Maya Guncheva

Modification of Rapana thomasiana hemocyanin with choline amino acid salts significantly enhances its antiproliferative activity against MCF-7 human breast cancer cells

This is the first study on the interactions of ionic liquids with large metalloproteins, in particular hemocyanins (Hcs). At first, complexes of a Hc from Rapana thomasiana (RtH) with a series of biocompatible choline amino acid salts [Chol][AA] were obtained. Applying UV-vis spectroscopy, Fourier-transformed infrared spectroscopy and differential scanning calorimetry the effect of these organic salts on the structure and thermal stability of RtH was assessed. Then, the cytotoxic effect of RtH–[Chol][AA] on breast cancer cells (MCF-7) and 3T3 fibroblast cells (non cancerous) was evaluated. We found that all [Chol][AA] induced clear time- and concentration-dependent alterations in the RtH conformation. The conformation and the thermal stability of IL-modified RtH depend strongly on the type of the anion of the tested compounds. All [Chol][AA]-modified RtHs exhibited lower thermal stability than the native RtH. At the same time, we established a good correlation between the structure of RtH and its antitumor activity. Namely, RtH–[Chol][AA] complexes exhibited enhanced antiproliferative activity toward the MCF-7 cell line. The observed antiproliferative effect was cell specific and the compounds have no effect or in some cases have stimulatory effect on fibroblasts.

Do N-terminal nucleophile hydrolases indeed have a single amino acid catalytic center?

A new set of experimental kinetic data on the hydrolysis of a series of phenylacetyl p‐substituted anilides catalyzed by penicillin G acylase from Escherichia coli (PGA) is presented in this article. The Hammett plot of log(kcat,R/kcat,H) versus σp− has three linear segments, which distinguishes the enzyme from the other N‐terminal nucleophile hydrolases for which data are available. Three amino acids in the vicinity of the catalytic SerB1 (AsnB241, AlaB69, and GlnB23) were included in the quantum mechanical model. The stable structures and the transition states for acylation were optimized by molecular mechanical modeling and at the AM1 level of theory for three model substrates (with H, a methoxy group or a nitro group in the para position in the leaving group). Intrinsic interactions of several functional groups at the active site of PGA are discussed in relation to the catalytic efficiency of the enzyme. The energy barrier computed for the first step of acylation (the nucleophilic attack of SerB1) is lower than that for the second step (the collapse of the tetrahedral intermediate). However, the electronic properties of the substituent on the leaving group affect the structure of the second transition state. It is shown that the main chain carbonyl group of GlnB23 forms a hydrogen bond with the leaving group nitrogen, thus influencing the hydrolysis rate. On the basis of our computations, we propose an interpretation of the complex character of the Hammett plot for the reaction catalyzed by PGA. We suggest a modified scheme of the catalytic mechanism in which some of the intramolecular interactions essential for catalysis are included.

Thermal and conformational stability of insulin in the presence of imidazolium-based ionic liquids

Abstract Stabilization of the monomeric form of insulin (In) under condition of low pH has been a recent challenge. This research aims to reveal the effect of a series of 1-butyl-3-methylimidazolium-based ionic liquids (ILs) on the stability of In dissolved in highly acidic solution (pH 2.0). Differential scanning calorimetry was applied to assess the thermal stability of In in the presence of these ILs. In addition, we monitored the IL-induced changes in the In secondary structure using Fourier transformed infrared spectroscopy. The peak of In thermal denaturation was shifted to higher temperatures in the presence of the tested acetate, trifluoroacetate and dicyanamide salts. At the same time, chloride and thiocyanate ILs had no effect on the thermal stability of the insulin, while the tricyanomethanide salt slightly destabilized the protein. The change in the In conformation affected not only the position but also the sharpness and the shape of the transition peak. As a whole, those ILs which were able to preserve or enhance helical structure of In produced stabilizing effect and those which stimulated the formation of unordered and random-coiled structures deteriorated its thermal stability. No aggregation of In in the presence of the imidazolium-based ILs was observed under the tested acidic media.

Rapana thomasiana hemocyanin modified with ionic liquids with enhanced anti breast cancer activity.

This is the first study on the surface modification of a hemocyanin from marine snail Rapana thomasiana (RtH) with series of imidazolium-based amino acid ionic liquids [emim][AA]. We monitored the induced by [emim][AA] conformational changes in RtH molecule and evaluated the effect of these ionic liquids (ILs) on the protein thermal stability. The cytotoxicity of all obtained RtH-[emim][AA] complexes was assessed toward breast cancer cells (MCF-7) and murine fibroblasts (3T3). As a whole, even small amounts of the tested ILs altered the secondary structure of RtH. The thermal denaturation of RtH in presence of [emim][AA] displayed multi-component transitions, which were shifted toward lower temperatures in comparison to those estimated for the native RtH. The profiles of the RtH-IL calorimetric curves show a clear dependence on the structure of the added salts. In addition, all RtH-[emim][AA] complexes exhibited an enhanced antiprofilerative activity of toward MCF-7 cells in comparison to that of the native RtH. The best results are observed for RtH-[emim][Leu], RtH-[emim][Trp] or RtH-[emim][Ile], which applied in concentration of 700 μg/mL inhibited the MCF-7 cell viability (for 24h) by 66, 63 and 53%, respectively. In addition, these IL-RtH complexes were less cytotoxic to 3T3 cells, i.e. they exhibited some cell specificity.

ExcEllEnt StabIlIty and SynthEtIc actIvIty of lIpaSE from B. stearothermophilus mc7 ImmobIlIzEd on tIn dIoxIdE In EnvIronmEntally frIEndly mEdIum

ABSTRACT A thermostable lipase from B. strearothermophilus MC7 was stabilized via immobilization on nanostructured tin dioxide (nanoSnO2-MC7). The synthetic activity of nanoSnO2-MC7 in two ionic liquids containing chloride anions was assessed. In the presence of 3-methyl-1-octyl imidazolium chloride, the stearyl stearate production proceeded at a much higher reaction rate than in solvent-free medium and 95 % conversion of the substrates was achieved for 5 h at 65°C. Our results showed that it also had a beneficial effect on the operational and thermal stability of the immobilized lipase MC7. In this green solvent, the biocatalyst was almost fully active after eight synthetic cycles and up to 60% of the initial activity of nanoSnO2-MC7 lipase was preserved after long heating at 75°C.

Arylamidase activity of neutral proteinase from Saccharomonospora canescens. Comparison with other Zn-proteinases that exhibit the same activity

The arylamidase activity of Zn-proteinase from Saccharomonospora canescens (NPS) was studied with series of peptide nitroanilides of varying amino acid sequence and N-acyl blocking groups. The partial mapping of the enzyme S(1), S(2), S(3), S(4) subsites shows that variations in all positions P(1) to P(4) in the substrate structure affect the catalytic efficiency. The importance of P(4)-S(4) and P(1)-S(1) interactions, which is a characteristic feature of the serine proteinases, is evidenced for the studied Zn-proteinases NPS and serralysin too. The presence of arylamidase activity in the case of Zn-proteinases-astacin EC 3.4.24.21 and serralysin EC 3.4.24.40 is correlated with some specific characteristics of their active site structure: penta-coordinated Zn(2+) and a tyrosyl residue as a fifth ligand to the Zn(2+). It is assumed that this tyrosyl residue plays a role in the productive binding and stabilization of the tetrahedral adduct formed during the reaction of enzyme-catalysed hydrolysis of peptide arylamides of corresponding length and sequence.

Structure and properties of a series of 2-cinnamoyl-1,3-indandiones and their metal complexes

A series of seven 2-cinnamoyl-1,3-indandiones and their metal(II) complexes were synthesized and characterized by means of spectroscopic (IR, NMR, electron absorption and emission spectroscopy) and/or single-crystal X-ray diffraction methods. The optical spectra of the organic compounds show very strong absorption in the visible region and weak fluorescence with moderate to strong Stokes shift. The effect of concentration, water addition and metal ion complexation on the optical properties was also studied. In search of potential practical application, the complexation of 2-cinnamoyl-1,3-indandiones with metal(II) ions was investigated. A series of non-charged complexes with Cu(II), Cd(II), Zn(II), Co(II) and Ni(II) was isolated and analyzed by elemental analyses and IR. Most of the complexes show presence of water molecules, most probably coordinated to the metal ion, thus forming octahedral geometry. For the paramagnetic Cu(II) complexes a distorted, flattened tetrahedral structure is proposed, basing on the EPR data. The optical properties of the metal complexes, however, do not differ appreciably from those of the free ligands.

Evaluation of the inhibitory potential of five squaric acid derivatives against pancreatic lipase

A series of 4-alkylamino-2-ethoxycyclobut-3en-1,2-diones has been synthesized, characterized and their inhibitory effect on pancreatic lipase (PL) was evaluated. The compound 1 has shown relatively high potency (IC50 = 0.11 mM) compared with the most effective anti-obesity drug, tetrahydrolipstatin (Orlistat) (IC50 value = 0.08 mM). The compounds have showed good selectivity toward PL and did not affect the activity of trypsin, another digestive enzyme.