Enantio-selectivity of human nucleoside monophosphate kinases

Over recent years, there has been a renewed interest in the development of L-nucleosides as safe and efficacious drugs for the treatment of viral infections. Biological activity of these compounds requires phosphorylation to their triphosphate form, involving nucleoside monophosphate kinases in the second step. In order to characterize the activation pathway of L-nucleosides of the pyrimidine series, we studied the enantio-selectivity of human uridylate-cytidylate and thymidylate kinases. The results showed that these enzymes are only weakly enantio-selective and are thus probably involved in the activation of L-nucleosides in vivo. An activation pathway for telbivudine (L-dT) was therefore proposed.     

L-Purine Nucleosides as Selective Antimalarials

Abstract

L-nucleosides selectively enter malaria infected erythrocytes and have the unique ability to be metabolised by the malarial adenosine deaminase. This has allowed us to design novel L-nucleosides as potential anti-malarials.     

Cyclopentane-nucleobase coupling in the synthesis of carbocyclic L-nucleosides: is a SN2-reaction an alternative to the Mitsunobu-reaction?

Several carbocyclic L-nucleosides have been synthesized by coupling a cyclopentane-system with heterocycles according to a modified Mitsunobu-protocol. This reaction gave two regioisomers, the N1-alkylated product and an unwanted O(2)-product. A simple S(N)2-reaction has been investigated as an alternative for such couplings.     

L-nucleosides containing modified nucleobases

The synthesis of base modified L-nucleosides is described with pyrrolo[2,3-d]pyrimidines, pyrazolo[3,4-d]pyrimidines, benzimidazoles, and imidazo[1,2-a]-s-triazines as nucleobases. The conformation of the nucleosides is studied and the antiviral activity is evaluated.     

New classes of fluorinated L-nucleosides; synthesis and antiviral activity.

The synthesis of 3'-fluorinated apionucleosides 7 and 2'-fluoro-2',3'-unsaturated L-nucleosides 8 via common synthon, 2-fluoro-butenolide 2, has been described. Among the newly synthesized nucleosides, L-2'-F-d4C, L-2'-F-d4FC and L-2'-F-d4A exhibit significant anti-HIV and anti-HBV activities.     

Structural basis for activation of the therapeutic L-nucleoside analogs 3TC and troxacitabine by human deoxycytidine kinase

L-nucleoside analogs represent an important class of small molecules for treating both viral infections and cancers. These pro-drugs achieve pharmacological activity only after enzyme-catalyzed conversion to their tri-phosphorylated forms. Herein, we report the crystal structures of human deoxycytidine kinase (dCK) in complex with the L-nucleosides (-)-beta-2',3'-dideoxy-3'-thiacytidine (3TC)--an approved anti-human immunodeficiency virus (HIV) agent--and troxacitabine (TRO)--an experimental anti-neoplastic agent. The first step in activating these agents is catalyzed by dCK. Our studies reveal how dCK, which normally catalyzes phosphorylation of the natural D-nucleosides, can efficiently phosphorylate substrates with non-physiologic chirality. The capability of dCK to phosphorylate both D- and L-nucleosides and nucleoside analogs derives from structural properties of both the enzyme and the substrates themselves. First, the nucleoside-binding site tolerates substrates with different chiral configurations by maintaining virtually all of the protein-ligand interactions responsible for productive substrate positioning. Second, the pseudo-symmetry of nucleosides and nucleoside analogs in combination with their conformational flexibility allows the L- and D-enantiomeric forms to adopt similar shapes when bound to the enzyme. This is the first analysis of the structural basis for activation of L-nucleoside analogs, providing further impetus for discovery and clinical development of new agents in this molecular class.     

2'-Fluoro-4'-thio-2',3'-unsaturated nucleosides: anti-HIV activity, resistance profile, and molecular modeling studies

Both D- and L-2'-fluoro-4'-thio-2',3'-unsaturated nucleosides were synthesized and their anti-HIV activity against the drug sensitive virus and lamivudine-resistant mutant (M184V) were evaluated. In vitro antiviral evaluation indicated that the L-isomers are more potent than the D-isomers, but unfortunately all were cross-resistant with 3TC. Molecular modeling studies revealed that the unnatural sugar moiety of the L-nucleosides as well as 4'-sulfur atom of the D-isomer has a steric conflict with the bulky side chain of valine 184, resulting in cross-resistance.     

