Silent information regulators are NAD(+)-dependent enzymes that display differential specificity toward acetylated substrates. This report provides first evidence for deacetylation activity of CobB1 in Streptomyces coelicolor. The protein is highly conserved in streptomycetes. The CobB1 protein catalytically removes the acetyl group from acetylated bovine serum albumin. In the absence of NAD+ or when NAD+ was substituted with nicotinamide, deacetylation was stopped. We isolated gene encoding AcetylCoA synthetaseA. The recombinant enzyme produces Acetyl-CoA from acetate. The highest acsA-mRNA level was detected in cells from the exponential phase of growth, and then decreased in transition and stationary phases of growth. Acetylated acsA loses the ability to transfer acetate to CoA. Deacetylation of the enzyme required CobB1, ATP-Mg2, and NAD+. Using specific antibodies against acetylated lys, CobB1, and acsA, we found relationship between level of CobB1 and acetylation of acsA, indicating that CobB1 is involved in regulating the acetylation level of acsA and consequently its activity. It was found that 1-acetyl-tetrahydroxy and 1-acetyl pentahydroxy antraquinone inhibit the deacetylation activity of CobB1.

• MeSH
• acetát-CoA-ligasa biosyntéza   chemie   genetika   MeSH
• acetylace MeSH
• anthrachinony chemie   MeSH
• bakteriální proteiny biosyntéza   chemie   izolace a purifikace   MeSH
• genetická transkripce MeSH
• inhibitory enzymů chemie   MeSH
• katalytická doména MeSH
• konzervovaná sekvence MeSH
• molekulární sekvence - údaje MeSH
• posttranslační úpravy proteinů MeSH
• regulace genové exprese u bakterií MeSH
• rekombinantní proteiny antagonisté a inhibitory   biosyntéza   chemie   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• sirtuiny antagonisté a inhibitory   biosyntéza   chemie   MeSH
• Streptomyces coelicolor enzymologie   růst a vývoj   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The purine salvage enzyme, hypoxanthine-guanine-(xanthine) phosphoribosyltransferase [HG(X)PRT], catalyses the synthesis of the purine nucleoside monophosphates, IMP, GMP or XMP essential for DNA/RNA production. In protozoan parasites, such as Plasmodium, this is the only route available for their synthesis as they lack the de novo pathway which is present in human cells. Acyclic nucleoside phosphonates (ANPs), analogs of the purine nucleoside monophosphates, have been found to inhibit Plasmodium falciparum (Pf) HGXPRT and Plasmodium vivax (Pv) HGPRT with K(i) values as low as 100 nM. They arrest parasitemia in cell based assays with IC(50) values of the order of 1-10 μM. ANPs with phosphonoalkyl and phosphonoalkoxyalkyl moieties linking the purine base and phosphonate group were designed and synthesised to evaluate the influence of this linker on the potency and/or selectivity of the ANPs for the human and malarial enzymes. This data shows that variability in the linker, as well as the positioning of the oxygen in this linker, influences binding. The human enzyme binds the ANPs with K(i) values of 0.5 μM when the number of atoms in the linker was 5 or 6 atoms. However, the parasite enzymes have little affinity for such long chains unless oxygen is included in the three-position. In comparison, all three enzymes have little affinity for ANPs where the number of atoms linking the base and the phosphonate group is of the order of 2-3 atoms. The chemical nature of the purine base also effects the K(i) values. This data shows that both the linker and the purine base play an important role in the binding of the ANPs to these three enzymes.

