PNP Inhibitors Research Articles

Purine nucleoside phosphorylase: A new pharmacological target in sickle cell disease and hemolytic vasculopathy

Under hemolytic conditions, there is a significant interaction among red blood cells (RBCs), endothelial cells (ECs), and platelets in the injured vasculature. In sickle cell disease (SCD), this interaction leads to painful vaso-occlusive crisis and end organ damage, results in poor quality of life, and unacceptably short life expectancy. We propose that these cells share two biochemical properties that are highly relevant to SCD. First, per cell volume, RBCs have the highest level of purine nucleoside phosphorylase (PNP, a key enzyme in purine metabolism), and ECs and platelets are very rich in PNP. Second, RBCs have to, ECs choose to, and platelets, when activated, switch to using glycolysis as a major pathway for intermediate phosphates and ATP production. We propose that these similarities are highly relevant to sickle cell disease (SCD) and hemolytic vascular injury. Human PNP catalyzes the phosphorolysis of guanosine/ deoxyguanosine and inosine /deoxyinosine into guanine and hypoxanthine, respectively, and generates ribose-1-phosphate and deoxyribose-1-phosphate. This results in forming a large amount of downstream pro-oxidant and vasculotoxic xanthines.Recently, we have detected that in SCD, hemolysis is associated with increased activity of extracellular PNP, accelerated guanosine and inosine metabolism, and augmented extracellular production of vasculotoxic hypoxanthine and xanthine. We identify PNP as a critical pathogenic mechanism in SCD and have proposed a link between PNP activity and the glycolytic production of intermediate phosphates and ATP. This link promotes (i) RBC sickling/hemolysis, (ii.) hemolytic angioproliferation, (iii.) oxidative vascular injury, and (iv.) platelets activation/aggregation.Furthermore, PNP accelerates guanosine/inosine metabolism and may diminish their well-documented anti-inflammatory, anti-ischemic, anti-thrombotic, and anti-nociceptive effects. We hypothesize that inhibition of PNP may lessen those four (i.to iv.) deleterious events and may potentiate the beneficial effects of guanosine/inosine, reducing the risk of vaso-occlusion and organ damage in SCD patients. Thereby, the PNP inhibitors may serve as a multipronged treatment approach for SCD.

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.

Abstract 3034: Purine nucleoside phosphorylase regulates metabolic and immune checkpoints

Abstract Purine nucleoside phosphorylase (PNP) functions as a central regulator of nucleotide metabolism by catalyzing the degradation of purine nucleosides guanosine and deoxyguanosine. PNP inactivation has been paradoxically associated with both immunodeficiency and autoimmunity in humans but the genetic and molecular determinants of these divergent outcomes are poorly defined and whether they reflect cell type specific mechanisms is currently unknown. Moreover, the mechanisms underlying the clinical efficacy of PNP inhibitors in a subset of patients with hematological malignancies are yet to be determined, thereby severely limiting the potential of this promising new class of metabolic targeted therapies. To address these gaps in knowledge we performed a systematic evaluation of the metabolic, immunological and cell-context phenotypic effects of targeting PNP using a clinical-stage inhibitor with picomolar affinity and excellent oral bioavailability. Here we show that across cancer types PNP inhibition is synthetically lethal with sterile alpha motif and HD domain-containing protein 1 (SAMHD1) deficiency by triggering massive dGTP pool expansion, and impairing DNA replication. These cytotoxic effects require the expression of deoxycytidine kinase (dCK) which mediates the intracellular trapping of deoxyguanosine and expansion of dGTP pools, and are opposed by environmental deoxycytidine produced by bone marrow stromal cells in a SAMHD1 and nucleoside transporter dependent manner. This resistance mechanism to PNP inhibition can be counteracted by the deoxycytidine catabolizing enzyme cytidine deaminase (CDA). We further show that in addition to, and independent of its dCK and CDA-dependent cytotoxic effects in SAMHD1 deficient cells, PNP inhibition elevates serum inflammatory cytokines and cytokine transcript levels in secondary lymphoid organs. Using scRNAseq we identify CD19+ B-cells and CSF1R+ macrophages, which express the pattern recognition receptor TLR7, as the drivers of these alterations. TLR7 activation in these cell types is promoted by guanosine accumulation which functions as a ligand for TLR7 alongside uridine-containing ssRNA. Importantly, TLR7 stimulation achieved by PNP inhibition initiates transcriptional alterations that are quantitatively and qualitatively unique from synthetic TLR7 agonists, such as the production of type I interferons. We additionally demonstrate that PNP inactivation stimulates germinal center responses in secondary lymphoid organs via the expansion of activated B-cells and T follicular helper cells. In summary, we demonstrate that by modulating multiple metabolic and immune checkpoints in a multigenic, cell type and environment dependent manner, pharmacological PNP inhibition elicits specific outcomes which can be leveraged for the development of novel therapeutic applications. Citation Format: Evan R. Abt, Khalid Rashid, Thuc M. Le, Hailey R. Lee, Amanda L. Creech, Ting-Ting Wu, Thomas Mehrling, Shanta Bantia, Caius G. Radu. Purine nucleoside phosphorylase regulates metabolic and immune checkpoints [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3034.

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.

Purine nucleoside phosphorylase inhibitors - an immunotherapy with novel mechanism of action for the treatment of melanoma

Meeting abstracts Contrary to expectations based on the immuno-compromised clinical phenotype of the PNP-deficient patients, the present study demonstrates that PNP inhibitors (PNPi) can activate immune cells that can help mount a robust antitumor response. PNP deficiency in humans lead to

Evaluation of capillary chromatographic supports for immobilized human purine nucleoside phosphorylase in frontal affinity chromatography studies

The aim of this work was to optimize the preparation of a capillary human purine nucleoside phosphorylase (HsPNP) immobilized enzyme reactor (IMER) for characterization and affinity screening studies of new inhibitors by frontal affinity chromatography coupled to mass spectrometry (FAC-MS). For this purpose two monolithic supports, a Chromolith Speed Rod (0.1mm I.D.×5cm) and a methacrylate-based monolithic epoxy polymeric capillary column (0.25mm I.D.×5cm) with epoxy reactive groups were considered and compared to an IMER previously developed using an open fused silica capillary. Each HsPNP-IMER was characterized in terms of catalytic activity using Inosine as standard substrate. Furthermore, they were also explored for affinity ranking experiments. Kd determination was carried out with the based fused silica HsPNP-IMER and the results are herein discussed.

Enzyme kinetics, structural analysis and molecular modeling studies on a series of Schistosoma mansoni PNP inhibitors

The enzyme purine nucleoside phosphorylase from Schistosoma mansoni (SmPNP) is an attractive molecular target for the development of novel drugs against schistosomiasis, a neglected tropical disease that affects about 200 million people worldwide. In the present work, enzyme kinetic studies were carried out in order to determine the potency and mechanism of inhibition of a series of SmPNP inhibitors. In addition to the biochemical investigations, crystallographic and molecular modeling studies revealed important molecular features for binding affinity towards the target enzyme, leading to the development of structure-activity relationships (SAR).

ChemInform Abstract: Synthesis and Biological Activity of a Novel Class of Purine Nucleoside Phosphorylase Inhibitors.

Abstract Purine nucleoside phosphorylase.(PNP, EC. 2.4.2.1) functions as a salvage enzyme in the purine and is important in the T-cell portion of the immune system As such, PNP is an important therapeutic target for diseases which are T-cell mediated. The biochemical basis for using PNP inhibitors as well as the various classes of inhibitors developed has been recently reviewed. Using X-ray crystallography we have designed and synthesized a series of 7-substituted 2-amino-1,5-dihydroHpyrrolo[3,2-djpyrimidin-4-ones which are potent PNP inhibitors .2-5 The 3-(pyridinylmethyl) derivative 1 (BCX-34, peldesine, IC50 = 0.036 ± 0.003 μM) is in clinical trials for the treatment of cutaneous T-cell Ivqphoma. acute lymphoblastic leukemia and HIV infections.

