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Find video protocols related to scientific articles indexed in Pubmed.
Inhibition of the Escherichia coli 6-oxopurine phosphoribosyltransferases by nucleoside phosphonates: potential for new antibacterial agents.
J. Med. Chem.
PUBLISHED: 08-27-2013
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Escherichia coli (Ec) cells possess two purine salvage enzymes: xanthine-guanine phosphoribosyltransferase (XGPRT) and hypoxanthine phosphoribosyltransferase (HPRT). EcXGPRT shares a common structural feature with other members of this family, a flexible loop that closes over the active site during catalysis. The replacement of six of these amino acids by alanine has no effect on the Km for the two substrates. However, the Ki for the nucleoside monophosphate increases by 27-fold, and the kcat is reduced by ?200-fold. Nucleoside phosphonates (NP) are good inhibitors of EcXGPRT and EcHPRT, with Ki values as low as 10 nM. In the absence of the flexible loop, these values increase by 5- to 30-fold, indicating the importance of the loop for high-affinity inhibition. Crystal structures of two NPs in complex with EcXGPRT explain the tight binding. Prodrugs of NPs with low Ki values for EcXGPRT or EcHPRT exhibit IC50 values between 5 and 23 ?M against Mycobacterium tuberculosis in cell-based assays, suggesting that these compounds are therapeutic leads against pathogenic bacteria.
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Role of human hypoxanthine guanine phosphoribosyltransferase in activation of the antiviral agent T-705 (favipiravir).
Mol. Pharmacol.
PUBLISHED: 08-01-2013
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6-Fluoro-3-hydroxy-2-pyrazinecarboxamide (T-705) is a novel antiviral compound with broad activity against influenza virus and diverse RNA viruses. Its active metabolite, T-705-ribose-5-triphosphate (T-705-RTP), is recognized by influenza virus RNA polymerase as a substrate competing with GTP, giving inhibition of viral RNA synthesis and lethal virus mutagenesis. Which enzymes perform the activation of T-705 is unknown. We here demonstrate that human hypoxanthine guanine phosphoribosyltransferase (HGPRT) converts T-705 into its ribose-5-monophosphate (RMP) prior to formation of T-705-RTP. The anti-influenza virus activity of T-705 and T-1105 (3-hydroxy-2-pyrazinecarboxamide; the analog lacking the 6-fluoro atom) was lost in HGPRT-deficient Madin-Darby canine kidney cells. This HGPRT dependency was confirmed in human embryonic kidney 293T cells undergoing HGPRT-specific gene knockdown followed by influenza virus ribonucleoprotein reconstitution. Knockdown for adenine phosphoribosyltransferase (APRT) or nicotinamide phosphoribosyltransferase did not change the antiviral activity of T-705 and T-1105. Enzymatic assays showed that T-705 and T-1105 are poor substrates for human HGPRT having Km(app) values of 6.4 and 4.1 mM, respectively. Formation of the RMP metabolites by APRT was negligible, and so was the formation of the ribosylated metabolites by human purine nucleoside phosphorylase. Phosphoribosylation and antiviral activity of the 2-pyrazinecarboxamide derivatives was shown to require the presence of the 3-hydroxyl but not the 6-fluoro substituent. The crystal structure of T-705-RMP in complex with human HGPRT showed how this compound binds in the active site. Since conversion of T-705 by HGPRT appears to be inefficient, T-705-RMP prodrugs may be designed to increase the antiviral potency of this new antiviral agent.
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The effect of novel [3-fluoro-(2-phosphonoethoxy)propyl]purines on the inhibition of Plasmodium falciparum, Plasmodium vivax and human hypoxanthine-guanine-(xanthine) phosphoribosyltransferases.
Eur J Med Chem
PUBLISHED: 03-19-2013
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Protozoan parasites from the Plasmodiidae family are the causative agents of malaria. Inhibition of hypoxanthine-guanine-(xanthine) phosphoribosyltransferase (HG(X)PRT) has been suggested as a target for development of new anti-malarial therapeutics. Acyclic nucleoside phosphonates (ANPs) are potent and selective inhibitors of plasmodial HG(X)PRTs. A new series of ANPs, based on the chemical structure and inhibitory activity of three ANPs, 2-(phosphonoethoxy)ethyl with either guanine or hypoxanthine as the base (PEEG and PEEHx) and 3-hydroxy-2-(phosphonomethoxy)propyl with guanine as the base (HPMPG), were prepared. These compounds are stereoisomers of 3-fluoro-(2-phosphonoethoxy)propyl (FPEPs) and 3-fluoro-(2-phosphonomethoxy)propyl (FPMPs) analogues. Both the (R)- and (S)-isomers of these fluorinated derivatives have higher Ki values (by 10- to 1000-fold) for human HGPRT and Plasmodium falciparum HGXPRT than the non-fluorinated ANPs. Possible explanations for these changes in affinity are proposed based on docking studies using the known crystal structures of human HGPRT in complex with PEEG.
