Platelet aggregation inhibitors. 2. Inhibition of platelet aggregation by 5'-, 2-, 6-, and 8-substituted adenosines

Kikugawa, Kiyomi; Iizuka, Kazuhiro; Higuchi, Yoshiyuki; Hirayama, Hiroki; Ichino, Motonobu

doi:10.1021/jm00274a015

Cited by 35 publications

(21 citation statements)

References 8 publications

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“…MT-ImmH was synthesized by the desilylation of the previously reported N-tert-butoxycarbonyl-7-O-tert-butyldimethylsilyl-2,3,6-trideoxy-3,6-imino-4,5-O-isopropylidene-D-allo-heptononitrile followed by methanesulfonylation of the 7-hydroxyl and displacement of the resulting mesylate with thiomethoxide. The resultant compound was converted into MT-Immucillin-H in an analogous fashion to that previously described (9,10). Structure and stereochemistry of MT-ImmH was confirmed by mass spectrometry and NMR.…”

Section: Methodsmentioning

confidence: 92%

Plasmodium falciparum Purine Nucleoside Phosphorylase

Shi

Ting

Kicska

et al. 2004

Journal of Biological Chemistry

110

114

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Plasmodium falciparum is a purine auxotroph, and starvation of purines is known to cause purine-less death in cultured cells (1-3). Parasites cultured in human erythrocytes can be killed rapidly by blocking PNP 1 with Immucillin-H and related Immucillins; however, these agents inhibit both P. falciparum and human PNPs, albeit with higher efficiency for human PNP (2, 4). The biochemical link between PNP inhibition and purine-less death is the formation of hypoxanthine, previously identified as a primary source of purines for P. falciparum (1, 3). The amino acid sequence of P. falciparum PNP as well as its substrate specificity differs from mammalian and bacterial enzymes (4, 5). We investigated this difference by structural analysis using Immucillin-H as a ligand to stabilize the catalytic site and as a starting point for synthesis of inhibitors more specific to PfPNP.Immucillin-H is effective in causing P. falciparum cell death by purine starvation even in the purine-rich environment of human erythrocytes. Hypoxanthine but not inosine can prevent the effect (2). The transition state of PfPNP has oxacarbenium ion character and ImmH binds to PfPNP with a dissociation constant of 860 pM, making it a powerful inhibitor for this target (Refs. 4 and 6; Fig. 1). However, ImmH was designed to be a transition state analogue of mammalian PNPs and binds with a constant of 56 pM to human PNP (7). Therefore, addition of this compound to human erythrocytes infected with P. falciparum has two effects, inhibition of human PNP at lower concentrations and inhibition of both human and PfPNPs at higher concentrations.Comparison of the PfPNP amino acid sequence against the data base of all PNPs indicated that this enzyme differs from mammalian and bacterial PNPs (see below). The structure of PfPNP in complex with ImmH and SO 4 revealed that the catalytic site has capacity for binding 5Ј-substituted nucleosides. 5Ј-Methylthioinosine and inosine are good substrates for PfPNP. We hypothesized that 5Ј-methylthio-substituted Immucillin would bind preferentially to PfPNP. This hypothesis was used to design, produce, and characterize 5Ј-methylthio-Immucillin-H (MT-ImmH), a novel transition state analogue inhibitor with high specificity for the P. falciparum PNP. EXPERIMENTAL PROCEDURESPfPNP-The coding sequence for PfPNP (4) was amplified by PCR from P. falciparum 3D7 genomic DNA, placed into the pTrcHis2-TOPO vector, expressed in Escherichia coli TOP 10, and purified to Ͼ95% homogeneity by nickel affinity chromatography. ImmH was synthesized by the convergent route (8). MT-ImmH was synthesized by the desilylation of the previously reported N-tert-butoxycarbonyl-7-O-tert-butyldimethylsilyl-2,3,6-trideoxy-3,6-imino-4,5-O-isopropylidene-D-allo-heptononitrile followed by methanesulfonylation of the 7-hydroxyl and displacement of the resulting mesylate with thiomethoxide. The resultant compound was converted into MT-Immucillin-H in an analogous fashion to that previously described (9, 10). Structure and stereochemistry of MT-ImmH was confirmed ...

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Section: Methodsmentioning

confidence: 92%

Plasmodium falciparum Purine Nucleoside Phosphorylase

Shi

Ting

Kicska

et al. 2004

Journal of Biological Chemistry

110

114

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“…Infected erythrocytes and various concentrations of drugs (5,10,20,50,75, and 125 M) were aliquoted in triplicate wells of a 96-well microtiter plate (Corning). Each well contained a total volume of 200 l with a final hematocrit of 1.5% and parasitemia of 0.5 to 1%.…”

Section: Methodsmentioning

confidence: 99%

“…Reagents for the assay were supplied in kit form by Flow Incorporated (Portland, Oreg.). At 36 h after the addition of drug at various concentrations (5,10,20,50,75,125,250, 500, and 1,000 M), 20 l of erythrocyte suspension was withdrawn from each culture well, mixed with 100 l of Malstat (from the assay kit) in a 96-well enzyme-linked immunosorbent assay plate, and covered with aluminum foil. Then 20 l of an aqueous mixture (1:1 [vol/vol]) of nitroblue tetrazolium (2 mg/ml) and phenazine ethosulfate (0.1 mg/ml) was added to each well, and the plate was kept at room temperature for 20 min.…”

Section: Methodsmentioning

confidence: 99%

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Methionine recycling pathways and antimalarial drug design

Sufrin

Meshnick

Spiess

et al. 1995

Antimicrob Agents Chemother

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5-Deoxy-5-(methylthio)adenosine (MTA) is an S-adenosylmethionine metabolite that is generated as a by-product of polyamine biosynthesis. In mammalian cells, MTA undergoes a phosphorolytic cleavage catalyzed by MTA phosphorylase to produce adenine and 5-deoxy-5-(methylthio)ribose-1-phosphate (MTRP). Adenine is utilized in purine salvage pathways, and MTRP is subsequently recycled to methionine. Whereas some microorganisms metabolize MTA to MTRP via MTA phosphorylase, others metabolize MTA to MTRP in two steps via initial cleavage by MTA nucleosidase to adenine and 5-deoxy-5-(methylthio)ribose (MTR) followed by conversion of MTR to MTRP by MTR kinase. In order to assess the extent to which these pathways may be operative in Plasmodium falciparum, we have examined a series of 5-alkyl-substituted analogs of MTA and the related MTR analogs and compared their abilities to inhibit in vitro growth of this malarial parasite. The MTR analogs 5-deoxy-5-(ethylthio)ribose and 5-deoxy-5-(hydroxyethylthio)ribose were inactive at concentrations up to 1 mM, and 5-deoxy-5-(monofluoroethylthio)ribose was weakly active (50% inhibitory concentration ‫؍‬ 700 M). In comparison, the MTA analogs, 5-deoxy-5-(ethylthio)adenosine, 5-deoxy-5-(hydroxyethylthio) adenosine (HETA), and 5-deoxy-5-(monofluoroethylthio)adenosine, had 50% inhibitory concentrations of 80, 46, and 61 M, respectively. Extracts of P. falciparum were found to have substantial MTA phosphorylase activity. Coadministration of MTA with HETA partially protected the parasites against the growth-inhibitory effects of HETA. Results of this study indicate that P. falciparum has an active MTA phosphorylase that can be targeted by analogs of MTA.Inhibitors of polyamine metabolism have emerged recently as promising agents for the chemotherapy of cancer (16) and of parasitic diseases (1). Polyamine biosynthetic enzymes which serve as drug targets include ornithine decarboxylase, S-adenosylmethionine decarboxylase, spermidine synthase, and spermine synthase. In addition, enzymes which degrade the polyamine biosynthesis by-product, 5Ј-deoxy-5Ј-(methylthio)adenosine (MTA), are potentially exploitable for chemotherapy, since MTA metabolism in microorganisms (8,13,18) and tumor cells (20) differs in significant ways from MTA metabolism in normal mammalian cells.In mammalian cells, MTA is rapidly cleaved by a highly specific MTA phosphorylase to yield adenine and 5-deoxy-5-(methylthio)ribose-1-phosphate (MTRP) (Fig. 1). Adenine is then salvaged, and MTRP is converted into methionine. MTA phosphorylase is absent in many solid tumors and leukemias (6). It is also absent in microorganisms such as Giardia lamblia (17) and Klebsiella pneumoniae (14, 15), which utilize an alternative pathway not present in mammalian cells for metabolism of MTA to MTRP. In this alternate pathway, MTA is cleaved to adenine and 5-deoxy-5-(methylthio)ribose (MTR) by MTA nucleosidase. MTR, once formed, is phosphorylated to MTRP by MTR kinase (Fig. 1). It has previously been reported that Plasmodium falciparum utiliz...

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“…With appropriate modifications, a variety of analogs and homologs could be synthesized, including adenosylhomocysteine (53,54), which had been recognized in the meantime as the principal metabolite of AdoMet (55). The most significant simplification in the synthesis of these compounds (Scheme 1, reactions c and d) was achieved by Kikugawa et al (56,57) and by Borchardt et al (58), who demonstrated that treatment of adenosine with SOCl in hexamethylphosphoramide under N2 yields 5'-deoxy-5'-chloroadenosine in quantity. This product crystallizes readily and reacts almost quantitatively with sodium mercaptides in liquid ammonia.…”

Section: Structure and Chemical Synthesis Of Mtamentioning

confidence: 99%

Methylthioadenosine

Schlenk

1983

Advances in Enzymology - And Related Areas of Molecular Biology

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Platelet aggregation inhibitors. 2. Inhibition of platelet aggregation by 5'-, 2-, 6-, and 8-substituted adenosines

Cited by 35 publications

References 8 publications

Plasmodium falciparum Purine Nucleoside Phosphorylase

Plasmodium falciparum Purine Nucleoside Phosphorylase

Methionine recycling pathways and antimalarial drug design

Methylthioadenosine

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