Potent orally bioavailable purine nucleoside phosphorylase inhibitor BCX-4208 induces apoptosis in B- and T-lymphocytes—A novel treatment approach for autoimmune diseases, organ transplantation and hematologic malignancies

The profound suppression of T-cell immunity seen in purine nucleoside phosphorylase (PNP; EC 2.4.2.1) deficient patients supports potential application of inhibitors of PNP in the therapy of T-cell mediated diseases. BCX-4208 is a novel potent transition state analog inhibitor of human PNP with an IC 50 of 0.5 nM. PNP inhibition leads to elevation of dGuo which is converted to dGTP mainly in lymphocytes causing imbalance in deoxynucleotide (dNTP) pools and cell apoptosis. In in vitro studies, neither BCX-4208 nor dGuo alone inhibits proliferation of lymphocytes. BCX-4208 in the presence of 10 µM deoxyguanosine (dGuo) inhibits lymphocyte proliferation induced by MLR, IL-2 or Con A with IC 50s of 0.159, 0.26 and 0.73 µM, respectively. The IC 50 for dGuo in the presence of 1 µM BCX-4208 for the IL-2 stimulated lymphocytes was 3.12 µM. dGTP in human lymphocytes is elevated and a 3–5 fold increase in dGTP results in 50% inhibition after in vitro exposure to BCX-4208 and dGuo. Flow cytometric analyses of human lymphocytes using annexin V staining reveal that BCX-4208 in the presence of dGuo induces cellular apoptosis in T-cells (CD3+), B-cells (CD20+, CD19+) and NK (CD56+) cells. BCX-4208 is orally bioavailable in mice and elevates plasma dGuo levels to 3.7 µM (predose levels < 0.004 µM), similar to levels seen in PNP-deficient patients and levels needed to cause apoptosis in T and B-cells. These data support the evaluation of BCX-4208 in the treatment of T-cell and B-cell mediated diseases. BCX-4208 is currently undergoing early clinical investigation in psoriasis and gout.

9‐Deazaguanine derivatives connected by a linker to difluoromethylene phosphonic acid are slow‐binding picomolar inhibitors of trimeric purine nucleoside phosphorylase

Genetic deficiency of purine nucleoside phosphorylase (PNP; EC 2.4.2.1) activity leads to a severe selective disorder of T-cell function. Therefore, potent inhibitors of mammalian PNP are expected to act as selective immunosuppressive agents against, for example, T-cell cancers and some autoimmune diseases. 9-(5',5'-difluoro-5'-phosphonopentyl)-9-deazaguanine (DFPP-DG) was found to be a slow- and tight-binding inhibitor of mammalian PNP. The inhibition constant at equilibrium (1 mm phosphate concentration) with calf spleen PNP was shown to be = 85 +/- 13 pm (pH 7.0, 25 degrees C), whereas the apparent inhibition constant determined by classical methods was two orders of magnitude higher ( = 4.4 +/- 0.6 nm). The rate constant for formation of the enzyme/inhibitor reversible complex is (8.4 +/- 0.5) x 10(5) m(-1).s(-1), which is a value that is too low to be diffusion-controlled. The picomolar binding of DFPP-DG was confirmed by fluorimetric titration, which led to a dissociation constant of 254 pm (68% confidence interval is 147-389 pm). Stopped-flow experiments, together with the above data, are most consistent with a two-step binding mechanism: E + I <--> (EI) <--> (EI)*. The rate constants for reversible enzyme/inhibitor complex formation (EI), and for the conformational change (EI) <--> (EI)*, are k(on1) = (17.46 +/- 0.05) x 10(5) m(-1).s(-1), k(off1) = (0.021 +/- 0.003) s(-1), k(on2) = (1.22 +/- 0.08) s(-1) and k(off2) = (0.024 +/- 0.005) s(-1), respectively. This leads to inhibition constants for the first (EI) and second (EI)* complexes of K(i) = 12.1 nM (68% confidence interval is 8.7-15.5 nm) and = 237 pm (68% confidence interval is 123-401 pm), respectively. At a concentration of 10(-4) m, DFPP-DG exhibits weak, but statistically significant, inhibition of the growth of cell lines sensible to inhibition of PNP activity, such as human adult T-cell leukaemia and lymphoma (Jurkat, HuT78 and CCRF-CEM). Similar inhibitory activities of the tested compound were noted on the growth of lymphocytes collected from patients with Hashimoto's thyroiditis and Graves' disease. The observed weak cytotoxicity may be a result of poor membrane permeability.

