A new type of neuron-specific aminopeptidase NAP-2 in rat brain synaptosomes

A novel neutral aminopeptidase (NAP-2) was found exclusively in the rat central nervous system (CNS). It was separated from the ubiquitous puromycin-sensitive aminopeptidase (PSA) and the neuron-specific aminopeptidase (NAP) by an automated FPLC-aminopeptidase analyzer. The activity of the neuronal aminopeptidase enriched in the synaptosomes is different from NAP and PSA in distribution and during brain development. The enzyme was purified 2230-fold to apparent homogeneity from rat brain cytosol with 4% recovery by ammonium sulfate fractionation, followed by column chromatography successively on Phenyl-Sepharose, Q-Sepharose, Sephadex G-200, and Mono Q. The single-chain enzyme with a molecular mass of 110 kDa has an optimal pH of 7.0 and a pI of 5.6. It splits β-naphthylamides of amino acid with aliphatic, polar uncharged, positively charged, and aromatic side chain. Leucyl β-naphthylamide (Leu βNA) is the best substrate with the highest hydrolytic coefficiency followed by Met βNA = Arg βNA = Lys βNA > Ala βNA > Tyr βNA > Phe βNA. The cysteine-, metallo-, glyco-aminopeptidase releases the N-terminal Tyr from Leu-enkephalin with a K_m 82 μM and a k_cat of 1.08 s⁻¹, and Met-enkephalin with a K_m of 106 μM and a k_cat of 2.6 s⁻¹. The puromycin-sensitive enzyme is most susceptible to amastatin with an IC₅₀ of 0.05 μM. The data indicate that the enzyme is a new type of NAP found in rodent. Its possible function in neuron growth, neurodegeneration, and carcinomas is discussed.     

Purification and characterization of a novel carbonyl reductase involved in oxidoreduction of aromatic β-amino ketones/alcohols

Aromatic β-amino ketones/alcohols such as adrenalone play an important role in some stereoselective synthesis of pharmaceuticals. Unfortunately, the transformation of aromatic β-amino ketones to their chiral alcohols has been carried out chemically as no corresponding biocatalyst has been available. Here, a novel carbonyl reductase responsible for the reduction of adrenalone to (R)-(−)-epinephrine was identified and characterized from Kocuria rhizophila. This enzyme was purified to homogeneity by ammonium sulfate precipitation followed by ion-exchange column chromatography, hydrophobic chromatography and gel chromatography. The purified enzyme yielded pure (R)-enantiomer product with high activity and utilized NADH as the cofactor. The enzyme had special significance by showing selectivity for many aromatic β-amino ketones/alcohols such as 2-amino-acetophenone, 2-amino-4′-hydroxyacetophenone, isoproterenol and ephedrine. The maximum reaction rate (V_max) and apparent Michaelis–Menten constant (K_m) for adrenalone and NADH were 14.62 μmol/(min mg) protein and 0.189 mM, 11.66 μmol/(min mg) protein and 0.204 mM respectively. These properties ensure the enzyme a promising future for industrial application as a replacement of chemical synthesis of aromatic β-amino chiral alcohols.     

β-Keto and β-hydroxyphosphonate analogs of biotin-5′-AMP are inhibitors of holocarboxylase synthetase

Holocarboxylase synthetase (HLCS) catalyzes the covalent attachment of biotin to cytoplasmic and mitochondrial carboxylases, nuclear histones, and over a hundred human proteins.Nonhydrolyzable ketophosphonate (β-ketoP) and hydroxyphosphonate (β-hydroxyP) analogs of biotin-5′-AMP inhibit holocarboxylase synthetase (HLCS) with IC₅₀ values of 39.7 μM and 203.7 μM. By comparison, an IC₅₀ value of 7 μM was observed with the previously reported biotinol-5′-AMP. The K_i values, 3.4 μM and 17.3 μM, respectively, are consistent with the IC₅₀ results, and close to the K_i obtained for biotinol-5′-AMP (7 μM). The β-ketoP and β-hydroxyP molecules are competitive inhibitors of HLCS while biotinol-5′-AMP inhibited HLCS by a mixed mechanism.     

Kojic acid derived hydroxypyridinone–chloroquine hybrids: Synthesis,crystal structure,antiplasmodial activity and β-haematin inhibition

Aminochloroquinoline–kojic acid hybrids were synthesized and evaluated for β-haematin inhibition and antiplasmodial activity against drug resistant (K1) and sensitive (3D7) strains of Plasmodium falciparum. Compound 7j was the most potent compound in both strains (IC₅₀^3D7 = 0.004 μM; IC₅₀^K1 = 0.03 μM) and had the best β-haematin inhibition activity (0.07 IC₅₀ equiv vs 1.91 IC₅₀ equiv for chloroquine). One compound 8c was found to be equipotent in both strains (IC₅₀ = 0.04 μM).     

Synthesis and QSAR study of novel α-methylene-γ-butyrolactone derivatives as antifungal agents

Thirty-six new α-benzylidene-γ-lactone compounds based α-methylene-γ-butyrolactone substructure were prepared and characterized by spectroscopic analysis. All compounds were evaluated for antifungal activities in vitro against six plant pathogenic fungi and the half maximal inhibitory concentration (IC₅₀) against Botrytis cinerea and Colletotrichum lagenarium were investigated. Compounds 5c-3 and 5c-5 with the halogen atom exhibited excellent fungicidal activity against B. cinerea (IC₅₀ = 22.91, 18.89 μM). The structure-activity relationships (SARs) analysis indicated that the derivatives with electron-withdrawing substituents at the meta- or para-positions improves the activity. Via the heuristic method, the generated quantitative structure-activity relationship (QSAR) model (R² = 0.961) revealed a strong correlation of antifungal activity against B. cinerea with molecular structures of these compounds. Meanwhile, the cytotoxicity of 20 representative derivatives was tested in the human tumor cells line (HepG2) and the hepatic L02 cells line, the result indicated that the synthesized compounds showed significant inhibitory activity and limited selectivity. Compound 5c-5 has the highest fungicidal activity with IC₅₀ = 18.89 μM (against B. cinerea.) but low cytotoxicity with IC₅₀ = 35.4 μM (against HepG2 cell line) and IC₅₀ = 68.8 μM (against Hepatic L02 cell line). These encouraging results can be providing an alternative, promising use of α-benzylidene-γ-lactone through the design and exploration of eco-friendly fungicides with low toxicity and high efficiency.     

Characterization of a new acid stable exo-β-1,3-glucanase of Rhizoctonia solani and its action on microbial polysaccharides

A new acid stable exo-β-1,3-glucanase of Rhizoctonia solani purified from a commercial source ‘Kitarase-M’, by a combination of ammonium sulfate precipitation, ion-exchange and gel filtration methods, had specific activity of 0.26 U/mg protein, K_m and V_max values of 0.78 mg/ml and 0.27 mM/min/mg protein, respectively. It had molecular weight of 62 kDa with optimum activity at 40 °C temperature and pH 5.0, with high stability at pH of 3–7. Unique amino acid sequence was found at N-terminal end. The substrate specificity studies confirmed that it is an exo-β-1,3-glucanase. It could hydrolyze curdlan powder to release glucose.     

Isolation and characterization of Ts19 Fragment II,a new long-chain potassium channel toxin from Tityus serrulatus venom

Ts19 Fragment II (Ts19 Frag-II) was first isolated from the venom of the scorpion Tityus serrulatus (Ts). It is a protein presenting 49 amino acid residues, three disulfide bridges, M_r 5534 Da and was classified as a new member of class (subfamily) 2 of the β-KTxs, the second one described for Ts scorpion. The β-KTx family is composed by two-domain peptides: N-terminal helical domain (NHD), with cytolytic activity, and a C-terminal CSαβ domain (CCD), with Kv blocking activity. The extensive electrophysiological screening (16 Kv channels and 5 Nav channels) showed that Ts19 Frag-II presents a specific and significant blocking effect on Kv1.2 (IC₅₀ value of 544 ± 32 nM). However, no cytolytic activity was observed with this toxin. We conclude that the absence of 9 amino acid residues from the N-terminal sequence (compared to Ts19 Frag-I) is responsible for the absence of cytolytic activity. In order to prove this hypothesis, we synthesized the peptide with these 9 amino acid residues, called Ts19 Frag-III. As expected, Ts19 Frag-III showed to be cytolytic and did not block the Kv1.2 channel. The post-translational modifications of Ts19 and its fragments (I–III) are also discussed here. A mechanism of post-translational processing (post-splitting) is suggested to explain Ts19 fragments production. In addition to the discovery of this new toxin, this report provides further evidence for the existence of several compounds in the scorpion venom contributing to the diversity of the venom arsenal.     

Microbial transformation of β-lapachone to its glycosides by Cunninghamella elegans ATCC 10028b

β-lapachone (1) has entered phases I and II clinical trials for the treatment of solid tumors and the therapeutic efficacy of β-lapachone is closely related to its metabolic process. In order to contribute to a better understanding of human metabolism of β-lapachone, Cunninghamella elegans ATCC 10028b was used as a microbial model of mammalian metabolism to biotransform β-lapachone and two new glycosylated derivatives were produced. The chemical structures were elucidated as 6-hydroxy-2,2-dimethyl-3,4-dihydro-2H-naphtho[1,2-b]pyran-5-O-β-d-glucopyranoside (2) and 5-hydroxy-2,2-dimethyl-3,4-dihydro-2H-naphtho[1,2-b]pyran-6-O-β-d-glucopyranoside (3) by ¹H NMR, ¹³C NMR, HMBC, HMQC, COSY and HRMS analyses. The major derivative (3) displayed a lower activity against breast cancer cell line SKBR-3 (IC₅₀ = 312.5 μM) than β-lapachone (IC₅₀ = 5.6 μM), but did not show cytotoxicity against normal fibroblasts cell line GM07492-A, whereas β-lapachone was highly toxic (IC₅₀ = 7.25 μM). These metabolites were reported here for the first time and are similar to those that occur in phase II of human metabolism     

New antifungal scaffold derived from a natural pharmacophore: Synthesis of α-methylene-γ-butyrolactone derivatives and their antifungal activity against Colletotrichum lagenarium

Thirty new and thirty-four known analogues were designed and synthesized to improve the potential use of the α-methylene-γ-butyrolactone ring, a natural pharmacophore. All structures were confirmed by ¹H and ¹³C NMR, MS, and single-crystal X-ray diffraction analyses. The results of antifungal and cytotoxic activity indicated that the synthesized analogues showed significant inhibitory activity and limited selectivity. Compound 45 exhibited the highest antifungal activity with IC₅₀ = 22.8 μM but moderate cytotoxic activity with IC₅₀ = 28.5 μM (against BGC823 cell line) and 7.7 μM (against HeLa cell line). Analysis of structure–activity relationships revealed that the incorporation of an aromatic ring into the β, γ positions of the lactone ring improved antifungal activity, and that the introduction of electron-withdrawing groups into the aromatic rings increased the activity compared with electron-donating groups. The above results identified 4-phenyl-3-phenyl-2-methylenebutyrolactone (33) as a lead scaffold for discovering and developing novel and improved crop-protection agents.     

Triterpenes from the roots of Lantana montevidensis with antiprotozoal activity

Bioassay guided fractionation of the roots of Lantana montevidensis (Verbenaceae) has resulted in the isolation and identification of three new triterpenoids; 13β-hydroxy-3-oxo-olean-11-en-28-oic acid (1), 12β,13β-dihydroxyolean-3-oxo-28-oic acid (2) and 12β,13β,22β-trihydroxyolean-3-oxo-28-oic acid (3) in addition to nine known compounds: oleanonic acid (4), oleanolic acid (5), 3β,25β-dihydroxy-olean-12-en-28-oic acid (6), lantadene A (7), 19α-hydroxy-3-oxo-olean-12-en-28-oic acid (8) pomolic acid (9), camaric acid (10) together with β-sitosterol (11) and β-sitosterol-3-O-β-d-glucoside (12). The structures of the isolated metabolites were elucidated based on comprehensive 1D and 2D NMR spectroscopic data as well as HR-ESI–MS. The extracts and the isolated metabolites were evaluated for their antiprotozoal and antimicrobial activities. Compound 2 showed antibacterial activity against Staphylococcus aureus and methicillin resistant S. aureus with IC₅₀ values against both organisms of 2.1 μM and compound 10 showed activity against same organisms with IC₅₀ values 8.74 and 8.09 μM, respectively, compared to the positive control ciprofloxacin (IC₅₀ = 0.3 μM against S. aureus and MRSA). Compounds 1, 4, 5, 6, and 10 showed moderate antileishmanial activity with IC₅₀ values ranging between (2.54–14.95 μM) and IC₉₀ values ranging between (11.90–19.47 μM), using pentamidine as a control (IC₅₀ values 2.09 ≥ 16.8 μM) and IC₉₀ values ranging between (4.72 ≥ 16.8 μM). These compounds also showed highly potent antitrypanosomal activity with IC₅₀ values ranging between (0.39–7.12 μM) and IC₉₀ values ranging between (1.91–10.51 μM), which are more efficient than the DFMO, the antitrypanosomal drug employed as positive control (IC₅₀ and IC₉₀values 11.82 and 30.82 μM).     

