Hypoxanthine-guanine-xanthine Phosphoribosyltransferase Research Articles

Identification of new inhibitors of Plasmodium falciparum hypoxanthine–guanine–xanthine Phosphoribosyltransferase (HG(X)PRT): An outlook towards the treatment of malaria

Plasmodium, a protozoan parasite responsible for causing malaria relies on the purine salvage pathway to synthesize purine as they are incapable of synthesizing them de novo. This pathway is crucial for the survival of the parasite and hence enzymes of this pathway can serve as antimalarial drug targets. One of the enzymes of this pathway is hypoxanthine guanine (xanthine) phosphoribosyltransferase [HG(X)PRT] that serves as novel target, potentially less prone to existing resistance mechanisms seen with the use of traditional antimalarial drugs. HGXPRT inhibition disrupts the parasite's ability to synthesize nucleotides, essential for its growth and replication. In this regard, the current study was designed to identify the inhibitors of HGXPRT. For this purpose, the enzyme was produced through recombinant technology and purified with 10 mg/ L yield. Followed this, UV-based enzyme inhibition assay was optimized and >200 fully characterized compounds were evaluated for their HGXPRT inhibitory activity. Out of them fourteen compounds 1–14 showed significant to weak inhibition of HGXPRT enzyme with IC50 values in the range of 15.7 to 229.6 μM, as compared to the standard inhibitor i.e. 9-deazaguanine (IC50 = 12 ± 1.0 μM). In- silico and biophysical studies were further performed on active compounds to get structural insights into enzyme-inhibitor complex at the atomic level. Docking studies predicted that these inhibitors accommodate the purine binding site of enzyme and interacted with critical residues such as Asp148, Phe197, and Val198. Biophysical studies showed that these identified inhibitors interacted with HGXPRT enzyme in a non-ambiguous manner. Furthermore, these inhibitors were found to be non-cytotoxic against human fibroblast cell lines (BJ). Hence, this study identified 14 hits that could lead to further research towards anti-malarial drug design and development.

Inhibition and Mechanism of Plasmodium falciparum Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferase.

Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPRT) is essential for purine salvage of hypoxanthine into parasite purine nucleotides. Transition state analogue inhibitors of PfHGXPRT are characterized by kinetic analysis, thermodynamic parameters, and X-ray crystal structures. Compound 1, 9-deazaguanine linked to an acyclic ribocation phosphonate mimic, shows a kinetic Ki of 0.5 nM. Isothermal titration calorimetry (ITC) experiments of 1 binding to PfHGXPRT reveal enthalpically driven binding with negative cooperativity for the binding of two inhibitor molecules in the tetrameric enzyme. Crystal structures of 1 bound to PfHGXPRT define the hydrogen bond and ionic contacts to complement binding thermodynamics. Dynamics of ribosyl transfer from 5-phospho-α-d-ribosyl 1-pyrophosphate (PRPP) to hypoxanthine were examined by 18O isotope exchange at the bridging phosphoryl oxygen of PRPP pyrophosphate. Rotational constraints or short transition state lifetimes prevent torsional rotation and positional isotope exchange of bridging to nonbridging oxygen in the α-pyrophosphoryl group. Thermodynamic analysis of the transition state analogue and magnesium pyrophosphate binding reveal random and cooperative binding to PfHGXPRT, unlike the obligatory ordered reaction kinetics reported earlier for substrate kinetics.

Kinetic Characterization and Inhibition of Trypanosoma cruzi Hypoxanthine-Guanine Phosphoribosyltransferases.

Chagas disease, caused by the parasitic protozoan Trypanosoma cruzi, affects over 8 million people worldwide. Current antiparasitic treatments for Chagas disease are ineffective in treating advanced, chronic stages of the disease, and are noted for their toxicity. Like most parasitic protozoa, T. cruzi is unable to synthesize purines de novo, and relies on the salvage of preformed purines from the host. Hypoxanthine–guanine phosphoribosyltransferases (HGPRTs) are enzymes that are critical for the salvage of preformed purines, catalyzing the formation of inosine monophosphate (IMP) and guanosine monophosphate (GMP) from the nucleobases hypoxanthine and guanine, respectively. Due to the central role of HGPRTs in purine salvage, these enzymes are promising targets for the development of new treatment methods for Chagas disease. In this study, we characterized two gene products in the T. cruzi CL Brener strain that encodes enzymes with functionally identical HGPRT activities in vitro: TcA (TcCLB.509693.70) and TcC (TcCLB.506457.30). The TcC isozyme was kinetically characterized to reveal mechanistic details on catalysis, including identification of the rate-limiting step(s) of catalysis. Furthermore, we identified and characterized inhibitors of T. cruzi HGPRTs originally developed as transition-state analogue inhibitors (TSAIs) of Plasmodium falciparum hypoxanthine–guanine–xanthine phosphoribosyltransferase (PfHGXPRT), where the most potent compound bound to T. cruzi HGPRT with low nanomolar affinity. Our results validated the repurposing of TSAIs to serve as selective inhibitors for orthologous molecular targets, where primary and secondary structures as well as putatively common chemical mechanisms are conserved.

