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

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

Normal HPRT coding region in a male with gout due to HPRT deficiency (Brief Communication)

A deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT; EC 2.4.2.8) is associated with a spectrum of disease that ranges from gouty arthritis (OMIM 300323) to the more severe Lesch-Nyhan syndrome (OMIM 300322). To date, all cases of HPRT deficiency have shown a mutation within the HPRT cDNA. In the present study of an individual with gout due to HPRT deficiency, we found a normal HPRT cDNA sequence. This is the first study to provide an example of HPRT deficiency which appears to be due to a defect in the regulation of the gene. © 2005 Elsevier Inc. All rights reserved

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

Human hypoxanthine-guanine phosphoribosyltransferase. Development of a spectrophotometric assay and its use in detection and characterization of mutant forms

A simple and rapid spectrophotometric assay for the estimation of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity in human tissues is described. It is based on the increase in absorbance at 257.5 nm which occurs when the substrate guanine is converted to its 5'-mononucleotide, GMP. The assay has been developed to measure HGPRT activity in erythrocyte and lymphocyte lysates and in brain homogenates, and has been used in the screening of patients with hyperuricaemia and/or hyperuricosuria for HGPRT deficiency. It has also been used to determine the steady-state kinetic constants of a mutant form of the enzyme. The spectrophotometric assay is compared with the radioactive assay currently used to measure HGPRT activity

Synthesis of purine N-9-[2-hydroxy-3-O-(phosphonomethoxy)propyl] derivatives and their side-chain modified analogs as potential antimalarial agents

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 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-α-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 values of 2 μM and 5 μM for PfHGXPRT and PvHGPRT, respectively. The addition of pyrophosphate to the assay decreased the K 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

Crystal structures of Trypanosoma brucei hypoxanthine – guanine – xanthine phosphoribosyltransferase in complex with IMP , GMP and XMP

The 6-oxopurine phosphoribosyltransferases (PRTs) are drug targets for the treatment of parasitic diseases. This is due to the fact that parasites are auxotrophic for the 6-oxopurine bases relying on salvage enzymes for the synthesis of their 6-oxopurine nucleoside monophosphates. In Trypanosoma brucei, the parasite that is the aetiological agent for sleeping sickness, there are three 6-oxopurine PRT isoforms. Two are specific for hypoxanthine and guanine, whilst the third, characterized here, uses all three naturally occurring bases with similar efficiency. Here, we have determined crystal structures for TbrHGXPRT in complex with GMP, XMP and IMP to investigate the structural basis for substrate specificity. The results show that Y201 and E208, not commonly observed within the purine binding pocket of 6-oxopurine PRTs, contribute to the versatility of this enzyme. The structures further show that a nearby water can act as an adaptor to facilitate the binding of XMP and GMP. When GMP binds, a water can accept a proton from the 2-amino group but when XMP binds, the equivalent water can donate its proton to the 2-oxo group. However, when IMP is bound, no water molecule is observed at that location. DATABASE: Coordinates and structure factors were submitted to the Protein Data Bank and have accession codes of 6MXB, 6MXC, 6MXD and 6MXG for the TbrHGXPRT.XMP complex, TbrHGXPRT.GMP complex, TbrHGXPRT.IMP complex, and TbrHGPRT.XMP complex, respectively