Treatment of P190 Bcr/Abl lymphoblastic leukemia cells with inhibitors of the serine/threonine kinase CK2.
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Animals↗
Biomedical subjects
Z Kazimierczuk
Publications and source records attributed to Z Kazimierczuk.
Synthesis and antimycobacterial activity of 2-substituted halogenobenzimidazoles.
A series of substituted 2-polyfluoroalkyl and 2-nitrobenzylsulphanyl benzimidazoles was synthesized. The compounds were evaluated for their activity against four Mycobacterium strains; the activities were expressed as the minimum inhibitory concentration (MIC). The substances tested showed appreciable antimycobacterial activity, particularly 5,6-dichloro-2-nonafluorobutylbenzimidazole (2h), and 5-halogeno- (5a-c) and 4,6-dihalogeno- (5d and 5g) 2-(3,5-dinitrobenzylsulphanyl)benzimidazoles, whose MIC values for Mycobacterium kansasii and Mycobacterium avium exceeded that of isoniazide that was used as a reference compound. Relationships between structure and biological activity of the tested benzimidazole derivatives are discussed.
Anti-Bacterial Agents↗
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.
Antiviral Agents↗
Alkaline hydrolysis of cladribine.
The kinetics of hydrolysis of 2-chloro-2'-deoxyadenosine (cladribine) was studied at various sodium hydroxide concentrations and temperatures. HPLC analysis of reaction mixtures showed that the main products were 2'-deoxyisoguanosine and 2'-deoxyguanosine. The first one was the result of the hydroxyl anion attack, whereas the presence of the other nucleoside has evidenced the existence of hitherto undescribed rearrangement reaction in purine derivatives.
Adenosine↗
Adamantylaminopyrimidines and -pyridines are potent inducers of tumor necrosis factor-alpha.
A series of (1-adamantyl)aminopyrimidine and -pyridine derivatives was prepared by adamantyl cation attack on amino heterocycles. The adamantylated compounds, particularly 2-(1-adamantyl)amino-6-methylpyridine, were found to be potent TNF-alpha inducers in murine melanoma cells transduced with gene for human TNF-alpha.
Adamantane↗
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.
Enzyme Activation↗
2-aza-2'-deoxyadenosine: synthesis, base-pairing selectivity, and stacking properties of oligonucleotides.
2-Aza-2'-deoxyadenosine (2, z2Ad) is synthesized via its 1,N6-etheno derivative 7 and enzymatically deaminated to 2-aza-2'-deoxyinosine (3). Compound 2 is converted into the phosphoramidite building block 10b. This is employed in solid-phase oligonucleotide synthesis. The 2-azapurine base forms a strong base pair with guanine, but a much weaker one with adenine, thymine, and cytosine. Oligonucleotide duplexes with dangling nucleotide residues, such as 2-aza-2'-deoxyadenosine and 7-deaza-2'-deoxyadenosine (4, c7Ad), either on one or both termini, are synthesized, and the thermal stability of the duplexes is correlated with the hydrophobic properties of the dangling nucleotide residues.
Adenosine↗
Synthesis and antimicrobial activity of new adamantane derivatives I.
A series of fourteen derivatives of adamantane was synthesised. The new compound 4-(adamant-1-ylmethoxycarbonyl)phthalanhydride obtained from 1-adamantane-methanol and trimellitic anhydride chloride appeared very useful for preparation of a number of N-substituted phthalimides. Antimicrobial activity of the newly obtained derivatives such as, for example, 4-(adamant-1-ylmethoxycarbonyl)-N-(5-carboxypentamethylene)p hthalimide or 4-(adamant-1-ylmethoxycarbonyl)-N-(L-alanyl)phthalimide was tested against Staphylococcus aureus, Bacillus sp., Micrococcus flavus and Enterococcus faecium. The minimal inhibitory concentration (MIC) for these compounds against S. aureus were 0.022 and 0.05 microg/ml, respectively.
