Xanthine-guanine 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.

METABOLIC ACTIVATION OF 2,6-DIMETHYLANILINE: MUTATIONAL SPECIFICITY IN THE GPT GENE OF AS52 CELLS

Objective: The purpose of the current work was to characterize the mechanisms of cytotoxicity and mutagenesis of a potential human bladder carcinogen 2,6-dimethylaniline (2,6-DMA).Methods: Chinese hamster ovary (CHO) AS52 cells were exposed to either human S9 activated 2,6-DMA for 6 h or its N-hydroxylamine and aminophenol metabolites for 1 h in serum-free medium. Cell survival was determined by trypan blue exclusion 24 h after treatment, and 6-thioguanine-resistant mutants at the xanthine-guanine phosphoribosyl transferase (gpt) gene locus were assessed with doses, of which relative survival is 30% or more. Nested polymerase chain reaction-based deletion analysis was also performed.Results: AS52 cells exhibited a dose-dependent increase in cytotoxicity and mutant fraction on treatment of 2,6-DMA and its metabolites but show a considerable variation in potency with aminophenol metabolites having the highest potency and parent compound at least; at the highest 2,6-dimethylaminophenol dose (10 μM), the mutant fraction in AS52 cells was 8-fold (13.2×10−5) greater than the spontaneous fraction of 1.62×10−5. Total deletion of the gpt gene sequences was found in 1 (4%) spontaneous and 2 (6%) the 6-thioguanine mutants generated by N-hydroxy-2,6-DMA.Conclusions: These findings indicate the mutagenicity of 2,6-DMA at the gpt gene, which is mediated through hydroxylamine and aminophenol metabolites, and contribute to the elucidation of mechanisms through which 2,6-DMA may exert its effects in vivo.

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.

MUTATIONAL SPECIFICITY OF 2, 6-DIMETHYLANILINE WITH IN VITRO HUMAN S9 METABOLIC ACTIVATION IN THE GPT GENE OF AS52 CELLS

Objective: The purpose of the current work was to characterize mechanisms of cytotoxicity and mutagenesis of a potential human bladder carcinogen 2,6-dimethylaniline (2,6-DMA).Methods: Chinese hamster ovary (CHO) AS52 cells were exposed to either human S9 activated 2,6-DMA for 6 h or its N-hydroxylamine and aminophenol metabolites for 1 h in serum-free medium. Cell survival determined by trypan blue exclusion 24 h after treatment, and 6-thioguanine-resistant mutants at the xanthine-guanine phosphoribosyltransferase (gpt) gene locus were assessed with doses of which relative survival is 30% or more. Nested PCR-based deletion analysis was also performed.Results: AS52 cells exhibited a dose-dependent increase in cytotoxicity and mutant fraction upon treatment of 2,6-DMA and its metabolites, but showing considerable variation in potency with aminophenol metabolites having the highest potency and parent compound at least; at the highest 2,6-dimethyaminophenol dose (10 μM), the mutant fraction in AS52 cells was 8 fold (13.2×10-5) greater than the spontaneous fraction of 1.62×10-5. Total deletion of the gpt gene sequences was found in 1 (4%) of spontaneous and 2 (6%) of the 6-thioguanine mutants generated by N-hydroxy-2,6-DMA.Conclusion: These findings indicate the mutagenicity of 2,6-DMA at the gpt gene, which is mediated through hydroxylamine and aminophenol metabolites, and contribute to the elucidation of mechanisms through which 2,6-DMA may exert its effects in vivo.

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.

A proteomic approach to investigating gene cluster expression and secondary metabolite functionality in Aspergillus fumigatus.

