HMGR Inhibitors Research Articles

Inhibitors of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase Decrease the Growth, Ergosterol Synthesis and Generation of petite Mutants in Candida glabrata and Candida albicans

Candida glabrata and Candida albicans, the most frequently isolated candidiasis species in the world, have developed mechanisms of resistance to treatment with azoles. Among the clinically used antifungal drugs are statins and other compounds that inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), resulting in decreased growth and ergosterol levels in yeasts. Ergosterol is a key element for the formation of the yeast cell membrane. However, statins often cause DNA damage to yeast cells, facilitating mutation and drug resistance. The aim of the current contribution was to synthesize seven series of compounds as inhibitors of the HMGR enzyme of Candida ssp., and to evaluate their effect on cellular growth, ergosterol synthesis and generation of petite mutants of C. glabrata and C. albicans. Compared to the reference drugs (fluconazole and simvastatin), some HMGR inhibitors caused lower growth and ergosterol synthesis in the yeast species and generated fewer petite mutants. Moreover, heterologous expression was achieved in Pichia pastoris, and compounds 1a, 1b, 6g and 7a inhibited the activity of recombinant CgHMGR and showed better binding energy values than for α-asarone and simvastatin. Thus, we believe these are good candidates for future antifungal drug development.

Searching for Effective Treatments in HFpEF: Implications for Modeling the Disease in Rodents.

While the prevalence of heart failure with preserved ejection fraction (HFpEF) has increased over the last two decades, there still remains a lack of effective treatment. A key therapeutic challenge is posed by the absence of animal models that accurately replicate the complexities of HFpEF. The present review summarizes the effects of a wide spectrum of therapeutic agents on HF. Two online databases were searched for studies; in total, 194 experimental protocols were analyzed following the PRISMA protocol. A diverse range of models has been proposed for studying therapeutic interventions for HFpEF, with most being based on pressure overload and systemic hypertension. They have been used to evaluate more than 150 different substances including ARNIs, ARBs, HMGR inhibitors, SGLT-2 inhibitors and incretins. Existing preclinical studies have primarily focused on LV diastolic performance, and this has been significantly improved by a wide spectrum of candidate therapeutic agents. Few experiments have investigated the normalization of pulmonary congestion, exercise capacity, animal mortality, or certain molecular hallmarks of heart disease. The development of comprehensive preclinical HFpEF models, with multi-organ system phenotyping and physiologic stress-based functional testing, is needed for more successful translation of preclinical research to clinical trials.

A fungal tolerance trait and selective inhibitors proffer HMG-CoA reductase as a herbicide mode-of-action

Decades of intense herbicide use has led to resistance in weeds. Without innovative weed management practices and new herbicidal modes of action, the unabated rise of herbicide resistance will undoubtedly place further stress upon food security. HMGR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) is the rate limiting enzyme of the eukaryotic mevalonate pathway successfully targeted by statins to treat hypercholesterolemia in humans. As HMGR inhibitors have been shown to be herbicidal, HMGR could represent a mode of action target for the development of herbicides. Here, we present the crystal structure of a HMGR from Arabidopsis thaliana (AtHMG1) which exhibits a wider active site than previously determined structures from different species. This plant conserved feature enables the rational design of specific HMGR inhibitors and we develop a tolerance trait through sequence analysis of fungal gene clusters. These results suggest HMGR to be a viable herbicide target modifiable to provide a tolerance trait.

Potential dual inhibitors of PCSK-9 and HMG-R from natural sources in cardiovascular risk management.