• MeSH
• aktivace enzymů účinky léků   MeSH
• antimalarika chemická syntéza   chemie   farmakologie   MeSH
• kinetika MeSH
• lidé MeSH
• pentosyltransferasy antagonisté a inhibitory   genetika   metabolismus   MeSH
• Plasmodium falciparum účinky léků   enzymologie   MeSH
• Plasmodium vivax účinky léků   enzymologie   MeSH
• protozoální proteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• puriny chemická syntéza   chemie   farmakologie   MeSH
• rekombinantní proteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Betaine-homocysteine S-methyltransferase 2 (BHMT-2) catalyzes the transfer of a methyl group from S-methylmethionine to l-homocysteine, yielding two molecules of l-methionine. It is one of three homocysteine methyltransferases in mammals, but its overall contribution to homocysteine remethylation and sulfur amino acid homeostasis is not known. Moreover, recombinant BHMT-2 is highly unstable, which has slowed research on its structural and catalytic properties. In this study, we have prepared the first series of BHMT-2 inhibitors to be described, and we have tested them with human recombinant BHMT-2 that has been stabilized by copurification with human recombinant BHMT. Among the compounds synthesized, (2S,8RS,11RS)-5-thia-2,11-diamino-8-methyldodecanedioic acid (11) was the most potent (K(i)(app) ∼77 nM) and selective inhibitor of BHMT-2. Compound 11 only weakly inhibited human BHMT (IC(50) about 77 μM). This compound (11) may be useful in future in vivo studies to probe the physiological significance of BHMT-2 in sulfur amino acid metabolism.

• MeSH
• betain-homocystein-S-methyltransferasa antagonisté a inhibitory   chemie   MeSH
• enzymatické testy MeSH
• homocystein analogy a deriváty   chemická syntéza   chemie   MeSH
• kinetika MeSH
• lidé MeSH
• rekombinantní proteiny antagonisté a inhibitory   MeSH
• stereoizomerie MeSH
• sulfidy chemická syntéza   chemie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Inhibitors of acetylcholinesterase are compounds widely used in the treatment of various diseases, such as Alzheimer's disease, glaucoma and Myasthenia gravis (MG). Compounds used in the therapy of MG posses a positive charge in the molecule to ensure peripheral effect of action and minimal blood-brain barrier penetration. The most prescribed carbamate inhibitors are however known for many severe side effects related to the carbamylation of AChE. This paper describes preparation and in vitro evaluation of 20 newly prepared bis-isoquinolinium inhibitors of potential concern for MG. The newly prepared compounds were evaluated in vitro on human recombinant AChE and human plasmatic butyrylcholinesterase (BChE). Their inhibitory ability was expressed as IC50 and compared to chosen standards ambenonium dichloride, edrophonium chloride, BW284c51 and ethopropazine hydrochloride. Three novel compounds presented promising inhibition (in nM range) of both enzymes in vitro better or similar to edrophonium and BW284c51, but worse to ambenonium. The novel inhibitors did not present higher selectivity toward AChE or BChE. The kinetic assay confirmed non-competitive inhibition of hAChE by two selected promising novel compounds. Two newly prepared compounds were also chosen for docking studies that confirmed apparent π-π or π-cationic interactions aside the cholinesterases catalytic sites. The SAR findings were discussed.

• MeSH
• acetylcholinesterasa metabolismus   MeSH
• butyrylcholinesterasa krev   metabolismus   MeSH
• cholinesterasové inhibitory chemická syntéza   chemie   farmakologie   MeSH
• erytrocyty enzymologie   MeSH
• isochinoliny chemická syntéza   chemie   farmakologie   MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární struktura MeSH
• myasthenia gravis farmakoterapie   MeSH
• rekombinantní proteiny antagonisté a inhibitory   MeSH
• stereoizomerie MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

3- and 8-(8-phosphonooctyl)-8-aza-7,9-dideazaxanthine, and 1,8-bis(8-aza-7,9-dideazaxanthin-8-yl)octane were prepared and found to inhibit thymidine phosphorylase from Escherichia coli, human recombinant TP expressed in V79, and TP purified from human placenta. The IC(50) values ranged from 3.5 to 27μM.

• MeSH
• Escherichia coli enzymologie   MeSH
• inhibitory enzymů chemie   farmakologie   MeSH
• lidé MeSH
• placenta enzymologie   MeSH
• pyrimidinony chemie   farmakologie   MeSH
• pyrroly chemie   farmakologie   MeSH
• rekombinantní proteiny antagonisté a inhibitory   metabolismus   MeSH
• těhotenství MeSH
• thymidinfosforylasa antagonisté a inhibitory   metabolismus   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We report here results of screening directed to finding new anti-leishmanial drugs among 2,6-disubstituted purines and corresponding 3,7-disubstituted pyrazolo[4,3-d]pyrimidines. These compounds have previously been shown to moderately inhibit human cyclin-dependent kinases. Since some compounds reduced viability of axenic amastigotes of Leishmania donovani, we screened them for interaction with recombinant leishmanial cdc-2 related protein kinase (CRK3/CYC6), an important cell cycle regulator of the parasitic protozoan. Eighteen pairs of corresponding isomers were tested for viability of amastigotes and for inhibition of CRK3/CYC6 kinase activity. Some compounds (9A, 12A and 13A) show activity against amastigotes with EC(50) in a range 1.5-12.4μM. Structure-activity relationships for the tested compounds are discussed and related to the lipophilicity of the compounds.

