.

Restoring Tumour Selectivity of the Bioreductive Prodrug PR-104 by Developing an Analogue Resistant to Aerobic Metabolism by Human Aldo-Keto Reductase 1C3.

PR-104 is a phosphate ester pre-prodrug that is converted in vivo to its cognate alcohol, PR-104A, a latent alkylator which forms potent cytotoxins upon bioreduction. Hypoxia selectivity results from one-electron nitro reduction of PR-104A, in which cytochrome P450 oxidoreductase (POR) plays an important role. However, PR-104A also undergoes ‘off-target’ two-electron reduction by human aldo-keto reductase 1C3 (AKR1C3), resulting in activation in oxygenated tissues. AKR1C3 expression in human myeloid progenitor cells probably accounts for the dose-limiting myelotoxicity of PR-104 documented in clinical trials, resulting in human PR-104A plasma exposure levels 3.4- to 9.6-fold lower than can be achieved in murine models. Structure-based design to eliminate AKR1C3 activation thus represents a strategy for restoring the therapeutic window of this class of agent in humans. Here, we identified SN29176, a PR-104A analogue resistant to human AKR1C3 activation. SN29176 retains hypoxia selectivity in vitro with aerobic/hypoxic IC50 ratios of 9 to 145, remains a substrate for POR and triggers γH2AX induction and cell cycle arrest in a comparable manner to PR-104A. SN35141, the soluble phosphate pre-prodrug of SN29176, exhibited superior hypoxic tumour log cell kill (>4.0) to PR-104 (2.5–3.7) in vivo at doses predicted to be achievable in humans. Orthologues of human AKR1C3 from mouse, rat and dog were incapable of reducing PR-104A, thus identifying an underlying cause for the discrepancy in PR-104 tolerance in pre-clinical models versus humans. In contrast, the macaque AKR1C3 gene orthologue was able to metabolise PR-104A, indicating that this species may be suitable for evaluating the toxicokinetics of PR-104 analogues for clinical development. We confirmed that SN29176 was not a substrate for AKR1C3 orthologues across all four pre-clinical species, demonstrating that this prodrug analogue class is suitable for further development. Based on these findings, a prodrug candidate was subsequently identified for clinical trials.

Role of Human Aldo-Keto Reductases in the Metabolic Activation of the Carcinogenic Air Pollutant 3-Nitrobenzanthrone.

3-Nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen detected in diesel exhaust particulate and ambient air pollution. It requires metabolic activation via nitroreduction to promote DNA adduct formation and tumorigenesis. NAD(P)H:quinone oxidoreductase 1 (NQO1) has been previously implicated as the major nitroreductase responsible for 3-NBA activation, but it has recently been reported that human aldo-keto reductase 1C3 (AKR1C3) displays nitroreductase activity toward the chemotherapeutic agent PR-104A. We sought to determine whether AKR1C isoforms could display nitroreductase activity toward other nitrated compounds and bioactivate 3-NBA. Using discontinuous enzymatic assays monitored by UV-HPLC, we determined that AKR1C1-1C3 catalyze three successive two-electron nitroreductions toward 3-NBA to form the reduced product 3-aminobenzanthrone (3-ABA). Evidence of the nitroso- and hydroxylamino- intermediates were obtained by UPLC-HRMS. Km, kcat, and kcat/ Km values were determined for recombinant AKR1C and NQO1 and compared. We found that AKR1C1, AKR1C3, and NQO1 have very similar apparent catalytic efficiencies (8 vs 7 min-1 mM-1) despite the higher kcat of NQO1 (0.058 vs 0.012 min-1). AKR1C1-1C3 possess a Km much lower than that of NQO1, which suggests that they may be more important than NQO1 at the low concentrations of 3-NBA to which humans are exposed. Given that inhalation represents the primary source of 3-NBA exposure, we chose to evaluate the relative importance of AKR1C1-1C3 and NQO1 in human lung epithelial cell lines. Our data suggest that the combined activities of AKR1C1-1C3 and NQO1 contribute equally to the reduction of 3-NBA in A549 and HBEC3-KT cell lines and together represent approximately 50% of the intracellular nitroreductase activity toward 3-NBA. These findings have significant implications for the metabolism of nitrated polycyclic aromatic hydrocarbons and suggest that the hitherto unrecognized nitroreductase activity of AKR1C enzymes should be further investigated.

