Purine Nucleoside Phosphorylase Inhibitor Research Articles

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

The Deoxynucleoside Triphosphate Triphosphohydrolase Activity of SAMHD1 Protein Contributes to the Mitochondrial DNA Depletion Associated with Genetic Deficiency of Deoxyguanosine Kinase

The dNTP triphosphohydrolase SAMHD1 is a nuclear antiviral host restriction factor limiting HIV-1 infection in macrophages and a major regulator of dNTP concentrations in human cells. In normal human fibroblasts its expression increases during quiescence, contributing to the small dNTP pool sizes of these cells. Down-regulation of SAMHD1 by siRNA expands all four dNTP pools, with dGTP undergoing the largest relative increase. The deoxyguanosine released by SAMHD1 from dGTP can be phosphorylated inside mitochondria by deoxyguanosine kinase (dGK) or degraded in the cytosol by purine nucleoside phosphorylase. Genetic mutations of dGK cause mitochondrial (mt) DNA depletion in noncycling cells and hepato-cerebral mtDNA depletion syndrome in humans. We studied if SAMHD1 and dGK interact in the regulation of the dGTP pool during quiescence employing dGK-mutated skin fibroblasts derived from three unrelated patients. In the presence of SAMHD1 quiescent mutant fibroblasts manifested mt dNTP pool imbalance and mtDNA depletion. When SAMHD1 was silenced by siRNA transfection the composition of the mt dNTP pool approached that of the controls, and mtDNA copy number increased, compensating the depletion to various degrees in the different mutant fibroblasts. Chemical inhibition of purine nucleoside phosphorylase did not improve deoxyguanosine recycling by dGK in WT cells. We conclude that the activity of SAMHD1 contributes to the pathological phenotype of dGK deficiency. Our results prove the importance of SAMHD1 in the regulation of all dNTP pools and suggest that dGK inside mitochondria has the function of recycling the deoxyguanosine derived from endogenous dGTP degraded by SAMHD1 in the nucleus.

Tight binding enantiomers of pre-clinical drug candidates

MTDIA is a picomolar transition state analogue inhibitor of human methylthioadenosine phosphorylase and a femtomolar inhibitor of Escherichia coli methylthioadenosine nucleosidase. MTDIA has proven to be a non-toxic, orally available pre-clinical drug candidate with remarkable anti-tumour activity against a variety of human cancers in mouse xenografts. The structurally similar compound MTDIH is a potent inhibitor of human and malarial purine nucleoside phosphorylase (PNP) as well as the newly discovered enzyme, methylthioinosine phosphorylase, isolated from Pseudomonas aeruginosa. Since the enantiomers of some pharmaceuticals have revealed surprising biological activities, the enantiomers of MTDIH and MTDIA, compounds 1 and 2, respectively, were prepared and their enzyme binding properties studied. Despite binding less tightly to their target enzymes than their enantiomers compounds 1 and 2 are nanomolar inhibitors.

New medications in development for the treatment of hyperuricemia of gout.

To update recent developments in medications targeting hyperuricemia, but not including medications recently labelled in the European Union and the United States. A new xanthine oxidase inhibitor, Topiloric (Fujiyakuhin Co., Ltd. Japan) Uriadec (Sanwa Kagaku Kenkyusho Co., Ltd. Japan), has been developed and labelled in Japan. An inhibitor of purine nucleoside phosphorylase, Ulodesine, is in development in combination with allopurinol. The rest of the medications in the pipeline for hyperuricemia are targeting renal transporters of uric acid, mainly URAT1 and OAT4, acting as uricosuric agents. Most of them, such as lesinurad and arhalofenate, are being tested in trials in combination with allopurinol and febuxostat. The most potent RDEA3170 is being tested in monotherapy, but also associated with febuxostat. Recently, medications showing dual activity, inhibiting both xanthine oxidoreductase and URAT1, have been communicated or started exploratory clinical trials. There is no report of medications targeting other transporters such as Glut9 or ABCG2. There are a number of medications in the pipeline targeting hyperuricemia, mostly uricosurics in combination with xanthine oxidase inhibitors, but some targeting both xanthine oxidoreductase and URAT1. Increasing the number of available medications will ensure proper control of hyperuricemia to target serum urate levels in the near future for most, if not all, patients with hyperuricemia.

