Biomedical subjects

K Lesiak

Publications and source records attributed to K Lesiak.

At least 19 recordsLinked to original sources

PubMed1999

Synthesis of a novel C-nucleoside, 2-amino-7-(2-deoxy-beta-D-erythro- pentofuranosyl)-3H,5H-pyrrolo-[3,2-d]pyrimidin-4-one (2'-deoxy-9-deazaguanosine).

A synthesis of the C-nucleoside, 2-amino-7-(2-deoxy-beta-D-erythro- pentofuranosyl)-3H,5H-pyrrolo[3,2-d]pyrimidin-4-one (9-deaza-2'-deoxyguanosine) was achieved starting from 2-amino-6-methyl-3H-pyrimidin-4-one (5) and methyl 2-deoxy-3,5-di-O-(p-nitrobenzoyl)-D-erythro-pento-furanoside (11). The anomeric configuration of the C-nucleoside was established by 1H NMR, NOEDS and ROESY. This C-nucleoside did not inhibit the growth of T-cell lymphoma cells.

Animals↗

PubMed1999

Bicyclic monoterpene diols induce differentiation of S91 melanoma and PC12 pheochromocytoma cells by a cyclic guanosine-monophosphate-dependent pathway.

Previously, we showed that 5-norbornene-2,2-dimethanol (5-NBene-2,2-DM) is an effective inducer of melanogenesis in cultured cells and guinea-pig skin [Brown et al. (1998) J. Invest. Dermatol., 110:428-437]. This study shows that 2,3-cis/exo-pinanediol (2,3-cs/ex-PinD) is a more effective inducer of melanogenesis than 5-NBene-2,2-DM in S91 mouse melanoma cells. Furthermore, 2,3-cs/ex-PinD appears to penetrate guinea-pig skin better than 5-NBene-2,2-DM and to induce higher levels of pigmentation. Both 5-NBene-2,2-DM and 2,3-cs/ex-PinD induce synthesis of nitric oxide (NO) in S91 cells, and the melanogenic activity of both compounds is reduced by inhibitors of the NO/cyclic guanosine monophosphate (cGMP)/protein kinase(PK) G signaling pathway, but not by inhibitors of the PKC or PKA pathways. Thus, these bicyclic monoterpene diols appear to induce melanogenesis by the same pathway in S91 cells as that shown previously for ultraviolet radiation in melanocytes (Romero-Graillet et al. (1996) J. Biol. Chem., 271:28052-28056). These compounds also induce NO synthesis, neurite outgrowth, and tyrosine hydroxylase activity in PC12 pheochromocytoma cells. Neurite outgrowth in PC12 cells is blocked by the guanylate cyclase inhibitor, LY83583 (6-anilino-2,8-quinolinequinone), indicating that, similar to S91 cells, the induction of morphological differentiation of PC12 cells by bicyclic monoterpene diols is regulated by a cGMP-dependent pathway.

Animals↗

PubMed1998

A pseudosquare knot structure of DNA in solution.

We report a high-resolution NMR structure of a homodimer formed by a synthetic 25 residue DNA oligonucleotide GCTCCCATGGTTTTTGTGCACGAGC. This structure presents a novel structural motif for single-stranded nucleic acids, called a pseudosquare knot (PSQ). The oligonucleotide was originally designed to mimic a slipped-loop structure (SLS), another "unusual" DNA structure postulated as an alternative conformation for short direct repeats in double-stranded DNA. The design of the sequence is compatible with both SLS and PSQ structures, both of which possess identical sets of base-paired and unpaired nucleotides but different tertiary folds. We used deuteration of the H8 positions of purines to ascertain that the PSQ is actually formed under the conditions used. The PSQ structure was solved based on homonuclear proton nuclear Overhauser effect data using complete relaxation matrix methods. The structure essentially consists of two side-by-side helices connected by single-stranded loops. Each of the helices is well-defined; however, the relative orientation of the two remains undetermined by the NMR data. The sequences compatible with the PSQ formation are frequent in single-stranded genomes; this structure may play a role as a dimerization motif.

