Ketoreductase Research Articles

Thymol Mediates Bactericidal Activity against Staphylococcus aureus by Targeting an Aldo–Keto Reductase and Consequent Depletion of NADPH

Staphylococcus aureus is a common pathogen that can cause life-threatening infections. Treatment of antibiotic-resistant S. aureus infection needs effective antibacterial agents. Thymol, a generally recognized safe natural compound, has potential as an alternative to treat S. aureus infections. However, the targets and mechanisms of action of thymol were not fully understood. Bioinformatics analysis showed that IolS, a predicted aldo-keto reductase (AKR) in S. aureus, could be a potential target of thymol. Isothermal titration calorimetry (ITC) analysis demonstrated that thymol directly binds IolS and amino acid residues (Y30 and L33) are essential for such binding. Deletion of IolS or mutation of Y30A and L33A reduced the bactericidal activity of thymol at the concentration of 200 μg/mL, suggesting that thymol mediates bactericidal activity via binding with IolS. Biochemical analysis showed that addition of thymol significantly increased AKR activity of IolS from 1.6 ± 0.1 to 2.4 ± 0.2 U (p < 0.05). The content of NADPH within S. aureus cells decreased significantly from 105 ± 5 to 72 ± 3 pmol/108 cells (p < 0.05) following thymol treatment at the concentration of 200 μg/mL. Importantly, addition of NADPH could alleviate the bactericidal effect of thymol on S. aureus, indicating that the depletion of NADPH is responsible for thymol-mediated bactericidal activity. Overall, these results demonstrated that thymol could directly bind IolS and increase its AKR activity, leading to the depletion of NADPH and bactericidal effect. AKR activity of IolS could be a promising target for the development of new antimicrobials.

Chimeric 6-methylsalicylic acid synthase with domains of acyl carrier protein and methyltransferase from Pseudallescheria boydii shows novel biosynthetic activity.

SummaryPolyketides are important secondary metabolites, many of which exhibit potent pharmacological applications. Biosynthesis of polyketides is carried out by a single polyketide synthase (PKS) or multiple PKSs in successive elongations of enzyme‐bound intermediates related to fatty acid biosynthesis. The polyketide gene PKS306 from Pseudallescheria boydii NTOU2362 containing domains of ketosynthase (KS), acyltransferase (AT), dehydratase (DH), acyl carrier protein (ACP) and methyltransferase (MT) was cloned in an attempt to produce novel chemical compounds, and this PKS harbouring green fluorescent protein (GFP) was expressed in Saccharomyces cerevisiae. Although fluorescence of GFP and fusion protein analysed by anti‐GFP antibody were observed, no novel compound was detected. 6‐methylsalicylic acid synthase (6MSAS) was then used as a template and engineered with PKS306 by combinatorial fusion. The chimeric PKS containing domains of KS, AT, DH and ketoreductase (KR) from 6MSAS with ACP and MT from PKS306 demonstrated biosynthesis of a novel compound. The compound was identified with a deduced chemical formula of C7H10O3, and the chemical structure was named as 2‐hydroxy‐2‐(propan‐2‐yl) cyclobutane‐1,3‐dione. The novel compound synthesized by the chimeric PKS in this study demonstrates the feasibility of combinatorial fusion of PKS genes to produce novel polyketides.

Consecutive Prostate Cancer Specimens Revealed Increased Aldo⁻Keto Reductase Family 1 Member C3 Expression with Progression to Castration-Resistant Prostate Cancer.

Aldo-keto reductase family 1 member C3 (AKR1C3) is an enzyme in the steroidogenesis pathway, especially in formation of testosterone and dihydrotestosterone, and is believed to have a key role in promoting prostate cancer (PCa) progression, particularly in castration-resistant prostate cancer (CRPC). This study aims to compare the expression level of AKR1C3 between benign prostatic epithelium and cancer cells, and among hormone-naïve prostate cancer (HNPC) and CRPC from the same patients, to understand the role of AKR1C3 in PCa progression. Correlation of AKR1C3 immunohistochemical expression between benign and cancerous epithelia in 134 patient specimens was analyzed. Additionally, correlation between AKR1C3 expression and prostate-specific antigen (PSA) progression-free survival (PFS) after radical prostatectomy was analyzed. Furthermore, we evaluated the consecutive prostate samples derived from 11 patients both in the hormone-naïve and castration-resistant states. AKR1C3 immunostaining of cancer epithelium was significantly stronger than that of the benign epithelia in patients with localized HNPC (p < 0.0001). High AKR1C3 expression was an independent factor of poor PSA PFS (p = 0.032). Moreover, AKR1C3 immunostaining was significantly stronger in CRPC tissues than in HNPC tissues in the same patients (p = 0.0234). Our findings demonstrate that AKR1C3 is crucial in PCa progression.

Using Ketoreductases for the Chemoenzymatic Synthesis of a Stereotriad

Polyketides are diverse natural products of high biological activity produced from complex molecular assembly lines called polyketide synthases (PKSs). Some of the most important bioactive molecules are polyketides, including geldanamycin, doxycycline, tetracycline, and erythromycin. Other polyketides have been shown to have antifungal, antiparasitic, immunosuppressive, and even anticancer properties. Of particular focus here is the ketoreductase (KR), one of the many possible domains within PKSs. The KR reduces carbonyl groups stereoselectively and controls the majority of stereocenters in the polyketide product. KRs have been used in a bottom‐up approach here, harnessing their stereoselective reductions to produce products that would traditionally be difficult and time‐consuming to synthesize. A diketide was first synthesized chemically and reduced with a tylosin KR from Streptomyces fradiae (TylKR2). This reduced diketide was then extended into a triketide through the Masamune C‐acylation reaction. Finally, TylKR2 and a mycolactone KR from a bacterial artificial chromosome (MycKR6) were found to reduce S‐(2‐acetamidoethyl)(4R,5R)‐5‐methoxy‐4‐methyl‐3‐oxoheptanethioate to S‐(2‐acetamidoethyl) (3S,4S,5R)‐3‐hydroxy‐5‐methoxy‐4‐methylheptanethioate in the case of TylKR2 and to S‐(2‐acetamidoethyl) (3R,4S,5R)‐3‐hydroxy‐5‐methoxy‐4‐methylheptanethioate in the case of MycKR6. The reductions were performed in vitro using the glucose dehydrogenase regeneration system with NADP+. In this way, each of the 3 stereocenters was set chemoenzymatically using KRs to form a stereotriad. With similar KRs, it is likely that all 8 possible stereoisomers could be synthesized. Ultimately, these di‐ and triketide building blocks could be extremely useful functional groups to experiment with in drug synthesis and may result in more potent or effective medicines. Additionally, this work could be extended into a methodology for setting as many stereocenters as desired ‐ possibly one day allowing for customizable polyketide synthesis in the same way oligonucleotides and polypeptides can be made today.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Potent and Highly Selective Aldo-Keto Reductase 1C3 (AKR1C3) Inhibitors Act as Chemotherapeutic Potentiators in Acute Myeloid Leukemia and T-Cell Acute Lymphoblastic Leukemia.

Aldo-keto reductase 1C3 (AKR1C3) catalyzes the synthesis of 9α,11β-prostaglandin (PG) F2α and PGF2α prostanoids that sustain the growth of myeloid precursors in the bone marrow. The enzyme is overexpressed in acute myeloid leukemia (AML) and T-cell acute lymphoblastic leukemia (T-ALL). Moreover, AKR1C3 confers chemotherapeutic resistance to the anthracyclines: first-line agents for the treatment of leukemias. The highly homologous isoforms AKR1C1 and AKR1C2 inactivate 5α-dihydrotestosterone, and their inhibition would be undesirable. We report herein the identification of AKR1C3 inhibitors that demonstrate exquisite isoform selectivity for AKR1C3 over the other closely related isoforms to the order of >2800-fold. Biological evaluation of our isoform-selective inhibitors revealed a high degree of synergistic drug action in combination with the clinical leukemia therapeutics daunorubicin and cytarabine in in vitro cellular models of AML and primary patient-derived T-ALL cells. Our developed compounds exhibited >100-fold dose reduction index that results in complete resensitization of a daunorubicin-resistant AML cell line to the chemotherapeutic and >100-fold dose reduction of cytarabine in both AML cell lines and primary T-ALL cells.