• MeSH
• Leishmania donovani účinky léků   MeSH
• proteinkinasa CDC2 antagonisté a inhibitory   genetika   metabolismus   MeSH
• protozoální proteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• puriny chemická syntéza   chemie   farmakologie   MeSH
• pyrazoly chemie   MeSH
• pyrimidiny chemická syntéza   chemie   farmakologie   MeSH
• rekombinantní proteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• trypanocidální látky chemická syntéza   chemie   farmakologie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

D-eritadenine and (S)-DHPA are aliphatic adenosine analogues known to target S-adenosylhomocysteine hydrolase (SAHH) and potent antiviral compounds. In the present study, we demonstrate that these two compounds also display efficacy against recombinant SAHH enzyme of the protozoan parasite Cryptosporidium parvum, as well as inhibition of parasite growth in vitro. Our data confirm that SAHH could serve as a rational drug target in cryptosporidial infection and antiviral adenosine analogues are potential candidates for drug development against cryptosporidiosis.

• MeSH
• adenin analogy a deriváty   chemie   farmakologie   MeSH
• adenosylhomocysteinasa antagonisté a inhibitory   MeSH
• Cryptosporidium parvum účinky léků   enzymologie   genetika   růst a vývoj   MeSH
• inhibitory enzymů chemie   farmakologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• regresní analýza MeSH
• rekombinantní proteiny antagonisté a inhibitory   MeSH
• RNA protozoální analýza   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Ornithine-delta-aminotransferase (OAT, EC 2.6.1.13) catalyzes the transamination of L-ornithine to L-glutamate-gamma-semialdehyde. The physiological role of OAT in plants is not yet well understood. It is probably related to arginine catabolism resulting in glutamate but the enzyme has also been associated with stress-induced proline biosynthesis. We investigated the enzyme from pea (PsOAT) to assess whether diamines and polyamines may serve as substrates or they show inhibitory properties. First, a cDNA coding for PsOAT was cloned and expressed in Escherichia coli to obtain a recombinant protein with a C-terminal 6xHis tag. Recombinant PsOAT was purified under native conditions by immobilized metal affinity chromatography and its molecular and kinetic properties were characterized. Protein identity was confirmed by peptide mass fingerprinting after proteolytic digestion. The purified PsOAT existed as a monomer of 50 kDa and showed typical spectral properties of enzymes containing pyridoxal-5'-phosphate as a prosthetic group. The cofactor content of PsOAT was estimated to be 0.9 mol per mol of the monomer by a spectrophotometric analysis with phenylhydrazine. L-Ornithine was the best substrate (K(m)=15 mM) but PsOAT also slowly converted N(alpha)-acetyl-L-ornithine. In these reactions, 2-oxoglutarate was the exclusive amino group acceptor (K(m)=2mM). The enzyme had a basic optimal pH of 8.8 and displayed relatively high temperature optimum. Diamines and polyamines were not accepted as substrates. On the other hand, putrescine, spermidine and others represented weak non-competitive inhibitors. A model of the molecular structure of PsOAT was obtained using the crystal structure of human OAT as a template.

