Autophosphorylation of αCaMKII affects social interactions in mice.

The α-Ca2+ /calmodulin-dependent protein kinase II (αCaMKII), a key regulator of the glutamatergic synapse, has been implicated in many psychiatric disorders characterized by social impairments. Here we tested whether autophosphorylation of αCaMKII at threonine 286, which prolongs the activity of the enzyme, affects social behaviors in mice. We observed that autophosphorylation-deficient (αCaMKII-T286A) mutant female mice showed abnormal social behaviors characterized by decreased social preference and interest in conspecifics of the same sex, as compared to their wild-type littermates. Moreover, we developed a mathematical approach to analyze social interactions in group-housed mice in the automated IntelliCages. Using this approach we observed that αCaMKII-T286A mutants show decreased levels of social interactions in a social group, as compared with WT mice. WT mice increased the frequency of close social interactions when learning about the location of the food reward. This phenomenon was absent in the mutants. Overall, our data indicates that autophosphorylation of αCaMKII affects social interactions.

Trichinella spiralis infection enhances protein kinase C phosphorylation in guinea pig alveolar macrophages.

To learn more about the signalling pathways involved in superoxide anion production in guinea pig alveolar macrophages, triggered by Trichinella spiralis infection, protein level and phosphorylation of mitogen activated protein (MAP) kinases and protein kinase C (PKC) were investigated. Infection with T. spiralis, the nematode having 'lung phase' during colonization of the host, enhances PKC phosphorylation in guinea pig alveolar macrophages. Isoenzymes beta and delta of PKC have been found significantly phosphorylated, although their location was not changed as a consequence of T. spiralis infection. Neither in macrophages from T. spiralis-infected guinea pig nor in platelet-activating factor (PAF)-stimulated macrophages from uninfected animals, participation of MAP kinases in respiratory burst activation was statistically significant. The parasite antigens seem to act through macrophage PAF receptors, transducing a signal for enhanced NADPH oxidase activity, as stimulating effect of newborn larvae homogenate on respiratory burst was abolished by specific PAF receptor antagonist CV 6209. A suppressive action of T. spiralis larvae on host alveolar macrophage innate immunological response was reflected by diminished protein level of ERK2 kinase and suppressed superoxide anion production, in spite of high level of PKC phosphorylation.

A non-classical type of alveolar macrophage response to Trichinella spiralis infection.

Studies of arginase expression and activity in guinea pig alveolar macrophages during Trichinella spiralis infection, prompted by earlier observation of innate lung response to the parasite, showed the macrophages to express both activity and protein of arginase type I. In cultured macrophages part of the enzyme was found to be always released to the extracellular medium. Whereas BCG in vivo treatment, alone or preceded by T. spiralis infection, stimulated arginase activity, T. spiralis infection alone affected the enzyme distribution between intracellular and extracellular fractions, and properties (K(m) and V(max)), rather than total (intracellular + extracellular) activity, with TGF-beta apparently responsible for a part of the effect. Anti-TGF-beta antibody treatment of the animals influenced both arginase activation by Mn(2+) and dependence of the enzyme-catalysed reaction on pH. Whereas T. spiralis infection activated guinea pig alveolar macrophages by the type II macrophage activation, as indicated by constant arginase expression, associated with previously demonstrated lack of stimulation of nitric oxide production, BCG treatment invoked an alternative type of activation mechanism, reflected by stimulation of macrophage arginase, but not iNOS, activity.

Interaction of thymidylate synthase with the 5'-thiophosphates, 5'-dithiophosphates, 5'-H-phosphonates and 5'-S-thiosulfates of 2'-deoxyuridine, thymidine and 5-fluoro-2'-deoxyuridine.

New analogs of dUMP, dTMP and 5-fluoro-dUMP, including the corresponding 5'-thiophosphates (dUMPS, dTMPS and FdUMPS), 5'-dithiophosphates (dUMPS2, dTMPS2 and FdUMPS2), 5'-H-phosphonates (dUMP-H, dTMP-H and FdUMP-H) and 5'-S-thiosulfates (dUSSO3, dTSSO3 and FdUSSO3), have been synthesized and their interactions studied with highly purified mammalian thymidylate synthase. dUMPS and dUMPS2 proved to be good substrates, and dTMPS and dTMPS2 classic competitive inhibitors, only slightly weaker than dTMP. Their 5-fluoro congeners behaved as potent, slow-binding inhibitors. By contrast, the corresponding 5'-H-phosphonates and 5'-S-thiosulfates displayed weak activities, only FdUMP-H and FdUSSO3 exhibiting significant interactions with the enzyme, as weak competitive slow-binding inhibitors versus dUMR The pH-dependence of enzyme time-independent inhibition by FdUMP and FdUMPS was found to correlate with the difference in pKa values of the phosphate and thiophosphate groups, the profile of FdUMPS being shifted (approximately 1 pH unit) toward lower pH values, so that binding of dUMP and its analogs is limited by the phosphate secondary hydroxyl ionization. Hence, together with the effects of 5'-H-phosphonate and 5'-S-thiosulfate substituents, the much weaker interactions of the nucleotide analogs (3-5 orders of magnitude lower than for the parent 5'-phosphates) with the enzyme is further evidence that the enzyme's active center prefers the dianionic phosphate group for optimum binding.

5-Substituted N(4)-hydroxy-2'-deoxycytidines and their 5'-monophosphates: synthesis, conformation, interaction with tumor thymidylate synthase, and in vitro antitumor activity.

Convenient procedures are described for the synthesis of 5-substituted N(4)-hydroxy-2'-deoxycytidines 5a,b,d-h via transformation of the respective 5-substituted 3', 5'-di-O-acetyl-2'-deoxyuridines 1a-c,e-h. These procedures involved site-specific triazolation or N-methylimidazolation at position C(4), followed by hydroxylamination and deblocking with MeOH-NH(3). Nucleosides 5a,b,d-h were selectively converted to the corresponding 5'-monophosphates 6a,b,d-h with the aid of the wheat shoot phosphotransferase system. Conformation of each nucleoside in D(2)O solution, deduced from (1)H NMR spectra and confirmed by molecular mechanics calculations, showed the pentose ring to exist predominantly in the conformation S (C-2'-endo) and the N(4)-OH group as the cis rotamer. Cell growth inhibition was studied with two L5178Y murine leukemia cell lines, parental and 5-fluoro-2'-deoxyuridine (FdUrd)-resistant, the latter 70-fold less sensitive toward FdUrd than the former. With FdUrd-resistant L5178Y cells, 5-fluoro-N(4)-hydroxy-2'-deoxycytidine (5e) caused almost 3-fold stronger growth inhibition than FdUrd; 5e was only some 3-fold weaker growth inhibitor of the resistant cells than of the parental cells. Thymidylate synthase inhibition was studied with two forms of the enzyme differing in sensitivities toward 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP), isolated from parental and FdUrd-resistant L1210 cell lines. All N(4)-hydroxy-dCMP (6a,b,d-h) and dUMP analogues studied were competitive vs dUMP inhibitors of the enzyme. Analogues 6b,d-h and 5-hydroxymethyl-dUMP, similar to N(4)-hydroxy-dCMP (6a) and FdUMP, were also N(5), N(10)-methylenetetrahydrofolate-dependent, hence mechanism-based, slow-binding inhibitors. 5-Chloro-dUMP, 5-bromo-dUMP, and 5-iodo-dUMP, similar to dTMP, did not cause a time-dependent inactivation of the enzyme. Instead, they behaved as classic inhibitors of tritium release from [5-(3)H]dUMP. 5-Bromo-dUMP and 5-iodo-dUMP showed substrate activity independent of N(5), N(10)-methylenetetrahydrofolate in the thymidylate synthase-catalyzed dehalogenation reaction. The =N-OH substituent of the pyrimidine C(4) prevented the enzyme-catalyzed release from the C(5) of Br(-) and I(-) (the same shown previously for H(+)). While FdUMP and 6a showed a higher affinity and greater inactivation power with the parental cell than FdUrd-resistant cell enzyme, an opposite relationship could be seen with 5-hydroxymethyl-dUMP.

Crystal structures of rat thymidylate synthase inhibited by Tomudex, a potent anticancer drug.

Two crystal structures of rat thymidylate synthase (TS) complexed with dUMP and the anticancer drug Tomudex (ZD1694) have been determined to resolutions of 3.3 and 2.6 A. Tomudex is one of several new antifolates targeted to TS and the first to be approved for clinical use. The structures represent the first views of any mammalian TS bound to ligands and suggest that the rat protein undergoes a ligand-induced conformational change similar to that of the Escherichia coli protein. Surprisingly, Tomudex does not induce the "closed" conformation in rat TS that is seen on binding to E. coli TS, resulting in inhibitor atoms that differ in position by more than 1.5 A. Several species-specific differences in sequence may be the reason for this. Phe 74 shifts to a new position in the rat complex and is in van der Waals contact with the inhibitor, while in the E. coli protein the equivalent amino acid (His 51) hydrogen bonds to the glutamate portion of the inhibitor. Amino acids Arg 101, Asn 106, and Met 305 make no contacts with the inhibitor in the open conformation, unlike the equivalent residues in the E. coli protein (Thr 78, Trp 83, and Val 262). dUMP binding is similar in both proteins, except that there is no covalent adduct to the active site cysteine (Cys 189) in the rat structures. Two insertions in the rat protein are clearly seen, but the N-termini (residues 1-20) and C-termini (residues 301-307) are disordered in both crystal forms.

Acyclic analogues of 5-fluoro-dUMP and 5-fluoro-2'-deoxyuridine: synthesis and inhibition of thymidylate synthase and tumour cell growth.

1-[(2-Hydroxyethoxy)methyl]-5-fluorouracil (HEMFU) and 1-[(1,3-dihydroxy-2-propoxy)methyl]-5-fluorouracil (DHPFU) were prepared by alkylation of the di-O-TMS derivative of 5-fluorouracil and phosphorylated with the use of the wheat shoot phosphotransferase system to their monophosphates, HEMFUMP and DHPFUMP. 1-(2-Phosphonylmethoxyethyl)-5-fluorouracil (PMEFU) was obtained by condensation of diethyl-2-chloroethoxymethanephosphonate with 5-fluorouracil and cleavage of the alkylphosphoester with trimethylbromosilane. Inhibition of highly purified thymidylate synthase from mouse tumour Ehrlich carcinoma and leukemia L1210 cells by each of the nucleotide analogues, DHPFUMP, PMEFU and HEMFUMP, and of L5178Y mouse leukemia cell growth by the nucleoside (HEMFU) analogue, were studied. DHPFUMP proved to be the strongest inhibitor, non-competitive vs dUMP, with K(i)app 2.8 microM for time-independent interaction with the enzyme and N5,N10-methylenetetrahydrofolate (CH2H4PteGlu). In the presence of CH2H4PteGlu, DHPFUMP exhibited time-dependent inactivation of the enzyme, the inactivation rate plots being biphasic and pointing to Ki values in the microM range (10(3)-fold higher than for 5-fluoro-dUMP). HEMFUMP and PMEFU were much weaker inhibitors of the enzyme, with K(i)app values of 0.26 mM (non-competitive vs dUMP) and 30 mM (non-competitive vs dUMP), respectively. HEMFU, despite the weak interaction of its nucleotide analogue with the enzyme, proved to be a strong cell (L5178Y) growth inhibitor, with IC50 in the range 10(-5) M.

Synthesis and interactions with thymidylate synthase of 2,4-dithio analogues of dUMP and 5-fluoro-dUMP.

The 2,4-dithio analogues of 2'-deoxyuridine and 2'-deoxy-5-fluorouridine have been synthesized by thiation of the previously described 2-thio analogues, and then phosphorylated enzymatically or chemically to yield 2,4-dithio-dUMP and 2,4-dithio-5-fluoro-dUMP. In striking contrast to the 2-thio and 4-thio analogues of dUMP, which are good substrates of thymidylate synthase, 2,4-dithio-dUMP is not a substrate. But, surprisingly, it is a competitive inhibitor, relative to dUMP, of the purified enzymes from both parental and FdUrd-resistant L1210 cells, with K(i) values of 32 microM and 55 microM, respectively. Although 2,4-dithio-5-fluoro-dUMP behaved as a typical slow-binding inhibitor of the enzyme, its K(i) value was 10(3)-10(4)-fold higher than those for the corresponding 2-thio and 4-thio congeners. Similarly, 2,4-dithio-FdUrd was a much weaker inhibitor of tumour cell growth (IC50 approximately 10(-5)M) than FdUrd (IC50 approximately 10(-9)M), 2-thio-FdUrd(IC50 approximately 10(-7)M) or 4-thio-FdUrd (IC50 approximately 5x10(-8)M), while with 2,4-dithio-dUrd no influence on cell growth could be observed. Theoretical considerations, based on calculated aromaticities of the uracil and thiouracil rings, suggest that lack of substrate activity of 2,4-dithio-dUMP may result from increased pyrimidine ring aromaticity of the latter, leading to resistance of C(6) to nucleophilic attack by the enzyme active center cysteine.

Thymidylate synthases from Hymenolepis diminuta and regenerating rat liver: purification, properties, and inhibition by substrate and cofactor analogues.

Comparative studies of thymidylate synthases, isolated from the tapeworm, Hymenolepis diminuta, and regenerating liver of its host, rat, aimed at a possibility of specific inhibition of the helminthic enzyme, are presented. While similar in structure (dimers with monomer molecular masses of 33.7 kDa and 34.9 kDa, respectively) and parameters describing interactions with substrates and products, the tapeworm and rat enzymes differed in the dependences of reaction velocity on temperature (Arrhenius plots biphasic and linear, respectively). The tapeworm, compared with the host, enzyme was less sensitive to the competitive slow-binding inhibition by 5-fluoro-dUMP and its 2-thio congener, but equally sensitive to inhibition by 4-thio-5-fluoro-dUMP, N4-hydroxy-dCMP and N4-hydroxy-5-fluoro-dCMP, the latter being more potent inhibitor of the parasite enzyme than 5-fluoro-dUMP. alpha-Anomer of 5-fluoro-dUMP behaved as a very weak competitive slow-binding inhibitor of both enzymes. Both enzymes differed markedly in sensitivity to inhibition by 10-propargyl-5,8-dideazafolate and its di- and triglutamates (pddPteGlu1-3), with pddPteGlu1 being stronger inhibitor of the mammalian enzyme, but pddPteGlu3 showing opposite specificity. Sulfonamidobenzoylglutamate analogue of pddPteGlu (pddPteSO2Glu) and 2-desamino-2-methyl derivative of this analogue (CH3pddPteSO2Glu) were weaker inhibitors of both enzymes than the parent compound. Substitution of the glutamyl residue in CH3pddPteSO2Glu with either norvaline or alanine increased inhibition potency, whereas similar substitutions with glycine, valine or phenylglycine were without a distinct effect with the host enzyme but weakened inhibition of the tapeworm enzyme.

2-Thio derivatives of dUrd and 5-fluoro-dUrd and their 5'-monophosphates: synthesis, interaction with tumor thymidylate synthase, and in vitro antitumor activity.

A convenient synthesis of 5-fluoro-2-thiouracil (11) is based on hydrolytic deamination of 5-fluoro-2-thiocytosine (9). Lewis acid-catalyzed condensation of di-TMS-5-fluoro-2-thiouracil (13) or di-TMS-2-thiouracil (14) with 2-deoxy-3,5-di-O-p-toluyl-D-ribofuranosyl chloride (15) led to mixtures of the beta- and alpha-anomers of 3',5'-toluylated 2'-deoxy-5-fluoro-2-thiouridine (16 and 18) or 2'-deoxy-2-thiouridine (17 and 19), each of which was deblocked with MeOH-NH3 to give the desired free anomeric nucleoside pairs 1, 5 and 3, 7, respectively. These were selectively converted to the corresponding 5'-monophosphates 2, 6 and 4, 8, with the aid of the wheat shoot phosphotransferase system. Conformations of the nucleosides 1, 3, 5, 7 are deduced from 1H NMR spectra, and circular dichroism spectra for nucleotide anomeric pairs 2, 6 and 4, 8 are reported. Whereas beta-2-thio-dUMP (4) was a good substrate (Km approximately 10(-5) M), beta-5-fluoro-2-thio-dUMP (2) proved to be a potent competitive, slow-binding inhibitor (Ki approximately 10(-8) M) of the purified enzymes from Ehrlich ascites carcinoma and L1210 cells. The alpha-anomer 6 was a weak inhibitor, with Ki in the mM range, and its congener 8 hardly interacted with the enzyme. The beta-anomer 1 exhibited antitumor activity in a mouse leukemic cell line L5178Y (IC50 approximately 10(-6) M), hence 40-100-fold weaker than 5-fluoro-dUrd. Its alpha-anomer 5 was 10-fold less active, but exhibited at least 10-fold higher selectivity with respect to the tumor cells than the beta-anomer 1.

Interaction of 2-thio-5-fluoro-dUMP and 4-thio-5-fluoro-dUMP with mammalian normal and tumour and helminthic thymidylate synthases: influence of C(4)-substituents on specificity for enzyme inactivation.

To determine how 5-fluoro-dUMP modifications may affect its specificity, 2-thio-5-fluoro-dUMP and 4-thio-5-fluoro-dUMP were compared as inhibitors of thymidylate synthases isolated from parental and FdUrd-resistant mouse leukemia L1210 cells, human and rat colon adenocarcinomas, regenerating rat liver and the tapeworm, Hymenolepis diminuta, differing in sensitivity to time- and N5,10-methylenetetrahydrofolate-dependent inactivation by 5-fluoro-dUMP (Ki values ranging from 10(-9) to 10(-7) M). Inactivation by 2-thio-5-fluoro-dUMP, relative to 5-fluoro-dUMP, was 5-20-fold weaker, with specificity for inactivation of different thymidylate synthases paralleling that of 5-fluoro-dUMP. By contrast, 4-thio-5-fluoro-dUMP showed very different specificity, being as potent an inactivator for some enzymes as 5-fluoro-dUMP, but 45-85-fold weaker for others. The results suggest that an interplay between substituents at C(4) and C(5) of the pyrimidine ring may affect the specificity of thymidylate synthase inactivation.

Mechanism of inhibition of mammalian tumor and other thymidylate synthases by N4-hydroxy-dCMP, N4-hydroxy-5-fluoro-dCMP, and related analogues.

N4-Hydroxy-dCMP (N4-OH-dCMP), N4-methoxy-dCMP (N4-OMe-dCMP), and their 5-fluoro congeners (syntheses of which are described) were all slow-binding inhibitors of Ehrlich carcinoma thymidylate synthase (TS), competitive with respect to dUMP, and had differing kinetic constants describing interactions with the two TS binding sites. N4-OH-dCMP was not a substrate (no dihydrofolate produced; no tritium released with 5-3H-labeled molecule), and its inactivation of TS was methylenetetrahydrofolate-dependent, hence mechanism-based, with arrest of a step posterior to addition of cofactor and blocking abstraction of the C(5) hydrogen. Ki values for N4-OH-dCMP and its 5-fluoro analogue were in the range 10(-7) - 10(-8) M, 2-3 orders of magnitude higher for the corresponding N4-OMe analogues. The 5-methyl analogue of N4-OH-dCMP was 10(4)-fold less potent, pointing to the anti rotamer of the imino form of exocyclic N4-OH, relative to the ring N(3), as the active species. This is consistent with weaker slow-binding inhibition of the altered enzyme from 5-FdUrd-resistant, relative to parent, L1210 cells by both FdUMP and N4-OH-dCMP, suggesting interaction of both N4-OH and C(5)-F groups with the same region of the active center. Kinetic studies with purified enzyme from five sources, viz., Ehrlich carcinoma, L1210 parental, and 5-FdUrd-resistant cells, regenerating rat liver, and the tapeworm Hymenolepis diminuta, demonstrated that addition of a 5-fluoro substituent to N4-OH-dCMP increased its affinity from 2- to 20-fold for the enzyme from different sources.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of the 5'-phosphates of the anti-HIV agents, 3'-azido-3'-deoxythymidine and 3'-azido-2',3'-dideoxyuridine, with thymidylate synthase.

A study has been made of the interaction of 3'-azido-3'-deoxythymidine 5'-phosphate (AZTMP) and 3'-azido-2',3'-dideoxy-uridine 5'-phosphate (AZdUMP) with thymidylate synthase. With the enzyme from L1210 cells and the tapeworm Hymenolepis diminuta, AZTMP was a weak inhibitor competitive with respect to dUMP (Ki = 6.3 mM and 0.5 mM); hence cytotoxicity of AZT, in cells in which accumulation of AZTMP is not high, is not due to inhibition of cellular thymidylate synthase. AZdUMP, with the L1210 enzyme, was a weak substrate (competition with dUMP described by apparent Ki = 4.7 mM), excluding conversion of AZdUMP to AZTMP as a source of toxicity of 3'-azido-2',3'-dideoxyuridine. An efficient procedure is described for enzymatic phosphorylation on a preparative scale of dideoxynucleosides.

Interaction of 5-fluoro-4-thio-2'-deoxyuridine 5'-phosphate with mammalian tumour thymidylate synthase: role of the pyrimidine N(3)-H dissociation.

The role of the pyrimidine N(3)-H in binding of dUMP derivatives to thymidylate synthase was evaluated with the aid of a new dUMP analogue, 5-fluoro-4-thio-dUMP, synthesized by an improved thiation and enzymatic phosphorylation. The interaction of this analogue, and of 5-FdUMP, with the enzyme, and the pH-dependence of these interactions, were compared. Both were slow-binding competitive inhibitors of the enzyme from Ehrlich carcinoma, L1210 and CCRF-CEM cells, with Ki an order of magnitude higher for 5-fluoro-4-thio-dUMP than for 5-FdUMP. With both nucleotides, as well as the parent nucleosides, enzyme inactivation increased as the pH was lowered from 8 to 6. Maximum inactivation with 5-FdUrd was at pH 7.0, and with 5-fluoro-4-thio-dUrd at pH 6.0, in agreement with the higher pKa for the N(3)-H dissociation of the former, and pointing to participation of the N(3)-H as a hydrogen donor in binding to the enzyme.

The relationship between the rate of glutamate deamination and long-chain acyl-CoA accumulation in kidney cortex mitochondria of rabbit.

The effect of long-chain acyl-CoA on glutamate dehydrogenase activity was studied in uncoupled rabbit kidney cortex mitochondria incubated with glutamate and palmitoylcarnitine in the presence of arsenite. The mitochondrial long-chain acyl-CoA (about 2 nmol/mg of protein) accumulated in the presence of arsenite resulted in an inhibition of ammonia production from 4.1 to 1.2 nmol/min per mg of protein. Leucine and ADP, activators of glutamate dehydrogenase, did not release the inhibitory effect of long-chain acyl-CoA on glutamate deamination. In view of the presented data it seems that inhibitory effect of long-chain acyl-CoA on glutamate dehydrogenase activity may have a physiological significance.

Synthesis and antimetabolic properties of 4,5-substituted pyrimidine 2'-deoxynucleosides and their 5'-monophosphates.

Reaction of the reagent of Lawesson, 2,4-bis(p-methoxyphenyl)-1,3,4-dithiadiphosphatane-2,4-disulfide, with blocked uracil nucleosides in dioxane leads to quantitative thionation at C(4). With the bases, thionation occurs at C(4) and, with two equivalents of the reagent, at C(2) and C(4). Enzymatic phosphorylation of 4-thio-FdUrd gave the 5'-monophosphate, which was further converted with NH2OH to N4-hydroxy-FdCMP. Both nucleotides were examined as potential inhibitors of thymidylate synthase, and 4-thio-FdUrd for cytotoxic activities vs monkey and human leukemic cell lines.