Novel nucleotide analogues bearing (1H-1,2,3-triazol-4-yl)phosphonic acid moiety as inhibitors of Plasmodium and human 6-oxopurine phosphoribosyltransferases

A novel family of acyclic nucleoside phosphonates (ANPs) bearing a (1H-1,2,3-triazol-4-yl)phosphonic acid group in the acyclic side chain have been prepared in order to study the influence of the hetaryl rigidizing element on the biological properties of such compounds. The key synthetic step consisted of a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) between diethyl ethynylphosphonate and the corresponding azidoalkyl precursor. Two ANPs in this family, bearing a guanine base, exhibited the highest potency for the human 6-oxopurine phosphoribosyltransferase irrespective of the stereochemistry on the C-2′ atom. Four compounds inhibited Plasmodium falciparum 6-oxopurine phosphoribosyltransferase with little differences in their Kvalues irrespective of whether the base was guanine, hypoxanthine or xanthine but only two, with guanine as base, inhibited PvHGPRT

Oligomeric state of hypoxanthine-guanine phosphoribosyltransferase from Mycobacterium tuberculosis

Sedimentation equilibrium and size-exclusion chromatography experiments on Mycobacterium tuberculosis hypoxanthine-guanine phosphoribosyltransferase (MtHGPRT) have established the existence of this enzyme as a reversibly associating mixture of dimeric and tetrameric species in 0.1\ua0M Tris-HCl−0.012\ua0M MgCl, pH 7.4. Displacement of the equilibrium position towards the larger oligomer by phosphate signifies the probable existence of MtHGPRT as a tetramer in the biological environment. These data thus add credibility to the relevance of considering enzyme function in the light of a published tetrameric structure deduced from X-ray crystallography. Failure of 5-phospho-α-D-ribosyl-1-pyrophosphate (PRib-PP) to perturb the dimer−tetramer equilibrium position indicates the equivalence and independence of binding for this substrate (the first to bind in an ordered sequential mechanism) to the two oligomers. By virtue of the displacement of the equilibrium position towards dimer that is affected by removing MgCl from the Tris-HCl buffer, it can be concluded that divalent metal ions, as well as phosphate, can affect the oligomerization. These characteristics of MtHGPRT in solution are correlated with published crystal structures of four enzyme−ligand complexes

Antimalarial activity of prodrugs of N-branched acyclic nucleoside phosphonate inhibitors of 6-oxopurine phosphoribosyltransferases

Acyclic nucleoside phosphonates (ANPs) that contain a 6-oxopurine base are good inhibitors of the human and Plasmodium falciparum 6-oxopurine phosphoribosyltransferases (PRTs), key enzymes of the purine salvage pathway. Chemical modifications, based on the crystal structures of several inhibitors in complex with the human PRTase, led to the design of a new class of inhibitors - the aza-ANPs. Because of the negative charges of the phosphonic acid moiety, their ability to cross cell membranes is, however, limited. Thus, phosphoramidate prodrugs of the aza-ANPs were prepared to improve permeability. These prodrugs arrest parasitemia with IC values in the micromolar range against Plasmodium falciparum-infected erythrocyte cultures (both chloroquine-sensitive and chloroquine-resistant Pf strains). The prodrugs exhibit low cytotoxicity in several human cell lines. Thus, they fulfill two essential criteria to qualify them as promising antimalarial drug leads

Pyrrolidine nucleoside bisphosphonates as antituberculosis agents targeting hypoxanthine-guanine phosphoribosyltransferase

Therapeutic treatment of tuberculosis (TB) is becoming increasingly problematic due to the emergence of drug resistant Mycobacterium tuberculosis (Mt). Thus, new targets for anti-TB drug discovery need to be identified to combat and eradicate this disease. One such target is hypoxanthine-guanine phosphoribosyltransferase (HGPRT) which synthesises the 6-oxopurine nucleoside monophosphates essential for DNA/RNA production. [3R,4R]-4-Hypoxanthin-9-yl-3-( (S)-2-hydroxy-2-phosphonoethyl)oxy-1-N-(phosphonopropionyl)pyrrolidine and [3R,4R-4-guanin-9-yl-3-((S)-2-hydroxy-2-phosphonoethyl)oxy-1-N-(phosphonopropionyl)pyrrolidine (compound 6) are the most potent inhibitors of MtHGPRT yet discovered having K-i values of 60 nM. The crystal structure of the MtHGPRT.6 complex was obtained and compared with that of human HGPRT in complex with the same inhibitor. These structures provide explanations for the 60-fold difference in the inhibition constants between these two enzymes and a foundation for the design of next generation inhibitors. In addition, crystal structures of MtHGPRT in complex with two pyrrolidine nucleoside phosphosphonate inhibitors plus pyrophosphate provide insights into the final stage of the catalytic reaction. As the first step in ascertaining if such compounds have the potential to be developed as anti-TB therapeutics, the tetra-(ethyl L-phenylalanine) tetraamide prodrug of 6 was tested in cell based assays. This compound arrested the growth of virulent Mt not only in its replicating phase (IC50 of 14 mu M) but also in its latent phase (IC50 of 29 mu M). Furthermore, it arrested the growth of Mt in infected macrophages (MIC50 of 85 mu M) and has a low cytotoxicity in mammalian cells (CC50 of 132 +/- 20 mu M). These inhibitors are therefore viewed as forerunners of new anti-TB chemotherapeutics. (C) 2018 Elsevier Masson SAS. All rights reserved

Effects of dredging-related pressures on critical ecological processes for organisms other than fish or coral. Report of Theme 9 - Project 9.1 prepared for the Dredging Science Node

In November 2013 a workshop was held at CSIRO Floreat, which brought together national and international marine scientists. The workshop addressed two primary objectives: • identify the timing of critical ecological processes in tropical and temperate ecosystems with a focus on non-coral and non-fish biota (seagrass, seaweed, sponges, ascidians, bryozoans, molluscs, echinoderms, crustaceans and non-coral cnidarians); and • identify environmental windows for critical ecological processes identified in Objective 1. This will be achieved by compiling information on the timing of reproduction, release of propagules and recruitment for these organisms, as well as the temporal and spatial scales of reproduction and recruitment events. During Workshop 1 a conceptual diagram was developed to illustrate and guide the decision process behind the selection of environmental windows (EWs) (see Figure 1). The life histories of the biota investigated were then identified and listed in detailed tables with specific reference to potential effects of dredging at each life history stage..

Localization of the 5-phospho-α-d-ribosyl-1-pyrophosphate binding site of human hypoxanthine-guanine phosphoribosyltransferase

Human erythrocyte hypoxanthine-guanine phosphoribosyltransferase (HPRT) is inactivated by iodoacetate in the absence, but not in the presence, of the substrate, 5-phospho-α-d-ridosyl-1-pyrophosphate (PRib-PP). Treatment of HPRT with [C]iodoacetate followed by tryptic digestion, peptide separation and sequencing has shown that Cys-22 reacts with iodoacetate only in the absence of PRib-PP; this strongly suggests that Cys-22 is in or near the PRib-PP binding site. In contrast, Cys-105 reacts with [C]iodoacetate both in the presence and absence of PRib-PP. Carboxymethylation of Cys-22 resulted in an increase in the K for PRib-PP, but no change in V. Storage of HPRT also resulted in an increase in the K for PRib-PP and a decrease in its susceptibility to inactivation by iodoacetate. Dialysis of stored enzyme against 1 mM dithiothreitol resulted in a marked decrease in K for PRib-PP. The stoichiometry of the reaction of [C]iodoacetate with Cys-22 in HPRT leading to inactivation (approx. 1 residue modified per tetramer) showed that, in this preparation of HPRT purified from erythrocytes, only about 25% of the Cys-22 side chains were present as free and accessible thiols. Titration of thiol groups 5,5′-dithiobis(2-nitro-benzoic acid)] and the effect of dithiothreitol on K for PRib-PP indicate that oxidation of thiol groups occurs on storage of HPRT, even in the presence of 1mM β-mercaptoethanol

Structure-based development of Plasmodium hypoxanthine-guanine phosphoribosyltransferase inhibitors: a proof of concept study

Structure-based development of Plasmodium hypoxanthine-guanine phosphoribosyltransferase inhibitors: a proof of concept study Manon Laportea, Dana Hockováb, Dianne T. Keoughc, Luke W. Guddatc, Lieve Naesensa aRega Institute, KU Leuven, Belgium; bInstitute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic, cSchool of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Australia The malarial parasites Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) are purine auxotrophs that rely on the salvage of host purines for their survival and growth. A critical enzyme in this salvage pathway, hypoxanthine guanine phosphoribosyltransferase (HGPRT), is considered a prime target for antimalarial therapy. Our collaboration between the University of Queensland, the Institute of Organic Chemistry and Biochemistry (Prague) and the Rega institute (KU Leuven), is focussed on rational development of novel acyclic nucleoside phosphonates (ANPs) that are structural analogues of the HGPRT nucleotide reaction products IMP and GMP. Based on available HGPRT crystal structures, ANP derivatives with a second phosphonate group attached (bisANPs) were designed. These compounds proved to be particularly strong HGPRT inhibitors with Ki values as low as 30 nM, and displayed antimalarial activity in Pf-infected erythrocytes with IC50 values as low as 3.8 µM (Keough et al. 2013). However, since the Plasmodium parasite possesses several enzymes that could possibly serve as the target for inhibition by the (bis)ANPs, either directly or after metabolic conversion, it remained to be established whether the observed antiparasitic effect indeed results from HGPRT inhibition. We here present a target validation assay to assess, in a cellular environment, the inhibitory effect of (bis)ANPs towards HGPRT. This method complements the enzymatic assays (in which purified HGPRT enzymes are studied in a cell-free environment), and the Plasmodium cell culture assay (that involves replication of the whole Pf parasite). First, we created adenoviral (Ad) vectors containing the cDNA sequences encoding human, Pf or PvHGPRT. Transduction of these Ad vectors into HGPRT-deficient human 1306 cells generated high HGPRT expression levels, as estimated by a tritium release assay with [2,8-3H]hypoxantine (Balzarini and De Clercq, 1992). Several (bis)ANPs were shown to inhibit the HGPRT reaction by the human or Pv enzyme. Our novel assay allows to validate Plasmodium HGPRT inhibitors in cell culture and will be instrumental to guide further development of this new class of antimalarial drugs.status: submitte

The crystal structure of free human hypoxanthineguanine phosphoribosyltransferase reveals extensive conformational plasticity throughout the catalytic cycle

Human hypoxanthine-guanine phosphoribosyltransferase (HGPRT) catalyses the synthesis of the purine nucleoside monophosphates, IMP and GMP, by the addition of a 6-oxopurine base, either hypoxanthine or guanine, to the 1-beta-position of 5-phospho-U-D-ribosyl-1-pyrophosphate (PRib-PP). The mechanism is sequential, with PRib-PP binding to the free enzyme prior to the base. After the covalent reaction, pyrophosphate is released followed by the nucleoside monophosphate. A number of snapshots of the structure of this enzyme along the reaction pathway have been captured. These include the structure in the presence of the inactive purine base analogue, 7-hydroxy [4,3-d] pyrazolo pyrimidine (HPP) and PRib-PP. Mg2+, and in complex with IMP or GMP. The third structure is that of the immucillinHP.Mg2+.PPi complex, a transition-state analogue. Here, the first crystal structure of free human HGPRT is reported to 1.9 angstrom resolution, showing that significant conformational changes have to occur for the substrate(s) to bind and for catalysis to proceed. Included in these changes are relative movement of subunits within the tetramer, rotation and extension of an active-site alpha-helix (D137-D153), reorientation of key active-site residues K68, D137 and K165, and the rearrangement of three active-site loops (100-128, 165-173 and 186-196). Toxoplasina gondii HGXPRT is the only other 6-oxopurine phosphoribosyltransferase structure solved in the absence of ligands. Comparison of this structure with human HGPRT reveals significant differences in the two active sites, including the structure of the flexible loop containing K68 (human) or K79 (T gondii). (c) 2005 Elsevier Ltd. All rights reserved