Mycoplasma hyorhinis-encoded cytidine deaminase efficiently inactivates cytosine-based anticancer drugs

Mycoplasmas may colonize tumor tissue in patients. The cytostatic activity of gemcitabine was dramatically decreased in Mycoplasma hyorhinis-infected tumor cell cultures compared with non-infected tumor cell cultures. This mycoplasma-driven drug deamination could be prevented by exogenous administration of the cytidine deaminase (CDA) inhibitor tetrahydrouridine, but also by the natural nucleosides or by a purine nucleoside phosphorylase inhibitor. The M. hyorhinis-encoded CDAHyor gene was cloned, expressed as a recombinant protein and purified. CDAHyor was found to be more catalytically active than its human equivalent and efficiently deaminates (inactivates) cytosine-based anticancer drugs. CDAHyor expression at the tumor site may result in selective drug inactivation and suboptimal therapeutic efficiency.

WS3-4 - New Treatment Modalities for Atl

ATL is a distinct malignancy of Treg/TH2 cells with caused by HTLV-1. ATL still has a worse prognosis than other T-cell malignancies in general. The diversity in clinical features and prognosis of patients with this disease has led to its subtype-classification into acute, lymphoma, chronic, and smoldering types. In case acute, lymphoma or unfavorable chronic subtypes (aggressive ATL), and favorable chronic or smoldering subtypes (indolent ATL), intensive chemotherapy followed by allogeneic stem cell transplantation and watchful waiting until disease progression has been recommended, respectively in Japan. A retrospective analysis suggested that the combination of interferon alpha and zidovudine was promising for the treatment of ATL, especially for leukemic subtypes. There is no plateau with an initial steep slope and subsequent gentle slope in the survival curves for aggressive and indolent ATL treated with chemotherapy and watchful waiting, respectively, although the prognosis is much better in the latter (MST; 1 year vs. 5 years), suggesting the need of refinement of subtype-classification. Defucosylated humanized anti-CCR4 monoclonal antibody (mogamulizumab), which showed strengthened ADCC activity as compared to usual Ab in preclinical analysis, was approved for the treatment of relapsed/refractory ATL based on the results of phase I and II studies, with response rate of about 50% and manageable toxicities including moderate to severe skin reaction, in Japan. Recently, the results of the subsequent randomized phase II study of chemotherapy +/- mogamulizumab for untreated patients with aggressive ATL was reported. This combination is anticipated because the former was more effective for ATL cells in lymphnode than those in peripheral blood and the latter was vice versa. The combination of mLSG15 plus mogamulizumab was well tolerated and produced a higher CR rate. Several new agent trials for ATL are ongoing and in preparation after translational research, including histone deacetylase inhibitors, a purine nucleoside phosphorylase inhibitor, a proteasome inhibitor and lenalidomide.

Final results of a multicenter phase II study of the purine nucleoside phosphorylase (PNP) inhibitor forodesine in patients with advanced cutaneous t-cell lymphomas (CTCL) (Mycosis fungoides and Sézary syndrome)

Forodesine is a potent inhibitor of purine nucleoside phosphorylase (PNP) that leads to intracellular accumulation of deoxyguanosine triphosphate (dGTP) in T and B cells, resulting in apoptosis. Forodesine has demonstrated impressive antitumor activity in early phase clinical trials in cutaneous T-cell lymphoma (CTCL). In this phase II study, patients with CTCL who had already failed three or more systemic therapies were recruited. We investigated the response rate, safety and tolerability of oral forodesine treatment in subjects with cutaneous manifestations of CTCL, stages IB, IIA, IIB, III and IVA. The safety population encompassing all stages was used for analysis of accountability, demographics and safety. The efficacy population differed from the safety population by exclusion of stage IB and IIA patients. All 144 patients had performance status 0-2. The median duration of CTCL from diagnosis was 53 months (5-516 months). The median number of pretreatments was 4 (range: 3-15). No complete remissions were observed. In the efficacy group of patients, 11% achieved partial remission and 50% had stable disease. The median time to response was 56 days and the median duration of response was 191 days. A total of 96% of all treated patients reported one or more adverse events (AEs) and 33% reported a serious AE. The majority of AEs were classified as mild or moderate in severity. The most commonly reported AEs (>10%) were peripheral edema, fatigue, insomnia, pruritus, diarrhea, headache and nausea. Overall eight patients died during the study: five due to sepsis and infections, one due to a second malignancy (esophageal cancer), one due to disease progression and one due to liver failure. Oral forodesine at a dose of 200 mg daily is feasible and shows partial efficacy in this highly selected CTCL population and some durable responses.