Substituent Position‐Controlled Stereoselectivity in Enzymatic Reduction of Diaryl‐ and Aryl(heteroaryl)methanones

AbstractWe report here the discovery of a novel ketoreductase (KRED), named KmCR2, with a broad substrate spectrum on bioreduction of sterically bulky diaryl‐ and aryl(heteroaryl)methanones. The position of the substituent on aromatic rings (meta versus para or ortho) was revealed to control the stereospecificity of KmCR2. The stereoselective preparation of both enantiomers of diaryl‐ or aryl(heteroaryl)methanols using strategically engineered substrates with a traceless directing group (bromo group) showcased the potential application of this substrate‐controlled bioreduction reaction. The combined use of substrate engineering and protein engineering, was demonstrated to be a useful strategy in efficiently improving stereoselectivity or switching stereopreference of enzymatic processes.magnified image

Serum aldo–keto reductase family 1 member B10 predicts advanced liver fibrosis and fatal complications of nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is associated with liver inflammation in patients with nonalcoholic fatty liver disease, and it can progress to liver fibrosis at an advanced stage, as well as hepatocellular carcinoma (HCC) and portal hypertension. Although liver fibrosis is accurately diagnosed via biopsy, noninvasive methods are preferable. Aldo-keto reductase family 1 member B10 (AKR1B10) is associated with HCC and is secreted into the blood by liver cells via a lysosome-mediated nonclassical pathway. Accordingly, we analyzed whether secretion of AKR1B10 protein is associated with advanced NASH. We performed histological staging in 85 Matteoni classification type III and IV NASH patients and evaluated the incidence of HCC, formation of gastroesophageal varices, and prognosis according to serum AKR1B10 and Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA(+)-M2BP)(M2BPGi) and by comparison with conventional markers of fibrosis. A positive correlation was found between the Brunt classification and serum AKR1B10 level. In Brunt stage 4 patients, AKR1B10 levels were higher than those of other liver fibrosis markers, with higher specificity. The cutoff values for AKR1B10 and WFA(+)-M2BP for stage 4 fibrosis were 1.03 and 3.11, respectively. The rates of stage 4 fibrosis, HCC incidence, and gastroesophageal varix formation were significantly different between the two groups subdivided according to these cutoff levels. Moreover, the patients in the higher value group had significantly worse prognosis after NASH diagnosis CONCLUSION: AKR1B10 is a useful serum biomarker for advanced liver fibrosis in NASH and, combined with serum WFA(+)-M2BP, can predict HCC development, gastroesophageal varix formation, and poor prognosis.

Molecular basis of methylation and chain-length programming in a fungal iterative highly reducing polyketide synthase.

Exchange of 32 different sub-fragments of the C-methyltransferase (C-MeT), pseudo-ketoreductase (ΨKR) and ketoreductase (KR) catalytic domains of the tenellin iterative Type I polyketide synthase non ribosomal peptide synthetase (PKS-NRPS) TENS by homologous fragments from the desmethylbassianin (DMBS) and militarinone (MILS) PKS-NRPS led to the creation of chimeric synthetases in which programming fidelity was altered, resulting in the production of mixtures of products with different methylation patterns and chain lengths. Swap of KR domain subfragments with the homologous fragments from the KR of the heptaketide militarinone synthetase resulted in the synthesis of penta, hexa and heptaketides. The results of these and previous experiments are rationalised by considering the existence of competition for acyl-carrier protein (ACP) bound substrates between different catalytic domains of the PKS. In particular, competition between the C-MeT and ketoreductase domains (KR) can account for methylation programming, and competition between the KR and the off-loading NRPS accounts for chain-length selectivity.

Ketoreductase catalyzed stereoselective bioreduction of α-nitro ketones.

We report here the stereoselective bioreduction of α-nitro ketones catalyzed by ketoreductases (KREDs) with publicly known sequences. YGL039w and RasADH/SyADH were able to reduce 23 class I substrates (1-aryl-2-nitro-1-ethanone (1)) and ten class II substrates (1-aryloxy-3-nitro-2-propanone (4)) to furnish both enantiomers of the corresponding β-nitro alcohols, with good-to-excellent conversions (up to >99%) and enantioselectivities (up to >99% ee) being achieved in most cases. To the best of our knowledge, KRED-mediated reduction of class II α-nitro ketones (1-aryloxy-3-nitro-2-propanone (4)) is unprecedented. Select β-nitro alcohols, including the synthetic intermediates of bioactive molecules (R)-tembamide, (S)-tembamide, (S)-moprolol, (S)-toliprolol and (S)-propanolol, were stereoselectively synthesized in preparative scale with 42% to 90% isolated yields, showcasing the practical potential of our developed system in organic synthesis. Finally, the advantage of using KREDs with known sequence was demonstrated by whole-cell catalysis, in which β-nitro alcohol (R)-2k, the key synthetic intermediate of hypoglycemic natural product (R)-tembamide, was produced in a space-time yield of 178 g L-1 d-1 as well as 95% ee by employing the whole cells of a recombinant E. coli strain coexpressing RasADH and glucose dehydrogenase as the biocatalyst.

Exploration of [2 + 2 + 2] cyclotrimerisation methodology to prepare tetrahydroisoquinoline-based compounds with potential aldo-keto reductase 1C3 target affinity.

Tetrahydroisoquinoline (THIQ) is a key structural component in many biologically active molecules including natural products and synthetic pharmaceuticals. Here, we report on the use of transition-metal mediated [2 + 2 + 2] cyclotrimerisation of alkynes to generate tricyclic THIQs with potential to selectively inhibit AKR1C3.

Streptomyces avermitilis industrial strain as cell factory for Ivermectin B1a production

Ivermectin, a kind of valuable derivatives of Avermectin, is distinct from Avermectin due to the saturated bond at C22C23 position. Combinatorial biosynthesis of Ivermectins based on Avermectins biosynthetic gene cluster (ave) has been achieved recently, while the establishment of an Ivermectin homogeneous component producing strain is challenging because of the limited compatibility between the native and heterologous polyketide synthase (PKS) domains. In this study, the PKS module 2 Dehydratase (DH)-Enoylreductase (ER)-Ketoreductase (KR) domain set of Meilingmycin, which is another naturally occurring homologue of Avermectin, was employed to substitute the DH-KR domains of Avermectins PKS module 2 to generate an Ivermectin biosynthetic gene cluster (ive). Ivermectins B1a and A1a were heterologously biosynthesized in a classic actinomyces host Streptomyces lividans. The Avermectin B1a high-producing strain S. avermitilis 3-115 was genetically engineered to give an artificial host cell and Ivermectin B1a single component was effectively produced with a production of 1.25 ± 0.14 g/L.

Structural and Functional Studies of a gem-Dimethylating Methyltransferase from a trans-Acyltransferase Assembly Line.

The methyl substituents in products of trans-acyltransferase assembly lines are usually incorporated by S-adenosyl-methionine (SAM)-dependent methyltransferase (MT) domains. The gem-dimethyl moieties within the polyketide disorazol are installed through the iterative action of an MT in the third module of its assembly line. The 1.75-Å-resolution crystal structure of this MT helps elucidate how it catalyzes the addition of two methyl groups. Activity assays of point mutants on β-ketoacyl chains linked to an acyl carrier protein and N-acetylcysteamine provide additional insights into the roles of active site residues. The replacement of an alanine with a phenylalanine at an apparent gatekeeping position resulted in more monomethylation than dimethylation. MTs may form an interface with ketoreductases (KRs) and even mediate the docking of trans-acyltransferase assembly line polypeptides through this association.

Biochemical characterization of the minimal domains of an iterative eukaryotic polyketide synthase.

Iterative type I polyketide synthases (PKS) are megaenzymes essential to the biosynthesis of an enormously diverse array of bioactive natural products. Each PKS contains minimally three functional domains, β‐ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), and a subset of reducing domains such as ketoreductase (KR), dehydratase (DH), and enoylreductase (ER). The substrate selection, condensation reactions, and β‐keto processing of the polyketide growing chain are highly controlled in a programmed manner. However, the structural features and mechanistic rules that orchestrate the iterative cycles, processing domains functionality, and chain termination in this kind of megaenzymes are often poorly understood. Here, we present a biochemical and functional characterization of the KS and the AT domains of a PKS from the mallard duck Anas platyrhynchos (ApPKS). ApPKS belongs to an animal PKS family phylogenetically more related to bacterial PKS than to metazoan fatty acid synthases. Through the dissection of the ApPKS enzyme into mono‐ to didomain fragments and its reconstitution in vitro, we determined its substrate specificity toward different starters and extender units. ApPKS AT domain can effectively transfer acetyl‐CoA and malonyl‐CoA to the ApPKS ACP stand‐alone domain. Furthermore, the KS and KR domains, in the presence of Escherichia coli ACP, acetyl‐CoA, and malonyl‐CoA, showed the ability to catalyze the chain elongation and the β‐keto reduction steps necessary to yield a 3‐hydroxybutyryl‐ACP derivate. These results provide new insights into the catalytic efficiency and specificity of this uncharacterized family of PKSs.

In silico Prediction, Characterization, Molecular Docking, and Dynamic Studies on Fungal SDRs as Novel Targets for Searching Potential Fungicides Against Fusarium Wilt in Tomato.

Vascular wilt of tomato caused by Fusarium oxysporum f.sp. lycopersici (FOL) is one of the most devastating diseases, that delimits the tomato production worldwide. Fungal short-chain dehydrogenases/reductases (SDRs) are NADP(H) dependent oxidoreductases, having shared motifs and common functional mechanism, have been demonstrated as biochemical targets for commercial fungicides. The 1,3,6,8 tetra hydroxynaphthalene reductase (T4HNR) protein, a member of SDRs family, catalyzes the naphthol reduction reaction in fungal melanin biosynthesis. We retrieved an orthologous member of T4HNR, (complexed with NADP(H) and pyroquilon from Magnaporthe grisea) in the FOL (namely; FOXG_04696) based on homology search, percent identity and sequence similarity (93% query cover; 49% identity). The hypothetical protein FOXG_04696 (T4HNR like) had conserved T-G-X-X-X-G-X-G motif (cofactor binding site) at N-terminus, similar to M. grisea (1JA9) and Y-X-X-X-K motif, as a part of the active site, bearing homologies with two fungal keto reductases T4HNR (M. grisea) and 17-β-hydroxysteroid dehydrogenase from Curvularia lunata (teleomorph: Cochliobolus lunatus PDB ID: 3IS3). The catalytic tetrad of T4HNR was replaced with ASN115, SER141, TYR154, and LYS158 in the FOXG_04696. The structural alignment and superposition of FOXG_04696 over the template proteins (3IS3 and 1JA9) revealed minimum RMSD deviations of the C alpha atomic coordinates, and therefore, had structural conservation. The best protein model (FOXG_04696) was docked with 37 fungicides, to evaluate their binding affinities. The Glide XP and YASARA docked complexes showed discrepancies in results, for scoring and ranking the binding affinities of fungicides. The docked complexes were further refined and rescored from their docked poses through 50 ns long MD simulations, and binding free energies (ΔGbind) calculations, using MM/GBSA analysis, revealed Oxathiapiprolin and Famoxadone as better fungicides among the selected one. However, Famoxadone had better interaction of the docked residues, with best protein ligand contacts, minimum RMSD (high accuracy of the docking pose) and RMSF (structural integrity and conformational flexibility of docking) at the specified docking site. The Famoxadone was found to be acceptable based on in silico toxicity and in vitro growth inhibition assessment. We conclude that the FOXG_04696, could be employed as a novel candidate protein, for structure-based design, and screening of target fungicides against the FOL pathogen.

Membrane functionalization of polymersomes: alleviating mass transport limitations by integrating multiple selective membrane transporters for the diffusion of chemically diverse molecules

Recently, the interest in polymersomes as nanoreactors for synthetic applications has increased due to interesting proof-of-concept studies, indicating a versatile use of polymeric vesicles to compartmentalize complex reaction cascades. However, the low permeability of polymeric membranes and the requirement for a controlled mass transport across the compartment boundaries have posed a major limitation to the broad applicability of polymersomes for synthetic reactions. Current advances in the functional integration of membrane proteins (MPs) into poly(2-dimethylsiloxane)-based membranes have allowed the selective increase of the permeability for a controlled mass transport of the desired compounds across the membrane. Herein we demonstrate that polymer membranes are capable of harboring different MPs to alleviate the mass transport limitations of chemically diverse molecules, thereby enabling complex cascade reactions to be performed within the nanoreactors. The ability to functionalize the polymer membrane with multiple, highly selective MPs allows a reduction in mass transport limitations without abandoning compartmentalization of the reaction space on a low molecular mass level. As the model reaction, a two enzyme system consisting of a ketoreductase (KR) and a formate dehydrogenase was studied. For the transport of the hydrophobic substrate and product of the KR, the MPs AlkL, OmpW, OprG and TodX were investigated. For the transport of formate, OmpF, PhoE and FocA were used. AlkL showed the highest integration efficiency (39%) and a maximum of 120 AlkL molecules were successfully inserted into each polymersome. The highest channel-specific effects on the mass transfer were achieved using TodX and PhoE, respectively. The combination of both proteins led to an improvement of the space-time yield of the product (S)-pentafluorophenyl ethanol by 2.32-fold compared to nanoreactors without MPs.

Generation of novel family of reductases from PCR based library for the synthesis of chiral alcohols and amines

Biocatalysis has shown tremendous potential in the synthesis of drugs and drug intermediates in the last decade. Screening of novel biocatalysts from the natural genome space is the growing trend to replenish the harsh chemical synthetic routes, commonly used in the pharmaceutical and chemical industry. Here, we report a novel ketoreductase (KERD) and a nitrile reductase isolated from the PCR based library generated from the genome of Rhodococcus ruber and Bacillus subtilis, respectively. Both the proteins are hypothetical in nature as there is no putative homology found in the database, although both the enzymes have significant activity towards the synthesis of chiral alcohols and amines. Enzyme activity over a wide range of substrates (aromatic and aliphatic) for both the novel catalysts was observed. From the unique gene sequence to activity over a broad range of substrate and >99% conversion at higher concentrations (100 mM and above) entitles both the hypothetical enzymes as novel. The novel KERD has shown >99% selectivity for the synthesis of (S)-phenylethanol which makes it a potential candidate for industrial catalysis. The novel nitrile reductase has also shown promising activity for the synthesis of (R)-2-phenylethanolamine, which is a difficult moiety to synthesize chemically. In this report, starting from a homology based library, two highly potent whole cell biocatalysts are obtained.

Substrate-bound structures of a ketoreductase from amphotericin modular polyketide synthase.

Ketoreductase (KR) domains of modular polyketide synthases (PKSs) control the stereochemistry of C2 methyl and C3 hydroxyl substituents of polyketide intermediates. To understand the molecular basis of stereocontrol exerted by KRs, the crystal structure of a KR from the second module of the amphotericin PKS (AmpKR2) complexed with NADP+ and 2-methyl-3-oxopentanoyl-pantetheine was solved. This first ternary structure provides direct evidence to the hypothesis that a substrate enters into the active site of an A-type KR from the side opposite the coenzyme to generate an L-hydroxyl substituent. A comparison with the ternary complex of a G355T/Q364H mutant sheds light on the structural basis for stereospecificity toward the substrate C2 methyl substituent. Functional assays suggest the pantetheine handle shows obvious influence on the catalytic efficiency and the stereochemical outcome. Together, these findings extend our current understanding of the stereochemical control of PKS KR domains.

