Aldo-keto Reductase Research Articles

Screening recombinant and wildtype solventogenic Clostridium species for in vivo transformation of methylglyoxal and acetol to 1,2-propanediol

Bioproduction of industrially relevant chemicals such as 1,2-propanediol (1,2-PD) is central to decarbonization of the economy. Currently, 1,2-PD is produced from fossil-derived feedstocks, contributing to CO2 emission and generation of hazardous wastes. In this study, Clostridium beijerinckii NCIMB 8052 (Cbei), C. pasteurianum ATCC 6013 (Cpas), and Clostridium tyrobutyricum ATCC 25755 (Ctyro) were screened for the ability to transform methylglyoxal and acetol—precursors of 1,2-PD—to 1,2-PD in cultures exogenously supplemented with both compounds. Only Cbei transformed methylglyoxal to 1,2-PD (0.08 g/L). Subsequently, multiple recombinant strains of Cbei were engineered to express methylglyoxal-targeting aldo-keto reductases [mgR, yeaE, yhdN (from Cpas), yqhD, and ydjG (from Enterobacter hormaechei UW0SKVC1), and glycerol dehydrogenase (gldA; from Ctyro]. With exogenous supplementation of methylglyoxal, the growth of the control strain of Cbei was impaired. Conversely, Cbei_ydjG, Cbei_yqhD, and Cbei_gldA showed 19.3 %, 130 % and 162 % enhanced growth following methylglyoxal challenge, respectively, whilst producing 0.100, 0.032, and 0.042 g/L 1,2-PD. Wildtype or recombinant strains of Cbei, Cpas, and Ctyro transformed ∼100 % of exogenously supplemented acetol to 1,2-PD. Cbei_mgsA+mgR co-expressing methylglyoxal synthase (mgsA) and mgR (both from Cpas) produced up to 88 % more butanol on both glucose and lactose than the control strains.

Safety, tolerability, pharmacokinetics and clinical activity of AST-3424, an AKR1C3-activated bis-alkylating moiety prodrug, in subjects with advanced solid tumors in China: A phase I dose-escalation study.

3121 Background: Aldo-keto reductase family 1 member C3 (AKR1C3) modulates cellular differentiation and proliferation through indirect regulation of ligand access to hormone and nuclear receptor signaling. AKR1C3 is expressed at high levels in various human cancers, including hepatocellular carcinoma (HCC). AST-3424 is an AKR1C3-activated prodrug and releases a toxic bis-alkylating moiety (AST-2660) in the presence of AKR1C3, which forms intra- and inter-strand DNA crosslinks resulting in cell death. Here we report the results of a phase I dose escalation study of AST-3424 in patients with advanced solid tumors (NCT06239155). Methods: Patients with histologically and/or cytologically confirmed advanced solid tumors who failed standard treatment or no standard treatment, or not suitable for standard treatment were enrolled to receive AST-3424 intravenously (i.v.) at doses of 1, 2, 4, 6, 8 mg/m2 on Day 1 and Day 8 every 21 days. A parallel and accelerated titration for 1 and 2 mg/m2, and 3+3 dose escalation design for other cohorts were used to guide dose escalation and maximum tolerable dose (MTD) determination. Results: 21 subjects were enrolled and received at least one treatment (HCC=4, colorectal cancer=6, gastric cancer=4, pancreatic carcinoma=2, breast cancer=2, intrahepatic cholangiocarcinoma=1, prostate cancer=1, salivary gland cancer=1). 4 dose-limiting toxicities (DLTs) were observed (3 grade 4 platelet count decreased; 1 grade 2 gamma-glutamyltransferase increased, DLT reported due to dosing delayed more than 14 days). Safety Review Committee confirmed 6.0 mg/m2 as the MTD and recommended Phase 2 dose (RP2D). Grade≥3 adverse events (AEs) were observed in in 10 patients (47.6%). 8 patients (38.1%) had grade≥3 treatment-related AEs (TRAEs), being anemia (33.3%), platelet count decreased (19.0%), white blood cell count decreased (9.5%) and neutrophil count decreased (9.5%). Most of the grade≥3 AEs were observed in the 8.0 mg/m2 cohort. No patient had a fatal TRAE. 15 subjects with baseline target lesions had at least one post baseline assessment. 9 subjects (42.9%) had stable disease (SD); 1 subject (4.8%) had non-complete remission/non-disease progression (Non-CR/Non-PD); 5 subjects (23.8%) had progressive disease (PD). AST-3424 showed linear pharmacokinetic characteristics in the range of 1.0-8.0 mg/m2 with a half-life of 0.16-0.40 hours. AST-2660 showed linear pharmacokinetic characteristics in the range of 4.0-8.0 mg/m2 with a half-life of 1.59-2.08 hours. Conclusions: AST-3424 showed a tolerable safety profile and preliminary anti-tumor activity in advanced solid tumors. The phase II dose expansion phase of AST-3424 monotherapy is ongoing in subjects diagnosed as advanced HCC and with high AKR1C3 expression. Clinical trial information: NCT06239155 .

