Screening for <em>Thermotoga maritima</em> Membrane-Bound Pyrophosphatase Inhibitors

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave ...

University of Helsinki

Synthesis of reference standards is needed to determine the presence and function of steroid glucuronides in the brain or other tissues, because commercial sources of steroid glucuronide standards are limited or unavailable. In the present study porcine, rat, and bovine liver microsomes were tested to evaluate their ability to glucuronidate eight neurosteroids and neuroactive steroids of various types: dehydroepiandrosterone, pregnenolone, isopregnanolone, 5alpha-tetrahydrodeoxycorticosterone, corticosterone, cortisol, beta-estradiol, and testosterone. In general, the glucuronidation efficiency of rat liver was rather poor compared with that of bovine and porcine liver microsomes. Since porcine liver apparently has a relatively large amount of dehydrogenase, its microsomes also produced dehydrogenated steroids and their glucuronides, as well as various regioisomers in which the site of glucuronidation varied. In contrast, bovine liver microsomes produced mainly a single major glucuronidation product and few dehydrogenation products and gave the best overall yield for two-third of the steroids tested. The enzymatic synthesis of five glucuronides of four steroids was carried out and the conditions, purification, and analytical methods for the glucuronidation products were optimized. The steroid glucuronides synthesized were characterized by nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography-mass spectrometry (LC-MS). The stereochemically pure steroid glucuronide conjugates were recovered in milligram amounts (yield 10-78%) and good purity (>85-90%), which is sufficient for LC-MS/MS method development and analyses of steroid glucuronides in biological matrices such as brain, urine, or plasma.

Enzyme-assisted synthesis and characterization of glucuronide conjugates of neuroactive steroids.

As part of our continuing studies of polymer-supported pericyclic reactions for preparing biologically interesting heterocyclic compounds, we have introduced a traceless solid-phase synthesis of hexahydrocinnolines. We developed a method in which mild reaction conditions can be used for the hetero-Diels-Alder reactions on a polymeric support. The dienoic 3-vinyl-2-cyclohexenol attached to a Wang resin through an ether linkage undergoes [4 + 2] cycloaddition reaction with several azadienophiles. The highly stereoselective Diels-Alder reaction showed preferential formation of a single cycloadduct resulting from an anti attack of the dienophile on the polymer-bound diene. Trifluoroacetic acid-mediated cleavage of the polymer-bound cycloadducts yields fused nonaromatic hexahydrocinnolines in moderate yields in three steps.

Stereoselective aza Diels-Alder reaction on solid phase: a facile synthesis of hexahydrocinnoline derivatives.

Three angiotensin II receptor antagonists--losartan, candesartan, and zolarsartan--were investigated. All the compounds, which are structural analogues, are metabolized via conjugation to glucuronic acid. Interestingly, both O- and N-glucuronidation take place, so that regioisomers are formed. One ether O-glucuronide, two acyl O-glucuronides, and five tetrazole-N-glucuronides were biosynthesized, in milligram scale, from the three sartan aglycones. Liver microsomes from bovine, moose, rat, and pig and recombinant human UDP-glucuronosyltransferases were used as catalysts. The synthesized compounds were identified as sartan glucuronides by mass spectrometry, while the sites of glucuronidation were determined by nuclear magnetic resonance spectroscopy. Drug metabolites are needed as standards for pharmaceutical research and, as the present study shows, they can easily be produced with enzymes as catalyst.

Enzyme-assisted synthesis and structure characterization of glucuronic acid conjugates of losartan, candesartan, and zolarsartan.

Betulin, a naturally occurring abundant triterpene is converted in four steps to 3,28-di-O-acetyllupa-12,18-diene. When various 4-substituted urazoles were oxidized to the corresponding urazines with iodobenzene diacetate in the presence of 3,28-di-O-acetyllupa-12,18-diene, new heterocyclic betulin derivatives were produced. These betulin derivatives were examined in a microplate assay at 50 microM for their ability to inhibit the growth of Leishmania donovani axenic amastigotes, a species that causes the fatal visceral leishmaniasis. GI(50) (concentration for 50% growth inhibition) values of the most effective compounds were determined and their cytotoxicity on the human macrophage cell line THP-1 evaluated. The anti-leishmanial activity on L. donovani amastigotes growing in macrophages was also examined. The heterocycloadduct between 3,28-di-O-acetyllupa-12,18-diene and 4-methylurazine was the most effective derivative with an GI(50)=8.9 microM against L. donovani amastigotes.

Synthesis and anti-leishmanial activity of heterocyclic betulin derivatives.

Oncogenic Pim family kinases are often overexpressed in human hematopoietic malignancies as well as in solid tumours. These kinases contribute to tumorigenesis by promoting cell survival and by enhancing resistance against chemotherapy and radiation therapy. Furthermore, we have recently shown that they increase the metastatic potential of adherent cancer cells. Here we describe identification of tricyclic benzo[cd]azulenes and their derivatives as effective and selective inhibitors of Pim kinases. These compounds inhibit Pim autophosphorylation and abrogate the anti-apoptotic effects of Pim kinases. They also reduce cancer cell motility and suppress proliferation of lymphoblastoid cell lines infected and immortalized by the Epstein-Barr virus. Thus, these novel Pim-selective inhibitors provide promising compounds for both research and therapeutic purposes.