Crystal structure of Schistosoma purine nucleoside phosphorylase complexed with a novel monocyclic inhibitor

A novel inhibitor of Schistosoma PNP was identified using an “in silico” approach allied to enzyme inhibition assays. The compound has a monocyclic structure which has not been previously described for PNP inhibitors. The crystallographic structure of the complex was determined and used to elucidate the binding mode within the active site. Furthermore, the predicted pose was very similar to that determined crystallographically, validating the methodology. The compound Sm_VS1, despite its low molecular weight, possesses an IC 50 of 1.3 μM, surprisingly low when compared with purine analogues. This is presumably due to the formation of eight hydrogen bonds with key residues in the active site E203, N245 and T244. The results of this study highlight the importance of the use of multiple conformations for the target during virtual screening. Indeed the Sm_VS1 compound was only identified after flipping the N245 side chain. It is expected that the structure will be of use in the development of new highly active non-purine based compounds against the Schistosoma enzyme.

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.

Plasmodium falciparum Purine Nucleoside Phosphorylase Is Critical for Viability of Malaria Parasites

Human malaria infections resulting from Plasmodium falciparum have become increasingly difficult to treat due to the emergence of drug-resistant parasites. The P. falciparum purine salvage enzyme purine nucleoside phosphorylase (PfPNP) is a potential drug target. Previous studies, in which PfPNP was targeted by transition state analogue inhibitors, found that those inhibiting human PNP and PfPNPs killed P. falciparum in vitro. However, many drugs have off-target interactions, and genetic evidence is required to demonstrate single target action for this class of potential drugs. We used targeted gene disruption in P. falciparum strain 3D7 to ablate PNP expression, yielding transgenic 3D7 parasites (Deltapfpnp). Lysates of the Deltapfpnp parasites showed no PNP activity, but activity of another purine salvage enzyme, adenosine deaminase (PfADA), was normal. When compared with wild-type 3D7, the Deltapfpnp parasites showed a greater requirement for exogenous purines and a severe growth defect at physiological concentrations of hypoxanthine. Drug assays using immucillins, specific transition state inhibitors of PNP, were performed on wild-type and Deltapfpnp parasites. The Deltapfpnp parasites were more sensitive to PNP inhibitors that bound hPNP tighter and less sensitive to MT-ImmH, an inhibitor with 100-fold preference for PfPNP over hPNP. The results demonstrate the importance of purine salvage in P. falciparum and validate PfPNP as the target of immucillins.

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.

Crystal structure of calf spleen purine nucleoside phosphorylase complexed to a novel purine analogue

The combined use of a rapid virtual screen of a small fragment library together with a single point enzyme assay has been used for the discovery of novel PNP inhibitors. The availability of readily soakable crystals of bovine PNP has allowed the approach to be experimentally validated by determining the crystal structure of one of the inhibitor-PNP complexes. Comparison of the experimentally determined binding mode with that predicted by the virtual screening shows them to be similar. This represents a starting point for the growth of the ligand into a higher affinity inhibitor.

Immucillins as Antibiotics for T‐Cell Proliferation and Malaria

AbstractFor Abstract see ChemInform Abstract in Full Text.