Adamantane↗
Crystal structure of the purine nucleoside phosphorylase (PNP) from Cellulomonas sp. and its implication for the mechanism of trimeric PNPs.
The three-dimensional structure of the trimeric purine nucleoside phosphorylase (PNP) from Cellulomonas sp. has been determined by X-ray crystallography. The binary complex of the enzyme with orthophosphate was crystallized in the orthorhombic space group P212121 with unit cell dimensions a=64.1 A, b=108.9 A, c=119.3 A and an enzymatically active trimer in the asymmetric unit. X-ray data were collected at 4 degrees C using synchrotron radiation (EMBL/DESY, Hamburg). The structure was solved by molecular replacement, with the calf spleen PNP structure as a model, and refined at 2.2 A resolution. The ternary "dead-end" complex of the enzyme with orthophosphate and 8-iodoguanine was obtained by soaking crystals of the binary orthophosphate complex with the very weak substrate 8-iodoguanosine. Data were collected at 100 K with CuKalpha radiation, and the three-dimensional structure refined at 2.4 A resolution. Although the sequence of the Cellulomonas PNP shares only 33 % identity with the calf spleen enzyme, and almost no identity with the hexameric Escherichia coli PNP, all three enzymes have many common structural features, viz. the nine-stranded central beta-sheet, the positions of the active centres, and the geometrical arrangement of the ligands in the active centres. Some similarities of the surrounding helices also prevail. In Cellulomonas PNP, each of the three active centres per trimer is occupied by orthophosphate, and by orthophosphate and base, respectively, and small structural differences between monomers A, B and C are observed. This supports cooperativity between subunits (non-identity of binding sites) rather than existence of more than one binding site per monomer, as previously suggested for binding of phosphate by mammalian PNPs. The phosphate binding site is located between two conserved beta- and gamma-turns and consists of Ser46, Arg103, His105, Gly135 and Ser223, and one or two water molecules. The guanine base is recognized by a zig-zag pattern of possible hydrogen bonds, as follows: guanine N-1...Glu204 O(epsilon1)...guanine NH2...Glu204 O(epsilon2). The exocyclic O6 of the base is bridged via a water molecule to Asn246 N(delta), which accounts for the inhibitory, but lack of substrate, activity of adenosine. An alternative molecular mechanism for catalysis by trimeric PNPs is proposed, in which the key catalytic role is played by Glu204 (Glu201 in the calf and human enzymes), while Asn246 (Asn243 in the mammalian enzymes) supports binding of 6-oxopurines rather than catalysis. This mechanism, in contrast to that previously suggested, is consistent with the excellent substrate properties of N-7 substituted nucleosides, the specificity of trimeric PNPs versus 6-oxopurine nucleosides and the reported kinetic properties of Glu201/Ala and Asn243/Ala point variants of human PNP.
Amino Acid Sequence↗
Substrate/inhibitor properties of human deoxycytidine kinase (dCK) and thymidine kinases (TK1 and TK2) towards the sugar moiety of nucleosides, including O'-alkyl analogues.
Nucleoside analogues with modified sugar moieties have been examined for their substrate/inhibitor specificities towards highly purified deoxycytidine kinase (dCK) and thymidine kinases (tetrameric high-affinity form of TK1, and TK2) from human leukemic spleen. In particular, the analogues included the mono- and di-O'-methyl derivatives of dC, dU and dA, syntheses of which are described. In general, purine nucleosides with modified sugar rings were feebler substrates than the corresponding cytosine analogues. Sugar-modified analogues of dU were also relatively poor substrates of TK1 and TK2, but were reasonably good inhibitors, with generally lower Ki values vs TK2 than TK1. An excellent discriminator between TK1 and TK2 was 3'-hexanoylamino-2',3'-dideoxythymidine, with a Ki of approximately 600 microM for TK1 and approximately 0.1 microM for TK2. 3'-OMe-dC was a superior inhibitor of dCK to its 5'-O-methyl congener, consistent with possible participation of the oxygen of the (3')-OH or (3')-OMe as proton acceptor in hydrogen bonding with the enzyme. Surprisingly alpha-dT was a good substrate of both TK1 and TK2, with Ki values of 120 and 30 microM for TK1 and TK2, respectively; and a 3'-branched alpha-L-deoxycytidine analogue proved to be as good a substrate as its alpha-D-counterpart. Several 5'-substituted analogues of dC were good non-substrate inhibitors of dCK and, to a lesser extent, of TK2. Finally, some ribonucleosides are substrates of the foregoing enzymes; in particular C is a good substrate of dCK, and 2'-OMe-C is an even better substrate than dC.