Sulfide, sulfoxide and sulfone bridged acyclic nucleoside phosphonates as inhibitors of the Plasmodium falciparum and human 6-oxopurine phosphoribosyltransferases: Synthesis and evaluation

Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) is a recognized target for antimalarial chemotherapeutics. It synthesises all of the 6-oxopurine nucleoside monophosphates, IMP, GMP and XMP needed by the malarial parasite, Plasmodium falciparum (Pf). PfHGXPRT is also indirectly responsible for the synthesis of the adenosine monophosphate, AMP. The acyclic nucleoside phosphonates (ANPs) are a class of PfHGXPRT inhibitors. Prodrugs of these compounds are able to arrest the growth of Pf in cell culture. In the search for new inhibitors of PfHGXPRT, a series of sulfur containing ANPs (thia-ANPs) has been designed and synthesized. These compounds are based on the structure of 2-(phosphonoethoxy)ethylguanine (PEEG) and PEEHx which consist of a purine base (i.e. guanine or hypoxanthine) linked to a phosphonate group by five atoms i.e. four carbons and one oxygen. Here, PEEG and PEEHx were modified by substituting a sulfide, sulfoxide or a sulfone bridge for the oxygen atom in the linker. The effect of these substitutions on the Ki values for human HGPRT and PfHGXPRT was investigated and showed that most of the thia-ANPs distinctively favour PfHGXPRT. For example, the thia-analogue of PEEHx has a Ki value of 0.2 μM for PfHGXPRT, a value 25-fold lower than for the human counterpart. Prodrugs of these compounds have IC50 values in the 4–6 μM range in antimalarial cell-based assays, making them attractive compounds for further development as antimalarial drug leads.

Acyclic nucleoside phosphonates with unnatural nucleobases, favipiravir and allopurinol, designed as potential inhibitors of the human and Plasmodium falciparum 6-oxopurine phosphoribosyltransferases

Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) is a key enzyme of the purine salvage pathway and its activity is crucial for the survival of certain parasites e.g. Plasmodium falciparum (Pf). Acyclic nucleoside phosphonates (ANPs) containing either guanine or hypoxanthine as the purine base are inhibitors of this enzyme. In this part of a SAR study, these two naturally-occurring nucleobases attached to an acyclic moiety were replaced by allopurinol or favipiravir. Both allopurinol and favipiravir ANPs were prepared via Mitsunobu reaction. The alkylation of favipiravir was optimized to yield both N- and O- regioisomers but the N-regioisomers were unstable under deprotection conditions. Thus, only the ANPs containing the O-isomer of favipiravir and those containing allopurinol were evaluated as potential inhibitors of human HGPRT and PfHGXPRT. Two ANPs with allopurinol as the base have Ki values of 10 μM and 30 μM for PfHGXPRT but do not inhibit human HGPRT activity at concentrations of 100–150 μM.

One-Pot, One-Step Production of Dietary Nucleotides by Magnetic Biocatalysts

The enzymatic synthesis of nucleotides offers several advantages over traditional multistep chemical methods, such as stereoselectivity, regioselectivity, enantioselectivity, simple downstream processing, and the use of mild reaction conditions. However, in order to scale up these bioprocesses, several drawbacks, such as the low enzyme stability and recycling, must be considered. Enzyme immobilization may overcome these cost-related problems by enhancing protein stability and facilitating the separation of products. In this regard, tetrameric hypoxanthine–guanine–xanthine phosphoribosyltransferase (HGXPRT) from Thermus thermophilus HB8 was covalently immobilized onto glutaraldehyde-activated MagReSyn®Amine magnetic iron oxide porous microparticles (MTtHGXPRT). In this context, two different strategies were followed: (a) an enzyme immobilization through its N-terminus residues at pH 8.5 (derivatives MTtHGXPRT1-3); and (b) a multipoint covalent immobilization through the surface lysine residues at pH 10 (derivatives MTtHGXPRT4-5). The immobilized derivatives of MTtHGXPRT3 (activity 1581 international units per gram of support, IU/g; retained activity 29%) and MTtHGXPRT5 (activity 1108 IU/g; retained activity 23%) displayed the best wet biocatalyst activity, and retained activity values in the enzymatic synthesis of inosine-5′-monophosphate (IMP). In addition, the dependence of the activities and stabilities of both derivatives on pH and temperature was tested, as well as their reusability potential. Taking these results into account, MTtHGXPRT3 was chosen as the best biocatalyst (negligible loss of activity at 60 °C during 24 h; reusable up to seven cycles). Finally, as proof of concept, the enzymatic production of dietary nucleotides from high concentrations of low soluble bases was achieved.