Synthesis of enantiomerically pure D- and L-bicyclo[3.1.0]hexenyl carbanucleosides and their antiviral evaluation

Based upon the fact that L-nucleosides have been generally known to be less cytotoxic than D-counterparts, L-bicyclo[3.1.0]hexenyl carbanucleoside derivatives with a fixed north conformation were designed and synthesized by employing a novel synthetic strategy starting from (R)-epichlorohydrin in order to search for new anti-HIV agents with high potency and less cytotoxicity. A tandem alkylation, γ-lactonization, a chemoselective reduction of ester in the presence of γ-lactone functional group, a RCM reaction, and a Mitsunobu coupling reaction were used as key reactions. D-Counterpart nucleosides were also prepared according to the same synthetic method. Among the synthesized carbanucleosides, D-thymine nucleoside, D-2 and L-thymine nucleoside, L-2 exhibited excellent anti-HIV-1 and -2 activities, in MT-4 cells, which were higher than those of ddI, an anti-AIDS drug. Whereas D-2 exhibited high cytotoxicity in MT-4 cell lines, L-2 did not show any discernible cytotoxicity in all cell lines tested, reflecting that L-2 may be a good candidate for an anti-AIDS drug. L-2 also showed weak anti-HSV-2 activity without cytotoxicity. However, none of the synthesized nucleosides exhibited antiviral activities against RNA viruses including coxsakie, influenza, corona and polio viruses, maybe due to their 2',3'-dideoxy structure. Potent antiviral effects of D-2 and L-2 indicate that nucleosides belonging to a class of D4Ns can be an excellent candidate for anti-DNA virus agents. This research strongly supports L-nucleosides of a class of D4Ns to be a very promising candidate for antiviral agents due to its low cytotoxicity and a good antiviral activity.     

L-nucleoside analogues as potential antimalarials that selectively target Plasmodium falciparum adenosine deaminase

The L-stereoisomer analogues of D-coformycin selectively inhibited P. falciparum adenosine deaminase (ADA) in the picomolar range (L-isocoformycin, Ki 7 pM; L-coformycin, Ki 250 pM). While the L-nucleoside analogues, L-adenosine, 2,6-diamino-9-(L-ribofuranosyl)purine and 4-amino-1-(L-ribofuranosyl)pyrazolo[3,4-d]-pyrimidine were selectively deaminated by P. falciparum ADA, L-thioinosine and L-thioguanosine were not. This is the first example of 'non-physiological' L-nucleosides that serve as either substrates or inhibitors of malarial ADA and are not utilised by mammalian ADA.     

Anti-HBV specific beta-L-2'-deoxynucleosides

A unique series of simple unnatural L-nucleosides that specifically inhibit hepatitis B virus (HBV) replication has been discovered. These molecules have in common a hydroxyl group in the 3'-position (3'-OH) of the beta-L-2'-deoxyribose sugar that confers antiviral activity specifically against hepadnaviruses. Replacement of the 3'-OH broadens activity to other viruses. Substitution in the base decreases antiviral potency and selectivity. Human DNA polymerases and mitochondrial function are not effected. Plasma viremia is reduced up to 8 logs in a woodchuck model of chronic HBV infection. These investigational drugs, used alone or in combination, are expected to offer new therapeutic options for patients with chronic HBV infection.     

New Classes of Fluorinated L-Nucleosides; Synthesis and Antiviral Activity

Abstract

The synthesis of 3′-fluorinated apionucleosides 7 and 2′-fluoro-2′, 3′-unsaturated L-nucleosides 8 via common synthon, 2-fluoro-butenolide 2, has been described. Among the newly synthesized nucleosides, L-2′-F-d4C, L-2′-F-d4FC and L-2′-F-d4A exhibit significant anti-HIV and anti-HBV activities.     

Metabolism of pyrimidine L-nucleosides.

