Synthesis, ABTS-Radical Scavenging Activity, and Antiproliferative and Molecular Docking Studies of Novel Pyrrolo1,2-a]quinoline Derivatives

A new 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)-radical scavenging and antiproliferative agents of pyrrolo1,2-a]quinoline derivatives have been synthesized. An efficient method for the synthesis of 14 novel diversified pyrrolo1,2-a]quinoline derivatives has been described using 4-(1,3-dioxolan-2-yl)quinoline and different phenacyl bromides in acetone and followed by reacting with different acetylenes in dimethylformamide/K2CO3. The structure of the newly synthesized compounds was determined by infrared, H-1 NMR, C-13 NMR, mass spectrometry, and elemental analysis. The in vitro antioxidant activity revealed that among all the tested compounds 5n exhibited maximum scavenging activity with ABTS. Compound 5b has showed good antiproliferative activity as an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase.

Serine/Threonine/Tyrosine Protein Kinase Phosphorylates Oleosin, a Regulator of Lipid Metabolic Functions

Plant oils are stored in oleosomes or oil bodies, which are surrounded by a monolayer of phospholipids embedded with oleosin proteins that stabilize the structure. Recently, a structural protein, Oleosin3 (OLE3), was shown to exhibit both monoacylglycerol acyltransferase and phospholipase A(2) activities. The regulation of these distinct dual activities in a single protein is unclear. Here, we report that a serine/threonine/tyrosine protein kinase phosphorylates oleosin. Using bimolecular fluorescence complementation analysis, we demonstrate that this kinase interacts with OLE3 and that the fluorescence was associated with chloroplasts. Oleosin-green fluorescent protein fusion protein was exclusively associated with the chloroplasts. Phosphorylated OLE3 exhibited reduced monoacylglycerol acyltransferase and increased phospholipase A(2) activities. Moreover, phosphatidylcholine and diacylglycerol activated oleosin phosphorylation, whereas lysophosphatidylcholine, oleic acid, and Ca2+ inhibited phosphorylation. In addition, recombinant peanut (Arachis hypogaea) kinase was determined to predominantly phosphorylate serine residues, specifically serine-18 in OLE3. Phosphorylation levels of OLE3 during seed germination were determined to be higher than in developing peanut seeds. These findings provide direct evidence for the in vivo substrate selectivity of the dual-specificity kinase and demonstrate that the bifunctional activities of oleosin are regulated by phosphorylation.

Mycobacterium tuberculosis pantothenate kinase: possible changes in location of ligands during enzyme action

The crystal structures of complexes of Mycobacterium tuberculosis pantothenate kinase with the following ligands have been determined: (i) citrate; (ii) the nonhydrolysable ATP analogue AMPPCP and pantothenate (the initiation complex); (iii) ADP and phosphopantothenate resulting from phosphorylation of pantothenate by ATP in the crystal (the end complex); (iv) ATP and ADP, each with half occupancy, resulting from a quick soak of crystals in ATP (the intermediate complex); (v) CoA; (vi) ADP prepared by soaking and cocrystallization, which turned out to have identical structures, and (vii) ADP and pantothenate. Solution studies on CoA binding and catalytic activity have also been carried out. Unlike in the case of the homologous Escherichia coli enzyme, AMPPCP and ADP occupy different, though overlapping, locations in the respective complexes; the same is true of pantothenate in the initiation complex and phosphopantothenate in the end complex. The binding site of MtPanK is substantially preformed, while that of EcPanK exhibits considerabl plasticity. The difference in the behaviour of the E. coli and M. tuberculosis enzymes could be explained in terms of changes in local structure resulting from substitutions. It is unusual for two homologous enzymes to exhibit such striking differences in action. Therefore, the results have to be treated with caution. However, the changes in the locations of ligands exhibited by M. tuberculosis pantothenate kinase are remarkable and novel.

The Evolution of Guanylyl Cyclases as Multidomain Proteins: Conserved Features of Kinase-Cyclase Domain Fusions

Guanylyl cyclases (GCs) are enzymes that generate cyclic GMP and regulate different physiologic and developmental processes in a number of organisms. GCs possess sequence similarity to class III adenylyl cyclases (ACs) and are present as either membrane-bound receptor GCs or cytosolic soluble GCs. We sought to determine the evolution of GCs using a large-scale bioinformatic analysis and found multiple lineage-specific expansions of GC genes in the genomes of many eukaryotes. Moreover, a few GC-like proteins were identified in prokaryotes, which come fused to a number of different domains, suggesting allosteric regulation of nucleotide cyclase activity Eukaryotic receptor GCs are associated with a kinase homology domain (KHD), and phylogenetic analysis of these proteins suggest coevolution of the KHD and the associated cyclase domain as well as a conservation of the sequence and the size of the linker region between the KHD and the associated cyclase domain. Finally, we also report the existence of mimiviral proteins that contain putative active kinase domains associated with a cyclase domain, which could suggest early evolution of the fusion of these two important domains involved in signa transduction.

Effect of environmental stress of low pressure on tyrosine aminotransferase and phenylalanine 4-hydroxylase activities in the rat.

Tyrosine aminotransferase activity in the liver increased about fourfold after 9h, on exposure of rats to stress of low pressure. 2. The phenylalanine hydroxylase activity increased about 60% on exposure for 24h or more. 3. An environmental pressure decrease of about 0.033 MN/m2 is needed to increase the activity of tyrosine aminotransferase. 4. Adrenalectomy completely abolished the increase in activity of tyrosine aminotransferase obtained on exposure to low pressure. 5. Treatment with cycloheximide or actinomycin D prevented the increase in activity of tyrosine aminotransferase. 6. Treatment with cycloheximide at the early part of exposure to stress prevented the increase in activity of phenylalanine hydroxylase obtained after 24h.

New structural insights and molecular-modelling studies of 4-methyl-5-beta-hydroxyethylthiazole kinase from Pyrococcus horikoshii OT3 (PhThiK)

4-Methyl-5-beta-hydroxyethylthiazole kinase (ThiK) catalyses the phosphorylation of the hydroxyl group of 4-methyl-5-beta-hydroxyethylthiazole. This work reports the first crystal structure of an archaeal ThiK: that from Pyrococcus horikoshii OT3 (PhThiK) at 1.85 angstrom resolution with a phosphate ion occupying the position of the beta-phosphate of the nucleotide. The topology of this enzyme shows the typical ribokinase fold of an alpha/beta protein. The overall structure of PhThiK is similar to those of Bacillus subtilis ThiK (BsThiK) and Enterococcus faecalis V583 ThiK (EfThiK). Sequence analysis of ThiK enzymes from various sources indicated that three-quarters of the residues involved in interfacial regions are conserved. It also revealed that the amino-acid residues in the nucleotide-binding, magnesium ion-binding and substrate-binding sites are conserved. Binding of the nucleotide and substrate to the ThiK enzyme do not influence the quaternary association (trimer) as revealed by the crystal structure of PhThiK.

Light- and cytokinin-induced changes in the levels of leucine and tyrosine isoaccepting tRNA species and modified nucleotide contents of total tRNA in cucumber seedlings

We have determined relative levels of chloroplast leucine and tyrosine isoaccepting tRNAs and modified nucleotide contents from total tRNAs isolated from dark-grown, light-grown, N6-isopentenyladenine (i6A)-treated dark-grown and i6A-treated light-grown cucumber seedlings. Significant increases in the relative amounts of tRNA(Leu)2 and tRNA(Leu)3 were observed in the i6A-treated dark-grown seedlings compared to dark-grown, light-grown and i6A-treated light-grown seedlings. On the other hand, i6A-treated light-grown seedlings tRNA(Tyr)1 increased to 85% of total tRNAs(Tyr) from about 9% in light-grown seedlings and tRNA(Tyr)2 decreased to 15% compared with 91% in light-grown seedlings. Analysis of modified nucleotide of total tRNAs indicated that pT, pI, pm1A, pm5C, pGm, pm1G, pm2G and pm7G contents were significantly higher in the total tRNA of i6A-treated dark-grown seedlings than those from untreated dark-grown seedlings. Illumination of 8-day-old dark-grown seedlings for 12 h increased the contents of pT, pI, pGm and pm1G when compared to 8-day-old dark-grown seedlings with extended growth for 12 h in dark. On the contrary, i6A had no stimulatory effect in the contents of modified nucleotide in the light-grown seedlings.

Computer-Aided Drug Design of Pyranopyrazoles and Related Compounds for Checkpoint Kinase-1

Checkpoint-1 kinase plays an important role in the G(2)M cell cycle control, therefore its inhibition by small molecules is of great therapeutic interest in oncology. In this paper, we have reported the virtual screening of an in-house library of 2499 pyranopyrazole derivatives against the ATP-binding site of Chk1 kinase using Glide 5.0 program, which resulted in six hits. All these ligands were docked into the site forming most crucial interactions with Cys87, Glu91 and Leu15 residues. From the observed results these ligands are suggested to be potent inhibitors of Chk1 kinase with sufficient scope for further elaboration.

The Role of UPF0157 in the Folding of M-tuberculosis Dephosphocoenzyme A Kinase and the Regulation of the Latter by CTP

Background: Targeting the biosynthetic pathway of Coenzyme A (CoA) for drug development will compromise multiple cellular functions of the tubercular pathogen simultaneously. Structural divergence in the organization of the penultimate and final enzymes of CoA biosynthesis in the host and pathogen and the differences in their regulation mark out the final enzyme, dephosphocoenzyme A kinase (CoaE) as a potential drug target. Methodology/Principal Findings: We report here a complete biochemical and biophysical characterization of the M. tuberculosis CoaE, an enzyme essential for the pathogen's survival, elucidating for the first time the interactions of a dephosphocoenzyme A kinase with its substrates, dephosphocoenzyme A and ATP; its product, CoA and an intrinsic yet novel inhibitor, CTP, which helps modulate the enzyme's kinetic capabilities providing interesting insights into the regulation of CoaE activity. We show that the mycobacterial enzyme is almost 21 times more catalytically proficient than its counterparts in other prokaryotes. ITC measurements illustrate that the enzyme follows an ordered mechanism of substrate addition with DCoA as the leading substrate and ATP following in tow. Kinetic and ITC experiments demonstrate that though CTP binds strongly to the enzyme, it is unable to participate in DCoA phosphorylation. We report that CTP actually inhibits the enzyme by decreasing its Vmax. Not surprisingly, a structural homology search for the modeled mycobacterial CoaE picks up cytidylmonophosphate kinases, deoxycytidine kinases, and cytidylate kinases as close homologs. Docking of DCoA and CTP to CoaE shows that both ligands bind at the same site, their interactions being stabilized by 26 and 28 hydrogen bonds respectively. We have also assigned a role for the universal Unknown Protein Family 0157 (UPF0157) domain in the mycobacterial CoaE in the proper folding of the full length enzyme. Conclusions/Significance: In view of the evidence presented, it is imperative to assign a greater role to the last enzyme of Coenzyme A biosynthesis in metabolite flow regulation through this critical biosynthetic pathway.

Functional diversity of human protein kinase splice variants marks significant expansion of human kinome

Background: Protein kinases are involved in diverse spectrum of cellular processes. Availability of draft version of the human genomic data in the year 2001 enabled recognition of repertoire of protein kinases. However, over the years the human genomic data is being refined and the current release of human genomic data has helped us to recognize a larger repertoire of over 900 human protein kinases represented mainly by splice variants. Results: Many of these identified protein kinases are alternatively spliced products. Interestingly, some of the human kinase splice variants appear to be significantly diverged in terms of their functional properties as represented by incorporation or absence of one or more domains. Many sets of protein kinase splice variants have substantially different domain organization and in a few sets of splice variants kinase domains belong to different subfamilies of kinases suggesting potential participation in different signal transduction pathways. Conclusions: Addition or deletion of a domain between splice variants of multi-domain kinases appears to be a means of generating differences in the functional features of otherwise similar kinases. It is intriguing that marked sequence diversity within the catalytic regions of some of the splice variant kinases result in kinases belonging to different subfamilies. These human kinase splice variants with different functions might contribute to diversity of eukaryotic cellular signaling.

Characterization of mouse neuronal Ca2+/calmodulin kinase II inhibitor alpha

We have overexpressed an 8.5-kDa mouse Ca2+/calmodulin kinase II inhibitor a protein (mCaMKIIN alpha) in Escherichia coli and demonstrate that the recombinant protein is a potent inhibitor of Ca2+/calmodulin kinase 11 (CaMKII) in vitro. However, antibodies raised against recombinant mCaMKIIN alpha. react with an similar to 37-kDa protein present in mouse brain. The pattern of expression of the similar to 37-kDa protein is similar to that of mCaMKIIN alpha mRNA as both are expressed in normal but not Japanese encephalitis virus (JEV)-infected mouse brain. Subcellular localization studies indicate that the similar to 37-kDa protein is present in the post-synaptic density (PSD) where mCaMKII alpha is known to perform key regulatory functions. We conclude that the similar to 37-kDa protein identified in this study is mCaMKIIN alpha. and its localization in the PSD indicates a novel role for this protein in the regulation of neuronal CaMKII alpha. (c) 2007 Elsevier B.V. All rights reserved.

Antithyroid Drugs and their Analogues Protect Against Peroxynitrite-Mediated Protein Tyrosine Nitration-A Mechanistic Study

In this paper, the effect of some commonly used antithyroid drugs and their analogues on peroxynitrite-mediated nitration of proteins is described. The nitration of tyrosine residues in bovine serum albumin (BSA) and cytochromec was studied by Western blot analysis. These studies reveal that the antithyroid drugs methimazole (MMI), 6-n-propyl-2-thiouracil (PTU), and 6-methyl-2-thiouracil (MTU), which contain thione moieties, significantly reduce the tyrosine nitration of both BSA and cytochrome c. While MMI exhibits good peroxynitrite (PN) scavenging activity, the thiouracil compounds PTU and MTU are slightly less effective than MMI. The S- and Se-methylated compounds show a weak inhibitory effect in the nitration of tyrosine, indicating that the presence of a thione or selone moiety is important for an efficient inhibition. Similarly, the replacement of N-H moiety in MMI by N-methyl or N-m-methoxybenzyl substituents dramatically reduces the antioxidant activity of the parent compound. Theoretical studies indicate that the substitution of N-H moiety by N-Me significantly increases the energy required for the oxidation of sulfur center by PN. However, such substitution in the selenium analogue of MMI increases the activity of parent compound. This is due to the facile oxidation of the selone moiety to the corresponding selenenic and seleninic acids. Unlike N,N'-disubstituted thiones, the corresponding selones efficiently scavenge PN, as they predominantly exist in their zwitterionic forms in which the selenium atom carries a large negative charge.

Inhibition of a Protein Tyrosine Phosphatase Using Mesoporous Oxides

The feasibility of utilizing mesoporous matrices of alumina and silica for the inhibition of enzymatic activity is presented here. These studies were performed on a protein tyrosine phosphatase by the name chick retinal tyrosine phosphotase-2 (CRYP-2), a protein that is identical in sequence to the human glomerular epithelial protein-1 and involved in hepatic carcinoma. The inhibition of CRYP-2 is of tremendous therapeutic importance. Inhibition of catalytic activity was examined using the Sustained delivery of p-nitrocatechol sulfate (pNCS) from bare and amine functionalized mesoporous silica (MCM-48) and mesoporous alumina (Al2O3). Among the various mesoporous matrices employed, amine functionalized MCM-48 exhibited the best release of pNCS and also inhibition of CRYP-2. The maximum speed of reaction nu(max) (= 160 +/- 10 mu mol/mnt/mg) and inhibition constant K-i (=85.0 +/- 5.0 mu mol) estimated using a competitive inhibition model were Found to be very similar to inhibition activities of protein tyrosine phosphatases using other methods.

Importance of tyrosine residues of Bacillus stearothermophilus serine hydroxymethyltransferase in cofactor binding and l-allo-Thr cleavage

Serine hydroxymethyltransferase (SHMT) from Bacillus stearothermophilus (bsSHMT) is a pyridoxal 5'-phosphate-dependent enzyme that catalyses the conversion of l-serine and tetrahydrofolate to glycine and 5,10-methylene tetrahydrofolate. In addition, the enzyme catalyses the tetrahydrofolate-independent cleavage of 3-hydroxy amino acids and transamination. In this article, we have examined the mechanism of the tetrahydrofolate-independent cleavage of 3-hydroxy amino acids by SHMT. The three-dimensional structure and biochemical properties of Y51F and Y61A bsSHMTs and their complexes with substrates, especially l-allo-Thr, show that the cleavage of 3-hydroxy amino acids could proceed via Cα proton abstraction rather than hydroxyl proton removal. Both mutations result in a complete loss of tetrahydrofolate-dependent and tetrahydrofolate-independent activities. The mutation of Y51 to F strongly affects the binding of pyridoxal 5'-phosphate, possibly as a consequence of a change in the orientation of the phenyl ring in Y51F bsSHMT. The mutant enzyme could be completely reconstituted with pyridoxal 5'-phosphate. However, there was an alteration in the λmax value of the internal aldimine (396 nm), a decrease in the rate of reduction with NaCNBH3 and a loss of the intermediate in the interaction with methoxyamine (MA). The mutation of Y61 to A results in the loss of interaction with Cα and Cβ of the substrates. X-Ray structure and visible CD studies show that the mutant is capable of forming an external aldimine. However, the formation of the quinonoid intermediate is hindered. It is suggested that Y61 is involved in the abstraction of the Cα proton from 3-hydroxy amino acids. A new mechanism for the cleavage of 3-hydroxy amino acids via Cα proton abstraction by SHMT is proposed.