Kinetic Isotope Effects and Transition State Structure for Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferase from Plasmodium falciparum.

Plasmodium falciparum parasites are purine auxotrophs that rely exclusively on the salvage of preformed purines from their human hosts to supply the requirement for purine nucleotides. Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) catalyzes the freely reversible Mg2+-dependent conversion of 6-oxopurine bases to their respective nucleotides and inorganic pyrophosphate. The phosphoribosyl group is derived from 5-phospho-α-d-ribosyl 1-pyrophosphate (PRPP). The enzyme from malaria parasites (PfHGXPRT) is essential as hypoxanthine is the major precursor in purine metabolism. We used specific heavy atom labels in PRPP and hypoxanthine to measure primary (1-14C and 9-15N) and secondary (1-3H and 7-15N) intrinsic kinetic isotope effect (KIE) values for PfHGXPRT. Intrinsic isotope effects contain information for understanding enzymatic transition state properties. The transition state of PfHGXPRT was explored by matching KIE values predicted from quantum mechanical calculations to the intrinsic values determined experimentally. This approach provides information about PfHGXPRT transition state bond lengths, geometry, and atomic charge distribution. The transition state structure of PfHGXPRT was determined in the physiological direction of addition of ribose 5-phosphate to hypoxanthine by overcoming the chemical instability of PRPP. The transition state for PfHGXPRT forms nucleotides through a well-developed and near-symmetrical DN*AN, SN1-like transition state.

Slow ligand-induced conformational switch increases the catalytic rate in Plasmodium falciparum hypoxanthine guanine xanthine phosphoribosyltransferase.

P. falciparum (Pf) hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) exhibits a unique mechanism of activation where the enzyme switches from a low activity (unactivated) to a high activity (activated) state upon pre-incubation with substrate/products. Xanthine phosphoribosylation by unactivated PfHGXPRT exhibits a lag phase, the duration of which reduces with an increase in concentration of the enzyme or substrate, PRPP·Mg(2+). Activated PfHGXPRT does not display the lag phase and exhibits a ten-fold drop in the Km value for PRPP·Mg(2+). These observations suggest the involvement of ligand-mediated oligomerization and conformational changes in the process of activation. The dipeptide Leu-Lys in the PPi binding site of human and T. gondii HG(X)PRT that facilitates PRPP·Mg(2+) binding by isomerization from trans to cis conformation is conserved in PfHGXPRT. Free energy calculations using the well-tempered metadynamics technique show the ligand-free enzyme to be more stable when this dipeptide is in the trans conformation than in the cis conformation. The high rotational energy barrier observed for the conformational change from experimental and computational studies permits delineation of the activation mechanism.

Acyclic phosph(on)ate inhibitors of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase

The pathogenic protozoa responsible for malaria lack enzymes for the de novo synthesis of purines and rely on purine salvage from the host. In Plasmodium falciparum (Pf), hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) converts hypoxanthine to inosine monophosphate and is essential for purine salvage making the enzyme an anti-malarial drug target. We have synthesized a number of simple acyclic aza-C-nucleosides and shown that some are potent inhibitors of Pf HGXPRT while showing excellent selectivity for the Pf versus the human enzyme.

Still Seeking an Effective “One–Two” Malaria Vaccine Punch

Still Seeking an Effective “One–Two” Malaria Vaccine Punch

Acyclic Immucillin Phosphonates: Second-Generation Inhibitors of Plasmodium falciparum Hypoxanthine- Guanine-Xanthine Phosphoribosyltransferase

Plasmodium falciparum, the primary cause of deaths from malaria, is a purine auxotroph and relies on hypoxanthine salvage from the host purine pool. Purine starvation as an antimalarial target has been validated by inhibition of purine nucleoside phosphorylase. Hypoxanthine depletion kills Plasmodium falciparum in cell culture and in Aotus monkey infections. Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) from P. falciparum is required for hypoxanthine salvage by forming inosine 5'-monophosphate, a branchpoint for all purine nucleotide synthesis in the parasite. Here, we present a class of HGXPRT inhibitors, the acyclic immucillin phosphonates (AIPs), and cell permeable AIP prodrugs. The AIPs are simple, potent, selective, and biologically stable inhibitors. The AIP prodrugs block proliferation of cultured parasites by inhibiting the incorporation of hypoxanthine into the parasite nucleotide pool and validates HGXPRT as a target in malaria.