Immucillins as Antibiotics for T‐Cell Proliferation and Malaria

The genetic deficiency of human PNP causes a specific immunodeficiency by inducing apoptosis in dividing T‐cells. Powerful inhibitors of PNP have been designed from the experimental determination of the transition state structure of PNPs. The Immucillins are transition state analogue inhibitors with K d values as low as 7 pM. In the presence of deoxyguanosine the Immucillins kill activated human T‐cells but not other cell types. The Immucillins are orally available and of low toxicity to mice. Immucillins also inhibit PNP from Plasmodium falciparum. Parasites cultured in human erythrocytes are killed by purine starvation in the presence of Immucillins and can be rescued by hypoxanthine.

The Chemical Development of CI-972 and CI-1000: A Continuous Nitration, A MgCl2/Et3N-Mediated C-Alkylation of a Chloronitropyrimidine, A Catalytic Protodediazotization of a Diazonium Salt, and an Air Oxidation of an Amine

Efficient, large-scale processes were developed for the preparation of the potent PNP inhibitors 2,6-diamino-3,5-dihydro-7-(3-thienylmethyl)-4H-pyrrolo[3,2-d]pyrimidin-4-one hydrochloride, monohydrate (1) and 2-amino-3,5-dihydro-7-(3-thienylmeth-yl)-4H-pyrrolo[3,2-d]pyrimidin-4-one hydrochloride, monohydrate (2). We report (1) a safe, continuous nitration process for the preparation of 2-amino-6-chloro-5-nitro-4-pyrimidinol (8a) and its stable diisopropylamine salt (8b), (2) the first MgCl2/Et3N-mediated C-alkylation of a chloronitropyrimidine, (3) a rare catalytic protodediazotization of the diazonium salt 2-amino-4-oxo-7-thiophen-3-ylmethyl-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidine-6-diazonium chloride (14), (4) a single-step process to prepare 2 directly from 2-amino-6-hydroxy-5-nitro-α-(3-thienylmethyl)-4-pyrimidineacetonitrile (12) using a sponge nickel-catalyzed reduction, and (5) a method to convert the over-reduction by-product 2,5-diamino-6-(1-aminomethyl-2-thiophen-3-yl-ethyl)-pyrimidin-4-ol (16) ...

Synthesis and Biological Activity of a Novel Class of Purine Nucleoside Phosphorylase Inhibitors

Purine nucleoside phosphorylase.(PNP, EC. 2.4.2.1) functions as a salvage enzyme in the purine and is important in the T-cell portion of the immune system As such, PNP is an important therapeutic target for diseases which are T-cell mediated. The biochemical basis for using PNP inhibitors as well as the various classes of inhibitors developed has been recently reviewed. Using X-ray crystallography we have designed and synthesized a series of 7-substituted 2-amino-1,5-dihydroHpyrrolo[3,2-djpyrimidin-4-ones which are potent PNP inhibitors .2-5 The 3-(pyridinylmethyl) derivative 1 (BCX-34, peldesine, IC50 = 0.036 ± 0.003 μM) is in clinical trials for the treatment of cutaneous T-cell Ivqphoma. acute lymphoblastic leukemia and HIV infections.

Antiviral acyclic nucleoside phosphonate analogues as inhibitors of purine nucleoside phosphorylase.

Purine nucleoside phosphorylase (PNP, E.C.2.4.2.1) catalyses reversible phosphoro-lysis of purine ribo- and deoxyribonucleosides. Mammalian phosphorylases are specific for 6-ketopurines while some bacterial PNPs (e.g. from E. coli and S. typhimurium) have very broad specificty.l–4 Genetic deficiency of PNP leads to a loss of cellular immunity.5 The potential clinical applications of PNP inhibitors include treatment of T-cell leukemias, suppression of the host-vs-graft response in organ transplantation and potentation of the chemotherapeutic action of PNP-cleavable nucleosides.6