Base Composition↗

PubMed1998

Nuclear magnetic resonance structure of d(GCATATGATAG). d(CTATCATATGC): a consensus sequence for promoters recognized by sigma K RNA polymerase.

The three-dimensional structure of d(GCATATGATAG).d(CTATCATATGC), from the promoter region of a gene regulating sporulation in Bacillus subtilis mother cells, was determined utilizing two-dimensional nuclear Overhauser effect (2D NOE) and double-quantum-filtered COSY (2QF-COSY) spectra. To minimize the effect of methods used to obtain restraints and refine structure, several variables were studied. Interproton distance bounds were calculated very conservatively by running the complete relaxation matrix program MARDIGRAS hundreds of times using 2D NOE spectra for exchangeable and for nonexchangeable protons at different mixing times, assuming different overall correlation times and different starting structures. The 435 distance restraints were used with two different structural refinement methods: restrained molecular dynamics (rMD) and restrained Monte Carlo calculations (rMC). Refinement using different procedures and starting structures resulted in essentially the same structure (<0.8 A rmsd), indicating that the structure is defined by experimental restraints and not the refinement method or variables used. R factors indicate the structures fit the experimental NOE data very well. Some helical parameters, notably large negative X displacement, are characteristic of A-DNA, but others are characteristic of B-DNA. As with TG.CA steps in other duplex DNA sequences studied in our laboratory, the two TG.CA steps have a positive roll, with T6-G7 exhibiting the largest, and consequently a bent helix axis. The converged structure represents a time-averaged structure. However, multiple conformations, especially in deoxyriboses, were evident from vicinal coupling constants obtained from quantitative simulations of 2QF-COSY cross-peaks and from persistent inconsistencies in experimental distances due to nonlinear conformational averaging.

Base Sequence↗

PubMed1998

Synthesis of a methylenebis(phosphonate) analogue of mycophenolic adenine dinucleotide: a glucuronidation-resistant MAD analogue of NAD.

Mycophenolic alcohol (MPAlc), obtained by reduction of the carboxylic group of mycophenolic acid (MPA), was coupled with 2',3'-O-isopropylideneadenosine 5'-methylenebis(phosphonate) (4) in the presence of diisopropylcarbodiimide (DIC) to give P1-(2',3'-O-isopropylideneadenosin-5'-yl)-P2-(mycophenolic alcohol-6'-yl)methylenebis(phosphonate) (8) in 32% yield. Deisopropy-lidenation of 8 with CF3COOH/H2O afforded the methylenebis(phosphonate) analogue 3 of mycophenolic adenine dinucleotide (MAD). Compound 3, beta-methylene-MAD, was found to be a potent inhibitor of inosine monophosphate dehydrogenase (IMPDH) type II (Ki = 0.3 microM) as well as an inhibitor of growth of K562 cells (IC50 = 1.5 microM). In contrast to MPA and mycophenolic alcohol, beta-methylene-MAD was not converted into the glucuronide when incubated with uridine 5'-diphosphoglucuronyltransferase.

Adenine Nucleotides↗

PubMed1998

Aliphatic and alicyclic diols induce melanogenesis in cultured cells and guinea pig skin.

We have found that several aliphatic and alicyclic diols induce melanogenesis in cultured S91 mouse melanoma cells and normal human epidermal melanocytes (NHEM). In addition, these compounds induce melanogenesis when applied to guinea pig skin, with transfer of melanin to keratinocytes and formation of "supranuclear caps," as occurs in naturally pigmented skin. The relative order of potency of some of these diols in NHEM is 5-norbornene-2,2-dimethanol > 3,3-dimethyl-1,2-butanediol > cis-1,2-cyclopentanediol > 2,3-dimethyl-2,3-butanediol > 1,2-propanediol. Following treatment with these diols or 3-isobutyl-1-methylxanthine, melanin and tyrosinase activity are increased within S91 cells and NHEM; however, for cultured NHEM, the largest increases of melanin and tyrosinase occur in an extracellular particulate fraction, shown by electron microscopy to consist almost entirely of stage III and IV melanosomes. These results indicate that cultured NHEM treated with diols export melanosomes in a fashion that is commensurate with natural melanogenic processes. In contrast, S91 mouse melanoma cells exhibit aberrant melanosomal trafficking, in accordance with the known defect in myosin-V mediated melanosomal transport. Both S91 cells and NHEM exhibit morphologic changes and growth arrest indicative of differentiation following treatment with diols. The diols described in this report are candidates for use as cosmeceutical tanning agents.