Synthesis of purine N9-[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 N9 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 Ki values of 2μM and 5μM for PfHGXPRT and PvHGPRT, respectively. The addition of pyrophosphate to the assay decreased the Ki 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.

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 μM. The data showed that both the position and nature of the hydrophobic substituent change the potency and selectivity of the ANPs.

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.

Human T cell recognition of the blood stage antigen Plasmodium hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT) in acute malaria.

BackgroundThe Plasmodium purine salvage enzyme, hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT) can protect mice against Plasmodium yoelii pRBC challenge in a T cell-dependent manner and has, therefore, been proposed as a novel vaccine candidate. It is not known whether natural exposure to Plasmodium falciparum stimulates HGXPRT T cell reactivity in humans.MethodsPBMC and plasma collected from malaria-exposed Indonesians during infection and 7–28 days after anti-malarial therapy, were assessed for HGXPRT recognition using CFSE proliferation, IFNγ ELISPOT assay and ELISA.ResultsHGXPRT-specific T cell proliferation was found in 44% of patients during acute infection; in 80% of responders both CD4+ and CD8+ T cell subsets proliferated. Antigen-specific T cell proliferation was largely lost within 28 days of parasite clearance. HGXPRT-specific IFN-γ production was more frequent 28 days after treatment than during acute infection. HGXPRT-specific plasma IgG was undetectable even in individuals exposed to malaria for at least two years.ConclusionThe prevalence of acute proliferative and convalescent IFNγ responses to HGXPRT demonstrates cellular immunogenicity in humans. Further studies to determine minimal HGXPRT epitopes, the specificity of responses for Plasmodia and associations with protection are required. Frequent and robust T cell proliferation, high sequence conservation among Plasmodium species and absent IgG responses distinguish HGXPRT from other malaria antigens.

Erythrocytic Adenosine Monophosphate as an Alternative Purine Source in Plasmodium falciparum

Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum.

Lead Compounds for Antimalarial Chemotherapy: Purine Base Analogs Discriminate between Human and P. Falciparum 6-Oxopurine Phosphoribosyltransferases

The malarial parasite Plasmodium falciparum depends on the purine salvage enzyme hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) to convert purine bases from the host to nucleotides needed for DNA and RNA synthesis. An approach to developing antimalarial drugs is to use HGXPRT to convert introduced purine base analogs to nucleotides that are toxic to the parasite. This strategy requires that these compounds be good substrates for the parasite enzyme but poor substrates for the human counterpart, HGPRT. Bases with a chlorine atom in the 6-position or a nitrogen in the 8-position exhibited strong discrimination between P. falciparum HGXPRT and human HGPRT. The k(cat)/K(m) values for the Plasmodium enzyme using 6-chloroguanine and 8-azaguanine as substrates were 50 - 80-fold and 336-fold higher than for the human enzyme, respectively. These and other bases were effective in inhibiting the growth of the parasite in vitro, giving IC(50) values as low as 1 microM.

Purification of human malaria parasite hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) using immobilized Reactive Red 120

Malaria is caused by Plasmodium parasite infection. The human malarial parasite does not have a de novo pathway for synthesis of nucleotides and the purine salvage pathway enzyme hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) is critical for survival. In our efforts to find inhibitors of the malarial parasite HGXPRT, we have developed a simple but effective purification protocol for this protein expressed in Escherichia coli without an affinity tag. The protocol consists of tandem columns of anion exchange and immobilized Reactive Red 120 resins. The enzyme is inactive as isolated but can be activated by incubation with substrate(s).

A Comparative QM/MM Simulation Study of the Reaction Mechanisms of Human and Plasmodium falciparum HG(X)PRTases

QM/MM hybrid potential free-energy simulations are performed to compare the reaction mechanisms of human hypoxanthine guanine phosphoribosyl transferase (HGPRTase) and the corresponding enyzme from Plasmodium falciparum (Pf), hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRTase). These enzymes share 44% of sequence identity but display very different affinities for xanthine. The calculations show that in both enzymes phosphoribosyl transfer proceeds via a dissociative mechanism from an anionic form of the substrate. Nevertheless, there are significant differences in the geometries of critical structures along the reaction paths which it may be possible to exploit for the design of specific inhibitors against the Pf enzyme.

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-gamma, and tumor necrosis factor-alpha, 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.