Antimetabolites, Antineoplastic↗
Synthesis of 6-aryloxy- and 6-arylalkoxy-2-chloropurines and their interactions with purine nucleoside phosphorylase from Escherichia coli.
The phase transfer method was applied to perform the nucleophilic substitution of 2,6-dichloropurines by modified arylalkyl alcohol or phenols. Since under these conditions only the 6-halogen is exchanged, this method gives 2-chloro-6-aryloxy- and 2-chloro-6-arylalkoxy-purines. 2-Chloro-6-benzylthiopurine was synthesized by alkylation of 2-chloro-6-thiopurine with benzyl bromide. The stereoisomers of 2-chloro-6-(1-phenyl-1-ethoxy)purine were obtained from R- and S-enantiomers of sec.-phenylethylalcohol and 2,6-dichloropurine. All derivatives were tested for inhibition with purified hexameric E. coli purine nucleoside phosphorylase (PNP). For analogues showing IC50 < 10 microM, the type of inhibition and inhibition constants were determined. In all cases the experimental data were best described by the mixed-type inhibition model and the uncompetitive inhibition constant, Kiu, was found to be several-fold lower than the competitive inhibition constant, Kic. This effect seems to be due to the 6-aryloxy- or 6-arylalkoxy substituent, because a natural PNP substrate adenine, as well as 2-chloroadenine, show mixed type inhibition with almost the same inhibition constants Kiu and Kic. The most potent inhibition was observed for 6-benzylthio-2-chloro-, 6-benzyloxy-2-chloro-, 2-chloro-6-(2-phenyl-1-ethoxy), 2-chloro-6-(3-phenyl-1-propoxy)- and 2-chloro-6-ethoxypurines (Kiu = 0.4, 0.6, 1.4, 1.4 and 2.2 microM, respectively). The R-stereoisomer of 2-chloro-6-(1-pheny-1-ethoxy)purine has Kiu = 2.0 microM, whereas inhibition of its S counterpart is rather weak (IC50 > 12 microM). More rigid (e.g. phenoxy-), non-planar (cyclohexyloxy-), or more bulky (2,4,6-trimethylphenoxy-) substituents at position 6 of the purine base gave less potent inhibitors (IC50 = 26, 56 and > 100 microM, respectively). The derivatives are selective inhibitors of hexameric "high-molecular mass" PNPs because no inhibitory activity vs. trimeric Cellulomonas sp. PNP was detected. By establishing the ligand-dependent stabilization pattern of the E. coli PNP it was shown that the new derivatives, similarly as the natural purine bases, are able to form a dead-end ternary complex with the enzyme and orthophosphate. It was also shown that the derivatives are substrates in the reverse synthetic direction catalyzed by E. coli PNP.
Enzyme Inhibitors↗
Antimicrobial activity of substituted azoles and their nucleosides.
Four new 2'-deoxynucleosides of benzimidazole derivatives were prepared. Antimicrobial activity of many indazole, benzotriazole, benzimidazole derivatives and their nucleosides were tested by the agar diffusion method. Among the investigated compounds, dinitro- and trifluoromethyl-substituted benzimidazoles and their nucleosides were the most potent.
Anti-Bacterial Agents↗
Substrate/inhibitor specificities of human deoxycytidine kinase (dCK) and thymidine kinases (TK1 and TK2).