Adenosine A1 receptor-dependent antinociception induced by inosine in mice: pharmacological, genetic and biochemical aspects.

Inosine is an endogenous nucleoside that has anti-inflammatory and antinociceptive properties. Inosine is a metabolite of adenosine, and some of its actions suggest the involvement of adenosine A1 receptors (A1Rs). The purpose of this study was to better understand mechanisms of inosine-induced antinociception by investigating the role of A1Rs and purine metabolism inhibitors. Inosine antinociception was evaluated using the formalin test in mice. An A1R-selective antagonist (DPCPX), A1R knockout mice (gene deletion) and mice with A1R reduced expression (antisense oligonucleotides) were used to assess the role of A1Rs in the antinociceptive action of inosine. Binding assays were performed to compare the affinity of inosine and adenosine for A1Rs. Finally, the role of adenosine and inosine breakdown was assessed using deoxycoformycin (DCF) and forodesine (FDS) as enzymatic inhibitors of adenosine deaminase and purine nucleoside phosphorylase, respectively. Inosine induced antinociception in the formalin test when given by systemic, spinal and peripheral routes. Systemically, inosine exhibited a potency similar to adenosine, and its effects were inhibited by DPCPX. Inosine did not induce antinociception in A1R knockout mice or in mice with reduced A1R expression. In binding studies, inosine bound to A1Rs with an affinity similar to adenosine. DCF had no effect on inosine actions. FDS augmented the antinociceptive effect of a low systemic dose of inosine and, at a higher dose, induced antinociception by itself. Collectively, these data indicate that inosine is an agonist for A1Rs with antinociceptive properties and a potency similar to adenosine and can be considered another endogenous ligand for this receptor.

Administration of deoxyribonucleosides or inhibition of their catabolism as a pharmacological approach for mitochondrial DNA depletion syndrome

Mitochondrial DNA (mtDNA) depletion syndrome (MDS) is characterized by a reduction in mtDNA copy number and consequent mitochondrial dysfunction in affected tissues. A subgroup of MDS is caused by mutations in genes that disrupt deoxyribonucleotide metabolism, which ultimately leads to limited availability of one or several deoxyribonucleoside triphosphates (dNTPs), and subsequent mtDNA depletion. Here, using in vitro experimental approaches (primary cell culture of deoxyguanosine kinase-deficient cells and thymidine-induced mtDNA depletion in culture as a model of mitochondrial neurogastrointestinal encephalomyopathy, MNGIE), we show that supplements of those deoxyribonucleosides (dNs) involved in each biochemical defect (deoxyguanosine or deoxycytidine, dCtd) prevents mtDNA copy number reduction. Similar effects can be obtained by specific inhibition of dN catabolism using tetrahydrouridine (THU; inhibitor of cytidine deaminase) or immucillin H (inhibitor of purine nucleoside phosphorylase). In addition, using an MNGIE animal model, we provide evidence that mitochondrial dNTP content can be modulated in vivo by systemic administration of dCtd or THU. In spite of the severity associated with diseases due to defects in mtDNA replication, there are currently no effective therapeutic options available. Only in the case of MNGIE, allogeneic hematopoietic stem cell transplantation has proven efficient as a long-term therapeutic strategy. We propose increasing cellular availability of the deficient dNTP precursor by direct administration of the dN or inhibition of its catabolism, as a potential treatment for mtDNA depletion syndrome caused by defects in dNTP metabolism.

Distortional binding of transition state analogs to human purine nucleoside phosphorylase probed by magic angle spinning solid-state NMR.

Transition state analogs mimic the geometry and electronics of the transition state of enzymatic reactions. These molecules bind to the active site of the enzyme much tighter than substrate and are powerful noncovalent inhibitors. Immucillin-H (ImmH) and 4'-deaza-1'-aza-2'-deoxy-9-methylene Immucillin-H (DADMe-ImmH) are picomolar inhibitors of human purine nucleoside phosphorylase (hPNP). Although both molecules are electronically similar to the oxocarbenium-like dissociative hPNP transition state, DADMe-ImmH is more potent than ImmH. DADMe-ImmH captures more of the transition state binding energy by virtue of being a closer geometric match to the hPNP transition state than ImmH. A consequence of these similarities is that the active site of hPNP exerts greater distortional forces on ImmH than on DADMe-ImmH to "achieve" the hPNP transition state geometry. By using magic angle spinning solid-state NMR to investigate stable isotope-labeled ImmH and DADMe-ImmH, we have explored the difference in distortional binding of these two inhibitors to hPNP. High-precision determinations of internuclear distances from NMR recoupling techniques, rotational echo double resonance, and rotational resonance, have provided unprecedented atomistic insight into the geometric changes that occur upon binding of transition state analogs. We conclude that hPNP stabilizes conformations of these chemically distinct analogs having distances between the cation and leaving groups resembling those of the known transition state.

Preclinical and Clinical Evaluation of Forodesine in Pediatric and Adult B-Cell Acute Lymphoblastic Leukemia

The discovery that purine nucleoside phosphorylase (PNP) deficiency leads to T-cell lymphopenia was the basis for introducing PNP inhibitors for T-cell leukemias. Forodesine is an orally bioavailable PNP inhibitor with picomolar potency. Because T lymphoblasts and indolent chronic lymphocytic leukemia (CLL) B cells inherently elicit favorable pharmacokinetics to accumulate deoxyguanosine triphosphate (dGTP), forodesine demonstrated promising activity in preclinical and clinical settings for patients with T-cell acute lymphoblastic leukemia (T-ALL) and B-cell CLL (B-CLL). However, the use of forodesine in B-cell ALL (B-ALL) is unknown. Leukemic blasts obtained from pediatric patients with de novo B-ALL (n= 10) were incubated with forodesine and deoxyguanosine (dGuo), and the biological end points of apoptosis, intracellular dGTP accumulation, and inhibition of RNA and DNA synthesis were measured. Additionally, adult patients with B-ALL (n= 2) were intravenously infused with 80 mg/m(2)/d dailyfor 5 days. After therapy, clinical response, toxicity, laboratory biomarkers including PNP enzyme inhibition, and plasma forodesine, dGuo, and intracellular dGTP levels were analyzed. Our invitro investigations demonstrated that forodesine treatment inhibited proliferation and induced modest apoptosis in de novo B-ALL lymphoblasts. There was time-dependent accumulation of dGTP and inhibition of RNA and DNA synthesis. During therapy, neither patient achieved a complete response (CR), but there was disease stabilization for several weeks in both patients. There was significant maintained inhibition of PNP enzyme in red blood cells, accumulation of forodesine and dGuo in plasma, and intracellular dGTP accumulation in both patients. Our preclinical and clinical investigations suggest that forodesine has activity in B-ALL. However, it needs to be either infused with dGuo or combined with established chemotherapeutic agents based on mechanistic rationale.

OP0106 Effect of BCX4208 add-on therapy to allopurinol 300 mg on plasma hypoxanthine and xanthine concentrations in gout patients

BackgroundBCX4208 is an oral, once-daily, purine nucleoside phosphorylase (PNP) inhibitor in clinical development as add-on therapy for the chronic management of hyperuricemia in patients with gout. Based on the role...

FRI0367 BCX4208 added to allopurinol increases response rates in patients with GOUT who fail to reach goal range serum uric acid on allopurinol alone: A randomized, double-blind, placebo-controlled trial

BackgroundBCX4208, a novel purine nucleoside phosphorylase inhibitor, blocks uric acid production at a step preceding xanthine oxidase and synergistically reduces serum urate concentration (sUA) in gout patients when combined with...

FRI0380 Long-term safety of BCX4208 added to allopurinol in the chronic management of GOUT: Results of a phase 2 24-week blinded safety extension and vaccine challenge study

BackgroundMost gout patients treated with 300 mg/d allopurinol do not reach the therapeutic goal range serum uric acid concentration (sUA) of <6.0 mg/dL (360 mmol/L).1 BCX4208, a purine nucleoside phosphorylase...

FRI0401 BCX4208, a novel enzyme inhibitor for chronic management of GOUT, shows a low risk of potential drug-drug interactions

BackgroundCommon comorbidities associated with gout, including obesity, hypertension, diabetes, and chronic kidney disease, may confer a greater risk of drug-drug interaction (DDI) through both polypharmacy and disease-associated alterations in drug...

The transition state analog inhibitor of Purine Nucleoside Phosphorylase (PNP) Immucillin-H arrests bone loss in rat periodontal disease models

Purine nucleoside phosphorylase (PNP) is a purine-metabolizing enzyme that catalyzes the reversible phosphorolysis of 6-oxypurine (deoxy)nucleosides to their respective bases and (deoxy)ribose-1-phosphate. It is a key enzyme in the purine salvage pathway of mammalian cells. The present investigation sought to determine whether the PNP transition state analog inhibitor (Immucillin-H) arrests bone loss in two models of induced periodontal disease in rats. Periodontal disease was induced in rats using ligature or LPS injection followed by administration of Immucillin-H for direct analysis of bone loss, histology and TRAP staining. In vitro osteoclast differentiation and activation of T CD4+ cells in the presence of Immucillin-H were carried out for assessment of RANKL expression, PNP and Cathepsin K activity. Immucillin-H inhibited bone loss induced by ligatures and LPS, leading to a reduced number of infiltrating osteoclasts and inflammatory cells. In vitro assays revealed that Immucillin-H could not directly abrogate differentiation of osteoclast precursor cells, but affected lymphocyte-mediated osteoclastogenesis. On the other hand, incubation of pre-activated T CD4+ with Immucillin-H decreased RANKL secretion with no compromise of cell viability. The PNP transition state analog Immucillin-H arrests bone loss mediated by T CD4+ cells with no direct effect on osteoclasts. PNP inhibitor may have an impact in the treatment of diseases characterized by the presence of pathogens and imbalances of bone metabolism.

Natural products as inhibitors of purine nucleoside phosphorylase, a target against Plasmodium spp

Natural products as inhibitors of purine nucleoside phosphorylase, a target against Plasmodium spp

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.

Design and Divergent Synthesis of Aza Nucleosides from a Chiral Imino Sugar

Several novel nucleoside analogues as potential inhibitors of glycosidases and purine nucleoside phosphorylase (PNP) have been synthesized via selective coupling of an appropriate nucleobase at different positions of an orthogonally protected imino sugar as a common precursor. This synthetic strategy offers a straightforward protocol for the assembly of imino sugar containing nucleosides, establishing a new repertoire of molecules as potential therapeutics.

Phase I study of BCX1777 (forodesine) in patients with relapsed or refractory peripheral T/natural killer‐cell malignancies

BCX1777 (forodesine), a novel purine nucleoside phosphorylase inhibitor, induces apoptosis, mainly in T cells. To evaluate the safety, tolerability, and pharmacokinetics of BCX1777, we conducted a phase I study in patients with relapsed or refractory peripheral T/natural killer-cell malignancies. Eligible patients had relapsed or refractory peripheral T/natural killer-cell malignancies without any major organ dysfunction. BCX1777 was administered orally once daily (dose escalation: 100, 200, and 300 mg) until disease progression requiring new therapy or unacceptable adverse events occurred. A total of 13 patients were enrolled and treated in three dose cohorts (100 mg/day, five patients; 200 mg/day, three patients; 300 mg/day, five patients). Although none of the patients developed dose-limiting toxicities, further dose escalation was not performed based on data from overseas. Therefore, the maximum tolerated dose was not determined. Adverse events of grade 3 or greater (≥2 patients) included lymphopenia (62%), anemia (15%), leukopenia (8%), and pyrexia (8%). Plasma pharmacokinetics parameter of BCX1777 (area under the plasma concentration-time curve) at day 1 in each cohort was 1948 ± 884, 4608 ± 1030, and 4596 ± 939 ng•h/mL, respectively. Disease control was achieved in approximately half of patients. One patient with anaplastic large cell lymphoma, which was negative for anaplastic lymphoma kinase, achieved a complete response, and two patients with cutaneous T-cell lymphoma achieved partial responses. BCX1777 was well tolerated at doses up to 300 mg once daily and showed preliminary evidence of activity in relapsed or refractory peripheral T/natural killer-cell malignancies, warranting further investigation.