A phase I/II study of enzalutamide in combination with indomethacin in men with castration-resistant prostate cancer (CRPC).

e17027 Background: Metastatic castration resistant prostate cancer (mCRPC) remains an incurable disease despite recent advances in therapy. In patients (pts) receiving the second-generation androgen receptor antagonist enzalutamide (enza), primary or adaptive resistance can occur due to upregulation of the steroidogenesis pathway, including aldo-keto reductase 1C3 (AKR1C3), which can also upregulate intratumoral androgen synthesis. Indomethacin, a non-steroidal anti-inflammatory drug and selective inhibitor of AKR1C3, demonstrates synergistic activity when combined with enza against enza-resistant CRPC in vitro and in vivo. We thus sought to study this combination in a clinical trial. Methods: This investigator-initiated Phase I/II trial enrolled pts with enza-naive mCRPC on androgen deprivation therapy. Prior abiraterone was allowed. Pts must have adequate organ function and ECOG performance status 0-2. In the phase I lead-in, a 3+3 dose de-escalation design was used to identify the recommended Phase II dose (RP2D) of indomethacin in combination with enza 160 mg oral (PO) once daily, after which a Phase II expansion proceeded. Pts continued treatment until disease progression or unacceptable toxicity. Co-primary endpoints were safety and PSA response, defined as 50% or more reduction from baseline. Secondary endpoints included overall response as determined by Prostate Cancer Working Group 2 criteria (PCWG2). Results: From 2017 - 2022, 26 pts were enrolled, with a median age of 71 (range 50-88), primarily white (n=16, 62%), median baseline PSA 19.45 (range 1.5-210), ECOG 1 (n=12, 46%), and most received prior abiraterone (n=24, 92%). In 6 pts enrolled to the phase I cohort, there were no grade 3 adverse events (AEs); RP2D of indomethacin was 50mg PO twice daily. All 26 pts were evaluable for safety and efficacy. Most common treatment-related AEs (n; %) were nausea (12; 46%), fatigue (11; 42%), diarrhea (8; 31%), hypertension (7; 27%), anorexia (6; 23%). Most common ≥ grade 3 treatment-related AEs were acute kidney injury (3; 12%), hypertension (3; 12%), and anemia (2; 8%). PSA response was observed in 8 pts (31%). By PCWG2, 14 pts (54%) experienced stable disease, while 10 pts (38%) had progressive disease. Conclusions: The combination of enza and indomethacin was safe, clinically feasible, and had clinical activity in pts with abiraterone-pretreated mCRPC. Further correlative studies are needed to identify pts who may benefit from AKR1C3 inhibition. Clinical trial information: NCT02935205 .

AKR1B10 expression characteristics in hepatocellular carcinoma and its correlation with clinicopathological features and immune microenvironment

Hepatocellular carcinoma (HCC) represents a major global health threat with diverse and complex pathogenesis. Aldo–keto reductase family 1 member B10 (AKR1B10), a tumor-associated enzyme, exhibits abnormal expression in various cancers. However, a comprehensive understanding of AKR1B10's role in HCC is lacking. This study aims to explore the expression characteristics of AKR1B10 in HCC and its correlation with clinicopathological features, survival prognosis, and tumor immune microenvironment, further investigating its role and potential regulatory mechanisms in HCC. This study conducted comprehensive analyses using various bioinformatics tools and databases. Initially, differentially expressed genes related to HCC were identified from the GEO database, and the expression of AKR1B10 in HCC and other cancers was compared using TIMER and GEPIA databases, with validation of its specificity in HCC tissue samples using the HPA database. Furthermore, the relationship of AKR1B10 expression with clinicopathological features (age, gender, tumor size, staging, etc.) of HCC patients was analyzed using the TCGA database's LIHC dataset. The impact of AKR1B10 expression levels on patient prognosis was evaluated using Kaplan–Meier survival analysis and the Cox proportional hazards model. Additionally, the correlation of AKR1B10 expression with tumor biology-related signaling pathways and tumor immune microenvironment was studied using databases like GSEA, Targetscan, and others, identifying microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) that regulate AKR1B10 expression to explore potential regulatory mechanisms. Elevated AKR1B10 expression was significantly associated with gender, primary tumor size, and fibrosis stage in HCC tissues. High AKR1B10 expression indicated poor prognosis and served as an independent predictor for patient outcomes. Detailed mechanism analysis revealed a positive correlation between high AKR1B10 expression, immune cell infiltration, and pro-inflammatory cytokines, suggesting a potential DANCR-miR-216a-5p-AKR1B10 axis regulating the tumor microenvironment and impacting HCC development and prognosis. The heightened expression of AKR1B10 in HCC is not only related to significant clinical-pathological traits but may also influence HCC progression and prognosis by activating key signaling pathways and altering the tumor immune microenvironment. These findings provide new insights into the role of AKR1B10 in HCC pathogenesis and highlight its potential as a biomarker and therapeutic target.

Pregnane X receptor (PXR) deficiency promotes hepatocarcinogenesis via induction of Akr1c18 expression and prostaglandin F2α (PGF2α) levels

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Pregnane X receptor (PXR), a xenobiotic-sensing nuclear receptor, plays a critical role in the metabolism of endogenous and exogenous substances in the liver. Here, we investigate whether PXR plays a role in pathogenesis of HCC. We show that liver tumors were developed in diethylnitrosamine (DEN)-treated in PXR knockout (KO) mice. Hepatic levels of prostaglandin F2α (PGF2α) and aldo–keto reductase family 1 member C18 (Akr1c18), a prostaglandin synthase of catalyzing reduction of PGH2 to PGF2α, were significantly elevated in DEN-treated PXR KO mice. Hepatic mRNA levels of alpha fetoprotein (AFP), cyclin D1 (Ccnd1), fibroblast growth factor 21 (FGF21), and inflammatory cytokine interleukin 6 (IL-6) were significantly increased in DEN-treated PXR KO mice. Other members of Akr1c family, liver metabolizing enzymes including Cyp1a2, Cyp2b10 and Cyp3a11, and bile acid synthesis enzyme Cyp7a1 mRNA levels were significantly decreased in DEN-treated PXR KO mice. Our findings revealed that PXR deficiency promoted DEN-induced HCC in mice via induction of Akr1c18 expression and PGF2α levels and the increased PGF2α levels synthetized by Akr1c18 enhanced hepatocytes proliferation and induced inflammatory cytokine production, which accelerated liver tumor development after DEN treatment, suggesting that PXR deficiency may create a microenvironment that is more prone to DEN-induced liver tumors and targeting PXR and Akr1c18 to reduce PGF2α biosynthesis may be a potential and novel therapeutic strategy for HCC.

Role of AKR1B10 in inflammatory diseases.

Inflammation is an important pathophysiological process in many diseases; it has beneficial and harmful effects. When exposed to various stimuli, the body triggers an inflammatory response to eliminate invaded pathogens and damaged tissues to maintain homeostasis. However, uncontrollable persistent or excessive inflammatory responses may damage tissues and induce various diseases, such as metabolic diseases (e.g. diabetes), autoimmune diseases, nervous system-related diseases, digestive system-related diseases, and even tumours. Aldo-keto reductase 1B10 (AKR1B10) is an important player in the development and progression of multiple diseases, such as tumours and inflammatory diseases. AKR1B10 is upregulated in solid tumours, such as hepatocellular carcinoma (HCC), non-small cell lung carcinoma, and breast cancer, and is a reliable serum marker. However, information on the role of AKR1B10 in inflammation is limited. In this study, we summarized the role of AKR1B10 in inflammatory diseases, including its expression, functional contribution to inflammatory responses, and regulation of signalling pathways related to inflammation. We also discussed the role of AKR1B10 in glucose and lipid metabolism and oxidative stress. This study provides novel information and increases the understanding of clinical inflammatory diseases.

Berberine directly targets AKR1B10 protein to modulate lipid and glucose metabolism disorders in NAFLD

Ethnopharmacological relevanceBerberine (BBR) is the main active component from Coptidis rhizome, a well-known Chinese herbal medicine used for metabolic diseases, especially diabetes for thousands of years. BBR has been reported to cure various metabolic disorders, such as nonalcoholic fatty liver disease (NAFLD). However, the direct proteomic targets and underlying molecular mechanism of BBR against NAFLD remain less understood. Aim of the studyTo investigate the direct target and corresponding molecular mechanism of BBR on NAFLD is the aim of the current study. Materials and methodsHigh-fat diet (HFD)-fed mice and oleic acid (OA) stimulated HepG2 cells were utilized to verify the beneficial impacts of BBR on glycolipid metabolism profiles. The click chemistry in proteomics, DARTS, CETSA, SPR and fluorescence co-localization analysis were conducted to identify the targets of BBR for NAFLD. RNA-seq and shRNA/siRNA were used to investigate the downstream pathways of the target. ResultsBBR improved hepatic steatosis, ameliorated insulin resistance, and reduced TG levels in the NAFLD models. Importantly, Aldo-keto reductase 1B10 (AKR1B10) was first proved as the target of BBR for NAFLD. The gene expression of AKR1B10 increased significantly in the NAFLD patients' liver tissue. We further demonstrated that HFD and OA increased AKR1B10 expression in the C57BL/6 mice's liver and HepG2 cells, respectively, whereas BBR decreased the expression and activities of AKR1B10. Moreover, the knockdown of AKR1B10 by applying shRNA/siRNA profoundly impacted the beneficial effects on the pathogenesis of NAFLD by BBR. Meanwhile, the changes in various proteins (ACC1, CPT-1, GLUT2, etc.) are responsible for hepatic lipogenesis, fatty acid oxidation, glucose uptake, etc. by BBR were reversed by the knockdown of AKR1B10. Additionally, RNA-seq was used to identify the downstream pathway of AKR1B10 by examining the gene expression of liver tissues from HFD-fed mice. Our findings revealed that BBR markedly increased the protein levels of PPARα while downregulating the expression of PPARγ. However, various proteins of PPAR signaling pathways remained unaffected post the knockdown of AKR1B10. ConclusionsBBR alleviated NAFLD via mediating PPAR signaling pathways through targeting AKR1B10. This study proved that AKR1B10 is a novel target of BBR for NAFLD treatment and helps to find new targets for the treatment of NAFLD by using active natural compounds isolated from traditional herbal medicines as the probe.

The CfKOB1 gene related to cell apoptosis is required for pathogenicity and involved in mycovirus-induced hypovirulence in Colletotrichum fructicola

Colletotrichum fructicola is a globally significant phytopathogenic fungus. Mycovirus-induced hypovirulence has great potential for biological control and study of fungal pathogenic mechanisms. We previously reported that the mycovirus Colletotrichum alienum partitivirus 1 (CaPV1) is associated with the hypovirulence of C. fructicola, and the present study aimed to further investigate a host factor and its roles in mycovirus-induced hypovirulence. A gene named CfKOB1, which encodes putative protein homologous to the β-subunit of voltage-gated potassium channels and aldo–keto reductase, is downregulated upon CaPV1 infection and significantly upregulated during the early infection phase of Nicotiana benthamiana by C. fructicola. Deleting the CfKOB1 gene resulted in diminished vegetative growth, decreased production of asexual spores, hindered appressorium formation, reduced virulence, and altered tolerance to abiotic stresses. Transcriptome analysis revealed that CfKOB1 regulates many metabolic pathways as well as the cell cycle and apoptosis. Furthermore, enhanced apoptosis was observed in the ΔCfKOB1 mutants. Viral RNA accumulation was significantly increased in the CfKOB1 deletion mutant. Additionally, our findings demonstrated that CaPV1 infection in the WT strain also induced cell apoptosis. Collectively, these results highlight the diverse biological roles of the CfKOB1 gene in the fungus C. fructicola, while it also participates in mycovirus-induced hypovirulence by regulating apoptosis.

Development of new steroid-based hydrazide and (thio)semicarbazone compounds with anticancer properties

Most breast and prostate cancers are caused by abnormal production or action of steroidal hormones. Hormonal drugs based on steroid scaffolds represent a significant class of chemotherapeutics that are routinely used in chemotherapy. In this study, the synthesis of new 17a-homo lactone and 17α-(pyridine-2-ylmethyl) androstane derivatives with hydrazide and semicarbazone motifs is presented. All compounds were screened for their effect on cell viability against a panel of five cancer cell lines and one healthy cell line. Two compounds showed significant cytotoxicity against cancer cells, with low toxicity against healthy cells. The relative binding affinities of compounds for the ligand-binding domains of estrogen receptor α, estrogen receptor β, androgen receptor and glucocorticoid receptor were tested using a fluorescence screen in yeast. Potential for inhibition of aldo-keto reductase 1C3 and 1C4 activity was measured in vitro. Experimental results are analyzed in the context of molecular docking simulations. Our results could help guide design of steroid compounds with improved anticancer properties against androgen- and estrogen-dependent cancers.

Machine learning-based algorithm identifies key mitochondria-related genes in non-alcoholic steatohepatitis

BackgroundEvidence suggests that hepatocyte mitochondrial dysfunction leads to abnormal lipid metabolism, redox imbalance, and programmed cell death, driving the onset and progression of non-alcoholic steatohepatitis (NASH). Identifying hub mitochondrial genes linked to NASH may unveil potential therapeutic targets.MethodsMitochondrial hub genes implicated in NASH were identified via analysis using 134 algorithms.ResultsThe Random Forest algorithm (RF), the most effective among the 134 algorithms, identified three genes: Aldo–keto reductase family 1 member B10 (AKR1B10), thymidylate synthase (TYMS), and triggering receptor expressed in myeloid cell 2 (TREM2). They were upregulated and positively associated with genes promoting inflammation, genes involved in lipid synthesis, fibrosis, and nonalcoholic steatohepatitis activity scores in patients with NASH. Moreover, using these three genes, patients with NASH were accurately categorized into cluster 1, exhibiting heightened disease severity, and cluster 2, distinguished by milder disease activity. ConclusionThese three genes are pivotal mitochondrial genes implicated in NASH progression.

Transcriptome-wide association mapping provides insights into the genetic basis and candidate genes governing flowering, maturity and seed weight in rice bean (Vigna umbellata)

BackgroundRice bean (Vigna umbellata), an underrated legume, adapts to diverse climatic conditions with the potential to support food and nutritional security worldwide. It is used as a vegetable, minor food crop and a fodder crop, being a rich source of proteins, minerals, and essential fatty acids. However, little effort has been made to decipher the genetic and molecular basis of various useful traits in this crop. Therefore, we considered three economically important traits i.e., flowering, maturity and seed weight of rice bean and identified the associated candidate genes employing an associative transcriptomics approach on 100 diverse genotypes out of 1800 evaluated rice bean accessions from the Indian National Genebank.ResultsThe transcriptomics-based genotyping of one-hundred diverse rice bean cultivars followed by pre-processing of genotypic data resulted in 49,271 filtered markers. The STRUCTURE, PCA and Neighbor-Joining clustering of 100 genotypes revealed three putative sub-populations. The marker-trait association analysis involving various genome-wide association study (GWAS) models revealed significant association of 82 markers on 48 transcripts for flowering, 26 markers on 22 transcripts for maturity and 22 markers on 21 transcripts for seed weight. The transcript annotation provided information on the putative candidate genes for the considered traits. The candidate genes identified for flowering include HSC80, P-II PsbX, phospholipid-transporting-ATPase-9, pectin-acetylesterase-8 and E3-ubiquitin-protein-ligase-RHG1A. Further, the WRKY1 and DEAD-box-RH27 were found to be associated with seed weight. Furthermore, the associations of PIF3 and pentatricopeptide-repeat-containing-gene with maturity and seed weight, and aldo–keto-reductase with flowering and maturity were revealed.ConclusionThis study offers insights into the genetic basis of key agronomic traits in rice bean, including flowering, maturity, and seed weight. The identified markers and associated candidate genes provide valuable resources for future exploration and targeted breeding, aiming to enhance the agronomic performance of rice bean cultivars. Notably, this research represents the first transcriptome-wide association study in pulse crop, uncovering the candidate genes for agronomically useful traits.

CHES1 modulated tumorigenesis and senescence of pancreas cancer cells through repressing AKR1B10

Pancreatic ductal adenocarcinoma (PDAC), is characteristic by a heterogeneous tumor microenvironment and gene mutations, conveys a dismal prognosis and low response to chemotherapy and immunotherapy. Here, we found that checkpoint suppressor 1 (CHES1) served as a tumor repressor in PDAC and was associated with patient prognosis. Functional experiments indicated that CHES1 suppressed the proliferation and invasion of PDAC by modulating cellular senescence. To further identify the downstream factor of CHES1 in PDAC, label-free quantitative proteomics analysis was conducted, which showed that the oncogenic Aldo-keto reductase 1B10 (AKR1B10) was transcriptionally repressed by CHES1 in PDAC. And AKR1B10 facilitated the malignant activity and repressed senescent phenotype of PDAC cells. Moreover, pharmaceutical inhibition of AKR1B10 with Oleanolic acid (OA) significantly induced tumor regression and sensitized PDAC cells to gemcitabine, and this combined therapy did not cause obvious side effects. Rescued experiments revealed that CHES1 regulated the tumorigenesis and gemcitabine sensitivity through AKR1B10-mediated senescence in PDAC. In summary, this study revealed that the CHES1/AKR1B10 axis modulated the progression and cellular senescence in PDAC, which might provide revenues for drug-targeting and senescence-inducing therapies for PDAC.

