Human Purine Research Articles

Capillary bioreactors based on human purine nucleoside phosphorylase: A new approach for ligands identification and characterization

The enzyme purine nucleoside phosphorylase (PNP) is a target for the discovery of new lead compounds employed on the treatment severe T-cell mediated disorders. Within this context, the development of new, direct, and reliable methods for ligands screening is an important task. This paper describes the preparation of fused silica capillaries human PNP (HsPNP) immobilized enzyme reactor (IMER). The activity of the obtained IMER is monitored on line in a multidimensional liquid chromatography system, by the quantification of the product formed throughout the enzymatic reaction. The KM value for the immobilized enzyme was about twofold higher than that measured for the enzyme in solution (255±29.2μM and 133±14.9μM, respectively). A new fourth-generation immucillin derivative (DI4G; IC50=40.6±0.36nM), previously identified and characterized in HsPNP free enzyme assays, was used to validate the IMER as a screening method for HsPNP ligands. The validated method was also used for mechanistic studies with this inhibitor. This new approach is a valuable tool to PNP ligand screening, since it directly measures the hypoxanthine released by inosine phosphorolysis, thus furnishing more reliable results than those one used in a coupled enzymatic spectrophotometric assay.

The human caveolin 1 gene upstream purine complex and neurodegeneration—A common signature

The caveolin 1 gene ( CAV1) is over-expressed in experimental animal models of multiple sclerosis (MS). Increased expression of this gene has also been reported in the Alzheimer's disease (AD) brain. Loss of this gene, on the other hand, has recently been reported to be associated with neruodegeneration. We have recently reported skew in the homozygote haplotypes of the human CAV1 gene − 1.5 kb upstream purine complex in patients afflicted with MS and late-onset AD vs. controls. In order to examine reproducibility of those findings, we sequenced the region in independent groups of MS patients (n = 120) and controls (n = 150). We report two novel extreme homozygote haplotypes at 86-bp and 142-bp in the patients vs. controls. The above haplotypes were also detected in the previously reported cases of late-onset AD. The range of homozygote haplotypes in the controls was detected at between 106-bp to 122-bp. Following pooling of the neurodegenerative (n = 486) and non-neurodegenerative (n = 610) subjects studied for the human CAV1 purine complex to date, twenty haplotypes were found to be homozygous in the neurodegenerative, and not in the control pool (p < 0.000001). Six overlapping haplotypes were detected in the MS and AD patients (p < 0.007), strengthening the role of this region as a common etiological factor in the pathophysiology of neurodegenerative disorders, possibly through inflammatory mechanisms. Those overlapping haplotypes contained motif lengths that were non-existent in the control homozygote pool. The CAV1 purine complex GGAA and GAAA motifs are binding sites for numerous inflammatory transcription factors including the Ets, STAT, and IRF family members. Further work on the functionality of this region will shed light on the downstream events to the disease-linked haplotypes.

Unraveling the structural and functional differences between purine nucleoside phosphorylase and 5′-deoxy-5′-methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus

Purine nucleoside metabolism in the archaeon Pyrococcus furiosus is catalyzed by purine nucleoside phosphorylase (PfPNP) and 5′-deoxy-5′-methylthioadenosine phosphorylase (PfMTAP). These enzymes, characterized by 50% amino acid sequence identity, show non-common features of thermophilicity and thermostability and are stabilized by intramolecular disulfide bonds. PfPNP is highly specific for 6-oxopurine nucleosides while PfMTAP is characterized by a broad substrate specificity with 6-aminopurine nucleosides as preferred substrates. Amino acid sequence comparison clearly shows that the hypothetical active sites of PfPNP and PfMTAP are almost identical and that, in analogy with human 5′-deoxy-5′-methylthioadenosine phosphorylase and human purine nucleoside phosphorylase, residue changes at level of the same crucial positions could be responsible for the switch of substrate specificity. To validate this hypothesis we changed the putative active site of PfPNP by site-directed mutagenesis. Substrate specificity and catalytic efficiency of PfPNP mutants were then analyzed by kinetic studies and compared with the wild-type enzyme. We carried out the molecular modeling of PfPNP and PfMTAP to obtain a picture of the overall enzyme structure and to identify structural features as well as interactions playing critical roles in thermostability. Finally, we utilized the structural models of mutant enzyme–substrate complex to rationalize the functional effects of the mutations.