Animals↗

PubMed1997

Synthesis of nonhydrolyzable analogues of thiazole-4-carboxamide and benzamide adenine dinucleotide containing fluorine atom at the C2' of adenine nucleoside: induction of K562 differentiation and inosine monophosphate dehydrogenase inhibitory activity.

Thiazole-4-carboxamide adenine dinucleotide (TAD) analogue 7 containing a fluorine atom at the C2' arabino configuration of the adenine nucleoside moiety was found to be a potent inducer of differentiation of K562 erythroid leukemia cells. This finding prompted us to synthesize its hydrolysis-resistant methylenebis(phosphonate) and difluoromethylenebis(phosphonate) analogues 8 and 9, respectively. Since both TAD and benzamide adenine dinucleotide (BAD) are potent inhibitors of inosine monophosphate dehydrogenase (IMPDH), the corresponding fluorine-substituted methylenebis(phosphonate) analogue 12 of BAD was also synthesized. Thus, 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine (13) was converted in five steps into the corresponding methylenebis(phosphonate) analogue 18. Dehydration of 18 with DCC led to the formation of the bicyclic trisanhydride intermediate 19a, which upon reaction with 2',3'-O-isopropylidenetiazofurin (20) or -benzamide riboside (21) followed by hydrolysis and deprotection afforded the desired methylene-bridged dinucleotides 8 and 12, respectively. The similar displacement of the 5'-mesyl function of 2',3'-O-isopropylidene-5'-O-mesyltiazofurin (24) with the difluoromethylenebis(phosphonic acid) derivative gave the phosphonate 25 which was coupled with 13 to afford 26. The desired difluoromethylenebis(phosphonate) analogue 9 was obtained by deprotection with Dowex 50/H+. This compound as well as beta-CF2-TAD (4) showed improved differentiation-inducing activity over beta-CH2-TAD (3), whereas analogues containing the -CH2-linkage (8 and 12) were inactive.

Adenine Nucleotides↗

PubMed1997

The practical synthesis of a methylenebisphosphonate analogue of benzamide adenine dinucleotide: inhibition of human inosine monophosphate dehydrogenase (type I and II).

beta-Methylene-BAD (8), a nonhydrolyzable analogue of benzamide adenine dinucleotide (BAD), was synthesized as potential inhibitor of human inosine monophosphate dehydrogenase (IMPDH). Treatment of 2',3'-O-isopropylideneadenosine 5'-methylenebisphosphonate (15) with DCC afforded P1,P4-bis(2',3'-O-isopropylideneadenosine) 5'-P1,P2:P3,P4-dimethylenetetrakisphosphonate (17). This compound was further converted with DCC to an active intermediate 18 which upon reaction with 3-(2',3'-O-isopropylidene-beta-D-ribofuranosyl)benzamide (19) gave, after hydrolysis and deisopropylidenation, the desired beta-methylene-BAD (8) in 95% yield. In a similar manner, treatment of 18 with 2',3'-O-isopropylidenetiazofurin (21) followed by hydrolysis and deprotection afforded beta-methylene-TAD (5) in 91% yield. Compound 8 (IC50 = 0.665 microM) was found to be a 6-8 times less potent inhibitor of IMPDH than 5 (IC50 = 0.107 microM) and was almost equally potent against IMPDH type I and type II. Although TAD and beta-methylene-TAD were bound by LADH with the same affinity, the binding affinity of 8 toward LADH (Ki = 333 microM) was found to be 50-fold lower than that of the parent pyrophosphate 7 (Ki = 6.3 microM).

Adenine Nucleotides↗

PubMed1996

Synthesis and characterization of composite nucleic acids containing 2', 5'-oligoriboadenylate linked to antisense DNA.