A combined proteomics and metabolomics approach was utilised to advance the identification and characterisation of secondary metabolites in Aspergillus fumigatus. Here, implementation of a shotgun proteomic strategy led to the identification of non-redundant mycelial proteins (n = 414) from A. fumigatus including proteins typically under-represented in 2-D proteome maps: proteins with multiple transmembrane regions, hydrophobic proteins and proteins with extremes of molecular mass and pI. Indirect identification of secondary metabolite cluster expression was also achieved, with proteins (n = 18) from LaeA-regulated clusters detected, including GliT encoded within the gliotoxin biosynthetic cluster. Biochemical analysis then revealed that gliotoxin significantly attenuates H2O2-induced oxidative stress in A. fumigatus (p>0.0001), confirming observations from proteomics data. A complementary 2-D/LC-MS/MS approach further elucidated significantly increased abundance (p<0.05) of proliferating cell nuclear antigen (PCNA), NADH-quinone oxidoreductase and the gliotoxin oxidoreductase GliT, along with significantly attenuated abundance (p<0.05) of a heat shock protein, an oxidative stress protein and an autolysis-associated chitinase, when gliotoxin and H2O2 were present, compared to H2O2 alone. Moreover, gliotoxin exposure significantly reduced the abundance of selected proteins (p<0.05) involved in de novo purine biosynthesis. Significantly elevated abundance (p<0.05) of a key enzyme, xanthine-guanine phosphoribosyl transferase Xpt1, utilised in purine salvage, was observed in the presence of H2O2 and gliotoxin. This work provides new insights into the A. fumigatus proteome and experimental strategies, plus mechanistic data pertaining to gliotoxin functionality in the organism.

Structural insights into the interactions of xpt riboswitch with novel guanine analogues: a molecular dynamics simulation study

Ligand recognition in purine riboswitches is a complex process requiring different levels of conformational changes. Recent efforts in the area of purine riboswitch research have focused on ligand analogue binding studies. In the case of the guanine xanthine phosphoribosyl transferase (xpt) riboswitch, synthetic analogues that resemble guanine have the potential to tightly bind and subsequently influence the genetic expression of xpt mRNA in prokaryotes. We have carried out 25 ns Molecular Dynamics (MD) simulation studies of the aptamer domain of the xpt G-riboswitch in four different states: guanine riboswitch in free form, riboswitch bound with its cognate ligand guanine, and with two guanine analogues SJ1 and SJ2. Our work reveals novel interactions of SJ1 and SJ2 ligands with the binding core residues of the riboswitch. The ligands proposed in this work bind to the riboswitch with greater overall stability and lower root mean square deviations and fluctuations compared to guanine ligand. Reporter gene assay data demonstrate that the ligand analogues, upon binding to the RNA, lower the genetic expression of the guanine riboswitch. Our work has important implications for future ligand design and binding studies in the exciting field of riboswitches.

Inhibition of the Escherichia coli 6-Oxopurine Phosphoribosyltransferases by Nucleoside Phosphonates: Potential for New Antibacterial Agents

Escherichia coli (Ec) cells possess two purine salvage enzymes: xanthine-guanine phosphoribosyltransferase (XGPRT) and hypoxanthine phosphoribosyltransferase (HPRT). EcXGPRT shares a common structural feature with other members of this family, a flexible loop that closes over the active site during catalysis. The replacement of six of these amino acids by alanine has no effect on the Km for the two substrates. However, the Ki for the nucleoside monophosphate increases by 27-fold, and the kcat is reduced by ∼200-fold. Nucleoside phosphonates (NP) are good inhibitors of EcXGPRT and EcHPRT, with Ki values as low as 10 nM. In the absence of the flexible loop, these values increase by 5- to 30-fold, indicating the importance of the loop for high-affinity inhibition. Crystal structures of two NPs in complex with EcXGPRT explain the tight binding. Prodrugs of NPs with low Ki values for EcXGPRT or EcHPRT exhibit IC50 values between 5 and 23 μM against Mycobacterium tuberculosis in cell-based assays, suggesting that these compounds are therapeutic leads against pathogenic bacteria.