Atherosclerotic cardiovascular disease (ASCVD) stands amongst the leading causes of mortality worldwide and has attracted the attention of world's leading pharmaceutical companies in order to tackle such mortalities. The low-density lipoprotein-cholesterol (LDL-C) is considered the most prominent biomarker for the assessment of ASCVD risk. Distinct inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-R), the chief hepatic cholesterogenic enzyme, are being used since last seven decades to manage hypercholesterolemia. On the other hand, discovery and the association of proprotein convertase subtilisin/kexin type-9 (PCSK-9) with increased ASCVD risk have established PCSK-9 as a novel therapeutic target in cardiovascular medicine. PCSK-9 is well reckoned to facilitate the LDL-receptor (LDL-R) degradation and compromised LDL-C clearance leading to the arterial atherosclerotic plaque formation. The currently available HMG-R inhibitors (statins) and PCSK-9 inhibitors (siRNA, anti-sense oligonucleotides, and monoclonal antibodies) have shown great promises in achieving LDL-C lowering goals, however, their life long prescriptions have raised significant concerns. These deficits associated with the synthetic HMG-R and PCSK-9 inhibitors called for the discovery of alternative therapeutic candidates with potential dual HMG-R and PCSK-9 inhibitory activities from natural origins. Therefore, this report firstly describes the mechanistic insights into the cholesterol homeostasis through HMG-R, PCSK-9, and LDL-R functionality and then compiles the pharmacological effects of natural secondary metabolites with special emphasis on their dual HMG-R and PCSK-9 inhibitory action. In conclusion, various natural products exhibit atheroprotective effects via targeting HMG-R and PCSK-9 activities and lipoprotein metabolism, however, further clinical assessments are still warranted prior their approval for ASCVD risk management in hypercholesterolemic patients.

Potential natural inhibitors of xanthine oxidase and HMG-CoA reductase in cholesterol regulation: in silico analysis

BackgroundHypercholesterolemia has posed a serious threat of heart diseases and stroke worldwide. Xanthine oxidase (XO), the rate-limiting enzyme in uric acid biosynthesis, is regarded as the root of reactive oxygen species (ROS) that generate atherosclerosis and cholesterol crystals. β-Hydroxy β-methylglutaryl-coenzyme A reductase (HMGR) is a rate-limiting enzyme in cholesterol biosynthesis. Although some commercially available enzyme inhibiting drugs have effectively reduced cholesterol levels, most of them have failed to meet potential drug candidates’ requirements. Here, we have carried out an in-silico analysis of secondary metabolites that have already shown good inhibitory activity against XO and HMGR in a wet lab setup.MethodsOut of 118 secondary metabolites reviewed, sixteen molecules inhibiting XO and HMGR were selected based on the IC50 values reported in in vitro assays. Further, receptor-based virtual screening was carried out against secondary metabolites using GOLD Protein-Ligand Docking Software, combined with subsequent post-docking, to study the binding affinities of ligands to the enzymes. In-silico ADMET analysis was carried out to explore their pharmacokinetic properties, followed by toxicity prediction through ProTox-II.ResultsThe molecular docking of amentoflavone (GOLD score 70.54, ∆G calc. = − 10.4 Kcal/mol) and ganomycin I (GOLD score 59.61, ∆G calc. = − 6.8 Kcal/mol) displayed that the drug has effectively bound at the competitive site of XO and HMGR, respectively. Besides, 6-paradol and selgin could be potential drug candidates inhibiting XO. Likewise, n-octadecanyl-O-α-D-glucopyranosyl (6′ → 1″)-O-α-D-glucopyranoside could be potential drug candidates to maintain serum cholesterol. In-silico ADMET analysis has shown that these sixteen metabolites were optimal within the categorical range compared to commercially available XO and HMGR inhibitors, respectively. Toxicity analysis through ProTox-II revealed that 6-gingerol, ganoleucoin K, and ganoleucoin Z are toxic for human use.ConclusionThis computational analysis supports earlier experimental evidence towards the inhibition of XO and HMGR by natural products. Further study is necessary to explore the clinical efficacy of these secondary molecules, which might be alternatives for the treatment of hypercholesterolemia.