In situ proteolysis of an N-terminal His tag with thrombin improves the diffraction quality of human aldo-keto reductase 1C3 crystals.

Human aldo-keto reductase 1C3 (AKR1C3) stereospecifically reduces steroids and prostaglandins and is involved in the biotransformation of xenobiotics. Its role in various cancers makes it a potential therapeutic target for the development of inhibitors. Recombinant AKR1C3 with a thrombin-cleavable N-terminal His6 tag was expressed from a pET-28(+) vector for structural studies of enzyme-inhibitor complexes. A modified in situ proteolysis approach was applied to specifically remove the His tag by thrombin cleavage during crystallization screening trials. This improved the morphology and diffraction quality of the crystals and allowed the acquisition of high-resolution diffraction data and structure solution. This approach may be generally applicable to other proteins expressed using the pET-28(+) vector.

Abstract 4580A: Metabolic activation of 3-nitrobenzanthrone by human aldo-keto reductases (AKR1C1-AKR1C4)

Abstract In 2012, the International Agency for Research on Cancer (IARC) classified diesel exhaust as a Group 1 carcinogen due to sufficient evidence that exposure is associated with increased risk for lung cancer in humans. However, only a subset of individuals exposed to diesel exhaust develops cancer, indicating the need to identify the genes involved in metabolic activation of these compounds and their genetic variants. Nitro-polycyclic aromatic hydrocarbons (NO2-PAH) are a major component of diesel exhaust and require metabolic activation to exert their carcinogenic activity. A representative NO2-PAH, 3-nitrobenzanthrone (3-NBA), is metabolically activated to 3-aminobenzanthrone (3-ABA) via a 6-electron nitroreduction catalyzed by NQO1 and POR. The reaction leads to the formation of 3-aminobenzanthrone (3-ABA) derived DNA adducts which promote G to T transversions. Building upon previous data that shows human aldo-keto reductase 1C3 (AKR1C3) contains nitroreductase activity towards chemotherapeutic agents (Guise, C.P., Abbattista, M.R., et al., Cancer Res, 70(4), 2010), we chose to examine the nitroreductase activity of AKR1C1-AKR1C4 towards NO2-PAH. We have demonstrated here for the first time that AKR1C enzymes catalyze the nitroreduction of 3-NBA to 3-ABA. We monitored reactions with reverse phase HPLC coupled to in-line photo-diode-array detection (PDA) and fluorescence detection (FLD) to quantify 3-NBA and 3-ABA levels. Fluorescence and UV spectroscopy were used to validate the identity of the compounds. This method was adapted for discontinuous enzymatic assays to measure steady state kinetic parameters for the nitoreductase activity of AKR1C1-AKR1C4 and NQO1. Results indicate that the NQO1 catalyzed reduction of 3-NBA has a higher specific activity, but the combined specific activities of AKR1C catalyzed reduction may play a more significant role in the overall production of 3-ABA. These results suggest that the relative expression of NQO1 and AKR1C enzymes will determine their respective contribution to 3-NBA reduction, especially since all the aforementioned enzymes are inducible by the Nrf2-Keap1 system. This work is supported by P30E513508 and RO1 CA39504 to TMP. Citation Format: Jessica R. Murray, Meng Huang, Tianzhu Zang, Volker M. Arlt, Heinz H. Schmeiser, Trevor M. Penning. Metabolic activation of 3-nitrobenzanthrone by human aldo-keto reductases (AKR1C1-AKR1C4). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4580A. doi:10.1158/1538-7445.AM2015-4580A

ASP9521, a novel, selective, orally bioavailable AKR1C3 (type 5, 17ß-hydroxysteroid dehydrogenase) inhibitor: In vitro and in vivo characterization.

5046 Background: Aldo–keto reductase 1C3 (AKR1C3), also known as type 5, 17β-hydroxysteroid dehydrogenase, is reported to be highly expressed in human normal prostate and prostate cancer (PC) and the expression increases along with increasing malignancy or grade of PC. Since AKR1C3 converts the adrenal androgen, androstenedione (AD) into testosterone (T), combination with a GnRH analogue and AKR1C3 inhibitor would be expected to provide total androgen blockade in the treatment of castration-resistant prostate cancer (CRPC). We obtained a lead compound having a non-steroidal scaffold by high throughput screening (HTS) approaches for targeting enzyme activity of AKR1C3. After optimization of the lead compound, we found ASP9521 as a potent, selective, and orally bioavailable AKR1C3 inhibitor. Methods: The inhibitory effect of ASP9521 on enzymatic conversion from AD to T by AKR1C3 was evaluated in vitro, and in CWR22R xenograft models. Effect of PSA production and proliferation on LNCaP cells stably expressing AKR1C3 was examined. Pharmacokinetics in various animals were also investigated. Results: ASP9521 showed potent inhibitory effect on enzymatic conversion from AD to T by both human AKR1C3 and cynomolgus monkey homologues in a concentration-dependent manner, with IC50 values of 11 and 49 nmol/L, respectively ASP9521 suppressed both AD-dependent PSA production and cell proliferation in LNCaP cells exogenously expressing AKR1C3 in vitro. The bioavailability of ASP9521after oral administration of 1mg/kg were 30% and 78% in rat and dog, respectively. Furthermore, ASP9521 single oral administration of 3 mg/kg suppressed AD-induced intratumoral T production in CWR22R xenografted castrate nude mice, and this inhibitory effect was maintained for 24 h. In addition, ASP9521 was rapidly eliminated from plasma after oral administration while its intratumoral concentration remained high in tumors expressing AKR1C3. Conclusions: These preclinical in vitro and in vivo data are consistent with a potent inhibition of 17β-hydroxysteroid dehydrogenase. The results suggest that ASP9521 should be investigated further to elucidate its role as treatment for PC.

Aldo-keto reductase 1C3 may be a new radioresistance marker in non-small-cell lung cancer

Human aldo-keto reductase 1C3, type 2 3α-hydroxysteroid dehydrogenase (HSD)/type 5 17β-HSD (AKR1C3) is known to be involved in steroid, prostaglandin and lipid aldehyde metabolism. The role of AKR1C3 in the radiosensitivity to X-rays of human non-small-cell lung cancer (NSCLC) cells was explored. In this study, a specific small interfering RNA (siRNA) to target the AKR1C3 gene was used. A suite of readouts including cell survival were determined using a colony formation assay; apoptosis evaluated by Annexin V expression levels, irradiation-induced cytotoxicity established using a MTT cell viability assay and cell cycle distribution measured by flow cytometry were used in characterizing the role of the AKR1C3 gene. Although AKR1C3 was significantly overexpressed in both our radioresistant subclone cells and NSCLC tissues, a specific AKR1C3 siRNA significantly enhanced cell radiosensitivity and was concomitant with decreased expression of this gene. Furthermore, reduced interleukin-6 (IL-6)-mediated radioresistance was observed when siRNA was used to knock down AKR1C3 activity. This AKR1C3-mediated radioresistance was correlated with an arrest in the G2/M cell cycle and a decreased induction of apoptosis. AKR1C3 may present a potential therapeutic target in addressing radioresistance of NSCLC, and in particular in IL-6-mediated radioresistance.

Abstract SY34-01: New prodrugs for targeting tumor hypoxia

Abstract SY34-01: New prodrugs for targeting tumor hypoxia

The Bioreductive Prodrug PR-104A Is Activated under Aerobic Conditions by Human Aldo-Keto Reductase 1C3

PR-104, currently in phase II clinical trials, is a phosphate ester pre-prodrug which is converted in vivo to its cognate alcohol, PR-104A, a prodrug designed to exploit tumor hypoxia. Bioactivation occurs via one-electron reduction to DNA crosslinking metabolites in the absence of oxygen. However, certain tumor cell lines activate PR-104A in the presence of oxygen, suggesting the existence of an aerobic nitroreductase. Microarray analysis identified a cluster of five aldo-keto reductase (AKR) family members whose expressions correlated with aerobic metabolism of PR-104A. Plasmid-based expression of candidate genes identified aldo-keto reductase 1C3 as a novel nitroreductase. AKR1C3 protein was detected by Western blot in 7 of 23 cell lines and correlated with oxic PR-104A metabolism, an activity which could be partially suppressed by Nrf2 RNAi knockdown (or induced by Keap1 RNAi), indicating regulation by the ARE pathway. AKR1C3 was unable to sensitize cells to 10 other bioreductive prodrugs and was associated with single-agent PR-104 activity across a panel of 9 human tumor xenograft models. Overexpression in two AKR1C3-negative tumor xenograft models strongly enhanced PR-104 antitumor activity. A population level survey of AKR1C3 expression in 2,490 individual cases across 19 cancer types using tissue microarrays revealed marked upregulation of AKR1C3 in a subset including hepatocellular, bladder, renal, gastric, and non-small cell lung carcinoma. A survey of normal tissue AKR1C3 expression suggests the potential for tumor-selective PR-104A activation by this mechanism. These findings have significant implications for the clinical development of PR-104.

Developmental Evaluation of Aldo-keto Reductase 1C3 Expression in the Cryptorchid Testis

To characterize aldo-keto reductase 1C3 (AKR1C3) expression in surgically-removed cryptorchid testes. Human AKR1C3 is a monomeric, cytoplasmic, multifunctional enzyme that reduces ketosteroids, ketoprostaglandins, and lipid aldehydes. AKR1C3 expression has been demonstrated in normal adult Leydig cells and peritubular fibromyocytes. No prior studies have evaluated AKR1C3 expression patterns across different age groups. Surgically excised cryptorchid testes were subjected to hematoxylin-eosin evaluation to review overall testicular pathology. These samples were then evaluated for AKR1C3 distribution via immunohistochemical staining with a monospecific AKR1C3 monoclonal antibody. Single-pathologist grading of AKR1C3 expression was correlated with clinical presentations. A total of 16 cryptorchid testes were identified from the 2000 to present. Median age at the time of surgery was 9 years (range, 1-17). The testes were excised due to atrophy, poor tissue consistency, and short spermatic cord length. AKR1C3 expression was identified at all ages in fibromyocytes surrounding the testicular tubules. Leydig cell expression was absent at Tanner stages 1 and 2, while strong expression was found at Tanner stage 3 and beyond. No AKR1C3 expression was seen in germ cells at any stage. AKR1C3 expression was identified in 18%-26% of Sertoli cells in patients at Tanner stages 2 and beyond. AKR1C3 expression occurs in a Tanner stage dependent-fashion. Pre- and peri-pubertal changes appear to promote expression of the enzyme in Leydig cells. In contrast to the normal adult testis, the pediatric cryptorchid testis reveals AKR1C3 expression in Sertoli cells. Additional studies are warranted to determine the involvement of AKR1C3 in testicular descent and development.

473 POSTER The bioreductive prodrug PR-104 is activated under aerobic conditions by human aldo-keto reductase 1C3 (prostaglandin F synthase)

473 POSTER The bioreductive prodrug PR-104 is activated under aerobic conditions by human aldo-keto reductase 1C3 (prostaglandin F synthase)

Regulation of human aldoketoreductase 1C3 (AKR1C3) gene expression in the adipose tissue

Aldoketoreductase 1C3 (AKR1C3) is a functional prostaglandin F synthase and a negative modulator of the availability of ligands for the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARγ). AKR1C3 expression is known to be associated with adiposity, one of the components of the metabolic syndrome. The aim of this study was to characterize the expression of AKR1C3 in the adipose tissue and adipocytes and to investigate its potential role in the metabolic syndrome. Using microarray analysis and realtime PCR, we studied the expression of AKR1C3 in adipose tissue samples from obese subjects with or without metabolic complications, during very low calorie diet-induced weight loss, and its expression in isolated human adipocytes of different sizes. The adipose tissue AKR1C3 expression levels were marginally lower in obese subjects with the metabolic syndrome compared with the levels in healthy obese subjects when analyzed using microarray (p = 0.078) and realtime PCR (p < 0.05), suggesting a secondary or compensatory effect. The adipose tissue mRNA levels of AKR1C3 were reduced during and after dietinduced weight-loss compared to the levels before the start of the diet (p < 0.001 at all time-points). The gene expression of AKR1C3 correlated with both adipose tissue mRNA levels and serum levels of leptin before the start of the diet (p < 0.05 and p < 0.01, respectively). Furthermore, large adipocytes displayed a higher expression of AKR1C3 than small adipocytes (1.5-fold, p < 0.01). In conclusion, adipose tissue AKR1C3 expression may be affected by metabolic disease, and its levels are significantly reduced in response to dietinduced weight loss and correlate with leptin levels.