Human Purine Research Articles

Evaluation of Lipid profile and Uric acid level in Type II Diabetes mellitus

T2DM is characterized primarily by pancreatic beta cell loss, inadequate insulin production, or insulin resistance in body cells, among other causes. T2DM is related to a variety of other indications and symptoms including coronary heart disease, kidney illness, eye abnormalities, peripheral vascular disease, digestive system diseases, high blood pressure and depression. Lipid abnormalities are common in persons with type 2 diabetes and prediabetes, although the pattern of the different lipids may vary between ethnic groups, economic levels and access to health care. Uric acid is produced during nucleotide and adenosine triphosphate (ATP) metabolism and is the end product of human purine. Biologically, uric acid plays a significant role in developing insulin resistance in animal models by reducing the bioavailability of nitric oxide which is required for insulin-stimulated glucose uptake. This study aimed to evaluate the lipid profile and uric acid level in patients with Type 2 diabetes mellitus. Blood samples were collected from 50 control and diabetic patients. The lipid profile and uric acid level were estimated by the kit method. Significantly higher levels of total cholesterol, triglycerides, HDL and LDL were observed in the diabetic group compared to the controls. Significantly low levels of serum uric acid and higher blood glucose levels were observed between normal and diabetic subjects. Diabetic dyslipidemia is curable if identified early. It can be improved by regulating glycemic (glucose) levels, lowering free fatty acids and increasing VLDL production by the liver. It can also be treated with weight loss, exercise, smoking cessation, a nutritious diet and pharmaceutical therapy.

Design, synthesis, and evaluation of chalcone derivatives as xanthine oxidase inhibitors

Xanthine oxidase (XO) is an important enzyme that catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid in the catabolism of purines in humans. This makes XO a well-recognized target in alleviating hyperuricemia. The present study adapted a structure-based drug discovery approach to develop potent and low-toxicity XO inhibitors with the chalcone skeleton. We introduced a carboxyl group and a hydroxyl group to the B ring and modified the A ring. 35 chalcone derivatives were designed and synthesized. All the 35 derivatives exhibited higher XO inhibition activities (IC50 = 0.064–0.559 μM) compared with allopurinol (IC50 = 2.588 μM). Their high affinity was attributed to strong hydrogen bond interactions formed between the introduced carboxyl and hydroxyl groups with key amino acid residues in XO. SAR analysis disclosed that carboxyl, hydroxyl, ethyl (12c), methylamino (12h), dimethylamino (12i), indolin (13k), and indol (13l) groups played important roles in improving the whole molecules’ inhibition potency against XO. ADME predictions and cytotoxicity assays suggested their pharmacokinetic characteristics and biocompatibility were desirable. Additionally, 12c exhibited a significant hypouricemic effect on potassium oxonate-induced hyperuricemia rats after orally administrated at a dose range of 10–40 mg/kg, representing a promising anti-hyperuricemia potential for further optimization and development.

Correlation of serum uric acid levels with certain anthropometric parameters in prediabetic and drug-naive diabetic subjects.

Uric acid is produced during the metabolism of nucleotide and adenosine triphosphate and contains the final product of human purine metabolism. It acts both as an antioxidant and pro-inflammatory marker and has a positive association with visceral fat in overweight subjects. The aim of the present study is to find an association of uric acid level with certain anthropometric parameters in subjects having type 2 diabetes. The study included 124 urban drug-naive diabetic Indian subjects above 18 years of age from the general population of the city of North India. Uric acid concentrations were estimated by the uricase method. Fasting plasma glucose (FPG) concentrations were estimated by the glucose oxidase-peroxidase method. Anthropometric measurements and information on lifestyle factors and disease history were collected through in-person meeting. All participants of the study subjects had a body mass index (BMI) of more than 23.5. BMI, waist-to-hip ratio (WHR), waist-to-height ratio, waist circumference, neck circumference, weight, age, sagittal abdominal diameter (SAD), skinfold thickness, and body roundness index were positively correlated with the serum uric acid level. The correlation of weight, BMI, SAD, and WHR was statistically significant. We found that serum uric acid level increases as body fat content increases. Statistical data show remarkable results for a significant correlation of uric acid level with BMI, WHR, SAD, and FPG. Hypertrophy occurs as a result of inflammatory processes and oxidative stress when the supply of energy starts to exceed the storage capacity of adipocytes, as a result, adipokines such as interleukin (IL)-1, IL-6, and tumor-necrosis factor-alpha are released more frequently which lead to low-grade chronic inflammation. Uric acid levels are much lean toward visceral obesity than overall body fat content.

Synthesis of metal–organic nanofiber/rGO nanocomposite as the sensing element for electrochemical determination of hypoxanthine

Abstract In this study, metal–organic nanofibers (MONFs) and reduced graphite oxide (rGO) nanocomposite were used to modify the surface of glassy carbon electrode, and the electrochemical sensor was applied to the differential pulse voltammetry determination of hypoxanthine, the oxidation intermediate of human purine degradation metabolism. The preparation of MONFs/rGO nanocomposite is simple, efficient, and environmentally friendly. The morphology and structure of MONFs/rGO nanocomposite were characterized by field emission scanning electron microscopy and X-ray diffraction. The results show that the improved sensor has a significant increase in current density, with linear ranges of 0.1–10 and 20–100 μM. Detection limit 0.01 μM (S/N = 3). Under the optimized conditions, the improved sensor shows very good stability, selectivity, and improved accuracy.

Structure-Guided Discovery of N5-CAIR Mutase Inhibitors.

Because purine nucleotides are essential for all life, differences between how microbes and humans metabolize purines can be exploited for the development of antimicrobial therapies. While humans biosynthesize purine nucleotides in a 10-step pathway, most microbes utilize an additional 11th enzymatic activity. The human enzyme, aminoimidazole ribonucleotide (AIR) carboxylase generates the product 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) directly. Most microbes, however, require two separate enzymes, a synthetase (PurK) and a mutase (PurE), and proceed through the intermediate, N5-CAIR. Toward the development of therapeutics that target these differences, we have solved crystal structures of the N5-CAIR mutase of the human pathogens Legionella pneumophila (LpPurE) and Burkholderia cenocepacia (BcPurE) and used a structure-guided approach to identify inhibitors. Analysis of the structures reveals a highly conserved fold and active site architecture. Using this data, and three additional structures of PurE enzymes, we screened a library of FDA-approved compounds in silico and identified a set of 25 candidates for further analysis. Among these, we identified several new PurE inhibitors with micromolar IC50 values. Several of these compounds, including the α1-blocker Alfuzosin, inhibit the microbial PurE enzymes much more effectively than the human homologue. These structures and the newly described PurE inhibitors are valuable tools to aid in further studies of this enzyme and provide a foundation for the development of compounds that target differences between human and microbial purine metabolism.

Connection between slow and fast dynamics in human purine nucleoside phosphorylase.

Protein motions occur across a variety of timescales, from vibrational fluctuations in femtoseconds, to correlated motions in picoseconds, all the way to milliseconds. There have been numerous studies that show these correlated motions may assist in catalysis, protein folding, and substrate specificity. It is also known through transition path sampling studies that rapid promoting vibrations contribute to enzyme catalysis. Human PNP (hPNP) is one enzyme that contains both a correlated motion and a rapid promoting vibration. The slower motion in this enzyme is associated with a particular loop motion that when open allows substrate entry and product release, but closes over the active site during catalysis. The work we report shows a connection between the two motions. We examine the differences between an unconstrained PNP structure, and a PNP structure with constraints on the loop motion. We employ Transition Path Sampling, reaction coordinate identification, electric field calculations, and free energy calculations to illustrate this coupling between the slow and fast protein dynamics.

Design and Synthesis of New Modified Flexible Purine Bases as Potential Inhibitors of Human PNP.

The great interest in studying the structure of human purine nucleoside phosphorylase (hPNP) and the continued search for effective inhibitors is due to the importance of the enzyme as a target in the therapy of T-cell proliferative diseases. In addition, hPNP inhibitors are used in organ transplant surgeries to provide immunodeficiency during and after the procedure. Previously, we showed that members of the well-known fleximer class of nucleosides are substrates of E. coli PNP. Fleximers have great promise as they have exhibited significant biological activity against a number of viruses of pandemic concern. Herein, we describe the synthesis and inhibition studies of a series of new fleximer compounds against hPNP and discuss their possible binding mode with the enzyme. At a concentration of 2 mM for the flex-7-deazapurines 1-4, a decrease in enzymatic activity by more than 50% was observed. 4-Amino-5-(1H-pyrrol-3-yl)pyridine 2 was the best inhibitor, with a Ki = 0.70 mM. Docking experiments have shown that ligand 2 is localized in the selected binding pocket Glu201, Asn243 and Phe200. The ability of the pyridine and pyrrole fragments to undergo rotation around the C-C bond allows for multiple binding modes in the active site of hPNP, which could provide several plausible bioactive conformations.

Connecting Conformational Motions to Rapid Dynamics in Human Purine Nucleoside Phosphorylase.

The influence of protein motions on enzyme catalysis remains a topic of active discussion. Protein motions occur across a variety of time scales, from vibrational fluctuations in femtoseconds, to collective motions in milliseconds. There have been numerous studies that show conformational motions may assist in catalysis, protein folding, and substrate specificity. It is also known through transition path sampling studies that rapid promoting vibrations contribute to enzyme catalysis. Human purine nucleoside phosphorylase (PNP) is one enzyme that contains both an important conformational motion and a rapid promoting vibration. The slower motion in this enzyme is associated with a loop motion, that when open allows substrate entry and product release but closes over the active site during catalysis. We examine the differences between an unconstrained PNP structure and a PNP structure with constraints on the loop motion. To investigate possible coupling between the slow and fast protein dynamics, we employed transition path sampling, reaction coordinate identification, electric field calculations, and free energy calculations reported here.

Connecting rapid promoting vibrations to conformational transitions in human purine nucleoside phosphorylase

One pathway to understanding the process by which enzymes catalyze chemical reactions is through the examination of internal correlated motions. There have been numerous studies that show these correlated motions may assist in catalysis, protein folding, and substrate specificity. It is also known through transition path sampling studies that rapid promoting vibrations contribute to enzyme catalysis. Human PNP is an important enzyme in purine metabolism, and it is the only purine salvage enzyme that catalyzes the reversible phosphorolysis of 6-oxopurine nucleosides to from a-D-ribose-1-phosphate.

Quantitative determination of uric acid using paper-based biosensor modified with graphene oxide and 5-amino-1,3,4-thiadiazole-2-thiol.

Uric acid is the primary end product of human purine metabolism and has been regarded as a key parameter in urine and blood for monitoring physiological conditions. This paper presents a paper-based biosensor for a quantitative determination of uric acid using electrochemical detection. The working electrode of the biosensor is modified with graphene oxide (GO) and 5-amino-1,3,4-thiadiazole-2-thiol (ATT) by electropolymerizing ATT on the surface of graphene oxide. In this study, cyclic voltammetry (CV) measurements required only 200 μL of analyte solution. The experimental results showed that the oxidation peak current increased as the concentration of uric acid become higher and exhibited a linear relationship in the concentration range of 0.1-10 mM, indicating that this proposed biosensor has high sensitivity. In addition, this biosensor has good selectivity to detect uric acid because ATT has a specific binding with it. In human blood and body fluids, nitrites may be the only factor that can interfere with the detection of uric acid using this proposed biosensor. Nevertheless, uric acid can be discriminated from nitrite in the CV measurement due to different oxidation potentials. Thus, this proposed paper-based biosensor is a promising tool for detecting uric acid in biological samples.