Tricyclic Benzo[cd]azulenes selectively inhibit activities of Pim kinases and restrict growth of Epstein-Barr virus-transformed cells.

Currently, nanosystems have been developed and applied as promising vehicles for different biomedical applications. We have developed three lignin nanoparticles (LNPs): pure lignin nanoparticles (pLNPs), iron(III)-complexed lignin nanoparticles (Fe-LNPs), and FeO-infused lignin nanoparticles (FeO-LNPs) with round shape, narrow size distribution, reduced polydispersity and good stability at pH 7.4. The LNPs showed low cytotoxicity in all the tested cell lines and hemolytic rates below 12% after 12 h of incubation. Additionally, they induced hydrogen peroxide production in a small extent and time-dependent manner, and the interaction with the cells increased over time, exhibiting a dose-dependent cell uptake. Concerning the drug loading, pLNPs showed the capacity to efficiently load poorly water-soluble drugs and other cytotoxic agents, e.g. sorafenib and benzazulene (BZL), and improve their release profiles at pH 5.5 and 7.4 in a sustained manner. Furthermore, the BZL-pLNPs presented an enhanced antiproliferation effect in different cells compared to the pure BZL and showed a maximal inhibitory concentration ranging from 0.64 to 12.4 μM after 24 h incubation. Overall, LNPs are promising candidates for drug delivery applications, and the superparamagnetic behavior of FeO-LNPs makes them promising for cancer therapy and diagnosis, such as magnetic targeting and magnetic resonance imaging.

In vitro evaluation of biodegradable lignin-based nanoparticles for drug delivery and enhanced antiproliferation effect in cancer cells.

The built-in o- and p-QM (QM = quinone methide) moieties in benzo[cd]azulen-3-ones account for an easy switch between the bridged 10π- and 6π-aromatic systems in organic synthesis. We report conjugate additions, oxidative nucleophilic substitutions of hydrogen, and reversible Michael additions under very mild conditions. In the presence of thiol nucleophiles, the protonated σ-adducts could be isolated and characterized. The typical preference for either the o- or p-QM moiety led to high regioselectivity. Furthermore, the inhibitory potency of the novel benzo[cd]azulenes against the human Pim-1 kinase was evaluated.

Nucleophilic Substitution of Hydrogen Facilitated by Quinone Methide Moieties in Benzo[cd]azulen-3-ones.

To carboxylate kraft lignin toward the functionalization of carboxylated lignin nanoparticles (CLNPs) with a block copolymer made of PEG, poly(histidine) and a cell-penetrating peptide and then evaluate the chemotherapeutic potential of the innovative nanoparticles.

Functionalization of carboxylated lignin nanoparticles for targeted and pH-responsive delivery of anticancer drugs.

The surface modification of nanoparticles (NPs) using different ligands is a common strategy to increase NP-cell interactions. Here, dentin phosphophoryn-derived peptide (DSS) lignin nanoparticles (LNPs) are prepared and characterized, the cellular internalization of the DSS-functionalized LNPs (LNPs-DSS) into three different cancer cell lines is evaluated, and their efficacy with the widely used iRGD peptide is compared. It is shown that controlled extent of carboxylation of lignin improves the stability at physiological conditions of LNPs formed upon solvent exchange. Functionalization with DSS and iRGD peptides maintains the spherical morphology and moderate polydispersity of LNPs. The LNPs exhibit good cytocompatibility when cultured with PC3-MM2, MDA-MB-231, and A549 in the conventional 2D model and in the 3D cell spheroid morphology. Importantly, the 3D cell models reveal augmented internalization of peptide-functionalized LNPs and improve antiproliferative effects when the LNPs are loaded with a cytotoxic compound. Overall, LNPs-DSS show equal or even superior cellular internalization than the LNPs-iRGD, suggesting that DSS can also be used to enhance the cellular uptake of NPs into different types of cells, and release different cargos intracellularly.

Preparation and Characterization of Dentin Phosphophoryn-Derived Peptide-Functionalized Lignin Nanoparticles for Enhanced Cellular Uptake.

Membrane-bound pyrophosphatases are homodimeric integral membrane proteins that hydrolyze pyrophosphate into orthophosphates, coupled to the active transport of protons or sodium ions across membranes. They are important in the life cycle of bacteria, archaea, plants, and parasitic protists, but no homologous proteins exist in vertebrates, making them a promising drug target. Here, we report the first nonphosphorus allosteric inhibitor of the thermophilic bacterium  membrane-bound pyrophosphatase and its bound structure together with the substrate analog imidodiphosphate. The unit cell contains two protein homodimers, each binding a single inhibitor dimer near the exit channel, creating a hydrophobic clamp that inhibits the movement of β-strand 1-2 during pumping, and thus prevents the hydrophobic gate from opening. This asymmetry of inhibitor binding with respect to each homodimer provides the first clear structural demonstration of asymmetry in the catalytic cycle of membrane-bound pyrophosphatases.

Alexandros Kiriazis

Drug Research Program,
Division of Pharmaceutical Chemistry and Technology,
Faculty of Pharmacy,
Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy

University of Helsinki

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Alexandros Kiriazis

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University of Helsinki

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Drug Research Program,
Division of Pharmaceutical Chemistry and Technology,
Faculty of Pharmacy,
Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy