Nicked-site substrates for a serine recombinase reveal enzyme–DNA communications and an essential tethering role of covalent enzyme–DNA linkages

To analyse the mechanism and kinetics of DNA strand cleavages catalysed by the serine recombinase Tn3 resolvase, we made modified recombination sites with a single-strand nick in one of the two DNA strands. Resolvase acting on these sites cleaves the intact strand very rapidly, giving an abnormal half-site product which accumulates. We propose that these reactions mimic second-strand cleavage of an unmodified site. Cleavage occurs in a synapse of two sites, held together by a resolvase tetramer; cleavage at one site stimulates cleavage at the partner site. After cleavage of a nicked-site substrate, the half-site that is not covalently linked to a resolvase subunit dissociates rapidly from the synapse, destabilizing the entire complex. The covalent resolvase–DNA linkages in the natural reaction intermediate thus perform an essential DNA-tethering function. Chemical modifications of a nicked-site substrate at the positions of the scissile phosphodiesters result in abolition or inhibition of resolvase-mediated cleavage and effects on resolvase binding and synapsis, providing insight into the serine recombinase catalytic mechanism and how resolvase interacts with the substrate DNA.     

Biochemical studies of the chromaffin granule. III. Redistribution of lipid phosphate,dopamine-β-hydroxylase and chromogranin a after freezing and thawing of the isolated granule membranes

Freezing and thawing a dialysed suspension of lysed chromaffin granules and sedimented membrane preparations resulted in redistribution of lipid phosphate and protein. By this treatment the high ratios of lipid phosphate/protein in the membrane fragments, isolated on sucrose density gradient from the dialysed suspensions and the sedimented membrane preparation, were reduced from 1.56 to 1.03 μmoles/mg and from 1.97 to 0.83 μmoles/mg, respectively.Multilamellar, liposomal structures could be isolated from the frozen and thawed membrane preparations and were found to sediment in the 0.4 M sucrose layer by density gradient centrigugation. This fraction was without morphological resemblance to the intact chromaffin granules or their membranes and was found to account for 53% of the lipid phosphate, 35% of chromogranin A, 21% of dopamine-β-hydroxylase activity and 12% of the protein of the total preparation. The specific activities of chromogranin A and dopamine-β-hydroxylase in the artificially formed liposomal structures closely resembled that of the solubilized protein and was significantly higher than in the lipid phosphate-depleted membrane fragments recovered in the 1.1 M sucrose layers.It is concluded that freezing and thawing as a means of purifying the isolated granule membranes lead not only to the solubilization of chromogranin A, but also to removal of dopamine-β-hydroxylase activity and lipid phosphate from the labile membrane fragments.     

“TRPV1 is a component of the atrial natriuretic signaling complex,and using orally delivered antagonists,presents a valid therapeutic target in the longitudinal reversal and treatment of cardiac hypertrophy and heart failure”

Activation of the atrial natriuretic signaling pathway is intrinsic to the pathological responses associated with a range of cardiovascular diseases that stress the heart, especially those involved in sustained cardiac pressure overload which induces hypertrophy and the pathological remodeling that frequently leads to heart failure. We identify transient receptor potential cation channel, subfamily V, member 1, as a regulated molecular component, and therapeutic target of this signaling system. Data show that TRPV1 is a physical component of the natriuretic peptide A, cGMP, PKG signaling complex, interacting with the Natriuretic Peptide Receptor 1 (NPR1), and upon binding its ligand, Natriuretic Peptide A (NPPA, ANP) TRPV1 activation is subsequently suppressed through production of cGMP and PKG mediated phosphorylation of the TRPV1 channel. Further, inhibition of TRPV1, with orally delivered drugs, suppresses chamber and myocyte hypertrophy, and can longitudinally improve in vivo heart function in mice exposed to chronic pressure overload induced by transverse aortic constriction, reversing pre-established hypertrophy induced by pressure load while restoring chamber function. TRPV1 is a physical and regulated component of the natriuretic peptide signaling system, and TRPV1 inhibition may provide a new treatment strategy for treating, and reversing the loss of function associated with cardiac hypertrophy and heart failure.     

A glycan of Ψ-factor from Dictyostelium discoideum contains a bisecting-GlcNAc, an intersecting-GlcNAc, and a core α-1,6-fucose

A secretory glycoprotein named Ψ-factor that we have purified and cloned from Dictyostelium discoideum is prespore cell-inducing factor. To address its functional significance, it is necessary to examine the attached sites and structures of its glycans as well as its protein structure. Here we identified and isolated a tryptic glycosylated peptide with the 71st to 89th amino acids of Ψ-factor that contained the consensus amino acid sequence for an N-linked glycan (N-T-T). MALDI-TOF mass spectrometry indicated that the major protonated molecular ions, [M+H](+), of the glycopeptide were present at m/z 3,806, the minor m/z 3,603 and 3,400 ions corresponding to the loss of one and two N-acetylhexosamines respectively. Digestion of it with N-glycosidase F gave a molecular mass of 1,766.9 for the whole glycan moiety, which accounts for its composition of five hexoses, four N-acetylhexosamines, and a deoxyhexose. Further digestion experiments on the basis of the substrate specificity of α-mannosidase and β-N-acetylhexosaminidase allowed us to elucidate the unique structure of the glycan, which contains a bisecting and an intersecting GlcNAc and a core α1,6-fucosyl moiety.     

Design of novel and potent cPLA2α inhibitors containing an α-methyl-2-ketothiazole as a metabolically stable serine trap

We report the design of novel, potent cPLA₂α inhibitors that possess an α-methyl-2-ketothiazole that acts as a serine-reactive moiety. We describe the optimization of the series for potency and metabolic stability towards ketone reduction. This was achieved by attenuating the reactivity of the ketone using a combination of electronic and steric effects.     

Synthesis,enzyme kinetics and computational evaluation of N-(β-d-glucopyranosyl) oxadiazolecarboxamides as glycogen phosphorylase inhibitors

All possible isomers of N-β-d-glucopyranosyl aryl-substituted oxadiazolecarboxamides were synthesised. O-Peracetylated N-cyanocarbonyl-β-d-glucopyranosylamine was transformed into the corresponding N-glucosyl tetrazole-5-carboxamide, which upon acylation gave N-glucosyl 5-aryl-1,3,4-oxadiazole-2-carboxamides. The nitrile group of the N-cyanocarbonyl derivative was converted to amidoxime which was ring closed by acylation to N-glucosyl 5-aryl-1,2,4-oxadiazole-3-carboxamides. A one-pot reaction of protected β-d-glucopyranosylamine with oxalyl chloride and then with arenecarboxamidoximes furnished N-glucosyl 3-aryl-1,2,4-oxadiazole-5-carboxamides. Removal of the O-acetyl protecting groups by the Zemplén method produced test compounds which were evaluated as inhibitors of glycogen phosphorylase. Best inhibitors of these series were N-(β-d-glucopyranosyl) 5-(naphth-1-yl)-1,2,4-oxadiazol-3-carboxamide (K_i = 30 μM), N-(β-d-glucopyranosyl) 5-(naphth-2-yl)-1,3,4-oxadiazol-2-carboxamide (K_i = 33 μM), and N-(β-d-glucopyranosyl) 3-phenyl-1,2,4-oxadiazol-5-carboxamide (K_i = 104 μM). ADMET property predictions revealed these compounds to have promising oral drug-like properties without any toxicity.     

The synthesis and biological evaluation of 1-C-alkyl-L-arabinoiminofuranoses, a novel class of α-glucosidase inhibitors

The asymmetric synthesis of 1-C-alkyl-l-arabinoiminofuranoses 1 was achieved by asymmetric allylic alkylation (AAA), ring closing metathesis (RCM), and Negishi cross coupling as key reactions. Some of the prepared compounds showed potent inhibitory activities towards intestinal maltase, with IC₅₀ values comparable to those of commercial drugs such as acarbose, voglibose, and miglitol, which are used in the treatment of type 2 diabetes. Among them, the inhibitory activity (IC₅₀ = 0.032 μM) towards intestinal sucrase of 1c was quite strong compared to the above commercial drugs.     

Activity of steroid 4 and derivatives 4a–4f as inhibitors of the enzyme 5α-reductase 1

It is known that the growth of prostate metastatic bone tumor depends on androgens, and tumor formation can start from migratory malignant cells produced in that organ. These cells exhibit grater type 1 5α-reductase (5α-R1) activity than type 2 5α-reductase. Noteworthy, both isozymes convert testosterone (T) to the more active androgen dihydrotestosterone (DHT) in the target tissues.Thus, in order to potentially improve the prognosis of this disease, in this work, seven derivatives of 17-(1H-benzimidazol-1-yl)-16-formillandrosta-5,16-dien-3β-yl benzoate (4a–f) and 17-(1H-benzimidazol-1-yl)-3-hydroxy-16-formylandrost-5,16-diene (4) were synthesized, characterized and identified as inhibitors of type 1 5α-reductase (5αR1). These derivatives having the advantage of improved plasma half-life.The inhibitory activity of the compounds towards 5α-R1 isoenzyme was determined by conversion of T into DHT in the presence or absence of compounds 4, 4a–f. Further, in vivo experiments were also carried out, treating gonadectomized hamsters with T and/or 4, 4a–f and evaluating their effect on the diameter of hamster flank organs and on the weight of the prostatic and seminal vesicles. Results indicated that compounds 4, 4b, 4c, served as in vitro inhibitors of the enzyme 5α-R1 and pharmacological experiments showed that 4 and derivatives 4a–f decreased the diameter of the flank glands, the weight of the prostate and seminal vesicles of treated hamsters without any appreciable toxicity during observation. Noteworthy the fact that compound 4 is the product, in all cases, of the hydrolysis of the series of esters 4a–f, thus they can serve as precursors (prodrugs) of the active form 4.     

Design,synthesis, and biological evaluation of a novel series of peripheral-selective noradrenaline reuptake inhibitors – Part 3

Peripheral-selective inhibition of noradrenaline reuptake is a novel mechanism for the treatment of stress urinary incontinence to overcome adverse effects associated with central action. Here, we describe our medicinal chemistry approach to discover a novel series of highly potent, peripheral-selective, and orally available noradrenaline reuptake inhibitors with a low multidrug resistance protein 1 (MDR1) efflux ratio by cyclization of an amide moiety and introduction of an acidic group. We observed that the MDR1 efflux ratio was correlated with the pK_a value of the acidic moiety. The resulting compound 9 exhibited favorable PK profiles, probably because of the effect of intramolecular hydrogen bond, which was supported by a its single-crystal structure. The compound 9, 1-{[(6S,7R)-7-(4-chloro-3-fluorophenyl)-1,4-oxazepan-6-yl]methyl}-2-oxo-1,2-dihydropyridine-3-carboxylic acid hydrochloride, which exhibited peripheral NET-selective inhibition at tested doses in rats by oral administration, increased urethral resistance in a dose-dependent manner.     

Design,synthesis, and biological evaluation of a novel series of peripheral-selective noradrenaline reuptake inhibitors—Part 2

Peripherally selective inhibition of noradrenaline reuptake is a novel mechanism for the treatment of stress urinary incontinence to overcome adverse effects associated with central action. Herein, we describe our medicinal chemistry approach to discover peripheral-selective noradrenaline reuptake inhibitors to avert the risk of P-gp-mediated DDI at the blood–brain barrier. We observed that steric shielding of the hydrogen-bond acceptors and donors (HBA and HBD) of compound 1 reduced the multidrug resistance protein 1 (MDR1) efflux ratio; however, the resulting compound 6, a methoxyacetamide derivative, was mainly metabolized by CYP2D6 and CYP2C19 in the in vitro phenotyping study, implying the risk of PK variability based on the genetic polymorphism of the CYPs. Replacement of the hydrogen atom with a deuterium atom in a strategic, metabolically hot spot led to compound 13, which was mainly metabolized by CYP3A4. To our knowledge, this study represents the first report of the effect of deuterium replacement for a major metabolic enzyme. The compound 13, N-{[(6S,7R)-7-(4-chloro-3-fluorophenyl)-1,4-oxazepan-6-yl]methyl}-2-[(2H³)methyloxy]acetamide hydrochloride, which exhibited peripheral NET selective inhibition at tested doses in rats, increased urethral resistance in a dose-dependent manner.     

Role of the Na,K-ATPaseβ-subunit in the cellular accumulation and maturation of the enzyme as assessed by glycosylation inhibitors

Summary No functional role could yet be established for the glycosylated -subunit of the Na,K-ATPase. In this study, we describe the intracellular processing of the -subunit as a glycoprotein in toad bladder cells and the consequences of its structural perturbation with glycosylation inhibitors on the cellular expression of the - and -subunits and on the structural and functional maturation of the enzyme. Controlled trypsinolysis of homogenates from pulse-labeled cells reveals that the -subunit is subjected to glycosylation-dependent structural rearrangements during its intracellular routing. Inhibition of correct terminal glycosylation of the -subunit with deoxynojirimycin or swainsonine has no effect on the trypsin sensitivity of the -subunit, its ability to perform cation-dependent conformation changes or the cellular Na,K-ATPase activity. Acquisition of core-sugars is sufficient for the enzyme to assume its catalytic functions. On the other hand, complete inhibition of glycosylation with tunicamycin leads to a destabilization of both the - and the -subunits as judged by their higher trypsin sensitivity. In addition, tunicamycin treatment results in a decrease of the amount of newly synthesized - and -subunit indicating that a glycoprotein, possibly the -subunit itself, plays a role in the efficient accumulation of the -subunit in the endoplasmic reticulum.     

Direct and indirect regulation of derrière,a Xenopus mesoderm-inducing factor,by VegT

One candidate for an endogenous mesoderm-inducing factor in Xenopus is derrière, a member of the TGFbeta family closely related to Vg1. In this paper we first show that derrière is able to exert long-range effects in the early Xenopus embryo, reinforcing the view that it functions as a secreted factor required for proper formation of posterior structures. Analysis of the derrière promoter shows that expression of the gene is controlled through a complex inductive network involving VegT and TGFbeta-related molecules and also, perhaps, FGF family members. The work confirms that derrière plays an important role in mesoderm formation and it illustrates the complex regulation to which inducing factors are subject.     

Evaluation of sulphonamide derivatives acting as inhibitors of human carbonic anhydrase isoforms I,II and Mycobacterium tuberculosis β-class enzyme Rv3273

A series of novel sulphonamide derivatives was obtained from sulphanilamide which was N4-alkylated with ethyl bromoacetate followed by reaction with hydrazine hydrate. The hydrazide obtained was further reacted with various aromatic aldehydes. The novel sulphonamides were characterised by infrared, mass spectrometry, ¹H- and ¹³C-NMR and purity was determined by high-performance liquid chromatography (HPLC). Human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I and II and Mycobacterium tuberculosis β-CA encoded by the gene Rv3273 (mtCA 3) inhibition activity was investigated with the synthesised compounds which showed promising inhibition. The K_Is were in the range of 54.6?nM–1.8?µM against hCA I, in the range of 32.1?nM–5.5?µM against hCA II and of 127?nM–2.12?µM against mtCA 3.     

Localization,induction and characterization of a novel carbon–carbon double bond reductase from Aspergillus versicolor

Abstract

A novel reductase has been detected in cell-free extracts from growing/resting cultures of the fungus Aspergillus versicolor D-1, which specifically catalyzes NADPH-dependent reduction of the γ,δ-double bond of the lactone-conjugated unsaturated system in securinine to form 14,15-dihydrosecurinine. The localization of the reductase has been investigated using differential centrifugation techniques. It was found that the securinine reductase is a cytosolic enzyme. The reductase was highly inducible in growing/resting cultures when securinine was used as the substrate and inducer. Optimal incubation conditions for assay of the securinine reductase were determined by using the enzyme preparation from resting cultures of A. versicolor D-1. The optimum temperature and pH for the reductase activity were in the range of 20–24°C and 8.0–8.5 in 0.05 M Tris–HCl buffer, respectively. The thermal stability of the securinine reductase was poor.     

Recognition and processing of double-stranded DNA by ExoX,a distributive 3′–5′ exonuclease

Members of the DnaQ superfamily are major 3′–5′ exonucleases that degrade either only single-stranded DNA (ssDNA) or both ssDNA and double-stranded DNA (dsDNA). However, the mechanism by which dsDNA is recognized and digested remains unclear. Exonuclease X (ExoX) is a distributive DnaQ exonuclease that cleaves both ssDNA and dsDNA substrates. Here, we report the crystal structures of Escherichia coli ExoX in complex with three different dsDNA substrates: 3′ overhanging dsDNA, blunt-ended dsDNA and 3′ recessed mismatch-containing dsDNA. In these structures, ExoX binds to dsDNA via both a conserved substrate strand-interacting site and a previously uncharacterized complementary strand-interacting motif. When ExoX complexes with blunt-ended dsDNA or 5′ overhanging dsDNA, a ‘wedge’ composed of Leu12 and Gln13 penetrates between the first two base pairs to break the 3′ terminal base pair and facilitates precise feeding of the 3′ terminus of the substrate strand into the ExoX cleavage active site. Site-directed mutagenesis showed that the complementary strand-binding site and the wedge of ExoX are dsDNA specific. Together with the results of structural comparisons, our data support a mechanism by which normal and mismatched dsDNA are recognized and digested by E. coli ExoX. The crystal structures also provide insight into the structural framework of the different substrate specificities of the DnaQ family members.     

Empowerment of coastal communities in cultivation and processing of Kappaphycus alvarezii��a case study at Vizhinjam village, Kerala, India

The Science & Society Division of Department of Science & Technology, Government of India sanctioned a project in 2005 under the Woman Scientist Program (DST WOS-B) with an objective to develop technologies for the cultivation of high-value seaweeds widely used for industrial purposes/human consumption with empowerment of the coastal communities in Kerala. The project was divided into two phases: an experimental/investigational phase and an extension phase. In the experimental phase, pilot-scale culture experiments were conducted to evaluate the possibility and feasibility of the cultivation of the red alga, Kappaphycus alvarezii, in the southwest coast of Kerala. Pilot-scale studies of seaweed culture were conducted in the shallow subtidal waters on the Vizhinjam Harbor area in two bamboo rafts tied with seeds of K. alvarezii (100?±?1.20 g) following accepted culture and growth monitoring procedures. The first harvest was carried out after 45 days and the growth was nearly eight times the initial biomass (826?±?2.80 g). A group of local fishermen were trained in the fabrication of culture rafts, implanting seed material, rearing the seedlings with periodical monitoring, harvesting, and post-harvest technologies like drying, sorting, packing, etc. as part of the pilot-scale study. The pilot-scale efforts showed good scope for the further expansion of the large-scale cultivation of K. alvarezii in the southwest coast of Kerala by imparting training and adopting fishermen families for their additional employment/alternative livelihood. Success in the pilot-scale cultivation led to the second phase of the project, the extension phase, which fulfilled the main objectives of the DST WOS-B programme. Vizhinjam Gramapanchayat (village), where the pilot-scale cultivation was carried out successfully and headed by a woman president, came forward to pool resources of the project to be implemented as a Model Seaweed Cultivation Programme. A training/workshop on ??seaweed farming technology?? was conducted and nearly 60 people were trained. Mangalam purusha sahaya sangham, a registered self-help group (SHG) of Vizhinjam, came forward to start the culture operations at Vizhinjam with technical backup from the DST-WOS-B Programme. Some seaweed entrepreneurs from inside and outside Kerala also came forward with a 100% buyback guarantee for the produce. Nationalised banks like the State Bank of India offered loans to trained SHGs for starting seaweed cultivation at commercial scale.     

Split target specificity of ResT: a design for protein delivery,site selectivity and regulation of enzyme activity?

The ResT telomere resolvase is responsible for maintaining the hairpin telomeres that cap the linear chromosome and minichromosomes of Borrelia burgdorferi. This enzyme acts at the tandem telomere junctions present within circular dimers resulting from DNA replication. ResT mediates the transesterification steps of resolution using a constellation of active site residues similar to that found in tyrosine recombinases and type IB topoisomerases. By combining this reaction mechanism with a hairpin binding module in its N-terminal domain, ResT reduces a fused telomere dimer into two hairpin monomers. ResT displays a split DNA binding specificity, with the N- and C-terminal domains targeting distinct regions of the telomere. This bi-specificity in binding is likely to be important in protein delivery, substrate selection and regulation of enzyme activity.     

Carbonic anhydrase inhibitors: Inhibition of the β-class enzyme from the pathogenic yeast Candida glabrata with sulfonamides,sulfamates and sulfamides

The fungal pathogen Candida glabrata encodes for a β-carbonic anhydrase (CA, EC 4.2.1.1), CgNce103, recently discovered. Only anions have been investigated as CgNce103 inhibitors up until now. Here we report the first sulfonamides inhibition study of this enzyme. Simple sulfonamides showed weak or moderate CgNce103 inhibitory properties, whereas acetazolamide, and a series of 4-substituted ureido-benzene-sulfonamides, sulfamates and sulfamides showed effective CgNce103 inhibitory properties, with K_Is in the range of 4.1–115 nM, being also ineffective as human CA II inhibitors. As there is significant resistance of C. glabrata clinical isolates to many classical antifungal agents, inhibition of the β-CA from this organism may allow an interesting means of controlling the pathogen growth, eventually leading to antifungals with a novel mechanism of action.     

Characterization of the murine Lbx2 promoter, identification of the human homologue, and evaluation as a candidate for Alström syndrome

The murine Lbx2 gene is a member of the ladybird family of homeobox genes, which is expressed in the developing urogenital system, eye, and brain. Using transgenic mice, we demonstrate that 9 kb of the 5' flanking region of mouse Lbx2 is able to direct expression of a reporter gene in a tissue-specific manner recapitulating the endogenous expression pattern. This regulatory region provides a novel reagent allowing for transgenic expression in the developing urogenital ridge. In addition, we describe the identification of the human homologue, LBX2. Comparison of the human LBX2 and mouse Lbx2 sequences upstream of the coding regions reveals sequence conservation suggesting conserved regulatory regions. Both the human LBX2 and the mouse Lbx2 genes have similar genomic structures and are composed of two exons separated by an intron. We mapped the mouse Lbx2 gene to 35 cM on chromosome 6 and the human LBX2 gene to a homologous region of chromosome 2p13. This is a candidate region for several inherited disorders, including Alstr?m syndrome, a disorder that includes ocular, urogenital, and renal abnormalities. Given the expression pattern of Lbx2, the chromosomal location in humans, and the potential function of mammalian ladybird genes, we have begun to analyze patients with ocular disorders and those with Alstr?m syndrome for mutations in LBX2. Although polymorphisms were identified, our results indicate that mutations in the coding region of LBX2 do not account for Alstr?m syndrome in the six kindreds analyzed.