Purification,modification and inhibition mechanism of angiotensin I-converting enzyme inhibitory peptide from silkworm pupa (Bombyx mori) protein hydrolysate

Angiotensin I-converting enzyme (ACE) inhibitory peptide from silkworm pupa (Bombyx mori) was purified, modified, as well as inhibition mechanism by using molecular docking analysis. Silkworm pupa protein was hydrolyzed by neutral protease and the obtained hydrolysate was subjected to various types of chromatography to acquire peptide isolate. Then the molecular mass and amino acid sequence of the peptide was determined by MALDI-TOF/TOF MS. Subsequently, thermal and digestive stability of the peptide were explored through a high temperature processing and a simulated gastrointestinal digestion. Finally, the peptide was modified to smaller peptides and investigated their potentiate activities. Results showed that the peptide from silkworm pupa was determined to be Gly-Asn-Pro-Trp-Met (603.7 Da) with IC₅₀ 21.70 μM. Stability testing showed that ACE inhibitory activities were not significantly changed at temperature from 40 to 80 °C as well as during in vitro gastrointestinal digestion. The inhibitory activity of four modified peptides were Trp-Trp > Gly-Asn-Pro-Trp-Trp > Asn-Pro-Trp-Trp > Pro-Trp-Trp, and the IC₅₀ of Trp-Trp was 10.76 μM Docking simulation revealed that the inhibitory activity was closely related to the spatial structure of peptide and zinc ions. The purified peptide and four modified peptides may be beneficial as functional food or drug for treating hypertension.     

Heterologous expression and characterisation of a biosynthetic thiolase from Clostridium butyricum DSM 10702

Biosynthetic thiolases (EC 2.3.1.9) are key enzymes in the branched catabolism of diverse clostridia as their activity and regulation influence the production of organic acids and solvents. In Clostridium butyricum, they are also involved in the production of hydrogen as a sustainable and environmentally benign energy source. In this study, the gene coding for thiolase from C. butyricum DSM 10702 was cloned by genome walking. It was found to consist of 1179 bp coding for a protein with 393 amino acids and a deduced molecular weight of 41.4 kDa. The enzyme was fused to an N-terminal his-tag, expressed in Escherichia coli, purified to near homogeneity and characterised for biochemical and kinetic properties. Gel filtration chromatography revealed that the catalytically active enzyme consists of a homotetramer. The enzyme showed a K_M of ～32 μM towards acetoacetyl-CoA and of ～21 μM towards CoASH at 30 °C and pH 8.0. Claisen condensation of acetyl-CoA by thiolase was analysed in a coupled enzyme assay, where β-hydroxybutyryl-CoA dehydrogenase was applied catalysing the subsequent NADH-dependant reduction of the formed condensation product acetoacetyl-CoA. For this purpose the latter enzyme was cloned from C. butyricum DSM 10702 and recombinantly expressed in E. coli. The K_M of thiolase towards acetyl-CoA was ～674 μM at 30 °C and pH 7.5. Acetyl-CoA condensation was inhibited even at micromolar concentrations of CoASH indicating that CoASH has an important regulatory function in vivo.     

Carbonic anhydrase activators: Activation of the β-carbonic anhydrase Nce103 from the yeast Saccharomyces cerevisiae with amines and amino acids

The protein encoded by the Nce103 gene of Saccharomyces cerevisiae, a β-carbonic anhydrase (CA, EC 4.2.1.1) designated as scCA, was investigated for its activation with amines and amino acids. scCA was poorly activated by amino acids such as l-/d-His, Phe, DOPA, Trp (K_As of 82–90 μM) and more effectively activated by amines such as histamine, dopamine, serotonin, pyridyl-alkylamines, aminoethyl-piperazine/morpholine (K_As of 10.2–21.3 μM). The best activator was l-adrenaline, with an activation constant of 0.95 μM. This study may help to better understand the catalytic/activation mechanisms of the β-CAs and eventually to design modulators of CA activity for similar enzymes present in pathogenic fungi, such as Candida albicans and Cryptococcus neoformans.     

Patent Reports

We synthesized a hydroquinone glucoside (HG) as a potential skin-whitening agent using Leuconostoc mesenteroides (B-1299CB BF563) dextransucrase with hydroquinone (HQ) as an acceptor and sucrose as a donor. The product was purified using butanol partitioning and silica gel column chromatography. The structure of the purified HG was determined by nuclear magnetic resonance and the ionic product was observed at m/z 295 (C12, H16, O7 Na)⁺. HG was identified as 4-hydroxyphenyl-α-d-glucopyranoside. The optimum condition of HG synthesis, determined using a response surface methodology, was 450 mM HQ, 215 mM sucrose, and 0.55 U/mL dextransucrase; the final HG produced was 544 mg/L. The IC₅₀ of diphenylpicryl-hydrazyl scavenging activity was 3.85 mM indicating a higher antioxidant activity compared to β-arbutin (IC₅₀ = 6.04 mM). HG-mediated inhibition of lipid peroxidation was 3.51% that of HQ (100%) and much higher than that of β-arbutin (0.81% of HQ). In addition the IC₅₀ value of nitrite-scavenging activity was 14.76 mM showing a superior scavenging activity to that of β-arbutin (IC₅₀ = 27.09 mM).     

Strategy for designing selective α-l-rhamnosidase inhibitors: Synthesis and biological evaluation of l-DMDP cyclic isothioureas

This study shows that the cyclization of l-DMDP thioureas to bicyclic l-DMDP isothioureas improved α-l-rhamnosidase inhibition which was further enhanced by increasing the length of the alkyl chain. The addition of a long alkyl chain, such as decyl or dodecyl, to the nitrogen led to the production of highly potent inhibitors of α-l-rhamnosidase; it also caused broad inhibition spectrum against β-glucosidase and β-galactosidase. In contrast, the corresponding N-benzyl-l-DMDP cyclic isothioureas display selective inhibition of α-l-rhamnosidase; 3′,4′-dichlorobenzyl-l-DMDP cyclic isothiourea (3r) was found to display the most potent and selective inhibition of α-l-rhamnosidase, with IC₅₀ value of 0.22 μM, about 46-fold better than the positive control 5-epi-deoxyrhamnojirimycin (5-epi-DRJ; IC₅₀ = 10 μM) and occupied the active-site of this enzyme (K_i = 0.11 μM). Bicyclic isothioureas of ido-l-DMDP did not inhibit α-l-rhamnosidase. These new mimics of l-rhamnose may affect other enzymes associated with the biochemistry of rhamnose including enzymes involved in progression of tuberculosis.     

Carbonic anhydrase activators: Activation of the β-carbonic anhydrase from the pathogenic yeast Candida glabrata with amines and amino acids

The protein encoded by the NCE103 gene of Candida glabrata, a β-carbonic anhydrase (CA, EC 4.2.1.1) designated as CgCA, was investigated for its activation with amines and amino acids. CgCA was weakly activated by amino acids such as l-/d-His, l-Phe, l-DOPA, and l-Trp and by histamine or dopamine (K_As of 21.2–37 μM) but more effectively activated by d-Phe, d-DOPA, d-Trp as well as serotonin, pyridyl-alkylamines, aminoethyl-piperazine/morpholine (K_As of 10.1–16.7 μM). The best activators were l-/d-Tyr, with activation constants of 7.1–9.5 μM. This study may bring a better understanding of the catalytic/activation mechanisms of β-CAs from pathogenic fungi.     

Antibacterial profiling of abietane-type diterpenoids

Abietic and dehydroabietic acid are interesting diterpenes with a highly diverse repertoire of associated bioactivities. They have, among others, shown antibacterial and antifungal activity, potentially valuable in the struggle against the increasing antimicrobial resistance and imminent antibiotic shortage. In this paper, we describe the synthesis of a set of 9 abietic and dehydroabietic acid derivatives containing amino acid side chains and their in vitro antimicrobial profiling against a panel of human pathogenic microbial strains. Furthermore, their in vitro cytotoxicity against mammalian cells was evaluated. The experimental results showed that the most promising compound was 10 [methyl N-(abiet-8,11,13-trien-18-yl)-d-serinate], with an MIC₉₀ of 60 μg/mL against Staphylococcus aureus ATCC 25923, and 8 μg/mL against methicillin-resistant S. aureus, Staphylococcus epidermidis and Streptococcus mitis. The IC₅₀ value for compound 10 against Balb/c 3T3 cells was 45 μg/mL.     

A long chain alkylated α-methylene-γ-butyrolactone from Artabotrys odoratissimus fruit

As part of our ongoing programme for isolation of bioactive molecules from the flora of the Indo-Burma biodiversity belt, an unusual long chain alkylated α-methylene-γ-butyrolactone was isolated from the juice of ripe fruit of Artabotrys odoratissimus R.Br. Its structure was determined as 3-methylene-4-pentadecyldihydrofuran-2-one by spectroscopic methods. It was found to have good antifungal activity against Alternaria tenuissima Kunze Ex Pers. isolated from solasodine producing plant Solanum khasianum Clarke. Minimum Inhibitory Concentration (MIC) and IC₅₀ for 3-methylene-4-pentadecyldihydrofuran-2-one were found as 300 and 51.37 μg/ml, respectively. The standard captan was found to have an MIC and IC₅₀ of 200 and 35.52 μg/ml, respectively.