Aldo-keto reductase family 1 member A1 contributes to increase the glycolytic metabolism and to accelerate cellular senescence of bone derived mesenchymal stem cells

Skeletal aging characterizes the important event where bone loss is accompanied by accumulation of marrow adipose tissues. This event may be due to dysfunctional differentiation of bone derived mesenchymal stem cells (BMSCs) switched from osteoblasts (OBs) to adipocytes (ADs). Our accomplished work demonstrated that Aldo-keto reductase family 1 member A1 (Akr1A1) might serve as an important control to modify the NAD+-dependent SIRT signaling pathway for regulating the adipo-osteogenic lineage differentiation of BMSCs. In this study, we established a senescence cell model for examining whether increased Akr1A1 expression in BMSCs contributes to preferential lineage differentiation to ADs at the expense of osteogenesis by switching on the glycolytic metabolism in senescent cells with D-galactose induction or natural aging in MSCs. Our result suggests that increased expression of the glycolytic enzyme, Akr1A1 contributes to increase the glycolytic metabolism and to accelerate cellular senescence of MSCs. Such information might be useful to help develop the novel therapeutic strategy for treating age-related bone diseases with “perspective Akr1A1 inhibitors”. Grants supported by NSTC (112-2314-B-005 -004 -, NSC-111-2314-B-005-003-), iEGG and Animal Biotechnology Center from the Feature Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE-112-S-0023-A) in Taiwan, and partially by the grants of CYCH-NCHULS110003, CYCH-NCHULS111003, CYCH-NCHULS112002, TCVGH-NCHU 112-7616 to Y-ML. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Enzymatic Function of Kvβ2 Controls Vascular Potassium Channel Activity

Voltage-gated potassium (Kv) channels expressed in vascular smooth muscle control membrane potential and thereby regulate vascular tone and organ perfusion. Native Kv1 channels in the resistance vasculature consist of an alpha pore domain in complex with intracellular beta (Kvβ) proteins, which are active aldo-keto reductases (AKR6A). Recent work suggests that the AKR function of Kvβ2 is required for vascular Kv1 channel NAD(P)(H) redox sensing and O2 sensitivity of resistance arterial tone. Nonetheless, whether acute changes in Kvβ2 AKR function impact vascular Kv1 activity is unknown. We propose that Kv1 gating is sensitive to changes in the levels of endogenous substrates and post-translational modification of Kvβ2. Here, we tested the hypothesis that the availability of the glycolytic byproduct and purported Kvβ substrate, methylglyoxal, and protein kinase C-dependent phosphorylation differentially regulate vascular Kvβ2 function. Using the inside-out configuration of the patch clamp technique, we measured the open probability (nPo) of Kv channels present in patches excised from human coronary smooth muscle cells. Perfusion of methylglyoxal (5 μM) resulted in significantly increased Kv channel nPo (3.3x10−5 ± 9.5x10−6 to 3.2x10−4 ± 2.1x10−4; n=5) and this effect was enhanced (~8-fold) in the presence of 100 μM NADPH, suggesting that Kv sensitivity to methylglyoxal is cofactor-dependent. Based on this, we next tested whether Kvβ phosphorylation regulates AKR catalysis and influences vascular Kv1 redox sensitivity. Using in situ proximity ligation, we found that serine phosphorylation of Kvβ2 in smooth muscle was increased upon treatment with the PKC activator phorbol 12-myristate 13-acetate (PMA; 100 nM), indicating that smooth muscle Kvβ2 is a phosphorylation target under these conditions. In vitro phosphorylation of purified Kvβ2 with PKCα significantly slowed catalysis (11.3 ± 0.3 vs. 6.5 ± 0.2 μmoles/min/mg protein, control and PKCα-treated rat Kvβ2.1, respectively). Pretreatment of cells with 10 nM PMA before excising inside-out membrane patches eliminated the increase in Kv channel nP(o) evoked by methylglyoxal (±NADPH). Moreover, ex vivo treatment of isolated coronary arteries with PMA (10 nM) abolished vasodilation in response to elevated NADH:NAD+ via application of 2–5 mM external L-lactate. Together, these results suggest that vascular smooth muscle Kv1 channels are functionally regulated by endogenous substrates and phosphorylation of intracellular Kvβ proteins. We propose that targeting vascular Kvβ AKR function may represent a novel strategy to modify K+ channel redox sensitivity and thus strengthen the coupling between metabolic demand and organ perfusion. T32-ES011564 and R01HL163818. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Quantification of total phenols in Coleus forskohlii and inhibition of aldose reductase by rosmarinic acid

Many complications of diabetes have been associated with the accumulation of intracellular sorbitol caused by excessive activity of aldose reductase (ALR2). ALR2 is a member of the aldo-keto reductases (AKR) superfamily and has gained significant attention due to its role in the development of diabetic complications. Inhibiting ALR2 could help prevent or delay some of these problems. Coleus forskohlii is a natural medicinal plant that is known for its medicinal effects and is also used as a beverage in several European countries. High-performance liquid chromatography (HPLC) was used to measure the polyphenol content of aqueous leaf extracts of C. forskohlii, and it was found that rosmarinic acid (RA) had the highest concentration (1.25 mg/g) compared to other polyphenols. In vitro bioassays were conducted to evaluate the inhibitory effect of RA on ALR2. RA significantly and dose-dependently reduced the build-up of sorbitol in human erythrocytes under high glucose conditions. Molecular docking studies suggest that RA, which is an essential polyphenol, could be a potential ALR2 inhibitor. This study is the first to show that RA has an IC50 value of 56 % to exhibit an inhibitory effect against ALR2.

Discovery of an Aldo-Keto reductase 1C3 (AKR1C3) degrader.

Aldo-keto reductase 1C3 (AKR1C3) is a protein upregulated in prostate cancer, hematological malignancies, and other cancers where it contributes to proliferation and chemotherapeutic resistance. Androgen receptor splice variant 7 (ARv7) is the most common mutation of the AR receptor that confers resistance to clinical androgen receptor signalling inhibitors in castration-resistant prostate cancer. AKR1C3 interacts with ARv7 promoting stabilization. Herein we report the discovery of the first-in-class AKR1C3 Proteolysis-Targeting Chimera (PROTAC) degrader. This first-generation degrader potently reduced AKR1C3 expression in 22Rv1 prostate cancer cells with a half-maximal degradation concentration (DC50) of 52 nM. Gratifyingly, concomitant degradation of ARv7 was observed with a DC50 = 70 nM, along with degradation of the AKR1C3 isoforms AKR1C1 and AKR1C2 to a lesser extent. This compound represents a highly useful chemical tool and a promising strategy for prostate cancer intervention.

AKR1B10 accelerates glycolysis through binding HK2 to promote the malignant progression of oral squamous cell carcinoma

BackgroundOral squamous cell carcinoma (OSCC) remains a rampant oral cavity neoplasm with high degree of aggressiveness. Aldo–keto reductase 1B10 (AKR1B10) that is an oxidoreductase dependent on nicotinamide adenine dinucleotide phosphate (NADPH) has been introduced to possess prognostic potential in OSCC. The present work was focused on specifying the involvement of AKR1B10 in the process of OSCC and its latent functional mechanism.MethodsAKR1B10 expression in OSCC tissues and cells were detected by RT-qPCR and Western blot analysis. CCK-8 method, EdU staining, wound healing and transwell assays respectively assayed cell viability, proliferation, migration and invasion. Immunofluorescence staining and Western blot evaluated epithelial mesenchymal transition (EMT). Adenosine triphosphate (ATP) contents, glucose consumption and extracellular acidification rate (ECAR) were measured by relevant commercially available kits and Seahorse XF96 Glycolysis Analyzer, severally. The expressions of proteins associated with metastasis and glycolysis were examined with Western blot. Co-IP assay confirmed the binding between AKR1B10 and hexokinase 2 (HK2).ResultsIt was observed that AKR1B10 expression was increased in OSCC tissues and cells. After AKR1B10 was knocked down, the proliferation, migration, invasion and EMT of OSCC cells were all hampered. Additionally, AKR1B10 silencing suppressed glycolysis and bound to HK2 in OSCC cells. Up-regulation of HK2 partially abolished the hampered glycolysis, proliferation, migration, invasion and EMT of AKR1B10-silenced OSCC cells.ConclusionTo sum up, AKR1B10 could bind to HK2 to accelerate glycolysis, thereby facilitating the proliferation, migration, invasion and EMT of OSCC cells.