Composite nucleic acids, known as 2-5A antisense chimeras, cause the 2-5A-dependent ribonuclease (RNase L) to catalyze the specific cleavage of RNA in cell free systems and in intact cells. Such 2-5A antisense chimeras are 5'-monophosphorylated, 2,'5'-linked oligoadenylates covalently attached to antisense 3',5'-oligodeoxyribonucleotides by means of a linker containing two residues of 1,4-butanediol phosphate. Here we report a fully automated synthesis of 2-5A antisense chimeras on a solid support using phosphoramidite methodology with specific coupling time modifications and their subsequent purification by reverse-phase ion-pair and anion exchange HPLC. Purified 2-5A antisense chimeras were characterized by [1H]NMR and [31P]NMR, MALDIMS, and capillary gel electrophoresis. The synthetic 2',5'-linked oligoadenylate showed no phosphodiester isomerization to 3',5' during or after synthesis. In addition, we have developed facile methodologies to characterize the chimeras using digestion with various hydrolytic enzymes including snake venom phosphodiesterase I and nuclease P1. Finally, Maxam-Gilbert chemical sequencing protocols have been developed to confirm the entire sequence of these chimeric oligonucleotides.

2',5'-Oligoadenylate Synthetase↗

PubMed1993

Targeting RNA for degradation with a (2'-5')oligoadenylate-antisense chimera.

Antisense oligonucleotides hold considerable promise both as research tools for inhibiting gene expression and as agents for the treatment of a myriad of human diseases. However, targeted destruction of RNA has been difficult to achieve in a versatile, efficient, and reliable manner. We have developed an effective strategy for cleaving unique RNA sequences with 2-5A-dependent RNase, an endoribonuclease that mediates inhibitory effects of interferon on virus infection and is activated by 5'-phosphorylated 2'-5'-linked oligoadenylates known as 2-5A [pn5' A2'(p5' A2')mp5'A], resulting in the cleavage of single-stranded RNA predominantly after UpUp and UpAp sequences. To direct 2-5A-dependent RNase to cleave unique RNA sequences, p5' A2' p5' A2'p5'A was covalently linked to an antisense oligonucleotide to yield a chimeric molecule (2-5A:AS). The antisense oligonucleotide component of 2-5A:AS bound a specific RNA sequence while the accompanying 2-5A component activated 2-5A-dependent RNase, thereby causing the cleavage of the RNA in the targeted sequence. This strategy was demonstrated by inducing specific cleavage within a modified human immunodeficiency virus type 1 vif mRNA in a cell-free system from human lymphoblastoid cells. Because 2-5A-dependent RNase is present in most mammalian cells, the control of gene expression based on this technology--including therapies for cancer, viral infections, and certain genetic diseases--can be envisioned.

Adenine Nucleotides↗

PubMed1992

A new and potent 2-5A analogue which does not require a 5'-polyphosphate to activate mouse L-cell RNase L.

In order to explore the possibility of supplanting the requirement of a 5'-triphosphate moiety for the activation of the 2-5A-dependent endonuclease (RNase L) of mouse L-cells, two new tetrameric analogues of 2-5A were synthesized. The first tetramer, obtained by both a modified prebiotic synthetic approach as well as a phosphite triester solid phase oligonucleotide synthesis method, was p5'A2'p5'A2'p5'(br8A)2'p5'(br8A). The second oligonucleotide was derived from the former by a sequence involving periodate oxidation, reaction with n-hexylamine, and cyanoborohydride reduction, resulting in conversion of the 2'-terminal adenosine residue to 9-(3'-aza-4'-hexyl-1',2',3',4'-tetradeoxyhexopyranos-1(1)-yl)-8-++ +bromoadenine. Both of these oligomers, bearing only 5'-monophosphate groups, were found to be as potent as 2-5A itself as activators of the RNase L of mouse L-cells.

Adenine Nucleotides↗

PubMed1992

Synthesis and biological activity of uronic acid analogues of 2-5A[5'-O-triphosphoryladenylyl(2----5')adenylyl-(2'----5')adenosine].