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Acyclic nucleoside phosphonates containing a second phosphonate group are potent inhibitors of 6-oxopurine phosphoribosyltransferases and have antimalarial activity.
J. Med. Chem.
PUBLISHED: 03-19-2013
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Acyclic nucleoside phosphonates (ANPs) that contain a 6-oxopurine base are good inhibitors of the Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) 6-oxopurine phosphoribosyltransferases (PRTs). Chemical modifications based on the crystal structure of 2-(phosphonoethoxy)ethylguanine (PEEG) in complex with human HGPRT have led to the design of new ANPs. These novel compounds contain a second phosphonate group attached to the ANP scaffold. {[(2-[(Guanine-9H-yl)methyl]propane-1,3-diyl)bis(oxy)]bis(methylene)}diphosphonic acid (compound 17) exhibited a Ki value of 30 nM for human HGPRT and 70 nM for Pf HGXPRT. The crystal structure of this compound in complex with human HGPRT shows that it fills or partially fills three critical locations in the active site: the binding sites of the purine base, the 5-phosphate group, and pyrophosphate. This is the first HG(X)PRT inhibitor that has been able to achieve this result. Prodrugs have been synthesized resulting in IC50 values as low as 3.8 ?M for Pf grown in cell culture, up to 25-fold lower compared to the parent compounds.
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Synthesis of purine N9-[2-hydroxy-3-O-(phosphonomethoxy)propyl] derivatives and their side-chain modified analogs as potential antimalarial agents.
Bioorg. Med. Chem.
PUBLISHED: 10-27-2011
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6-Oxopurine acyclic nucleoside phosphonates (ANPs) have been shown to be potent inhibitors of hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT), a key enzyme of the purine salvage pathway in human malarial parasites. These compounds also exhibit antimalarial activity against parasites grown in culture. Here, a new series of ANPs, hypoxanthine and guanine 9-[2-hydroxy-3-(phosphonomethoxy)propyl] derivatives with different chemical substitutions in the 2-position of the aliphatic chain were prepared and tested as inhibitors of Plasmodium falciparum (Pf) HGXPRT, Plasmodium vivax (Pv) HGPRT and human HGPRT. The attachment of an hydroxyl group to this position and the movement of the oxygen by one atom distal from N(9) in the purine ring compared with 2-(phosphonoethoxy)ethyl hypoxanthine (PEEHx) and 2-(phosphonoethoxy)ethyl guanine (PEEG) changes the affinity and selectivity for human HGPRT, PfHGXPRT and PvHGPRT. This is attributed to the differences in the three-dimensional structure of these inhibitors which affects their mode of binding. A novel observation is that these molecules are not always strictly competitive with 5-phospho-?-d-ribosyl-1-pyrophosphate. 9-[2-Hydroxy-3-(phosphonomethoxy)propyl]hypoxanthine (iso-HPMP-Hx) is a very weak inhibitor of human HGPRT but remains a good inhibitor of both the parasite enzymes with K(i) values of 2?M and 5?M for PfHGXPRT and PvHGPRT, respectively. The addition of pyrophosphate to the assay decreased the K(i) values for the parasite enzymes by sixfold. This suggests that the covalent attachment of a second group to the ANPs mimicking pyrophosphate and occupying its binding pocket could increase the affinity for these enzymes.
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Synthesis of 9-phosphonoalkyl and 9-phosphonoalkoxyalkyl purines: evaluation of their ability to act as inhibitors of Plasmodium falciparum, Plasmodium vivax and human hypoxanthine-guanine-(xanthine) phosphoribosyltransferases.
Bioorg. Med. Chem.
PUBLISHED: 08-09-2011
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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 ?M. 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 ?M 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.
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6-oxopurine phosphoribosyltransferase: a target for the development of antimalarial drugs.
Curr Top Med Chem
PUBLISHED: 05-31-2011
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Malaria remains the most serious parasitic diseases affecting humans in the world today, resulting in 1-2 million fatalities each year. Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) are the predominant causative agents. Both are responsible for widespread mortality and morbidity and are a serious socio-economic burden, especially for countries in the developing world. One of the most important defences against malaria has been the use of chemotherapeutic drugs (e.g. chloroquine, artemisinins, pyrimethamine) but these have mainly been found by serendipity. Their mechanisms was not understood at the time of their discovery and, even today, are still not unequivocal. For many of these compounds, the parasite is now resistant and, hence, there is an urgent need to develop new therapeutic drugs directed to validated targets. One metabolic pathway crucial for the survival and replication and survival of the parasite is the synthesis of the purine nucleoside monophosphates essential for the production of DNA/RNA molecules. A key enzyme in this pathway is the 6-oxopurine phosphoribosyltransferase (PRTase). The focus of this review is on the identification and characterization of inhibitors of the enzymes from both Pf and Pv as antimalarial drug leads. The acyclic nucleoside phosphonates (ANPs) appear to be excellent candidates because they are good inhibitors of the two Plasmodium enzymes, can be selective compared to the human enzyme, can arrest parasitemia in cell based assays, have low cytotoxicity to the human host cell and, because of their stable carbon-phosphorous bond, are stable within the cell.
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Plasmodium vivax hypoxanthine-guanine phosphoribosyltransferase: a target for anti-malarial chemotherapy.
Mol. Biochem. Parasitol.
PUBLISHED: 01-12-2010
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The malarial parasite, Plasmodium vivax (Pv), causes a serious infectious disease found primarily in Asia and the Americas. For protozoan parasites, 6-oxopurine phosphoribosyltransferases (PRTases) provide the only metabolic pathway to synthesize the purine nucleoside monophosphates essential for DNA/RNA production. We have purified the recombinant Pv 6-oxopurine (PRTase) and compared its properties with the human and Pf enzymes. The Pv enzyme uses hypoxanthine and guanine with similar catalytic efficiency to the Pf enzyme but xanthine is not a substrate, hence we identify this enzyme as PvHGPRT. Mass spectrometry suggests that PvHGPRT contains bound magnesium ions that are removed by EDTA resulting in loss of activity. However, the addition of Mg(2+) restores activity. Acyclic nucleoside phosphonates (ANPs) are good inhibitors of PvHGPRT having K(i) values as low as 3 microM. These compounds can form the basis for the design of new drugs aimed at combating malaria caused by Pv.
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Inhibition of hypoxanthine-guanine phosphoribosyltransferase by acyclic nucleoside phosphonates: a new class of antimalarial therapeutics.
J. Med. Chem.
PUBLISHED: 06-17-2009
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The purine salvage enzyme hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) is essential for purine nucleotide and hence nucleic acid synthesis in the malaria parasite, Plasmodium falciparum. Acyclic nucleoside phosphonates (ANPs) are analogues of the nucleotide product of the reaction, comprising a purine base joined by a linker to a phosphonate moiety. K(i) values for 19 ANPs were determined for Pf HGXPRT and the corresponding human enzyme, HGPRT. Values for Pf HGXPRT were as low as 100 nM, with selectivity for the parasite enzyme of up to 58. Structures of human HGPRT in complex with three ANPs are reported. On binding, a large mobile loop in the free enzyme moves to partly cover the active site. For three ANPs, the IC(50) values for Pf grown in cell culture were 1, 14, and 46 microM, while the cytotoxic concentration for the first compound was 489 microM. These results provide a basis for the design of potent and selective ANP inhibitors of Pf HGXPRT as antimalarial drug leads.
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Synthesis of branched 9-[2-(2-phosphonoethoxy)ethyl]purines as a new class of acyclic nucleoside phosphonates which inhibit Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase.
Bioorg. Med. Chem.
PUBLISHED: 06-16-2009
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The malarial parasite Plasmodium falciparum (Pf) lacks the de novo pathway and relies on the salvage enzyme, hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT), for the synthesis of the 6-oxopurine nucleoside monophosphates. Specific acyclic nucleoside phosphonates (ANPs) inhibit PfHGXPRT and possess anti-plasmodial activity. Two series of novel branched ANPs derived from 9-[2-(2-phosphonoethoxy)ethyl]purines were synthesized to investigate their inhibition of PfHGXPRT and human HGPRT. The best inhibitor of PfHGXPRT has a K(i) of 1 microM. The data showed that both the position and nature of the hydrophobic substituent change the potency and selectivity of the ANPs.
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Synthesis of novel N-branched acyclic nucleoside phosphonates as potent and selective inhibitors of human, Plasmodium falciparum and Plasmodium vivax 6-oxopurine phosphoribosyltransferases.
J. Med. Chem.
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Hypoxanthine-guanine-(xanthine) phosphoribosyltransferase (HG(X)PRT) is crucial for the survival of malarial parasites Plasmodium falciparum (Pf) and Plasmodium vivax (Pv). Acyclic nucleoside phosphonates (ANPs) are inhibitors of HG(X)PRT and arrest the growth of Pf in cell culture. Here, a novel class of ANPs containing trisubstituted nitrogen (aza-ANPs) has been synthesized. These compounds have a wide range of K(i) values and selectivity for human HGPRT, PfHGXPRT, and PvHGPRT. The most selective and potent inhibitor of PfHGXPRT is 9-[N-(3-methoxy-3-oxopropyl)-N-(2-phosphonoethyl)-2-aminoethyl]hypoxanthine (K(i) = 100 nM): no inhibition could be detected against the human enzyme. This compound exhibits the highest ever reported selectivity for PfHGXPRT compared to human HGPRT. For PvHGPRT, 9-[N-(2-carboxyethyl)-N-(2-phosphonoethyl)-2-aminoethyl]guanine has a K(i) of 50 nM, the best inhibitor discovered for this enzyme to date. Docking of these compounds into the known structures of human HGPRT in complex with ANP-based inhibitors suggests reasons for the variations in affinity, providing insights for the design of antimalarial drug candidates.
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