Arsenate and Phosphate as Nucleophiles at the Transition States of Human Purine Nucleoside Phosphorylase

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of 6-oxypurine (2'-deoxy)ribonucleosides, generating (2-deoxy)ribose 1-phosphate and the purine base. Transition-state models for inosine cleavage have been proposed with bovine, human, and malarial PNPs using arsenate as the nucleophile, since kinetic isotope effects (KIEs) are obscured on phosphorolysis due to high commitment factors. The Phe200Gly mutant of human PNP has low forward and reverse commitment factors in the phosphorolytic reaction, permitting the measurement of competitive intrinsic KIEs on both arsenolysis and phosphorolysis of inosine. The intrinsic 1'-(14)C, 1'-(3)H, 2'-(2)H, 9-(15)N, and 5'-(3)H(2) KIEs for inosine were measured for arsenolysis and phosphorolysis. Except for the remote 5'-(3)H(2), and some slight difference between the 2'-(2)H KIEs, all isotope effects originating in the reaction coordinate are the same within experimental error. Hence, arsenolysis and phosphorolysis proceed through closely related transition states. Although electrostatically similar, the volume of arsenate is greater than phosphate and supports a steric influence to explain the differences in the 5'-(3)H(2) KIEs. Density functional theory calculations provide quantitative models of the transition states for Phe200Gly human PNP-catalyzed arsenolysis and phosphorolysis, selected upon matching calculated and experimental KIEs. The models confirm the striking resemblance between the transition states for the two reactions.

Conformational Dynamics in Human Purine Nucleoside Phosphorylase with Reactants and Transition-State Analogues

Dynamic motions of human purine nucleoside phosphorylase (hPNP) in complex with transition-state analogues and reactants were studied using 10 ns explicit solvent molecular dynamics simulations. hPNP is a homotrimer that catalyzes the phosphorolysis of purine 6-oxynucleosides. The ternary complex of hPNP includes the binding of a ligand and phosphate to the active site. Molecular dynamics simulations were performed on the ternary complex of six ligands including the picomolar transition-state analogues, Immucillin-H (K(d) = 56 pM), DADMe-Immucillin-H (K(d) = 8.5 pM), DATMe-Immucillin-H (K(d) = 8.6 pM), SerMe-Immucillin-H (K(d) = 5.2 pM), the substrate inosine, and a complex containing only phosphate. Protein-inhibitor complexes of the late transition-state inhibitors, DADMe-Imm-H and DATMe-Imm-H, are inflexible. Despite the structural similarity of SerMe-Imm-H and DATMe-Imm-H, the protein complex of SerMe-Imm-H is flexible, and the inhibitor is highly mobile within the active sites. All inhibitors exhibit an increased number of nonbonding interactions in the active site relative to the substrate inosine. Water density within the catalytic site is lower for DADMe-ImmH, DATMe-Imm-H, and SerMe-Imm-H than that for the substrate inosine. Tight binding of the picomolar inhibitors results from increased interactions within the active site and a reduction in the number of water molecules organized within the catalytic site relative to the substrate inosine.

Modelling the effects of inhibitors of guanine nucleotide synthesis: implications for studies of cellular differentiation pathways

Mizoribine induces the differentiation of promyelocytes by an unknown mechanism that relies on compromised guanine nucleotide synthesis. I have found that mizoribine also perturbs adenosine nucleotide levels in HL-60 promyelocytes, particularly ATP. To reconcile these observations with the known actions of mizoribine I have adapted an existing model of human purine metabolism composed as an S-system familiar from Biochemical Systems Theory. Mizoribine's actions were then simulated and compared with experimental data.

Structural studies of tri-functional human GART

Human purine de novo synthesis pathway contains several multi-functional enzymes, one of which, tri-functional GART, contains three enzymatic activities in a single polypeptide chain. We have solved structures of two domains bearing separate catalytic functions: glycinamide ribonucleotide synthetase and aminoimidazole ribonucleotide synthetase. Structures are compared with those of homologous enzymes from prokaryotes and analyzed in terms of the catalytic mechanism. We also report small angle X-ray scattering models for the full-length protein. These models are consistent with the enzyme forming a dimer through the middle domain. The protein has an approximate seesaw geometry where terminal enzyme units display high mobility owing to flexible linker segments. This resilient seesaw shape may facilitate internal substrate/product transfer or forwarding to other enzymes in the pathway.

ChemInform Abstract: Certain 3,9-Dideazapurines as Inhibitors of Purine Nucleoside Phosphorylase.

Abstract The synthesis of four 3,9-dideazapurines is described. In order to achieve the desired substitution pattern, a new approach to the pyrrolo[2, 3-c]pyridine ring system was devised utilizing the Gassman reaction as the key step for its construction. The four target heterocycles were all evaluated for their ability to inhibit human erythrocytic purine nucleoside phosphorylase.

232. Ex Vivo Lentiviral Transduction of the Human Purine Nucleoside Phosphorylase (PNP) Gene in Bone Marrow Derived Hematopoietic Stem Cells: An In Vivo Model of Gene Therapy for PNP Deficient T-Cell Immunodeficiency

232. Ex Vivo Lentiviral Transduction of the Human Purine Nucleoside Phosphorylase (PNP) Gene in Bone Marrow Derived Hematopoietic Stem Cells: An In Vivo Model of Gene Therapy for PNP Deficient T-Cell Immunodeficiency

Effect of palmitoyl CoA on ADP‐evoked vasorelaxations in porcine isolated coronary and mesenteric arteries

Palmitoyl CoA (PaCoA) is an antagonist at human recombinant P2Y1 purine receptors (Coddou et al., 2003), HEK cells, human platelets (Manolopoulos et al., 2008), and rat thoracic aorta. The present study investigated the effect of PaCoA on vasorelaxation evoked by ADP in porcine isolated mesenteric and coronary arteries.In isometric tension recordings, ADP evoked concentration‐dependent relaxations of the pre‐contracted porcine mesenteric and coronary arteries with maximal responses of 40 ± 12 and 95 ± 7% and pEC50 values of 4.26 ± 0.33 and 3.27 ± 0.85 (n = 7–12), respectively. PaCoA (10 μM) had no significant effect on relaxations in the coronary artery but abolished relaxations in the mesenteric artery. Endothelium removal abolished ADP‐induced relaxations in the mesenteric but not coronary artery.The preferential inhibition by PaCoA of ADP‐evoked relaxations in the mesenteric, but not coronary, artery indicates the involvement of distinct mechanisms: endothelial P2Y1 receptors mediate vasorelaxation to ADP in the mesenteric artery while in the coronary artery ADP‐induced relaxation occurs via receptors on the smooth muscle that may involve adenosine release and activation of A2A receptors (Rayment et al., 2006).Coddou et al. (2003). FEBS Lett 536: 145–150Manolopoulos et al. (2008). Platelets 19: 134–145Rayment et al. (2007) FASEB J 21: 577–585Sponsored by the Jordanian government.

Phenotypes in utilization of formate for purine nucleotide biosynthesis de novo in adult humans

Folate coenzymes and their dependent enzymes introduce one‐carbon units at positions 2 (C2) and 8 (C8) of the purine ring during de novo biosynthesis. Formate is naturally occurring in humans and is one source of one‐carbon units. Although much is known about lower organisms, little data exist describing formate utilization for human purine biosynthesis. After IRB approval, mass spectrometric analysis of urinary uric acid, the final purine catabolite, following 1.0 g oral doses of [13C]sodium formate was performed in 6 healthy male adults. Using this method, we are can detect 13C enrichment at C2 and C8 independently. Our data suggested the presence of three phenotypes including: one phenotype where 13C was incorporated into C2 much greater than into C8, and in another, there was little or no 13C incorporation into the C2 or C8 position. In the third phenotype, 13C was incorporated into C8; but, C2 incorporation was greater. In subjects who incorporated [13C]formate into C2, peak enrichment occurred in voids from the 8–12 h period (24‐h clock). Our results are not similar to those in non‐mammalian organisms or cultured animal cells. The presence of these phenotypes may reflect genotypes in the purine biosynthesis pathway.

Four generations of transition-state analogues for human purine nucleoside phosphorylase

Inhibition of human purine nucleoside phosphorylase (PNP) stops growth of activated T-cells and the formation of 6-oxypurine bases, making it a target for leukemia, autoimmune disorders, and gout. Four generations of ribocation transition-state mimics bound to PNP are structurally characterized. Immucillin-H (K*i(1/4) 58 pM, first generation)contains an iminoribitol cation with four asymmetric carbons. DADMe-Immucillin-H (K*i(1/4) 9 pM, second-generation),uses a methylene-bridged dihydroxypyrrolidine cation with twoasymmetric centers.DATMe-Immucillin-H (K*i(1/4)9 pM, third-generation) contains an open-chain amino alcohol cation with two asymmetric carbons. SerMe-ImmH (K*i(1/4) 5 pM, fourth-generation) uses achiral dihydroxyaminoalcohol seramide as the ribocation mimic. Crystal structures of PNPs establish features of tight binding to be; 1) ion-pair formation between bound phosphate (or its mimic) and inhibitor cation, 2) leaving-group interactions to N1, O6, and N7 of 9-deazahypoxanthine, 3) interaction between phosphate and inhibitor hydroxyl groups, and 4) His257 interacting with the 5'-hydroxyl group. The first generation analogue is an imperfect fit to the catalytic site with a long ion pair distance between the iminoribitol and bound phosphate and weaker interactions to the leaving group. Increasing the ribocation to leaving-group distance in the second- to fourth-generation analogues provides powerful binding interactions and a facile synthetic route to powerful inhibitors. Despite chemical diversity in the four generations of transition-state analogues, the catalytic site geometry is almost the same for all analogues. Multiple solutions in transition-state analogue design are available to convert the energy of catalytic rate enhancement to binding energy in human PNP.

Conformational States of Human Purine Nucleoside Phosphorylase at Rest, at Work, and with Transition State Analogues

Human purine nucleoside phosphorylase (PNP) is a homotrimer binding tightly to the transition state analogues Immucillin-H (ImmH; K(d) = 56 pM) and DATMe-ImmH-Immucillin-H (DATMe-ImmH; K(d) = 8.6 pM). ImmH binds with a larger entropic penalty than DATMe-ImmH, a chemically more flexible inhibitor. The testable hypothesis is that PNP conformational states are more relaxed (dynamic) with DATMe-ImmH, despite tighter binding than with ImmH. PNP conformations are probed by peptide amide deuterium exchange (HDX) using liquid chromatography high-resolution Fourier transform ion cyclotron resonance mass spectrometry and by sedimentation rates. Catalytically equilibrating Michaelis complexes (PNP.PO(4).inosine <--> PNP.Hx.R-1-P) and inhibited complexes (PNP.PO(4).DATMe-ImmH and PNP.PO(4).ImmH) show protection from HDX at 9, 13, and 15 sites per subunit relative to resting PNP (PNP.PO(4)) in extended incubations. The PNP.PO(4).ImmH complex is more compact (by sedimentation rate) than the other complexes. HDX kinetic analysis of ligand-protected sites corresponds to peptides near the catalytic sites. HDX and sedimentation results establish that PNP protein conformation (dynamic motion) correlates more closely with entropy of binding than with affinity. Catalytically active turnover with saturated substrate sites causes less change in HDX and sedimentation rates than binding of transition state analogues. DATMe-ImmH more closely mimics the transition of human PNP than does ImmH and achieves strong binding interactions at the catalytic site while causing relatively modest alterations of the protein dynamic motion. Transition state analogues causing the most rigid, closed protein conformation are therefore not necessarily the most tightly bound. Close mimics of the transition state are hypothesized to retain enzymatic dynamic motions related to transition state formation.

Inhibition of human purine nucleoside phosphorylase by tenofovir phosphate congeners

The structure-activity study on the phosphates of phosphonomethoxypropyl derivatives of purine bases interacting with human purine nucleoside phosphorylase has shown that the most efficient inhibitors of the enzyme are (R)- and (S)-PMPGp with Ki ~ 1.9 × 10–8 and/or 2.2 × 10–8 mol/l. The kinetic experiments have proven, with the exception of both enantiomers of PMP-8-BrDAPp, strictly competitive character of inhibition for all ANP monophosphates tested. Bromine derivatives exhibited uncompetitive and mixed type of inhibition as well. These results were confirmed by docking studies. The substitution of purine moiety with the bromine at the position 8 lead to an allosteric binding of these compounds toward the enzyme.

Characterization of an engineered human purine nucleoside phosphorylase fused to an anti-her2/neu single chain Fv for use in ADEPT

BackgroundAntibody Directed Enzyme Prodrug Therapy (ADEPT) can be used to generate cytotoxic agents at the tumor site. To date non-human enzymes have mainly been utilized in ADEPT. However, these non-human enzymes are immunogenic limiting the number of times that ADEPT can be administered. To overcome the problem of immunogenicity, a fully human enzyme, capable of converting a non-toxic prodrug to cytotoxic drug was developed and joined to a human tumor specific scFv yielding a fully human targeting agent.MethodsA double mutant of human purine nucleoside phosphorylase (hDM) was developed which unlike the human enzyme can cleave adenosine-based prodrugs. For tumor-specific targeting, hDM was fused to the human anti-HER2/neu single chain Fv (scFv), C6 MH3B1. Enzymatic activity of hDM with its natural substrates and prodrugs was determined using spectrophotomeric approaches. A cell proliferation assay was used to assess the cytotoxicity generated following conversion of prodrug to drug as a result of enzymatic activity of hDM. Affinity of the targeting scFv, C6 MH3B1 fused to hDM to Her2/neu was confirmed using affinity chromatography, surface plasmon resonance, and flow-cytometry.ResultsIn vitro hDM-C6 MH3B1 binds specifically to HER2/neu expressing tumor cells and localizes hDM to tumor cells, where the enzymatic activity of hDM-C6 MH3B1, but not the wild type enzyme, results in phosphorolysis of the prodrug, 2-fluoro-2'-deoxyadenosine to the cytotoxic drug 2-fluoroadenine (F-Ade) causing inhibition of tumor cell proliferation. Significantly, the toxic small drug diffuses through the cell membrane of HER2/neu expressing cells as well as cells that lack the expression of HER2/neu, causing a bystander effect. F-Ade is toxic to cells irrespective of their growth rate; therefore, both the slowly dividing tumor cells and the non-dividing neighboring stromal cells that support tumor growth should be killed. Analysis of potential novel MHCII binding peptides resulting from fusion of hDM to C6 MH3B1 and the two mutations in hDM, and of the structure of hDM compared to the wild-type enzyme suggests that hDM-C6 MH3B1 should exhibit minimal immunogenicity in humans.ConclusionhDM-C6 MH3B1 constitutes a novel human based protein that addresses some of the limitations of ADEPT that currently preclude its successful use in the clinic.