Synthesis and anti-inflammatory activity of benzimidazole derivatives; an in vitro, in vivo and in silico approach

Many non-steroidal anti-inflammatory drugs (NSAIDs) concurrently inhibit both COX-1 and COX-2, with a preference for specifically targeting COX-2 due to its significant involvement in various pathologies. In addition to COX enzymes, several other targets, including Aldose reductase, Aldo-ketoreductase family 1-member C2, and Phospholipase A2, have been identified as contributors to inflammation and a myriad of other diseases.In this context, a series of 2-substituted benzimidazole derivatives was synthesized and assessed for their anti-inflammatory potential through both in vitro and in vivo assays. Molecular docking studies were conducted to elucidate the mechanism of action of these compounds against COX enzymes and other therapeutic targets associated with NSAIDs, such as Aldose reductase, AIKRC, and Phospholipase A2.Among the synthesized compounds, B2, B4, B7, and B8 demonstrated IC50 values lower than the standard ibuprofen, as determined by the Luminol-enhanced chemiluminescence assay. Validation of these findings was achieved through an in vivo carrageenan-induced mice paw edema model, confirming a comparable anti-inflammatory effect to diclofenac sodium observed in vitro. Notably, these compounds exhibited significant binding affinity with all therapeutic targets investigated in this study.These results suggest that the newly synthesized derivatives possess noteworthy anti-inflammatory potential, warranting further exploration for the development of novel multi-targeting inhibitors.

Precision Engineering of the Co-immobilization of Enzymes for Cascade Biocatalysis.

The design and orderly layered co-immobilization of multiple enzymes on resin particles remain challenging. In this study, the SpyTag/SpyCatcher binding pair was fused to the N-terminus of an alcohol dehydrogenase (ADH) and an aldo-keto reductase (AKR), respectively. A non-canonical amino acid (ncAA), p-azido-L-phenylalanine (p-AzF), as the anchor for covalent bonding enzymes, was genetically inserted into preselected sites in the AKR and ADH. Employing the two bioorthogonal counterparts of SpyTag/SpyCatcher and azide-alkyne cycloaddition for the immobilization of AKR and ADH enabled sequential dual-enzyme coating on porous microspheres. The ordered dual-enzyme reactor was subsequently used to synthesize (S)-1-(2-chlorophenyl)ethanol asymmetrically from the corresponding prochiral ketone, enabling the in situ regeneration of NADPH. The reactor exhibited a high catalytic conversion of 74 % and good reproducibility, retaining 80 % of its initial activity after six cycles. The product had 99.9 % ee, which that was maintained in each cycle. Additionally, the double-layer immobilization method significantly increased the enzyme loading capacity, which was approximately 1.7 times greater than that of traditional single-layer immobilization. More importantly, it simultaneously enabled both the purification and immobilization of multiple enzymes on carriers, thus providing a convenient approach to facilitate cascade biocatalysis.

Precision Engineering of the Co‐immobilization of Enzymes for Cascade Biocatalysis

AbstractThe design and orderly layered co‐immobilization of multiple enzymes on resin particles remain challenging. In this study, the SpyTag/SpyCatcher binding pair was fused to the N‐terminus of an alcohol dehydrogenase (ADH) and an aldo‐keto reductase (AKR), respectively. A non‐canonical amino acid (ncAA), p‐azido‐L‐phenylalanine (p‐AzF), as the anchor for covalent bonding enzymes, was genetically inserted into preselected sites in the AKR and ADH. Employing the two bioorthogonal counterparts of SpyTag/SpyCatcher and azide–alkyne cycloaddition for the immobilization of AKR and ADH enabled sequential dual‐enzyme coating on porous microspheres. The ordered dual‐enzyme reactor was subsequently used to synthesize (S)‐1‐(2‐chlorophenyl)ethanol asymmetrically from the corresponding prochiral ketone, enabling the in situ regeneration of NADPH. The reactor exhibited a high catalytic conversion of 74 % and good reproducibility, retaining 80 % of its initial activity after six cycles. The product had 99.9 % ee, which that was maintained in each cycle. Additionally, the double‐layer immobilization method significantly increased the enzyme loading capacity, which was approximately 1.7 times greater than that of traditional single‐layer immobilization. More importantly, it simultaneously enabled both the purification and immobilization of multiple enzymes on carriers, thus providing a convenient approach to facilitate cascade biocatalysis.