Immunoproteomic analysis of the protective response obtained with subunit and commercial vaccines against Glässer's disease in pigs

An immunoproteomic analysis of the protective response of subunit and commercial vaccines in colostrum-deprived pigs against Glässer's disease was carried out. A mixture of proteins with affinity to porcine transferrin (PAPT) from Haemophilus parasuis Nagasaki strain (serovar 5) was inoculated intramuscularly (PAPTM) and intratracheally (PAPTCp), along with a commercial bacterin. PAPT were separated using 2 dimensional electrophoresis (2DE) gels and with them, 2DE Western blots were carried out. A total of 17 spots were identified as positive with sera of pigs from any of the three vaccinated groups, the highest number of immunoreactive proteins being detected in those having received PAPTCp. Among them, six proteins (FKBP-type peptidyl-prolyl cis-trans isomerase, neuraminidase exo-α-sialidase, xanthine-guanine phosphoribosyl transferase, CMP-N-acetylneuraminic acid synthetase, phenylalanyl-tRNA synthetase and glyceraldehyde 3-phosphate dehydrogenase) were found to be novel immunogens in H. parasuis. These proteins showed a high potential as candidates in future subunit vaccines against Glässer's disease. The three experimental groups developed specific systemic total IgG (IgGt), IgG1, IgG2 and IgM antibodies after immunizations. In addition, those receiving PAPTCp yielded a serum IgA response.

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

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

Expression, purification and analysis of the activity of enzymes from the pentose phosphate pathway

RNAs, more than ever before, are increasingly viewed as biomolecules of the future, in the versatility of their functions and intricate three-dimensional folding. To effectively study them by nuclear magnetic resonance (NMR) spectroscopy, structural biologists need to tackle two critical challenges of spectral overcrowding and fast signal decay for large RNAs. Stable-isotope nucleotide labeling is one attractive solution to the overlap problem. Hence, developing effective methods for nucleotide labeling is highly desirable. In this work, we have developed a facile and streamlined source of recombinant enzymes from the pentose phosphate pathway for making such labeled nucleotides. The Escherichia coli (E. coli) genes encoding ribokinase (RK), adenine phosphoribosyltransferase (APRT), xanthine/guanine phosphoribosyltransferase (XGPRT), and uracil phosphoribosyltransferase (UPRT) were sub-cloned into pET15b vectors. All four constructs together with cytidine triphosphate synthetase (CTPS) and human phosphoribosyl pyrophosphate synthetase isoform 1 (PRPPS) were transformed into the E. coli BL21(AI) strain for protein over-expression. The enzyme preparations were purified to >90% homogeneity by a one-step Ni–NTA affinity chromatography, without the need of a further size-exclusion chromatography step. We obtained yields of 1530, 22, 482, 3120, 2120 and 2280 units of activity per liter of culture for RK, PRPPS, APRT, XGPRT, UPRT and CTPS, respectively; the specific activities were found to be 70, 22, 21, 128, 144 and 113U/mg, respectively. These specific activities of these enzyme constructs are comparable to or higher than those previously reported. In addition, both the growth conditions and purification protocols have been streamlined so that all the recombinant proteins can be expressed, purified and characterized in at most 2days. The availability and reliability of these constructs should make production of fully and site-specific labeled nucleotides for making labeled RNA accessible and straightforward, to facilitate high-resolution NMR spectroscopic and other biophysical studies.

Plasmodium falciparum: new molecular targets with potential for antimalarial drug development

Malaria remains one of the world’s most devastating infectious diseases. Drug resistance to all classes of antimalarial agents has now been observed, highlighting the need for new agents that act against novel parasite targets. The complete sequencing of the Plasmodium falciparum genome has allowed the identification of new molecular targets within the parasite that may be amenable to chemotherapeutic intervention. In this review, we investigate four possible targets for the future development of new classes of antimalarial agents. These targets include histone deacetylase, the aspartic proteases or plasmepsins, aminopeptidases and the purine salvage enzyme hypoxanthine–xanthine–guanine phosphoribosyltransferase.

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.

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.

Heterochromatinization as a Potential Mechanism of Nickel-Induced Carcinogenesis

Epigenetics refers to heritable patterns of gene expression that do not depend on alterations of the genomic DNA sequence. Nickel compounds have demonstrated carcinogenicity without any associated mutagenesis, suggesting that its mechanism of carcinogenesis is epigenetic in nature. One such potential mechanism is the heterochromatinization of chromatin within a region of the genome containing a gene sequence, inhibiting any further molecular interactions with that underlying gene sequence and effectively inactivating that gene. We report here the observation, by atomic force microscopy and circular dichroism spectropolarimetry, that nickel ion (Ni(2+)) condenses chromatin to a greater extent than the natural divalent cation of the cell, magnesium ion (Mg(2+)). In addition, we use a model experimental system that incorporates a transgene, the bacterial xanthine guanine phosphoribosyl transferase gene (gpt), differentially near, and far from, a heterochromatic region of the genome, in two cell lines, the Chinese hamster V79-derived G12 and G10 cells, respectively, to demonstrate by a DNase I protection assay that nickel treatement protects the gpt gene sequence from DNase I exonuclease digestion in the G12 cells, but not in the G10 cells. We conclude that condensation of chromatin by nickel is a potential mechanism of nickel-mediated gene regulation.

Synthesis of Polymerizable Protein Monomers for Protein-Acrylamide Hydrogel Formation

A novel method to produce protein polymer conjugates for protein-acrylamide hydrogel formation is described. Alkenes are incorporated onto the N-terminus of expressed proteins to produce polymerizable protein monomers that can be utilized in protein-acrylamide copolymerization. A 4-vinylbenzoic acid thioester was synthesized and attached to the N-termini of two protein models, the immunoglobulin-binding protein Protein G and the bacterial enzyme xanthine-guanine phosphoribosyltransferase (GPRT), utilizing native chemical ligation. N-terminal cysteine containing proteins utilized in native chemical ligation reactions were generated from His-tagged fusion proteins using tobacco etch virus NIa (TEV) protease cleavage. The 4-vinylbenzyl functionalized proteins were good substrates for immobilizing proteins into polyacrylamide hydrogels via free radical induced protein-acrylamide copolymerization. The protein copolymerization procedures developed in this report are mild enough to allow proteins to retain measurable biological activity as demonstrated by the retention of immunoglobulin binding ability by immobilized Protein G and enzymatic activity of immobilized GPRT.

Increasing Solubility of Proteins and Peptides by Site-Specific Modification with Betaine

Proteins and peptides with low solubility and which aggregate are often encountered in biochemical studies and in pharmaceutical applications of polypeptides. Here, we report a new strategy to improve solubility and prevent aggregation of polypeptides using site-specific modification with the small molecule betaine, which contains a quaternary ammonium moiety. Betaine was site-selectively attached to the N-termini of two aggregation-prone polypeptide models, the bacterial enzyme xanthine-guanine phosphoribosyltransferase (CG-GPRT) and the HIV entry inhibitor peptide CG-T20, utilizing native chemical ligation. N-terminal cysteines for the betaine ligation reactions were generated from His-tagged fusion proteins using TEV protease cleavage. Ligation of the betaine thioester (1) to the N-terminal cysteine-containing polypeptide models proceeded in high yield, though denaturing conditions were required for CG-T20 due to the hydrophobic nature of this peptide. CD spectroscopy and GPRT activity assays indicate that the betaine modification of CG-GPRT and CG-T20 does not significantly affect structure or activity of the polypeptides. Solubility and turbidity measurements of betaine-modified and unmodified polypeptides demonstrate that betaine modification can greatly increase solubility. Finally, it is shown that betaine-modified CG-T20 acts as an inhibitor of the aggregation of unmodified CG-T20.

Mutagenicity of 8-nitroguanosine, a product of nitrative nucleoside modification by reactive nitrogen oxides, in mammalian cells

8-Nitroguanosine is a nitratively modified nucleoside that is formed endogeneously under inflammatory conditions dependent on nitric oxide production, particularly associated with cancer risks. Here, we investigated the mutagenic potential of 8-nitroguanosine in mammalian cells. Treatment with 8-nitroguanosine (10–1000 μM) for 1 h significantly increased (by 6–8 times) the mutation frequency of the xanthine–guanine phosphoribosyltransferase ( gpt) gene in AS52 cells without cytotoxic effects. 8-Nitroguanosine treatment induced a G-to-T transversion in gpt gene at position 86. It also significantly increased levels of abasic sites in DNA. These observations suggest that formation of 8-nitroguanosine may contribute to the pathogenesis of inflammation-associated carcinogenesis.