The oligonucleotide ppp5'A2'p5'A2'p5'A, known as 2-5A, is a potent translational inhibitor involved in some aspects of interferon action. To explore the specific function of the charged 5'-triphosphate moiety, we prepared a series of congeners in which the 5' region was hypermodified. Thus, uronic acid derivatives were substituted for the 5' terminal adenosine residue of 2-5A. Compounds 9, 10, 11 and 12 carried adenosine 5'-uronic acid, ethyl adenosine 5'-uronate, adenosine 5'-uronamide, and adenosine 5'-(N-ethyl)uronamide, respectively, in place of the 5' terminal adenosine triphosphate moiety of 2-5A. While all the analogues showed some weak interaction with the 2-5A-dependent endonuclease (RNase L), compound 9 showed the strongest binding ability, and while unable to activate the mouse RNase L, could activate human RNase at a concentration 100-fold greater than that required for the parent 2-5A. This result suggests that the function of the 5'(poly)phosphate moiety of 2-5A may be fulfilled by some other anionic moiety.

Adenine Nucleotides↗

PubMed1991

2',5'-oligoadenylates inhibit relaxation of supercoiled DNA by calf thymus DNA topoisomerase I.

DNA topoisomerases interconvert various topological isomers of DNA and play key roles in replication and gene expression. The possible involvement of the 2',5'-oligoadenylates (2-5A) system in cell growth, regulation, and cell differentiation led us to investigate the effects of 2-5A on mammalian topoisomerases. We found that the calf thymus type I topoisomerase was inhibited by a variety of 2-5A compounds. The level of inhibition was dependent upon the number of residues and the degree of phosphorylation at the 5' terminus. The 5'-triphosphorylated 2',5' hexamer, ppp(Ap)5A, was the most effective, strongly reducing relaxation at less than micromolar concentrations. These results raise the possibility that physiological concentrations of 2-5A of sufficient chain length may be capable of regulating gene expression by virtue of a direct inhibition of DNA topoisomerase I.

Adenine Nucleotides↗

PubMed1990

Mechanism of HIV reverse transcriptase: enzyme-primer interaction as revealed through studies of a dNTP analogue, 3'-azido-dTTP.

Primer and dNTP recognition by purified HIV reverse transcriptase have been investigated. Earlier kinetic studies suggested that the reaction pathway for DNA synthesis is ordered, with template-primer and free enzyme combining to form the first complex in the reaction sequence [Majumdar et al. (1988) J. Biol. Chem. 263, 15657-15665], and through use of a particularly high affinity template-primer analogue [r(I)n.Sd(C)28], rate values for formation of the first complex were calculated [Majumdar et al. (1989) Biochemistry 28, 1340-1346]. We now report rate values for first complex formation in the usual model replication system with poly[r(A)].oligo [d(T)] as template-primer. We find that 3'-azido-dTTP (AZTTP) is a linear competitive inhibitor of DNA synthesis against the substrate dNTP (dTTP) in the poly[r(A)].oligo[d(T)] replication system. This suggests that 3'-azido-dTTP and dTTP combine with the same form of the enzyme in the reaction scheme, i.e., the enzyme-primer complex. This is not trivial, since a second analogue, 3'-amino-dTTP, also is an inhibitor against dTTP, but the mechanism in this case is linear noncompetitive. Because the inhibition by 3'-azido-dTTP is linear competitive, the KD for physical binding to the enzyme is assumed to be the same as the Ki for inhibition (20 nM). Substrate kinetic studies of DNA synthesis using 3'-azido-dTTP as substrate revealed that the Michaelis constant is 3 microM. Therefore, the Km for this substrate analogue is 100-fold higher than the KD for binding of the analogue to the enzyme-primer complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiviral Agents↗

PubMed1990

Photochemical crosslinking in oligonucleotide-protein complexes between a bromine-substituted 2-5A analog and 2-5A-dependent RNase by ultraviolet lamp or laser.