Substrate/inhibitor specificities of nucleoside analogues with modified sugar moieties toward highly purified deoxycytidine kinase (dCK) and thymidine kinases (TK1 and TK2) from human leukemic spleen have been examined. Substrate activities of cytosine nucleosides vs dCK were as follows: 2'-fluoro-dC > 2'-O-methyl-C > araC > 2'-fluoro-2'-deoxy-araC > 3'-O-methyl-dC = 3'-fluoro-2',3'-ddC > cytosine beta-L-riboside > 2',3'-ddC > C = 1-(4-hydroxy-1,2,-butadienyl)-cytosine (cytalene) = 2'-azido-dC. Modified purine nucleosides were only feeble substrates: ara-A > 2'-fluoro-2',3'-dideoxy-araA = 2'-O-methyl-A. With TK1 and TK2, similar sugar-modified analogues of dU and dT were feeble substrates. Surprisingly alpha-dT was a relatively good substrate, as well some beta-L-ribonucleo-sides. Several 5'-substituted analogues of dC were good non-substrate inhibitors of dCK and, to a lesser extent, of TK2. The overall data are relevant to the role of these enzymes in "activation" (by phosporylation) of nucleoside analogues with antiviral and antitumor activities.
Deoxycytidine Kinase↗
Antimicrobial activity of organic thiosulfates (Bunte salts).
A number of organic thiosulfates (Bunte salts) were prepared from appropriate primary bromides or iodides. In the case of substrates with long aliphatic chains, an addition of benzyltrimethylammonium chloride as phase transfer catalyst was very successful. The Bunte salts obtained were tested for antibacterial and fungicidal activity by means of the agar disc-diffusion method and by assignation of the minimum inhibitory concentrations (MIC). It was found that the microorganisms Proteus vulgaris, Candida albicans and Staphylococcus aureus showed the highest sensitivity. Biological activity of the compounds studied was dependent on the length of the aliphatic chain. Among the investigated compounds, aliphatic thiosulfates with 10-13 carbon atom chain were the most potent.
Anti-Bacterial Agents↗
7-Deazapurine 2'-deoxyribofuranosides are noncleavable competitive inhibitors of Escherichia coli purine nucleoside phosphorylase (PNP).
A series of 7-deazapurine 2'-deoxyribofuranosides were synthesized according to already known procedures and their substrate and inhibitor properties with purified E. coli purine nucleoside phosphorylase were examined. In agreement with previous findings, substrate activity was not detected for any of the compounds tested. Most of the nucleosides showed weak inhibition in the preliminary screening, i.e. at a concentration of about 100 microM. However some combinations of 6-chloro, 6-amino or 6-methoxy substituents with bulky hydrophobic groups at position 7 of the base and/or chloro, amino, methoxy or methylthio group at position 2 markedly enhanced affinity of such modified nucleosides for the E. coli enzyme. The most potent inhibition was observed for two nucleosides: 6-chloro- and 2-amino-6-chloro-7-deazapurine 2'-deoxyribofuranosides that show inhibition constants Ki = 2.4 and 2.3 microM, respectively. Several other compounds were also found to be good inhibitors, with inhibition constants in the range 5-50 microM. In all instances the inhibition was competitive vs. the nucleoside substrate 7-methylguanosine. Inhibition constants for 7-deazapurine nucleosides are in general several-fold lower than those observed for their purine counterparts. Therefore 7-deaza modification together with substitutions at positions 2, 6 and 7 of the base is a very promising approach to obtain competitive noncleavable inhibitors of E. coli PNP that may bind to the enzyme with inhibition constants in the microM range.
Deoxyadenosines↗
Human tonsillar lymphocytes as targets for immunosuppressive and anticancer drugs.
As has been shown earlier by us, the metabolism of extracellular deoxycytidine (dCyd) is 2-3 times higher in follicular and in PNA+ cells than in other cells. Deoxycytidine kinase (dCK) is one of the most important target enzymes for anti-proliferative drugs such as arabinosile-cytosine (ara-C), 2-Cl-deoxyadenosine (CdA). Neither the dCK activity nor the polypeptide correlates with the S phase of the cells, as thymidine kinase (TK1) does in tonsils. The newly developed anti-leukemic drug CdA, and also BrdA, are also phosphorylated by dCK and both effectively inhibit the 3H-dThd incorporation into DNA in tonsillar lymphocytes. A new molecular mechanism has been developed for CdA; it inhibits the interconversion of dCyd into dThd nucleotides. Analysis of the pools after 3H-dCyd labeling showed a decrease of the dUMP labeling. The inhibition of dCMP deaminase by the corresponding monophosphates (Cl-dAMP) in the cells has been suggested. CdA cannot be deaminated by adenosine deaminase (ADA), thus providing a good tool to investigate the importance of that enzyme during differentiation of the lymphoid cells. Elucidation of the nucleoside metabolism during the normal differentiation process might be the only way to get information about the same pathways in malignant transformations, i.e., in leukemias.
Adenosine Deaminase↗
2-Chloro-2'-deoxyadenosine (cladribine) and its analogues are good substrates and potent selective inhibitors of Escherichia coli purine-nucleoside phosphorylase.
2-Chloro-2'-deoxyadenosine (CldAdo), a nucleoside that has proven useful in the treatment of several chronic lymphoid malignancies, and its analogue, 2-bromo-2'-deoxyadenosine, are both effective inhibitors of the bacterial (Escherichia coli) purine-nucleoside phosphorylase (PNP), with Ki values of 4.5 microM and 6.3 microM, respectively. The examination of a series of base-modified analogues of CldAdo has shown that several other compounds have similar inhibitor properties, and has indicated that 6-benzyloxy-2-chloro-9-(2'-deoxy-beta-D-ribofuranosyl)purine is the most potent inhibitor with a Ki value of 0.5 microM, competitive with respect to inosine (Ino). CldAdo itself and its base-modified analogues, discounting those substituted at C(8), are also substrates for the E. coli PNP and undergo rapid glycosidic bond cleavage. CldAdo is degraded with substrate efficiency, i.e. Vmax/Km similar to that observed for Ino (130%), although the individual kinetic constants, Km and Vmax, are both approximately an order of magnitude lower than for Ino. All compounds tested are totally inactive as substrates and inhibitors for mammalian (calf spleen) PNP and therefore constitute a new class of potent selective, although cleavable, inhibitors of bacterial phosphorylases. 8-Bromo-2-chloro-2'-deoxyadenosine and 8-thio-2-chloro-2'-deoxyadenosine are the only base-modified CldAdo derivatives showing inhibitory activity against MOLT-3 (acute T-cell leukemia) and U-937 (histiocytic lymphoma) cells and, as shown in this study, are resistant to degradation by E. coli PNP. The above-mentioned results suggest that both analogues could be effective as oral cytotoxic agents that are noncleavable by enteric bacteria.
Cladribine↗
Spectrofluorimetric determination of 2-chloro-2'-deoxyadenosine (cladribine) in human plasma by photochemical reaction and chloroacetaldehyde derivatization.
A quantitative fluorescence assay for 2-chloro-2'-deoxyadenosine (cladribine, leustatin, 2-CdA) in human plasma is described. The drug was isolated from plasma by ethyl acetate extraction and derivatized by a two-step procedure in which 2'-deoxyisoguanosine (2'-diG) was first prepared by UV irradiation of 2-CdA and was then treated with chloroacetaldehyde to form the fluorescent derivative, 1,N6-etheno-2'-deoxyisoguanosine. Fluorescence intensity of the solutions was measured using an excitation wavelength of 275 nm and emission of 397 nm. The analytical measuring range of the method extends from about 1 microgram/l to at least 100 micrograms/l.
Acetaldehyde↗