The intraperitoneal application of L-nucleosides (L-Cyd, L-Urd, L-dThd) to mice results in distribution of these compounds into tissues of the organism and their gradual excretion in the unchanged form. The residual level has been observed with L-ribonucleosides only and contains in addition to the L-nucleoside its 5'-phosphate. The phosphorylation in vivo is catalyzed by nucleoside-kinase and utilizes ATP as the phosphate donor while glycerol 1-phosphate and creatine phosphate are inactive. The L-cytidine derivatives are in vivo deaminated to the derivatives of L-uridine. On the other hand, when L-uridine is applied in vivo, derivatives of L-cytidine are obtained on the level of both the nucleoside and 5'-ribonucleotide.     

ANTI-HBV SPECIFIC β-L-2′-DEOXYNUCLEOSIDES

A unique series of simple unnatural L-nucleosides that specifically inhibit hepatitis B virus (HBV) replication has been discovered. These molecules have in common a hydroxyl group in the 3′-position (3′-OH) of the β-L-2′-deoxyribose sugar that confers antiviral activity specifically against hepadnaviruses. Replacement of the 3′-OH broadens activity to other viruses. Substitution in the base decreases antiviral potency and selectivity. Human DNA polymerases and mitochondrial function are not effected. Plasma viremia is reduced up to 8 logs in a woodchuck model of chronic HBV infection. These investigational drugs, used alone or in combination, are expected to offer new therapeutic options for patients with chronic HBV infection.     

Molecular basis for the lack of enantioselectivity of human 3-phosphoglycerate kinase

Non-natural L-nucleoside analogues are increasingly used as therapeutic agents to treat cancer and viral infections. To be active, L-nucleosides need to be phosphorylated to their respective triphosphate metabolites. This stepwise phosphorylation relies on human enzymes capable of processing L-nucleoside enantiomers. We used crystallographic analysis to reveal the molecular basis for the low enantioselectivity and the broad specificity of human 3-phosphoglycerate kinase (hPGK), an enzyme responsible for the last step of phosphorylation of many nucleotide derivatives. Based on structures of hPGK in the absence of nucleotides, and bound to L and d forms of MgADP and MgCDP, we show that a non-specific hydrophobic clamp to the nucleotide base, as well as a water-filled cavity behind it, allows high flexibility in the interaction between PGK and the bases. This, combined with the dispensability of hydrogen bonds to the sugar moiety, and ionic interactions with the phosphate groups, results in the positioning of different nucleotides so to expose their diphosphate group in a position competent for catalysis. Since the third phosphorylation step is often rate limiting, our results are expected to alleviate in silico tailoring of L-type prodrugs to assure their efficient metabolic processing.     

Stereoisomeric selectivity of human deoxyribonucleoside kinases

Deoxynucleoside kinases catalyze the 5'-phosphorylation of 2'-deoxyribonucleosides with nucleoside triphosphates as phosphate donors. One of the cellular kinases, deoxycytidine kinase (dCK), has been shown to phosphorylate several L-nucleosides that are efficient antiviral agents. In this study we investigated the potentials of stereoisomers of the natural deoxyribonucleoside to serve as substrates for the recombinant cellular deoxynucleoside kinases. The cytosolic thymidine kinase exhibited a strict selectivity and phosphorylated only beta-D-Thd, while the mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK) as well as dCK all had broad substrate specificities. TK2 phosphorylated Thd and dCyd stereoisomers in the order: beta-D- > or = beta-L- > alpha-D- > or = alpha-L-isomer. dCK activated both enantiomers of beta-dCyd, beta-dGuo, and beta-dAdo with similar efficiencies, and alpha-D-dCyd also served as a substrate. dGK phosphorylated the beta-dGuo enantiomers with no preference for the ribose configuration; alpha-L-dGuo was also phosphorylated, and beta-L-dAdo and beta-L-dCyd were substrates but showed reduced efficiencies. The anomers of the 2',3'-dideoxy-D-nucleosides (ddNs) were tested, and TK2 and dCK retained their low selectivities. Unexpectedly, alpha-dideoxycytidine (ddC) was a 3-fold better substrate for dCK than beta-ddC. Similarly, alpha-dideoxythymidine (ddT) was a better substrate for TK2 than beta-ddT. dGK did not accept any D-ddNs. Thus, TK2, dCK, and dGK, similar to herpes simplex virus type 1 thymidine kinase (HSV-1 TK), showed relaxed stereoselectivities, and these results substantiate the functional similarities within this enzyme family. Docking simulations with the Thd isomers and the active site of HSV-1 TK showed that the viral enzyme may in some respects serve as a model for studying the substrate specificities of the cellular enzymes.     

Specific residues within the alpha 2 integrin subunit cytoplasmic domain regulate migration and cell cycle progression via distinct MAPK pathways.

The alpha(2) integrin subunit cytoplasmic domain is necessary for epidermal growth factor (EGF)-stimulated chemotactic migration and insulin-dependent entry into S-phase of mammary epithelial cells adherent to type I collagen. Truncation mutants revealed that the seven amino acids, KYEKMTK, in addition to the GFFKR motif were sufficient for these functions. Mutation of tyrosine 1134 to alanine inhibited the ability of the cells to phosphorylate p38 MAPK and to migrate in response to EGF but had only a modest effect on the ability of the cells to induce sustained phosphorylation of the ERK MAPK, to up-regulate cyclin E and cdk2 expression, and to enter S-phase when adherent to type I collagen. Conversely, mutation of the lysine 1136 inhibited the ability of the cells to increase cyclin E and cdk2 expression, to maintain long term phosphorylation of the ERK MAPK, and to enter S-phase but had no effect on the ability of the cells to phosphorylate the p38 MAPK or to migrate on type I collagen in response to EGF. Methionine 1137 was essential for both migration and entry into S-phase. Thus, distinctly different structural elements of the alpha(2) integrin cytoplasmic domain are required to engage the signaling pathways leading to cell migration or cell cycle progression.     

Localization of Escherichia coli RNA polymerase binding sites on bacteriophage S13 and 0X174 DNAs by electron microscopy.

Complexes between Escherichia coli RNA polymerase and bacteriophage S13 and phage phiX174 replicative form III DNAs have been shown to form at specific locations on the phage genomes. The major locations on S13 have been mapped at 8 to 10 and 92 to 96% of the genome length, starting from the unique Pst I cleavage site. The locations correspond to the beginnings of genes D and B, respectively. Four minor locations map at 18 to 22, 28 to 32, 50 to 56, and 70 to 74% of the genome. The 70 to 74% site corresponds to the beginning of the A gene. The major locations on phiX174 are at 8 to 10, 50 to 54, and 92 to 94% of the genome. The 50 to 54% site is at the start of the H gene and has an equivalent minor site on S13, but it is not a promoter site. Three minor sites on phiX174, at 20 to 24, 26 to 32, and 68 to 74% of the genome, correspond to sites on S13. The data confirm the locations of sites identified by restriction fragment binding experiments (E. Rassart and J. H. Spencer, J. Virol. 27:677--687, 1978) and the assignment of putative promoters at the start of genes A, B and D.     

Biosynthesis of mammalian glycoproteins. Glycosylation pathways in the synthesis of the nonreducing terminal sequences.

Six purified glycosyltransferase (a beta-galactoside alpha 2 leads to 6 sialyltransferase, a beta-galactoside alpha 2 leads to 3 sialyltransferase, an alpha-N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase, a beta-galactoside alpha 1 leads to 2 fucosyltransferase, a beta-N-acetylglucosaminide alpha 1 leads to 3 fucosyltransferase, and a (fucosyl alpha 1 leads to 2) galactoside alpha 1 leads to 3 N-acetyl-galactosaminyltransferase) have been used to study the biosynthetic pathways for formation of the nonreducing terminal oligosaccharide sequences in mammalian glycoproteins. The two glycoproteins used as model acceptor substrates in this study were human asialotransferrin, which contains the nonreducing terminal oligosaccharide sequence Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man, and antifreeze glycoprotein, which contains oligosaccarides with the structure, Gal beta 1 leads to 3GalNAc alph 1 leads O-Thr. Sequential action of the six glycosyltransferases on these model substrates led to the formation of previously described oligosaccharide structures. The studies reported here indicate that the substrate specificities of the individual enzymes dictate the structures that can be synthesized and the pathways by which they may be formed. The actions of a number of the transferasesare mutually exclusive, thereby prohibiting the formation of theoretically possible oligosaccharide structures. Oligosaccharides with the terminal sequence NeuAc alpha 2 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 3GalNAc and NeuAc alpha 2 leads to 6Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc cannot be formed because the prior incorporation of sialic acid by the sialyltransferases yields products that are not acceptor substrates for the fucosyltransferases, and vice versa. Synthesis of other products requires that the enzymes act sequentially in a specific order. The structures NeuAc alpha 2 leads to 6(Fuc alpha 1 leads to 2)Gal beta 1 leads to 4GlcNAc, Fuc alpha 1 leads to 2Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc, GalNAc alpha 1 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 4GlcNAc, and GalNAc alpha 1 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 3GalNAc can only be synthesized if the fucosyl alpha 1 leads to 2 galactose linkage is formed first. Synthesis of the pentasaccharide sequences GalNAc alpha 1 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GalNAc and GalNAc alpha 1 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc requires that the N-acetylgalactosaminyltransferase act last on the former structure and that the alpha 1 leads to 3 fucosyltransferase act last on the latter. In those instances where a product can be formed by one of two possible pathways, the comparisons of reaction rates indicate that one pathway is usually preferred...     

Transmitter-Induced Calcium Responses Differ in Astrocytes Acutely Isolated from Rat Brain and in Culture

Abstract: Glial fibrillary acid protein (GFAP)-positive astrocytes isolated from the cerebral cortices of 3–10-day-old rats frequently showed increased intracellular Ca²⁺ concentration responses to l -glutamate and glutamate analogues. However, few of the acutely isolated cells responded to ATP, and no such cells responded to serotonin [5-hydroxytryptamine (5-HT)]. The same cell that failed to respond to ATP or 5-HT often responded to glutamate. Culturing acutely isolated cells in media containing horse serum decreased Ca²⁺ responses to glutamate but increased the responses to ATP and induced responses to 5-HT. In primary cultures prepared from the cerebral cortices of 1-day-old rats and cultured in horse serum, fewer of the cells responded to glutamate, but almost all cells responded to ATP and 5-HT. The lack of, or limited response to, 5-HT or ATP in the acutely isolated cells seems unlikely to be due to selective damage to the respective receptors because acutely isolated GFAP-negative cells showed responses to ATP, several different proteases and mechanical dissociation yielded cells that also responded to glutamate but not to ATP, and exposure of primary cultures to papain did not abolish Ca²⁺ responses to several transmitters. The responses of the acutely isolated cells to glutamate but limited or lack of responses to ATP and 5-HT also correspond to what has been seen so far for astrocytes in situ. Thus, the present studies provide direct evidence that some of the receptors seen in primary astrocyte cultures may reflect a response to culture conditions and that, in the context of the relevant information so far available, acutely isolated astrocytes seem to reflect better the in vivo state.     

Adaptation to Delayed Force Perturbations in Reaching Movements

Adaptation to deterministic force perturbations during reaching movements was extensively studied in the last few decades. Here, we use this methodology to explore the ability of the brain to adapt to a delayed velocity-dependent force field. Two groups of subjects preformed a standard reaching experiment under a velocity dependent force field. The force was either immediately proportional to the current velocity (Control) or lagged it by 50 ms (Test). The results demonstrate clear adaptation to the delayed force perturbations. Deviations from a straight line during catch trials were shifted in time compared to post-adaptation to a non-delayed velocity dependent field (Control), indicating expectation to the delayed force field. Adaptation to force fields is considered to be a process in which the motor system predicts the forces to be expected based on the state that a limb will assume in response to motor commands. This study demonstrates for the first time that the temporal window of this prediction needs not to be fixed. This is relevant to the ability of the adaptive mechanisms to compensate for variability in the transmission of information across the sensory-motor system.