Inhibition mechanism of 3-hydroxy-3-methyl-glutaryl-CoA reductase by tocotrienol-rich rice bran fraction optimally extracted with ultrasonic energy

Tocotrienols (T3) are vitamin E components that inhibit 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), a primary target for cholesterol management. T3 was extracted from rice bran (RBE) using ultrasonic energy keeping solute: solvent ratio, power and time on specific energy and T3 concentration as responses as per Box–Behnken Design. The lowest specific energy (52.38 ± 0.14 J mL−1) uptake by the sample was most effective in enhancing the concentration of T3 in RBE (199.34 ± 0.63 μg mL−1). In vitro HMGR kinetics and in silico binding interactions of the identified α-, δ- and γ-T3 fractions were studied. Enzyme kinetic studies revealed an uncompetitive mode of inhibition by α-T3, γ-T3, and RBE and a mixed mode of inhibition for δ-T3. γ-T3 showed lowest IC50 concentration (11.33 μg mL−1) followed by α-T3 (16.73 μg mL−1), RBE (20.45 μg mL−1) and δ-T3 (23.16 μg mL−1). Molecular docking studies highlighted the hydrogen bonding of δ-T3 with Gln766 and α- and γ-T3 with Met655 and Val805 amino acid residues at the NADPH binding site of HMGR. Results indicate the potential use of T3 enriched RBE optimally extracted using ultrasound as potent HMGR inhibitor.

Inhibition of recombinant enzyme 3-hydroxy-3-methylglutaryl-CoA reductase from Candida glabrata by α-asarone-based synthetic compounds as antifungal agents

Due to increasing resistance of Candida species to antifungal drugs, especially azoles, new drugs are needed. The proposed compounds 3 and 4 are analogous to α-asarone (2), a naturally occurring potent inhibitor of HMGR with hypolipidemic and antifungal activity. We used the recombinant enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase of Candida glabrata (CgHMGR) as a model to test the effectiveness of the test compounds. Compounds 3 and 4 demonstrated inhibitory kinetics, having lower IC50 values (42.65 μM and 28.77 μM, respectively) than compound 2 (>100 μM). The docking studies showed better binding energies for compounds 3 and 4 (−5.35 and −6.1 kcal/mol, respectively) than for compound 2 (−4.53 kcal/mol). These findings suggest that the tested compounds are better than their natural analogue. Plaque assays were performed on the C. glabrata strain CBS138 by applying ergosterol or cholesterol to evaluate the possible reversal of the inhibition induced by compounds 2, 3 and 4. Inhibition was easily suppressed in all three cases, recovering the viability of C. glabrata. These results reveal that the CgHMGR model is excellent for testing antifungals. Compound 4 produced the best effect and is herein proposed as a new potent antifungal agent.

Epithelial-mesenchymal transition (EMT) beyond EGFR mutations per se is a common mechanism for acquired resistance to EGFR TKI.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) is a major advance in treating NSCLC with EGFR-activating mutations. However, acquired resistance, due partially to secondary mutations limits their use. Here we report that NSCLC cells with acquired resistance to gefitinib or osimertinib (AZD9291) exhibit EMT features, with a decrease in E-cadherin, and increases in vimentin and stemness, without possessing any EGFR secondary mutations. Knockdown of E-cadherin in parental cells increased gefitinib resistance and stemness, while knockdown of vimentin in resistant cells resulted in opposite effects. Src activation and Hakai upregulation were found in gefitinib-resistant cells. Knockdown of Hakai elevated E-cadherin expression, attenuated stemness, and resensitized the cells to gefitinib. Clinical cancer specimens with acquired gefitinib resistance also showed a decrease in E-cadherin and an increase in Hakai expression. The dual HDAC and HMGR inhibitor JMF3086 inhibited the Src/Hakai and Hakai/E-cadherin interaction to reverse E-cadherin expression, and attenuated vimentin and stemness to restore gefitinib sensitivity. The EMT features of AZD9291-resistant H1975 cells were related to the upregulation of Zeb1. Both gefitinib and AZD9291 sensitivity was restored by JMF3086 through reversing EMT. Our study not only revealed a common mechanism of EMT in both gefitinib and AZD9291 resistance beyond EGFR mutations per se, but also provides a new strategy to overcome it.

Antiglycation study of HMG-R inhibitors and tocotrienol against glycated BSA and LDL: A comparative study

Non-enzymatic glycation mediated advanced glycation end products (AGEs) generation results in the pathogenesis of diabetic complications and atherosclerotic cardiovascular disease (ASCVD) which is greatly influenced by 3-hydroxy-3-methyl-glutaryl Co-A reductase (HMG-R) activity. HMG-R inhibitors, statins, are well known for reducing mortality and morbidity of ASCVD in patients with diabetes due to their pleiotropic effects independent of cholesterol lowering. Due to distinct chemical structures, various statins may play important role in the inhibition of AGEs mediated pathologies. Herein, we evaluated the anti-glycating potential of atorvastatin (AT), rosuvastatin (RT), pitavastatin (PT), fluvastatin (FT), simvastatin (ST) alone as well in combination with ezetimibe (EZ) and tocotrienol (TT) against d-ribose mediated BSA and LDL glycation by various physicochemical approaches. Our data suggested that AT, TT, RT, EZ, EZ-AT, and EZ-RT were able to substantially inhibit the AGEs formation via modulation of hyperchromicity, fluorogenic AGEs, % contribution of α-helix and β-sheets to protein secondary structure, amide-I band stretching, carbonyl and HMF content in Gly-BSA as well as Gly-LDL. On the basis of above findings, we concluded that HMG-R inhibitors and TT, alone or in combination with EZ, may be established as terrific therapeutic agents for the patients suffering from AGEs induced diabetic cum ASCVD complications.

Volatiles Emitted at Different Flowering Stages of Jasminum sambac and Expression of Genes Related to α-Farnesene Biosynthesis.

Fresh jasmine flowers have been used to make jasmine teas in China, but there has been no complete information about volatile organic compound emissions in relation to flower developmental stages and no science-based knowledge about which floral stage should be used for the infusion. This study monitored volatile organic compounds emitted from living flowers of Jasminum sambac (L.) Ait. ‘Bifoliatum’ at five developmental stages and also from excised flowers. Among the compounds identified, α-farnesene, linalool, and benzyl acetate were most abundant. Since α-farnesene is synthesized through the Mevalonate pathway, four genes encoding 3-hydroxy-3-methylglutaryl coenzyme A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), farnesyl pyrophosphate synthase, and terpene synthase were isolated. Their expression patterns in living flowers at the five stages and in excised flowers coincided with the emission patterns of α-farnesene. Application of lovastatin, a HMGR inhibitor, significantly reduced the expression of the genes and greatly decreased the emission of α-farnesene. The sweet scent was diminished from lovastatin-treated flowers as well. These results indicate that α-farnesene is an important compound emitted from jasmine flowers, and its emission patterns suggest that flowers at the opening stage or flower buds 8 h after excision should be used for the infusion of tea leaves.

Mevalonate Cascade and Small Rho GTPase in Spinal Cord Injury.

The mevalonate pathway has been extensively studied for its involvement in cholesterol synthesis. Inhibition of this pathway using statins (3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors; HMGR inhibitors) is the primarily selected method due to its cholesterol-lowering effect, making statins the most commonly used (86-94%) cholesterol-lowering drugs in adults. This pathway has several other by-products that are affected by statins including GTPase molecules (guanine triphosphate-binding kinases), such as Rho/Rho-associated coiled kinase (ROCK) kinases, that are implicated in other diseases, including those of the central nervous system (CNS). These molecules control several aspects of neural cell life including axonal growth, cellular migration, and cell death, and therefore, are of increasing interest in the field of spinal cord injury (SCI). Limited regeneration capacity of nerve fibers in adult CNS has been considered the main obstacle for finding a SCI cure. Over the past two decades, the identity of inhibitory factors for regeneration has been widely investigated. It is well-established that the Rho/ROCK kinase system is specifically activated by the components of damaged spinal cord tissue, including oligodendrocytes and myelin, as well as extracellular matrix. This has led many groups to hypothesize that statin therapy may in fact enhance the current neurorestorative approaches. In this mini-review, a summary of SCI pathophysiology is discussed and the current literature targeting the regeneration obstacles in SCI are reviewed, with special attention to recent publications of the past decade. In addition, we focus on the current literature involving the use of pharmacological and molecular inhibitors of small GTPase molecules for treatment of neurotrauma. Inhibiting these molecules has been shown to increase neuroprotection, enhance axonal regeneration, and facilitate the implementation of cell replacement therapies. Based upon available literature, the need for clinical trials involving targeted inhibition of GTPase molecules remains strong. Some of these drugs are widely used for other diseases, and therefore re-purposing their application for neurotrauma can be fasttracked. These approaches can potentially modify the inhibitory environment of nervous tissue to allow the spontaneous repair capacity of injured tissue.

Recombinant 3-Hydroxy 3-Methyl Glutaryl-CoA Reductase from Candida glabrata (Rec-CgHMGR) Obtained by Heterologous Expression, as a Novel Therapeutic Target Model for Testing Synthetic Drugs.

The enzyme 3-hydroxy-3-methyl-glutaryl CoA reductase (HMGR) is a glycoprotein of the endoplasmic reticulum that participates in the mevalonate pathway, the precursor of cholesterol in human and ergosterol in fungi. This enzyme has three domains: transmembrane, binding, and soluble. In this study, we expressed and purified the soluble fraction of the HMGR enzyme from Candida glabrata (CgHMGR) in an Escherichia coli heterologous system and used it as a model for studying its inhibitory activity. The soluble fraction of CgHMGR was fused to the maltose binding protein (MBP), purified, and characterized. Optimal pH was 8.0, and its optimal temperature activity was 37°C. The k m and V max for the HMG-CoA were 6.5μM and 2.26×10-3μMmin-1, respectively. Recombinant CgHMGR was inhibited by simvastatin presenting an IC50 at 14.5μM. In conclusion, our findings suggest that the recombinant HMGR version from C. glabrata may be used as a study model system for HMGR inhibitors such as statins and newly synthesized inhibitor compounds that might be used in the treatment of hypercholesterolemia or mycosis.

Insight into the mechanism of polyphenols on the activity of HMGR by molecular docking.

Statins are hypolipidemic drugs that are effective in the treatment of hypercholesterolemia by attenuating cholesterol synthesis in the liver via competitive inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Recently, dietary changes associated with drug therapy have garnered attention as novel drugs to mitigate or ameliorate hypercholesterolemia. The present study was undertaken to observe different dietary polyphenols that can bind to the active site of HMGR and inhibit it. Results from the 12 dietary polyphenols tested reveal that polyphenols can bind to HMGR and block the binding of nicotinamide adenine dinucleotide phosphate (NADP+). We observed that the rigidity of phenolic rings prevents the polyphenols from docking to the enzyme activity site. The presence of an ester linkage between the phenolic rings in (–)-epigallocatechin-3-gallate (EGCG) and the alkyl chain in curcumin allows them to orient in the active site of the HMGR and bind to the catalytic residues. EGCG and curcumin showed binding to the active site residues with a low GRID score, which may be a potential inhibitor of HMGR. Kaempferol showed binding to HMG-CoA, but with low binding affinity. These observations provide a rationale for the consistent hypolipidemic effect of EGCG and curcumin, which has been previously reported in several epidemiological and animal studies. Therefore, this study substantiates the mechanism of polyphenols on the activity of HMGR by molecular docking and provides the impetus for drug design involving further structure–function relationship studies.

Molecular modeling studies of atorvastatin analogues as HMGR inhibitors using 3D-QSAR, molecular docking and molecular dynamics simulations

3-Hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR) is generally regarded as targets for the treatment of hypercholesterolemia. HMGR inhibitors (more commonly known as statins) are discovered as plasma cholesterol lowering molecules. In this work, 120 atorvastatin analogues were studied using a combination of molecular modeling techniques including three-dimensional quantitative structure–activity relationship (3D-QSAR), molecular docking and molecular dynamics (MD) simulation. The results show that the best CoMFA (comparative molecular field analysis) model has q2=0.558 and r2=0.977, and the best CoMSIA (comparative molecular similarity indices analysis) model has q2=0.582 and r2=0.919. Molecular docking and MD simulation explored the binding relationship of the ligand and the receptor protein. The calculation results indicated that the hydrophobic and electrostatic fields play key roles in QSAR model. After MD simulation, we found four vital residues (Lys735, Arg590, Asp690 and Asn686) and three hydrophobic regions in HMGR binding site. The calculation results show that atorvastatin analogues obtained by introduction of F atoms or gem-difluoro groups could obviously improve the inhibitory activity. The new HMGR inhibitor analogues design in this Letter had been submitted which is being currently synthesized by our laboratories.

Exploration of Virtual Candidates for Human HMG-CoA Reductase Inhibitors Using Pharmacophore Modeling and Molecular Dynamics Simulations

3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is a rate-controlling enzyme in the mevalonate pathway which involved in biosynthesis of cholesterol and other isoprenoids. This enzyme catalyzes the conversion of HMG-CoA to mevalonate and is regarded as a drug target to treat hypercholesterolemia. In this study, ten qualitative pharmacophore models were generated based on chemical features in active inhibitors of HMGR. The generated models were validated using a test set. In a validation process, the best hypothesis was selected based on the statistical parameters and used for virtual screening of chemical databases to find novel lead candidates. The screened compounds were sorted by applying drug-like properties. The compounds that satisfied all drug-like properties were used for molecular docking study to identify their binding conformations at active site of HMGR. The final hit compounds were selected based on docking score and binding orientation. The HMGR structures in complex with the hit compounds were subjected to 10 ns molecular dynamics simulations to refine the binding orientation as well as to check the stability of the hits. After simulation, binding modes including hydrogen bonding patterns and molecular interactions with the active site residues were analyzed. In conclusion, four hit compounds with new structural scaffold were suggested as novel and potent HMGR inhibitors.

The 3-hydroxy-3-methylglutaryl coenzyme-A reductases from fungi: A proposal as a therapeutic target and as a study model

The enzyme 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR) catalyzes the conversion of HMG-Co-A into mevalonate. This step is the limiting point for the synthesis of cholesterol in mammals and ergosterol in fungi. We describe in this article the genome organization of HMGR coding genes and those deduced from different fungi, recount the evidence showing statins as HMGR inhibitors for ergosterol synthesis and its effect in yeast viability, and propose fungal HMGR (HMGRf) as a model to study the use of pharmaceutical compounds to inhibit cholesterol and ergosterol synthesis. Bibliographical search and bioinformatic analyses were performed and discussed. HMGRfs belong to the class I with a high homology in the catalytic region. The sterol biosynthetic pathway in humans and fungi share many enzymes in the initial steps (such as the HMGR enzyme), but in the last steps enzymes are different rendering the two final products: cholesterol in mammals and ergosterol in fungi. With regards to inhibitors such as statins and other compounds, these affect also fungal viability. Since HMGR from Schizosaccharomyces pombe and Ustilago maydis are very similar to the human HMGR in the catalytic regions, we propose that fungal enzymes can be used to test inhibitors for a potential use in humans. We consider that HMGRf is a good therapeutic target to design and test new antifungal compounds.This manuscript is part of the series of works presented at the “V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi” (Oaxaca, Mexico, 2012).

The effects of statins on the mevalonic acid pathway in recombinant yeast strains expressing human HMG-CoA reductase

BackgroundThe yeast Saccharomyces cerevisiae can be a useful model for studying cellular mechanisms related to sterol synthesis in humans due to the high similarity of the mevalonate pathway between these organisms. This metabolic pathway plays a key role in multiple cellular processes by synthesizing sterol and nonsterol isoprenoids. Statins are well-known inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key enzyme of the cholesterol synthesis pathway. However, the effects of statins extend beyond their cholesterol-lowering action, since inhibition of HMGR decreases the synthesis of all products downstream in the mevalonate pathway. Using transgenic yeast expressing human HMGR or either yeast HMGR isoenzyme we studied the effects of simvastatin, atorvastatin, fluvastatin and rosuvastatin on the cell metabolism.ResultsStatins decreased sterol pools, prominently reducing sterol precursors content while only moderately lowering ergosterol level. Expression of genes encoding enzymes involved in sterol biosynthesis was induced, while genes from nonsterol isoprenoid pathways, such as coenzyme Q and dolichol biosynthesis or protein prenylation, were diversely affected by statin treatment. Statins increased the level of human HMGR protein substantially and only slightly affected the levels of Rer2 and Coq3 proteins involved in non-sterol isoprenoid biosynthesis.ConclusionStatins influence the sterol pool, gene expression and protein levels of enzymes from the sterol and nonsterol isoprenoid biosynthesis branches and this effect depends on the type of statin administered. Our model system is a cheap and convenient tool for characterizing individual statins or screening for novel ones, and could also be helpful in individualized selection of the most efficient HMGR inhibitors leading to the best response and minimizing serious side effects.

992 A PARACRINE HEDGEHOG SIGNALING MICROENVIRONMENT IN ADT-TREATED PROSTATE TUMORS INDUCES STEROIDOGENESIS FROM BENIGN PROSTATE STROMAL CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research (V)1 Apr 2013992 A PARACRINE HEDGEHOG SIGNALING MICROENVIRONMENT IN ADT-TREATED PROSTATE TUMORS INDUCES STEROIDOGENESIS FROM BENIGN PROSTATE STROMAL CELLS Amy Lubik, Emma Guns, Hans Adomat, and Ralph Buttyan Amy LubikAmy Lubik Vancouver, Canada More articles by this author , Emma GunsEmma Guns Vancouver, Canada More articles by this author , Hans AdomatHans Adomat Vancouver, Canada More articles by this author , and Ralph ButtyanRalph Buttyan Vancouver, Canada More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.574AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Androgen deprivation therapy (ADT) induces the expression and release of Hedgehog ligands (Hhs) from prostate cancer (PCa) cells. Previously we showed that treatment of primary human prostate stromal cells (PrSCs) with an HH agonist (SAG) increased the expression of steroidogenic genes and enabled them to significantly increase production of testosterone (T) when fed with DHEA, an adrenal precursor. We proposed that a paracrine HH microenvironment in ADT-treated PCas contribute to intratumoral steroidogenesis through this process. Here we show that steroidogenic activity of PrSCs is induced by alternate HH agonists and better describe the steroidogenic process leading to androgen production in PrSCs induced by paracrine HH. METHODS Human primary PrSCs (Clonetics) were grown in recommended medium. PrSCs were pre-starved of serum to induce primary cilia formation, then treated with 1 nM Ag1.5 (a HH agonist) for 48 hrs. Expression of steroidogenic genes was measured by quantitative PCR. Outputs of steroids from vehicle- or agonist-treated PrSC cells fed 22 OH-cholesterol were measured by LC/MS/MS. The HH antagonist, KAAD-cyclopamine (100 nM) or an HMGR inhibitor (simvastatin) was used to block the PrSC response to Ag1.5 or to inhibit active Hh release from Sonic Hh producing LNCaP cells. RESULTS Ag1.5 is as effective as 100 nM SAG in inducing expression of steroidogenic genes in PrSCs. Outputs of progesterone, DHEA, androsterone, T, or dihydrotestosterone (DHT) from 22 OH-cholesterol was significantly increased by Ag1.5. The effects of Ag1.5 on the expression of these genes was blocked by KAAD-cyclopamine. The ability of Ag1.5 to induce RDH5 expression and androsterone production show that the DHT output from PrSCs was a consequence of increased classical and backdoor pathway activities. Steroid outputs from HH-induced PrSCs was significantly higher (on a weight-per-weight basis) than production from parental LNCaP or the androgen growth-independent LNCaP-AI cells. Simvastatin reduced the release of active Hhs from LNCaP-Sonic Hh producing cells as measured by the reduced ability of conditioned medium from treated cells to induce steroidogenic genes in PrSCs. CONCLUSIONS Our results further confirm the potential of a paracrine HH signaling microenvironment of ADT-treated prostate tumor to induce steroidogenesis and androgen production from benign PrSCs. Outcomes show that a HH antagonist or cholesterol synthesis inhibitor might block the effects of paracrine HH in an ADT-treated tumor and delay progression to CRPC. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e407 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Amy Lubik Vancouver, Canada More articles by this author Emma Guns Vancouver, Canada More articles by this author Hans Adomat Vancouver, Canada More articles by this author Ralph Buttyan Vancouver, Canada More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

A Genetic and Pharmacological Analysis of Isoprenoid Pathway by LC-MS/MS in Fission Yeast

Currently, statins are the only drugs acting on the mammalian isoprenoid pathway. The mammalian genes in this pathway are not easily amenable to genetic manipulation. Thus, it is difficult to study the effects of the inhibition of various enzymes on the intermediate and final products in the isoprenoid pathway. In fission yeast, antifungal compounds such as azoles and terbinafine are available as inhibitors of the pathway in addition to statins, and various isoprenoid pathway mutants are also available. Here in these mutants, treated with statins or antifungals, we quantified the final and intermediate products of the fission yeast isoprenoid pathway using liquid chromatography-mass spectrometry/mass spectrometry. In hmg1-1, a mutant of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), ergosterol (a final sterol product), and squalene (an intermediate pathway product), were decreased to approximately 80% and 10%, respectively, compared with that of wild-type cells. Consistently in wild-type cells, pravastatin, an HMGR inhibitor decreased ergosterol and squalene, and the effect was more pronounced on squalene. In hmg1-1 mutant and in wild-type cells treated with pravastatin, the decrease in the levels of farnesyl pyrophosphate and geranylgeranyl pyrophosphate respectively was larger than that of ergosterol but was smaller than that of squalene. In Δerg6 or Δsts1 cells, mutants of the genes involved in the last step of the pathway, ergosterol was not detected, and the changes of intermediate product levels were distinct from that of hmg1-1 mutant. Notably, in wild-type cells miconazole and terbinafine only slightly decreased ergosterol level. Altogether, these studies suggest that the pleiotropic phenotypes caused by the hmg1-1 mutation and pravastatin might be due to decreased levels of isoprenoid pyrophosphates or other isoprenoid pathway intermediate products rather than due to a decreased ergosterol level.

3‐hydroxy 3‐methylglutaryl coenzyme a reductase inhibition impairs muscle regeneration

Skeletal muscle has the ability to regenerate new muscle fibers after injury. The process of new muscle formation requires that quiescent mononuclear muscle precursor cells (myoblasts) become activated, proliferate, differentiate, and fuse into multinucleated myotubes which, in turn, undergo further differentiation and mature to form functional muscle fibers. Previous data demonstrated the crucial role played by 3-hydroxy 3-methylglutaryl coenzyme A reductase (HMGR), the rate-limiting enzyme of cholesterol biosynthetic pathway, in fetal rat myoblast (L6) differentiation. This finding, along with epidemiological studies assessing the myotoxic effect of statins, HMGR inhibitors, allowed us to speculate that HMGR could be strongly involved in skeletal muscle repair. Thus, our research was aimed at evaluating such involvement: in vitro and in vivo experiments were performed on both mouse adult satellite cell derived myoblasts (SCDM) and mouse muscles injured with cardiotoxin. Results demonstrate that HMGR inhibition by the statin Simvastatin reduces SCDM fusion index, fast MHC protein levels by 60% and slow MHC by 40%. Most importantly, HMGR inhibition delays skeletal muscle regeneration in vivo. Thus, besides complaining of myopathies, patients given Simvastatin could also undergo an impairment in muscle repair.