• MeSH
• biokatalýza MeSH
• chromatografie afinitní MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• hrách setý enzymologie   MeSH
• molekulární sekvence - údaje MeSH
• molekulová hmotnost MeSH
• polyaminy farmakologie   MeSH
• rekombinantní proteiny antagonisté a inhibitory   chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• substrátová specifita MeSH
• transaminasa ornithin-oxokyselin antagonisté a inhibitory   chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The oxime reactivator K112 is a member of the new group of xylene linker-containing AChE reactivators. Its cholinergic properties could be of importance at OP poisoning and are not related to the AChE reactivation that has been studied. It has been found that, despite of reactivating potency, this compound has additional effects. These cholinergic effects include a weak inhibition of AChE (IC(50)=43.8 ± 4.88 μM), inhibition of binding to the porcine muscarinic M2 receptor (IC(50)=4.36 μM) and finally, the inhibition of HACU (68.4 ± 9.9%), a key regulatory step in the synthesis of ACh. The inhibition of the binding of (3H)-HC-3 (64.7 ± 4.7%) and the influence on the membrane fluidity have also been observed. Blocking properties of K112 on the muscarinic receptors have been revealed in the in vitro experiment (rat urinary bladder) and in the in vivo experiment (rat heart BPM) as well. All these cholinergic properties could significantly contribute to the antidotal effect of K112 at the poisoning by the organophosphates.

• MeSH
• acetylcholinesterasa metabolismus   MeSH
• cholinesterasové inhibitory chemie   farmakologie   MeSH
• fluidita membrány účinky léků   MeSH
• hipokampus účinky léků   enzymologie   MeSH
• hladké svalstvo účinky léků   enzymologie   MeSH
• krysa rodu rattus MeSH
• močový měchýř účinky léků   enzymologie   MeSH
• organofosforové sloučeniny MeSH
• otrava organofosfáty MeSH
• otrava farmakoterapie   enzymologie   MeSH
• oximy chemie   farmakologie   MeSH
• potkani Sprague-Dawley MeSH
• potkani Wistar MeSH
• prasata MeSH
• pyridinové sloučeniny chemie   farmakologie   MeSH
• reaktivátory cholinesterázy chemie   farmakologie   MeSH
• receptor muskarinový M2 antagonisté a inhibitory   MeSH
• rekombinantní proteiny antagonisté a inhibitory   MeSH
• srdce účinky léků   MeSH
• srdeční frekvence účinky léků   MeSH
• svalová kontrakce účinky léků   MeSH
• synaptozomy účinky léků   enzymologie   MeSH
• techniky in vitro MeSH
• vazba proteinů MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

OBJECTIVES: 2-Nitrophenol (2-NP) is the major detoxification metabolite of an important industrial pollutant and a potent carcinogen, 2-nitroanisole (2-NA). Characterization of the products of 2-NP metabolism by rat hepatic microsomes containing cytochromes P450 (CYPs) and identification of the major CYP enzymes participating in this process are aims of this study. METHODS: HPLC with UV detection was employed for the separation and characterization of 2-NP metabolites. Inducers and inhibitors of CYPs and rat recombinant CYPs were used to characterize the enzymes participating in 2-NP oxidation. RESULTS: Rat hepatic microsomes oxidize 2-NP to its hydroxylated metabolite, 2,5-dihydroxynitrobenzene (2,5-DNB). No nitroreductive metabolism leading to the formation of o-aminophenol was evident when using rat hepatic microsomes. Selective CYP inhibitors and hepatic microsomes of rats pre-treated with specific CYP inducers were used to characterize CYPs oxidizing 2-NP in rat livers. Based on these studies, we attribute most of 2-NP oxidation in rat liver to CYP2E1 and 3A, followed by CYP2D and 2C. Among recombinant rat CYP enzymes tested in this study, CYP2E1 and 2C11 were the most effective enzymes oxidizing 2-NP. Oxidation of 2-NP by rat CYP2E1 exhibits the Michaelis-Menten kinetics, having the Km value of 0.35 mM. CONCLUSION: The results found in this study, the first report on the metabolism of 2-NP by rat hepatic microsomes and rat CYP enzymes, demonstrate that CYP2E1 is the major enzyme oxidizing this compound in rat liver.

• MeSH
• anisoly metabolismus   MeSH
• inhibiční koncentrace 50 MeSH
• inhibitory cytochromu P450 MeSH
• jaterní mikrozomy enzymologie   metabolismus   MeSH
• kinetika MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• nitrofenoly metabolismus   MeSH
• oxidace-redukce MeSH
• rekombinantní proteiny antagonisté a inhibitory   metabolismus   MeSH
• systém (enzymů) cytochromů P-450 metabolismus   MeSH
• ultrafialové záření MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH