Mevalonate Kinase Deficiency and Squalene Synthase Inhibitor (TAK-475): The Balance to Extinguish the Inflammation.

Mevalonate Kinase Deficiency (MKD) is a rare inborn disease belonging to the family of periodic fever syndromes. The MKD phenotype is characterized by systemic inflammation involving multiple organs, including the nervous system. Current anti-inflammatory approaches to MKD are only partially effective and do not act specifically on neural inflammation. According to the new emerging pharmacology trends, the repositioning of drugs from the indication for which they were originally intended to another one can make mechanistic-based medications easily available to treat rare diseases. According to this perspective, the squalene synthase inhibitor Lapaquistat (TAK-475), originally developed as a cholesterol-lowering drug, might find a new indication in MKD, by modulating the mevalonate cholesterol pathway, increasing the availability of anti-inflammatory isoprenoid intermediates. Using an in vitro model for MKD, we mimicked the blockade of the cholesterol pathway and evaluated the potential anti-inflammatory effect of Lapaquistat. The results obtained showed anti-inflammatory effects of Lapaquistat in association with a low blockade of the metabolic pathway, while this effect did not remain with a tighter blockade. On these bases, Lapaquistat could be configured as an effective treatment for MKD's mild forms, in which the residual enzymatic activity is only reduced and not almost completely absent as in the severe forms.

Chemical Genetics Reveals a Role of Squalene Synthase in TGFß Signaling and Cardiomyogenesis.

KY02111 is a widely used small molecule that boosts cardiomyogenesis of the mesoderm cells derived from pluripotent stem cells, yet its molecular mechanism of action remains elusive. The present study resolves the initially perplexing effects of KY02111 on Wnt signaling and subsequently identifies squalene synthase (SQS) as a molecular target of KY02111 and its optimized version, KY-I. By disrupting the interaction of SQS with cardiac ER-membrane protein TMEM43, KY02111 impairs TGFß signaling, but not Wnt signaling, and thereby recapitulates the clinical mutation of TMEM43 that causes arrhythmogenic right ventricular cardiomyopathy (ARVC), an inherited heart disease that involves a substitution of myocardium with fatty tissue. These findings reveal a heretofore undescribed role of SQS in TGFß signaling and cardiomyogenesis. KY02111 may find its use in ARVC modeling as well as serve as a chemical tool for studying TGFß/SMAD signaling.

Bisphosphonate inhibitors of squalene synthase protect cells against cholesterol-dependent cytolysins.

Certain species of pathogenic bacteria damage tissues by secreting cholesterol-dependent cytolysins, which form pores in the plasma membranes of animal cells. However, reducing cholesterol protects cells against these cytolysins. As the first committed step of cholesterol biosynthesis is catalyzed by squalene synthase, we explored whether inhibiting this enzyme protected cells against cholesterol-dependent cytolysins. We first synthesized 22 different nitrogen-containing bisphosphonate molecules that were designed to inhibit squalene synthase. Squalene synthase inhibition was quantified using a cell-free enzyme assay, and validated by computer modeling of bisphosphonate molecules binding to squalene synthase. The bisphosphonates were then screened for their ability to protect HeLa cells against the damage caused by the cholesterol-dependent cytolysin, pyolysin. The most effective bisphosphonate reduced pyolysin-induced leakage of lactate dehydrogenase into cell supernatants by >80%, and reduced pyolysin-induced cytolysis from >75% to <25%. In addition, this bisphosphonate reduced pyolysin-induced leakage of potassium from cells, limited changes in the cytoskeleton, prevented mitogen-activated protein kinases cell stress responses, and reduced cellular cholesterol. The bisphosphonate also protected cells against another cholesterol-dependent cytolysin, streptolysin O, and protected lung epithelial cells and primary dermal fibroblasts against cytolysis. Our findings imply that treatment with bisphosphonates that inhibit squalene synthase might help protect tissues against pathogenic bacteria that secrete cholesterol-dependent cytolysins.

Inhibition of squalene synthase of rat liver by abietane diterpenes derivatives.

In the current study, chemical composition of cultivated Salvia canariensis L was determined. Carnosol was the main product isolated. We prepared more lipophilic derivatives from carnosol, and both isolated and semisynthetic abietane diterpenes were evaluated in vitro as inhibitors of squalene synthase. Among the compounds tested, carnosol was the most potent inhibitor (IC50 = 17.6 µM). These results highlight the great potential of this species for the production of new ingredients in nutritional supplements for the treatment of hyperlipidemia.

Roles of Farnesyl-Diphosphate Farnesyltransferase 1 in Tumour and Tumour Microenvironments.

Farnesyl-diphosphate farnesyltransferase 1 (FDFT1, squalene synthase), a membrane-associated enzyme, synthesizes squalene via condensation of two molecules of farnesyl pyrophosphate. Accumulating evidence has noted that FDFT1 plays a critical role in cancer, particularly in metabolic reprogramming, cell proliferation, and invasion. Based on these advances in our knowledge, FDFT1 could be a potential target for cancer treatment. This review focuses on the contribution of FDFT1 to the hallmarks of cancer, and further, we discuss the applicability of FDFT1 as a cancer prognostic marker and target for anticancer therapy.

Bavachinin inhibits cholesterol synthesis enzyme FDFT1 expression via AKT/mTOR/SREBP-2 pathway.

Non-alcoholic fatty liver disease (NAFLD) is a progressive and chronic liver disease. No effective drug is currently approved for the treatment of NAFLD. Traditionally it is thought that pathogenesis of NAFLD develops from some imbalance in lipid control, thereby leading to hepatotoxicity and disease development. Squalene synthase (SQS), encoded by FDFT1, is a key regulator in cholesterol synthesis and thus a potential target for the treatment of NAFLD. Here we could identify bavachinin, a component from traditional Chinese medicine Fructus Psoraleae (FP), which apparently protects HepaRG cells from palmitic acid induced death, suppressing lipid accumulation and cholesterol synthesis through inhibition of FDFT1 through the AKT/mTOR/SREBP-2 pathway. Over-expression of FDFT1 abolished bavachinin (BVC) -induced inhibition of cholesterol synthesis. The data presented here suggest that bavachinin acts as a cholesterol synthesis enzyme inhibitor, and might serve as a drug for treating NAFLD in the future.

New applications of squalene synthase inhibitors: Membrane cholesterol as a therapeutic target.

Squalene synthase (SQS) inhibitors, mostly known as antihyperlipidemic agents for controlling blood cholesterol levels, have been increasingly used to study alterations of the cholesterol content in cell membranes. As such, SQS inhibitors have been demonstrated to control cellular activities related to cancer cell proliferation and migration, neuron degeneration, and parasite growth. While the mechanisms behind the effects of cellular cholesterol are still being revealed in detail, the evidence for SQS as a therapeutic target for several seemingly unrelated diseases is increasing. SQS inhibitors may be the next promising candidates targeting the three remaining primary therapeutic areas, beyond cardiovascular disease, which still need to be addressed; their application as anticancer, antimicrobial, and antineurodegenerative agents appears promising for new drug discovery projects underway.

Discovery of a Small-Molecule-Dependent Photolytic Peptide.

We accidentally found that YM-53601, a known small-molecule inhibitor of squalene synthase (SQS), selectively depletes SQS from mammalian cells upon UV irradiation. Further analyses indicated that the photodepletion of SQS requires its short peptide segment located at the COOH terminus. Remarkably, when the 27 amino acid peptide was fused to green fluorescent protein or unrelated proteins at either the NH2 or COOH terminus, such fusion proteins were selectively depleted when the cells were treated with both YM-53601 and UV exposure. Product analysis and electron spin resonance experiments suggested that the UV irradiation promotes homolytic C-O bond cleavage of the aryl ether group in YM-53601. It is likely that the radical species generated from UV-activated YM-53601 abstract hydrogen atoms from the SQS peptide, leading to the photolysis of the entire protein. The pair of the SQS peptide and YM-53601 discovered in the present study paves the way for the design of a new small-molecule-controlled optogenetic tool.

[Development of Synthetic Strategies for Densely Oxygenated Natural Products: Total Synthesis of Lactacystin and Zaragozic Acid C Using Photochemical C(sp3)-H Functionalization].

This review describes two novel synthetic routes from (S)-pyroglutaminol to (+)-lactacystin, a potent inhibitor of the 20S proteasome and from d-gluconolactone derivative to zaragozic acid C, a potent squalene synthase inhibitor. In lactacystin synthesis, the photoinduced intermolecular C(sp3)-H alkynylation and intramolecular C(sp3)-H acylation chemoselectively and stereoselectively constructed the tetrasubstituted and trisubstituted carbon centers, respectively. In the synthesis of zaragozic acid C, the stereoselective installation of the two contiguous tetrasubstituted carbons was achieved by the photochemical intramolecular C(sp3)-H acylation of a densely oxygenated intermediate.

Evaluation of potential inhibitors of squalene synthase based on virtual screening and in vitro studies.

Squalene synthase (SQS) is a potential target for hyperlipidemia treatment. To identify novel chemical scaffolds of SQS inhibitors, we generated 3D-QSAR pharmacophore models using HypoGen. The best quantitative pharmacophore model, Hypo 1, was selected for virtual screening using two chemical databases, Specs and Traditional Chinese Medicine database (TCM). The best-mapped hit compounds were then subjected to filtering by Lipinski's rule of five and docking studies to refine the hits. Finally, five compounds were selected from the top-ranked hit compounds for SQS inhibitory assay in vitro. Three of these compounds could inhibit SQS in vitro, and should be further evaluated pre-clinically as a treatment for hyperlipidemia.

Aspergillus flavus squalene synthase as an antifungal target: Expression, activity, and inhibition.

Invasive aspergillosis (IA) is a life-threatening disease impacting immunocompromised individuals. Standard treatments of IA, including polyenes and azoles, suffer from high toxicity and emerging resistance, leading to the need to develop new antifungal agents with novel mechanisms of action. Ergosterol biosynthesis is a classic target for antifungals, and squalene synthase (SQS) catalyzes the first committed step in ergosterol biosynthesis in Aspergillus spp. making SQS of interest in the context of antifungal development. Here, we cloned, expressed, purified and characterized SQS from the pathogen Aspergillus flavus (AfSQS), confirming that it produced squalene. To identify potential leads targeting AfSQS, we tested known squalene synthase inhibitors, zaragozic acid and the phosphonosulfonate BPH-652, finding that they were potent inhibitors. We then screened a library of 744 compounds from the National Cancer Institute (NCI) Diversity Set V for inhibition activity. 20 hits were identified and IC50 values were determined using dose-response curves. 14 compounds that interfered with the assay were excluded and the remaining 6 compounds were analyzed for drug-likeness, resulting in one compound, celastrol, which had an AfSQS IC50 value of 830 nM. Enzyme inhibition kinetics revealed that celastrol binds to AfSQS in a noncompetitive manner, but did not bind covalently. Since celastrol is also known to inhibit growth of the highly virulent Aspergillus fumigatus by inhibiting flavin-dependent monooxygenase siderophore A (SidA, under iron starvation conditions), it may be a promising multi-target lead for antifungal development.

Unpacking the Pathogen Box-An Open Source Tool for Fighting Neglected Tropical Disease.

The Pathogen Box is a 400-strong collection of drug-like compounds, selected for their potential against several of the world's most important neglected tropical diseases, including trypanosomiasis, leishmaniasis, cryptosporidiosis, toxoplasmosis, filariasis, schistosomiasis, dengue virus and trichuriasis, in addition to malaria and tuberculosis. This library represents an ensemble of numerous successful drug discovery programmes from around the globe, aimed at providing a powerful resource to stimulate open source drug discovery for diseases threatening the most vulnerable communities in the world. This review seeks to provide an in-depth analysis of the literature pertaining to the compounds in the Pathogen Box, including structure-activity relationship highlights, mechanisms of action, related compounds with reported activity against different diseases, and, where appropriate, discussion on the known and putative targets of compounds, thereby providing context and increasing the accessibility of the Pathogen Box to the drug discovery community.

Studies with Guanidinium- and Amidinium-Based Inhibitors Suggest Minimal Stabilization of Allylic Carbocation Intermediates by Dehydrosqualene and Squalene Synthases.

Dehydrosqualene and squalene synthases catalyze the redox neutral and the reductive, head-to-head dimerization of farnesyl diphosphate, respectively. In each case, the reaction is thought to proceed via an initial dissociation of farnesyl diphosphate to form an allylic carbocation-pyrophosphate ion pair. This work describes the synthesis and testing of inhibitors in which a guanidinium or amidinium moiety is flanked by a phosphonylphosphinate group and a hydrocarbon tail. These functional groups bear a planar, delocalized, positive charge and therefore should act as excellent mimics of an allylic carbocation. An inhibitor bearing a neutral urea moiety was also prepared as a control. The positively charged inhibitors acted as competitive inhibitors against Staphylococcus aureus dehydrosqualene synthase with Ki values in the low micromolar range. Surprisingly, the neutral urea inhibitor was the most potent of the three. Similar trends were seen with the first half reaction of human squalene synthase. One interpretation of these results is that the active sites of these enzymes do not directly stabilize the allylic carbocation via electrostatic or π-cation interactions. Instead, it is likely that the enzymes use tight binding to the pyrophosphate and lipid moieties to promote catalysis and that electrostatic stabilization of the carbocation is provided by the bound pyrophosphate product. An alternate possibility is that these inhibitors cannot bind to the "ionization FPP-binding site" of the enzyme and only bind to the "nonionizing FPP-binding site". In either case, all reported attempts to generate potent inhibitors with cationic FPP analogues have been unsuccessful to date.

Rational Discovery of Novel Squalene Synthase Inhibitors through Pharmacophore Modelling.

INTRODUCTION: In the present research work, a pharmacophore based virtual screening was performed using Discovery Studio 2.1 for the discovery of some novel molecules as inhibitors of Squalene Synthase Enzyme, a key enzyme in cholesterol biosynthetic pathway. METHODS: A quantitative pharmacophore HypoGen was generated and the best HypoGen had two ring aromatic and one hydrogen bond acceptor lipid features. The best HypoGen showed a very good correlation coefficient (r = 0.901) with satisfactory cost analysis. Furthermore, the HypoGen was validated externally by predicting the activity of test set. The developed model was found to be predictive as it showed low error of prediction for test set molecules. The developed model was used as a search query for virtually screening two chemical databases: sample database from catalyst and minimaybridge. RESULTS AND DISCUSSION: The best hit with good fit value and low predicted activity was further modified to design novel drug-like molecules, which were able to bind to Squalene synthase enzyme active site. CONCLUSION: The best scoring molecule, compound 67 showed 53% inhibition of the human Squalene synthase enzyme, isolated from the cell lysates of Human Hepatoma Cell Line, at a dose of 10 mcg with an IC50 value of 9.43 µm.

Discovery of Potential Inhibitors of Squalene Synthase from Traditional Chinese Medicine Based on Virtual Screening and In Vitro Evaluation of Lipid-Lowering Effect.

Squalene synthase (SQS), a key downstream enzyme involved in the cholesterol biosynthetic pathway, plays an important role in treating hyperlipidemia. Compared to statins, SQS inhibitors have shown a very significant lipid-lowering effect and do not cause myotoxicity. Thus, the paper aims to discover potential SQS inhibitors from Traditional Chinese Medicine (TCM) by the combination of molecular modeling methods and biological assays. In this study, cynarin was selected as a potential SQS inhibitor candidate compound based on its pharmacophoric properties, molecular docking studies and molecular dynamics (MD) simulations. Cynarin could form hydrophobic interactions with PHE54, LEU211, LEU183 and PRO292, which are regarded as important interactions for the SQS inhibitors. In addition, the lipid-lowering effect of cynarin was tested in sodium oleate-induced HepG2 cells by decreasing the lipidemic parameter triglyceride (TG) level by 22.50%. Finally. cynarin was reversely screened against other anti-hyperlipidemia targets which existed in HepG2 cells and cynarin was unable to map with the pharmacophore of these targets, which indicated that the lipid-lowering effects of cynarin might be due to the inhibition of SQS. This study discovered cynarin is a potential SQS inhibitor from TCM, which could be further clinically explored for the treatment of hyperlipidemia.

Repositioning of Tak-475 In Mevalonate Kinase Disease: Translating Theory Into Practice.

BACKGROUND: Mevalonate Kinase Deficiency (MKD, OMIM #610377) is a rare autosomal recessive metabolic and inflammatory disease. In MKD, defective function of the enzyme mevalonate kinase, due to a mutation in the MVK gene, leads to the shortage of mevalonate- derived intermediates, which results in unbalanced prenylation of proteins and altered metabolism of sterols. These defects lead to a complex multisystem inflammatory and metabolic syndrome. OBJECTIVE: Although biologic therapies aimed at blocking the inflammatory cytokine interleukin- 1 can significantly reduce inflammation, they cannot completely control the clinical symptoms that affect the nervous system. For this reason, MKD can still be considered an orphan drug disease. The availability of MKD models reproducing the MKD-systematic inflammation, is crucial to improve the knowledge on its pathogenesis, which is still unknown. New therapies are also required in order to improve pateints' conditions and their quality of life. METHODS: MKD-cellular models can be obtained by biochemical inhibition of mevalonatederived isoprenoids. Of note, these cells present an exaggerated response to inflammatory stimuli that can be reduced by treatment with zaragozic acid, an inhibitor of squalene synthase, thus increasing the availability of isoprenoids intermediates upstream the enzymatic block. RESULTS: A similar action might be obtained by lapaquistat acetate (TAK-475, Takeda), a drug that underwent extensive clinical trials as a cholesterol lowering agent 10 years ago, with a good safety profile. CONCLUSIONS: Here we describe the preclinical evidence supporting the possible repositioning of TAK-475 from its originally intended use to the treatment of MKD and discuss its potential to modulate the mevalonate pathway in inflammatory diseases.

Enantiospecific total synthesis of the squalene synthase inhibitors (-)-CJ-13,982 and its enantiomer from a common intermediate.

The total syntheses of both the natural and unnatural enantiomers of the alkyl citrate natural product CJ-13,982 (1) from the common d-ribose-derived acid 6 are described.

Inhibiting mevalonate pathway enzymes increases stromal cell resilience to a cholesterol-dependent cytolysin.

Animal health depends on the ability of immune cells to kill invading pathogens, and on the resilience of tissues to tolerate the presence of pathogens. Trueperella pyogenes causes tissue pathology in many mammals by secreting a cholesterol-dependent cytolysin, pyolysin (PLO), which targets stromal cells. Cellular cholesterol is derived from squalene, which is synthesized via the mevalonate pathway enzymes, including HMGCR, FDPS and FDFT1. The present study tested the hypothesis that inhibiting enzymes in the mevalonate pathway to reduce cellular cholesterol increases the resilience of stromal cells to PLO. We first verified that depleting cellular cholesterol with methyl-ß-cyclodextrin increased the resilience of stromal cells to PLO. We then used siRNA to deplete mevalonate pathway enzyme gene expression, and used pharmaceutical inhibitors, atorvastatin, alendronate or zaragozic acid to inhibit the activity of HMGCR, FDPS and FDFT1, respectively. These approaches successfully reduced cellular cholesterol abundance, but mevalonate pathway enzymes did not affect cellular resilience equally. Inhibiting FDFT1 was most effective, with zaragozic acid reducing the impact of PLO on cell viability. The present study provides evidence that inhibiting FDFT1 increases stromal cell resilience to a cholesterol-dependent cytolysin.

Potential antimicrobial agents from triazole-functionalized 2H-benzo[b][1,4]oxazin-3(4H)-ones.

A series of substituted triazole functionalized 2H-benzo[b][1,4]oxazin-3(4H)-ones were synthesized by employing click chemistry and further characterized based on 1H NMR, 13C NMR, IR and mass spectral studies. All the synthesized derivatives were screened for their in vitro antimicrobial activities. Further, molecular docking studies were accomplished to explore the binding interactions between 1,2,3-triazol-4-yl-2H-benzo[b][1,4]oxazin-3(4H)-one and the active site of Staphylococcus aureus (CrtM) dehydrosqualene synthase (PDB ID: 2ZCS). These docking studies revealed that the synthesized derivatives showed high binding energies and strong H-bond interactions with the dehydrosqualene synthase validating the observed antimicrobial activity data. Based on antimicrobial activity and docking studies, the compounds 9c, 9d and 9e were identified as promising antimicrobial leads.

Developing potential agents against atherosclerosis: Design, synthesis and pharmacological evaluation of novel dual inhibitors of oxidative stress and Squalene Synthase activity.

For the treatment of multifactorial and complex diseases, it has become increasingly apparent that compounds acting at multiple targets often deliver superior efficacy compared to compounds with high specificity for only a single target. Based on previous studies demonstrating the important antioxidant and anti-hyperlipidemic effect of morpholine and 1,4-benzo(x/thi)azine derivatives (A-E), we hereby present the design, synthesis and pharmacological evaluation of novel dual-acting molecules as a therapeutic approach for atherosclerosis. Analogues 1-10 were rationally designed through structural modifications of their parent compounds (A-E) in order for structure-activity relationship studies to be carried out. Most compounds showed a significant inhibition against Squalene Synthase activity exhibiting at the same time a very potent multimodal antioxidant (against lipid peroxidation and as free-radical scavengers) effect, thus bringing to light the 2-aryl-1,4-benzo(x/thia)zin-2-ol scaffold as an outstanding pharmacophore for the design of potent antioxidants. Finally, the replacement of the octahydro-1,4-benzoxazine moiety of lead compound D with its respective 1,4-benzothiazine (compound 4), although conserved (anti-hypercholesterolemic) or even improved (anti-hyperlipidemic) activity, did not preserve the anti-diabetic effect of D.