Dianne T. Keough

Inhibition of hypoxanthine-guanine phosphoribosyltransferase by acyclic nucleoside phosphonates: a new class of antimalarial therapeutics.

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.

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

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.

Iron-containing acid phosphatases: Comparison of the enzymes from beef spleen and pig allantoic fluid

The iron-containing violet acid phosphatases from beef spleen and pig allantoic fluid have been purified to homogeneity. Molecular weight determinations by zonal gel filtration, SDS-gel electrophoresis, and ultracentrifugation support values close to 40,000 for both enzymes, necessitating reappraisal of literature values. Similarly, the equivalent weight for iron is close to 20,000 for both enzymes, indicating the presence of two iron atoms per molecule of enzyme. The enzymes also have very similar ultraviolet and visible spectra, with λmax values close to 550 nm, and e{lunate}550 values(in terms of iron) of 2.04 × 103 and 2.00 × 103 for the beef spleen and pig allantoic fluid enzymes respectively.

Enzymatically active zinc, copper and mercury derivatives of the one-iron form of pig allantoic fluid acid phosphatase

Derivatives of the violet, iron-containing acid phosphatase of pig allantoic fluid have been prepared in which one of the two iron atoms present in the native enzyme has been replaced by zinc, copper or mercury. The derivatives so formed are enzymatically active: the Zn-Fe, Cu-Fe and Hg-Fe enzymes have specific activities of about 80%, 25% and 17% respectively, of the maximum specific activity of the Fe-Fe enzyme in the standard assay at pH 4.9 with p-nitrophenyl phosphate as substrate. In contrast to the Fe-Fe enzyme, the mixed metal derivatives are not rapidly inactivated by H2O2. Visible absorption spectra of the derivatives confirm that all of the visible absorption of the Fe-Fe enzyme is due to one of the iron atoms. Attempts to prepare an active Cu-Cu enzyme were unsuccessful.

Mössbauer and EPR study of the binuclear iron centre in purple acid phosphatase

Mössbauer spectra have been determined on 57Fe-enriched samples of both pink (reduced) and purple (oxidized) forms of pig allantoic acid phosphatase (EC 3.1.3.2), and EPR spectra on corresponding unenriched samples. The spectra show unambiguously that both forms of the enzyme contain two distinct, antiferromagnetically coupled, high-spin iron atoms: a ferrous-ferric ion pair in the pink, reduced form, and a pair of ferric ions in the purple, oxidized form.

Synthesis of novel N-branched acyclic nucleoside phosphonates as potent and selective inhibitors of human, Plasmodium falciparum and Plasmodium vivax 6-oxopurine phosphoribosyltransferases.

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.

The purine salvage enzyme hypoxanthine guanine xanthine phosphoribosyl transferase is a major target antigen for cell-mediated immunity to malaria

Although there is good evidence that immunity to the blood stages of malaria parasites can be mediated by different effector components of the adaptive immune system, target antigens for a principal component, effector CD4+ T cells, have never been defined. We generated CD4+ T cell lines to fractions of native antigens from the blood stages of the rodent parasite, Plasmodium yoelii, and identified fraction-specific T cells that had a Th1 phenotype (producing IL-2, IFN-γ, and tumor necrosis factor-α, but not IL-4, after antigenic stimulation). These T cells could inhibit parasite growth in recipient severe combined immunodeficient mice. N-terminal sequencing of the fraction showed identity with hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT). Recombinant HGXPRT from the human malaria parasite, Plasmodium falciparum, activated the T cells in vitro, and immunization of normal mice with recombinant HGXPRT reduced parasite growth rates in all mice after challenge.

Crystal Structures of Free, Imp-, and Gmp- Bound Escherichia Coli Hypoxanthine Phosphoribosyltransferase

Crystal structures have been determined for free Escherichia coli hypoxanthine phosphoribosyltransferase (HPRT) (2.9 Å resolution) and for the enzyme in complex with the reaction products, inosine 5′‐monophosphate (IMP) and guanosine 5′‐monophosphate (GMP) (2.8 Å resolution). Of the known 6‐oxopurine phosphoribosyltransferase (PRTase) structures, E. coli HPRT is most similar in structure to that of Tritrichomonas foetus HGXPRT, with a rmsd for 150 Cα atoms of 1.0 Å. Comparison of the free and product bound structures shows that the side chain of Phe156 and the polypeptide backbone in this vicinity move to bind IMP or GMP. A nonproline cis peptide bond, also found in some other 6‐oxopurine PRTases, is observed between Leu46 and Arg47 in both the free and complexed structures. For catalysis to occur, the 6‐oxopurine PRTases have a requirement for divalent metal ion, usually Mg2+ in vivo. In the free structure, a Mg2+ is coordinated to the side chains of Glu103 and Asp104. This interaction may be important for stabilization of the enzyme before catalysis. E. coli HPRT is unique among the known 6‐oxopurine PRTases in that it exhibits a marked preference for hypoxanthine as substrate over both xanthine and guanine. The structures suggest that its substrate specificity is due to the modes of binding of the bases. In E. coli HPRT, the carbonyl oxygen of Asp163 would likely form a hydrogen bond with the 2‐exocyclic nitrogen of guanine (in the HPRT‐guanine‐PRib‐PP‐Mg2+ complex). However, hypoxanthine does not have a 2‐exocyclic atom and the HPRT‐IMP structure suggests that hypoxanthine is likely to occupy a different position in the purine‐binding pocket.

Plasmodium vivax hypoxanthine-guanine phosphoribosyltransferase: a target for anti-malarial chemotherapy

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.

Iron-containing acid phosphatases: Characterization of the metal-ion binding site of the enzyme from pig allantoic fluid

Abstract All of the iron can be removed from the violet acid phosphatase of pig allantoic fluid by treatment with sodium dithionite at pH 4.9. Of the two moles of iron present per mole of enzyme (40,000 g), half is lost rapidly, and the remainder much more slowly. Removal of half of the iron causes complete loss of acid phosphatase activity. Conditions have been defined for the isolation and complete reconstitution [by Fe(II) and β-mercaptoethanol] of two apoenzymes, designated “iron-free” and “one-iron” apoenzymes. Zn 2+ ions restore most of the acid phosphatase activity to the one-iron apoenzyme but not to the iron-free enzyme. No metal ions other than Fe(II) and Fe(III) restore significant activity to the iron-free apoenzyme, but Zn(II) and Ni(II) bind tightly to it.