Crystal structure and molecular dynamics studies of human purine nucleoside phosphorylase complexed with 7-deazaguanine

In humans, purine nucleoside phosphorylase (HsPNP) is responsible for degradation of deoxyguanosine, and genetic deficiency of this enzyme leads to profound T-cell mediated immunosuppression. HsPNP is a target for inhibitor development aiming at T-cell immune response modulation. Here we report the crystal structure of HsPNP in complex with 7-deazaguanine (HsPNP:7DG) at 2.75 A. Molecular dynamics simulations were employed to assess the structural features of HsPNP in both free form and in complex with 7DG. Our results show that some regions, responsible for entrance and exit of substrate, present a conformational variability, which is dissected by dynamics simulation analysis. Enzymatic assays were also carried out and revealed that 7-deazaguanine presents a lower inhibitory activity against HsPNP (K(i)=200 microM). The present structure may be employed in both structure-based design of PNP inhibitors and in development of specific empirical scoring functions.

Ribocation Transition State Capture and Rebound in Human Purine Nucleoside Phosphorylase

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of 6-oxy-purine nucleosides to the corresponding purine base and alpha-D-ribose 1-phosphate. Its genetic loss causes a lethal T cell deficiency. The highly reactive ribocation transition state of human PNP is protected from solvent by hydrophobic residues that sequester the catalytic site. The catalytic site was enlarged by replacing individual catalytic site amino acids with glycine. Reactivity of the ribocation transition state was tested for capture by water and other nucleophiles. In the absence of phosphate, inosine is hydrolyzed by native, Y88G, F159G, H257G, and F200G enzymes. Phosphorolysis but not hydrolysis is detected when phosphate is bound. An unprecedented N9-to-N3 isomerization of inosine is catalyzed by H257G and F200G in the presence of phosphate and by all PNPs in the absence of phosphate. These results establish a ribocation lifetime too short to permit capture by water. An enlarged catalytic site permits ribocation formation with relaxed geometric constraints, permitting nucleophilic rebound and N3-inosine isomerization.

Punching Holes in an Enzyme

Schramm and coworkers have punched holes into human purine nucleoside phosphorylase by substitution of glycine for aromatic amino acid residues at a protein lid. The results of studies on the enzymes with holes illuminate hidden chemistry that occurs at the enzyme active site.

Alternative route towards the convergent synthesis of a human purine nucleoside phosphorylase inhibitor—forodesine HCl

Forodesine HCl is being investigated as a potential target for the control of T-cell proliferation. Herein we present an alternative route for the synthesis of the target molecule with addition of lactam to the lithiated deazahypoxanthine (generated in situ). The lactam was synthesized in five steps starting from l-pyroglutamic acid.

Altered Enthalpy−Entropy Compensation in Picomolar Transition State Analogues of Human Purine Nucleoside Phosphorylase

Human purine nucleoside phosphorylase (PNP) belongs to the trimeric class of PNPs and is essential for catabolism of deoxyguanosine. Genetic deficiency of PNP in humans causes a specific T-cell immune deficiency, and transition state analogue inhibitors of PNP are in development for treatment of T-cell cancers and autoimmune disorders. Four generations of Immucillins have been developed, each of which contains inhibitors binding with picomolar affinity to human PNP. Full inhibition of PNP occurs upon binding to the first of three subunits, and binding to subsequent sites occurs with negative cooperativity. In contrast, substrate analogue and product bind without cooperativity. Titrations of human PNP using isothermal calorimetry indicate that binding of a structurally rigid first-generation Immucillin (K(d) = 56 pM) is driven by large negative enthalpy values (DeltaH = -21.2 kcal/mol) with a substantial entropic (-TDeltaS) penalty. The tightest-binding inhibitors (K(d) = 5-9 pM) have increased conformational flexibility. Despite their conformational freedom in solution, flexible inhibitors bind with high affinity because of reduced entropic penalties. Entropic penalties are proposed to arise from conformational freezing of the PNP.inhibitor complex with the entropy term dominated by protein dynamics. The conformationally flexible Immucillins reduce the system entropic penalty. Disrupting the ribosyl 5'-hydroxyl interaction of transition state analogues with PNP causes favorable entropy of binding. Tight binding of the 17 Immucillins is characterized by large enthalpic contributions, emphasizing their similarity to the transition state. Via introduction of flexibility into the inhibitor structure, the enthalpy-entropy compensation pattern is altered to permit tighter binding.