2',5'-oligoadenylates known as 2-5A [px(A2'p)nA; chi = 2 or 3, n greater than or equal to 2] are produced in interferon-treated cells in response to double-stranded RNA. 2-5A binds with high affinity to a 2-5A-dependent RNase resulting in the cleavage of single-stranded RNA. An efficient, rapid, and extremely sensitive photoaffinity labeling method was developed to facilitate detection of 2-5A-dependent RNase. A bromine-substituted and radioactive derivative of 2-5A, the 5'-monophosphate, p(A2'p)2(br8A2'p)2A3'-[32P]Cp, was synthesized as probe for 2-5A-dependent RNase. Even though this bromine-substituted analog of 2-5A bore no 5'-terminal triphosphate or diphosphate, it bound to 2-5A-dependent RNase with the same high affinity as did 2-5A per se but it was a less effective activator of the RNase under the present assay conditions. The presence of bromine atoms in the 2-5A analog enhanced by more than 200-fold crosslinking to 2-5A-dependent RNase under a uv lamp; many additional polypeptides were also labeled but at much lower levels. Furthermore, using high-intensity uv laser irradiation (308 nm) covalent attachment of the bromine-substituted 2-5A analog to 2-5A-dependent RNase was readily achieved within 10(-6) s.

Adenine Nucleotides↗

PubMed1990

Inhibition of the RNA polymerase of vesicular stomatitis virus by ppp5'A2'p5'A and related compounds.

The diadenylate triphosphates ppp5'A2'p5'A and ppp5'A3'p5'A were found to inhibit the purified RNA polymerase ('nucleocapsid') complex from vesicular stomatitis virus (VSV). The corresponding diadenylate monophosphate p5'A2'p5'A did not inhibit, nor did the triadenylate triphosphate ppp5'A2'p5'A2'p5'A; the diadenylate diphosphate pp5'A2'p5'A had intermediate inhibitory activity. Increasing the concentration of ATP, GTP or CTP in the reaction mixture decreased inhibition by ppp5'A2'p5'A, while UTP had minimal or no protective effect. ppp5'A2'p5'A did not protect the RNA polymerase from inactivation by N-ethylmaleimide. This suggests that the action of ppp5'A2'p5'A occurs at a site on the enzyme that is distinct from the N-ethylmaleimide-protecting, ATP-binding site characterized previously.

Adenine Nucleotides↗

PubMed1989

Conformational analysis of brominated pA2'-5'A2'-5'A analogs. An NMR and model-building study.

NMR and model-building studies were carried out on pA2'-5'A2'-5'A and analogs in which one or more of the A residues were replaced by 8-bromoadenosine. Chemical shifts, coupling constants and NOE data were used to obtain structural information. The N/S equilibrium constant of the ribose rings as well as the phase angles and puckering amplitudes were determined from the experimental coupling constants with the aid of an improved version of the PSEUROT program. Chemical shifts in combination with NOE data were used to monitor base-base interactions and the orientation of the bases (syn or anti). The combined data suggest that different types of stacking interactions are present in the various compounds. Bromination of the first or second residue in the trimers results in a preference for N-type sugar and syn orientation of the base in these residues. When A(3) is brominated, an S-type sugar conformation together with a syn orientation of the base is favoured at the 2' terminus. Energy-minimized models of the different stacking interactions are presented which fit the present collection of data. The possible correlation between biological activity of these compounds and their conformation is briefly discussed.

Adenosine↗

PubMed1988

AIDS dementia: synthesis and properties of a derivative of 3'-azido-3'-deoxythymidine (AZT) that may become 'locked' in the central nervous system.

In an attempt to provide a derivative of 3'-azido-3'-deoxythymidine (AZT) which might be sequestered in the central nervous system and release AZT, the dihydropyridine ester 5'-(1,4-dihydro-1-methyl-3-pyridinylcarbonyl)-3'-deoxythymidine, was synthesized in a three step sequence. This material showed potent anti-HIV-1 activity in MT-4 cells most probably by hydrolysis to the parent nucleoside, AZT. This dihydropyridine derivative of AZT could be easily oxidized to a positively charged pyridinium derivative of AZT in rat brain cytosol. In turn the pyridinium form could be hydrolyzed, non-enzymatically, to AZT.

Acquired Immunodeficiency Syndrome↗