Chemoenzymatic Approaches to the Synthesis of the Calcimimetic Agent Cinacalcet Employing Transaminases and Ketoreductases.

Several chemoenzymatic routes have been explored for the preparation of cinacalcet, a calcimimetic agent. Transaminases (TAs) and ketoreductases (KREDs) turned out to be useful biocatalysts for the preparation of key optically active precursors. Thus, the asymmetric amination of 1‐acetonaphthone yielded an enantiopure (R)‐amine, which can be alkylated in one step to yield cinacalcet. Alternatively, the bioreduction of the same ketone resulted in an enantiopure (S)‐alcohol, which was easily converted into the previous (R)‐amine. In addition, the reduction was efficiently performed with the KRED and its cofactor co‐immobilized on the same porous surface. This self‐sufficient heterogeneous biocatalyst presented an accumulated total turnover number (TTN) for the cofactor of 675 after 5 consecutive operational cycles. Finally, in a preparative scale synthesis the TA‐based approach was performed in aqueous medium and led to enantiopure cinacalcet in two steps and 50% overall yield.

Evaluation of A-ring fused pyridine d-modified androstane derivatives for antiproliferative and aldo-keto reductase 1C3 inhibitory activity.

New A-ring pyridine fused androstanes in 17a-homo-17-oxa (d-homo lactone), 17α-picolyl or 17(E)-picolinylidene series were synthesized and validated by X-ray crystallography, HRMS, IR and NMR spectroscopy. Novel compounds 3, 5, 8 and 12 were prepared by treatment of 4-en-3-one or 4-ene-3,6-dione d-modified androstane derivatives with propargylamine catalyzed by Cu(ii), and evaluated for potential anticancer activity in vitro using human cancer cell lines and recombinant targets of steroidal anti-cancer drugs. Pyridine fusion to position 3,4 of the A-ring may dramatically enhance affinity of 17α-picolyl compounds for CYP17 while conferring selective antiproliferative activity against PC-3 cells. Similarly, pyridine fusion to the A-ring of steroidal d-homo lactones led to identification of new inhibitors of aldo-keto reductase 1C3, an enzyme targeted in acute myeloid leukemia, breast and prostate cancers. One A-pyridine d-lactone steroid 5 also has selective submicromolar antiproliferative activity against HT-29 colon cancer cells. None of the new derivatives have affinity for estrogen or androgen receptors in a yeast screen, suggesting negligible estrogenicity and androgenicity. Combined, our results suggest that A-ring pyridine fusions have potential in modulating the anticancer activity of steroidal compounds.

Allele frequency and genotype distribution of aldo keto reductase 1D1 (AKR1D1) rs1872930 genetic variant in a Macedonian population

AKR1D1 has been recently recognized as novel candidate gene regulating CYP450 coexpression. The AKR1D1 3’-UTR SNP (rs1872930) functional genetic variation was considered as important source of phenotypic variation in the activity of CYP450. The minor allele frequency of rs1872930 varies among populations suggesting population specific pharmacogenetic implications. The aim of the present study was to determine the AKR1D1 (rs1872930) minor allele frequencies and genotypes distribution in a population from Republic of Macedonia. Four hundred and fifty unrelated Macedonian subjects were studied. AKR1D1 variant allele was detected by real time polymerase chain reaction. The distribution of AKR1D1*1/*1, *1/*36 and *36/*36 genotypes was 0.569, 0.360 and 0.071, respectively. The overall frequency of AKR1D1*36 variant allele was 0.251. Our study is the first to assess the frequency distribution of the AKR1D1*36 (rs1872930) genetic variant in population from Republic of Macedonia and the findings are in accordance with the frequency reported for the Caucasian population. Keywords: AKR1D1, rs1872930, CYP450, variability, gene expression, enzyme activity