On-flow magnetic particle activity assay for the screening of human purine nucleoside phosphorylase inhibitors

Human purine nucleoside phosphorylase (HsPNP) catalyzes reversible phosphorolysis of nucleosides and deoxynucleosides in the purine cascade. HsPNP has been a target on behalf of the development of new leads for the treatment of a variety of T-cell mediated disorders. Several studies on the HsPNP are focused on the identification of effective, safe, and selective inhibitors. Therefore, this study describes the development of direct, simple, reliable, and inexpensive enzymatic assays to screen HsPNP inhibitors. Initially, HsPNP was covalently immobilized on the surface of magnetic particles (MPs). Due to the versatility of the MPs as solid support for enzyme immobilization, two different methods to monitor the enzyme activity are presented. Firstly, the activity of HsPNP-MPs was assessed offline by HPLC-DAD quantifying the formed hypoxanthine. Then, HsPNP-MPs were trapped in a peek tube, furnishing a microreactor which was inserted on-flow in an HPLC-DAD system to monitor the enzyme activity by the hypoxanthine quantification. Kinetic assays provided KMapp values for the inosine substrate of 488.2 ± 49.1 and 1084 ± 111 µM for the offline and on-flow assays, respectively. For the first time, kinetic studies for Pi as substrate using the HsPNP-MPs exhibits a Michaelis-Menten kinetic, yielding KMapp values for offline and on-flow of 521.2 ± 62.9 µM and 601 ± 66.5 µM, respectively. Inhibition studies conducted with a fourth generation immucillin derivative (DI4G) were employed as proof of concept to validate the use of the HsPNP-MPs assays for screening purposes. Additionally, a small library containing 11 compounds was used to assess the selectivity of the developed assays. The results showed that both presented assays can be applied to selectively recognizing and characterizing HsPNP inhibitors. Particularly, the on-flow method exhibited a high throughput and performance because of its automation and represents an easy and practical approach to reuse the HsPNP-MPs. Besides, this novel enzyme activity assay model can be further applied to other biological targets.

Uric Acid as a Photosensitizer in the Reaction of Deoxyribonucleosides with UV Light of Wavelength Longer than 300 nm: Identification of Products from 2'-Deoxycytidine.

DNA reacts directly with UV light with a wavelength shorter than 300 nm. Although ground surface sunlight includes little of this short-wavelength UV light due to its almost complete absorption by the atmosphere, sunlight is the primary cause of skin cancer. Photosensitization by endogenous substances must therefore be involved in skin cancer development mechanisms. Uric acid is the final metabolic product of purines in humans, and is present at relatively high concentrations in cells and fluids. When a neutral mixed solution of 2'-deoxycytidine, 2'-deoxyguanosine, thymidine, and 2'-deoxyadenosine was irradiated with UV light with a wavelength longer than 300 nm in the presence of uric acid, all the nucleosides were consumed in a uric acid dose-dependent manner. These reactions were inhibited by the addition of radical scavengers, ethanol and sodium azide. Two products from 2'-deoxycytidine were isolated and identified as N4-hydroxy-2'-deoxycytidine and N4,5-cyclic amide-2'-deoxycytidine, formed by cycloaddition of an amide group from uric acid. A 15N-labeled uric acid, uric acid-1,3-15N2, having two 14N and two 15N atoms per molecule, produced N4,5-cyclic amide-2'-deoxycytidine containing both 14N and 15N atoms from uric acid-1,3-15N2. Singlet oxygen, hydroxyl radical, peroxynitrous acid, hypochlorous acid, and hypobromous acid generated neither N4-hydroxy-2'-deoxycytidine nor N4,5-cyclic amide-2'-deoxycytidine in the presence of uric acid. These results indicate that uric acid is a photosensitizer for the reaction of nucleosides by UV light with a wavelength longer than 300 nm, and that an unidentified radical derived from uric acid with a delocalized unpaired electron may be generated.

5'-Nucleotidase Plays a Key Role in Uric Acid Metabolism of Bombyx mori.

Uric acid (UA) is the end-product in the human purine metabolism pathway. The UA that accumulates in silkworm tissues is excreted as a nitrogen waste product. Here, we first validated that Bombyx mori has a homolog of the human gene that encodes the 5′-nucleotidase (5′N) involved in purine metabolism. The B. mori gene, Bm5′N, is located upstream of other genes involved in UA metabolism in the silkworm. Disruption of Bm5′N via the CRISPR/Cas9 system resulted in decreased UA levels in the silkworm epidermis and caused a translucent skin phenotype. When Bm5′N mutant silkworms were fed with the uric acid precursor inosine, the UA levels in the epidermis increased significantly. Furthermore, the metabolomic and transcriptomic analyses of Bm5′N mutants indicated that loss of the Bm5′N affected purine metabolism and the ABC transport pathway. Taken together, these results suggest that the UA pathway is conserved between the silkworm and humans and that the Bm5′N gene plays a crucial role in the uric acid metabolism of the silkworm. Thus, the silkworm may be a suitable model for the study of UA metabolism pathways relevant to human disease.

EPharmaLib: A Versatile Library of e-Pharmacophores to Address Small-Molecule (Poly-)Pharmacology.

Bioactive compounds oftentimes bind to several target proteins, thereby exhibiting polypharmacology. Experimentally determining these interactions is however laborious, and structure-based virtual screening (SBVS) of bioactive compounds could expedite drug discovery by prioritizing hits for experimental validation. Here, we present ePharmaLib, a library of 15,148 e-pharmacophores modeled from solved structures of pharmaceutically relevant protein-ligand complexes of the screening Protein Data Bank (sc-PDB). ePharmaLib can be used for target fishing of phenotypic hits, side effect predictions, drug repurposing, and scaffold hopping. In retrospective SBVS, a good balance was obtained between computational efficiency and predictive accuracy. As a proof of concept, we carried out prospective SBVS in conjunction with a photometric assay, which inferred that the mechanism of action of neopterin (an endogenous immunomodulator) putatively stems from its inhibition (IC50 = 18 μM) of the human purine nucleoside phosphorylase. This ready-to-use library is freely available at http://www.pharmbioinf.uni-freiburg.de/epharmalib.

Reduced purine biosynthesis in humans after their divergence from Neandertals.

We analyze the metabolomes of humans, chimpanzees, and macaques in muscle, kidney and three different regions of the brain. Although several compounds in amino acid metabolism occur at either higher or lower concentrations in humans than in the other primates, metabolites downstream of adenylosuccinate lyase, which catalyzes two reactions in purine synthesis, occur at lower concentrations in humans. This enzyme carries an amino acid substitution that is present in all humans today but absent in Neandertals. By introducing the modern human substitution into the genomes of mice, as well as the ancestral, Neandertal-like substitution into the genomes of human cells, we show that this amino acid substitution contributes to much or all of the reduction of de novo synthesis of purines in humans.

Hyperuricemia Drives Pancreatic β‐cell Death Facilitated by DEPTOR uratylation

Introduction Uric acid (UA) is the final metabolite of the human purine metabolism, also representing the main antioxidant in the blood. However, elevated serum uric acid (SUA) called hyperuricemia and causing gout has been linked to diabetes mellitus (DM). Loss of viable pancreatic β-cells is a hallmark of both development and progression of DM. We aimed to determine how elevated UA levels can induce a loss of cell viability through the AMPK-mTOR pathway, involving the uric acid transporter glucose transporter 9 (GLUT9). Material and methods We employed the human (1.1B4) and mouse (MIN6) pancreatic β-cell lines. Co-immunoprecipitation, siRNA knock-down, western blot analyses, autophagy, MTT and Caspase 3/7 assays were used to determine the effects of chronic hyperuricemia on molecular mechanisms of β-cell viability. Results Hyperuricemia reduced pancreatic β-cell viability by both reduced metabolic activity and increased autophagy/apoptosis, which could be reverted by the GLUT9 inhibitor benzbromarone as well as by GLUT9 knock-down. Phosphorylation of AMP-activated protein kinase (AMPK) was increased upon elevated UA conditions, whereas phosphorylation of mTOR complex-1 (mTORC1) activator Raptor was reduced. However, hyperuricemia increased expression of the mTORC1 negative regulator DEP domain-containing mTOR-interacting protein (DEPTOR). DEPTOR stabilization was mediated by changes in DEPTOR ubiquitination due to the down-regulation of the E3 ligase β-TrCP and upregulation of a newly identified DEPTOR-regulating ubiquitin-specific protease, USP3, in cells exposed to high UA conditions. In addition, we document for the first time that a protein is UA-modified (uratylated) in a cellular context. Interestingly, DEPTOR uratylation seems to contribute to the change of its ubiquitination. We observed this to be different between mouse and human β-cells, where in MIN6 cells only ubiquitin seems to be uratylated, whereas in 1.1B4 cells DEPTOR seems to be directly uratylated. This difference in uratylation of DEPTOR between mouse and human pancreatic β-cells possibly reflects a further evolved, more sophisticated post-transcriptional regulation of proteins based on the difference in UA handling between these species. Our observation also puts into question if mouse models are an accurate representation of the regulatory processes of basic physiological processes such as insulin secretion or β-cell viability in humans. Conclusion Hyperuricemic conditions resembling elevated SUA levels result in a decrease in pancreatic β-cell viability initiated by changes in DEPTOR ubiquitination/uratylation. This results in an increase of autophagy and ultimately apoptosis. Our findings thus suggest a causal basis for the connection between elevated SUA and diabetes mellitus.

The purine nucleoside phosphorylase pnp-1 regulates epithelial cell resistance to infection in C. elegans.

Intestinal epithelial cells are subject to attack by a diverse array of microbes, including intracellular as well as extracellular pathogens. While defense in epithelial cells can be triggered by pattern recognition receptor-mediated detection of microbe-associated molecular patterns, there is much to be learned about how they sense infection via perturbations of host physiology, which often occur during infection. A recently described host defense response in the nematode C. elegans called the Intracellular Pathogen Response (IPR) can be triggered by infection with diverse natural intracellular pathogens, as well as by perturbations to protein homeostasis. From a forward genetic screen, we identified the C. elegans ortholog of purine nucleoside phosphorylase pnp-1 as a negative regulator of IPR gene expression, as well as a negative regulator of genes induced by extracellular pathogens. Accordingly, pnp-1 mutants have resistance to both intracellular and extracellular pathogens. Metabolomics analysis indicates that C. elegans pnp-1 likely has enzymatic activity similar to its human ortholog, serving to convert purine nucleosides into free bases. Classic genetic studies have shown how mutations in human purine nucleoside phosphorylase cause immunodeficiency due to T-cell dysfunction. Here we show that C. elegans pnp-1 acts in intestinal epithelial cells to regulate defense. Altogether, these results indicate that perturbations in purine metabolism are likely monitored as a cue to promote defense against epithelial infection in the nematode C. elegans.

Binding thermodynamics and interaction patterns of human purine nucleoside phosphorylase-inhibitor complexes from extensive free energy calculations.

Human purine nucleoside phosphorylase (hPNP) plays a significant role in the catabolism of deoxyguanosine. The trimeric protein is an important target in the treatment of T-cell cancers and autoimmune disorders. Experimental studies on the inhibition of the hPNP observe that the first ligand bound to one of three subunits effectively inhibits the protein, while the binding of more ligands to the subsequent sites shows negative cooperativities. In this work, we performed extensive end-point and alchemical free energy calculations to determine the binding thermodynamics of the trimeric protein-ligand system. 13 Immucillin inhibitors with experimental results are under calculation. Two widely accepted charge schemes for small molecules including AM1-BCC and RESP are adopted for ligands. The results of RESP are in better agreement with the experimental reference. Further investigations of the interaction networks in the protein-ligand complexes reveal that several residues play significant roles in stabilizing the complex structure. The most commonly observed ones include PHE200, GLU201, MET219, and ASN243. The conformations of the protein in different protein-ligand complexes are observed to be similar. We expect these insights to aid the development of potent drugs targeting hPNP.

Symmetric Nucleosides as Potent Purine Nucleoside Phosphorylase Inhibitors.

Nucleic acids are one of the most enigmatic biomolecules crucial to several biological processes. Nucleic acid-protein interactions are vital for the coordinated and controlled functioning of a cell, leading to the design of several nucleoside/nucleotide analogues capable of mimicking these interactions and hold paramount importance in the field of drug discovery. Purine nucleoside phosphorylase is a well-established drug target due to its association with numerous immunodeficiency diseases. Here, we study the binding of human purine nucleoside phosphorylase (PNP) to some bidirectional symmetric nucleosides, a class of nucleoside analogues that are more flexible due to the absence of sugar pucker restraints. We compared the binding energies of PNP-symmetric nucleosides to the binding energies of PNP-inosine/Imm-H (a transition-state analogue), by means of 200 ns long all-atom explicit-solvent Gaussian accelerated molecular dynamics simulations followed by energetics estimation using the MM-PBSA methodology. Quite interestingly, we observed that a few symmetric nucleosides, namely, ν3 and ν4, showed strong binding with PNP (-14.1 and -12.6 kcal/mol, respectively), higher than inosine (-6.3 kcal/mol) and Imm-H (-9.6 kcal/mol). This is rationalized by an enhanced hydrogen-bond network for symmetric nucleosides compared to inosine and Imm-H while maintaining similar van der Waals contacts. We note that the chemical structures of both ν3 and ν4, due to an additional unsaturation in them, resemble enzymatic transition states and fall in the category of transition-state analogues (TSAs), which are quite popular.

Discovery of a Novel Inhibitor of Human Purine Nucleoside Phosphorylase by a Simple Hydrophilic Interaction Liquid Chromatography Enzymatic Assay.

Human purine nucleoside phosphorylase (HsPNP) belongs to the purine salvage pathway of nucleic acids. Genetic deficiency of this enzyme triggers apoptosis of activated T-cells due to the accumulation of deoxyguanosine triphosphate (dGTP). Therefore, potential chemotherapeutic applications of human PNP inhibitors include the treatment of T-cell leukemia, autoimmune diseases and transplant tissue rejection. In this report, we present the discovery of novel HsPNP inhibitors by coupling experimental and computational tools. A simple, inexpensive, direct and non-radioactive enzymatic assay coupled to hydrophilic interaction liquid chromatography and UV detection (LC-UV using HILIC as elution mode) was developed for screening HsPNP inhibitors. Enzymatic activity was assessed by monitoring the phosphorolysis of inosine (Ino) to hypoxanthine (Hpx) by LC-UV. A small library of 6- and 8-substituted nucleosides was synthesized and screened. The inhibition potency of the most promising compound, 8-aminoinosine (4), was quantified through Ki and IC50 determinations. The effect of HsPNP inhibition was also evaluated in vitro through the study of cytotoxicity on human T-cell leukemia cells (CCRF-CEM). Docking studies were also carried out for the most potent compound, allowing further insights into the inhibitor interaction at the HsPNP active site. This study provides both new tools and a new lead for developing novel HsPNP inhibitors.