Synthesis and structure-activity relationships of ticlopidine derivatives and analogs as inhibitors of ectonucleotidase CD39.

Ticlopidine is an antithrombotic prodrug of the thienotetrahydropyridine family. For platelet inhibition it has to undergo oxidative ring-opening by cytochrome P450 enzymes. The resulting thiol reacts with a cysteine residue of the purinergic P2Y12 receptor on thrombocytes resulting in covalent receptor blockade. Ticlopidine in its intact, not-metabolized form was previously shown to inhibit ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1, also known as cluster of differentiation (CD) 39). CD39 catalyzes the extracellular hydrolysis of ATP via ADP to AMP, which is further hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine. CD39 inhibition has been proposed as a novel strategy to increase the extracellular concentration of antiproliferative ATP, while decreasing immunosuppressive and cancer-promoting adenosine levels. In the present study, we performed an extensive structure-activity relationship (SAR) analysis of ticlopidine derivatives and analogs as CD39 inhibitors followed by an in-depth characterization of selected compounds. Altogether 74 compounds were synthesized, 41 of which are new, not previously described in literature. Benzotetrahydropyridines, in which the metabolically labile thiophene is replaced by a benzene ring, were discovered as a new class of allosteric CD39 inhibitors.

Sulfated Polysaccharides from Macroalgae Are Potent Dual Inhibitors of Human ATP-Hydrolyzing Ectonucleotidases NPP1 and CD39.

Extracellular ATP mediates proinflammatory and antiproliferative effects via activation of P2 nucleotide receptors. In contrast, its metabolite, the nucleoside adenosine, is strongly immunosuppressive and enhances tumor proliferation and metastasis. The conversion of ATP to adenosine is catalyzed by ectonucleotidases, which are expressed on immune cells and typically upregulated on tumor cells. In the present study, we identified sulfopolysaccharides from brown and red sea algae to act as potent dual inhibitors of the main ATP-hydrolyzing ectoenzymes, ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) and ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1, CD39), showing nano- to picomolar potency and displaying a non-competitive mechanism of inhibition. We showed that one of the sulfopolysaccharides tested as a representative example reduced adenosine formation at the surface of the human glioblastoma cell line U87 in a concentration-dependent manner. These natural products represent the most potent inhibitors of extracellular ATP hydrolysis known to date and have potential as novel therapeutics for the immunotherapy of cancer.

2-Substituted thienotetrahydropyridine derivatives: Allosteric ectonucleotidase inhibitors.

The antithrombotic prodrugs ticlopidine and clopidogrel are thienotetrahydro-pyridine derivatives that are metabolized in the liver to produce thiols that irreversibly block adenosine diphosphate (ADP)-activated P2Y12 receptors on thrombocytes. In their native, nonmetabolized form, both drugs were reported to act as inhibitors of ectonucleoside triphosphate diphosphohydrolase-1 (NTPDase1, CD39). CD39 catalyzes the extracellular hydrolysis of nucleoside tri- and diphosphates, mainly adenosine 5'-triphosphate (ATP) and ADP, yielding adenosine monophosphate, which is further hydrolyzed by ecto-5'-nucleotidase (CD73) to produce adenosine. While ATP has proinflammatory effects, adenosine is a potent anti-inflammatory, immunosuppressive agent. Inhibitors of CD39 and CD73 have potential as novel checkpoint inhibitors for the immunotherapy of cancer and infection. In the present study, we investigated 2-substituted thienotetrahydropyridine derivatives, structurally related to ticlopidine, as CD39 inhibitors. Due to their substituent on the 2-position, they will not be metabolically transformed into reactive thiols and can, therefore, be expected to be devoid of P2Y12 receptor-antagonistic activity in vivo. Several of the investigated 2-substituted thienotetrahydropyridine derivatives showed concentration-dependent inhibition of CD39. The most potent derivative, 32, showed similar CD39-inhibitory potency to ticlopidine, both acting as allosteric inhibitors. Compound 32 showed an improved selectivity profile: While ticlopidine blocked several NTPDase isoenzymes, 32 was characterized as a novel dual inhibitor of CD39 and CD73.

Hexosamine Biosynthetic Pathway-Derived O-GlcNAcylation Is Critical for RANKL-Mediated Osteoclast Differentiation.

O-linked-N-acetylglucosaminylation (O-GlcNAcylation) performed by O-GlcNAc transferase (OGT) is a nutrient-responsive post-translational modification (PTM) via the hexosamine biosynthetic pathway (HBP). Various transcription factors (TFs) are O-GlcNAcylated, affecting their activities and significantly contributing to cellular processes ranging from survival to cellular differentiation. Given the pleiotropic functions of O-GlcNAc modification, it has been studied in various fields; however, the role of O-GlcNAcylation during osteoclast differentiation remains to be explored. Kinetic transcriptome analysis during receptor activator of nuclear factor-kappaB (NF-κB) ligand (RANKL)-mediated osteoclast differentiation revealed that the nexus of major nutrient metabolism, HBP was critical for this process. We observed that the critical genes related to HBP activation, including Nagk, Gfpt1, and Ogt, were upregulated, while the global O-GlcNAcylation was increased concomitantly during osteoclast differentiation. The O-GlcNAcylation inhibition by the small-molecule inhibitor OSMI-1 reduced osteoclast differentiation in vitro and in vivo by disrupting the translocation of NF-κB p65 and nuclear factor of activated T cells c1 (NFATc1) into the nucleus by controlling their PTM O-GlcNAcylation. Furthermore, OSMI-1 had a synergistic effect with bone target therapy on osteoclastogenesis. Lastly, knocking down Ogt with shRNA (shOgt) mimicked OSMI-1's effect on osteoclastogenesis. Targeting O-GlcNAcylation during osteoclast differentiation may be a valuable therapeutic approach for osteoclast-activated bone diseases.

Koala and Wombat Gammaherpesviruses Encode the First Known Viral NTPDase Homologs and Are Phylogenetically Divergent from All Known Gammaherpesviruses.

There is a large taxonomic gap in our understanding of mammalian herpesvirus genetics and evolution corresponding to those herpesviruses that infect marsupials, which diverged from eutherian mammals approximately 150 million years ago (mya). We compare the genomes of two marsupial gammaherpesviruses, Phascolarctid gammaherpesvirus 1 (PhaHV1) and Vombatid gammaherpesvirus 1 (VoHV1), which infect koalas (Phascolarctos cinereus) and wombats (Vombatus ursinus), respectively. The core viral genomes were approximately 117 kbp and 110 kbp in length, respectively, sharing 69% pairwise nucleotide sequence identity. Phylogenetic analyses showed that PhaHV1 and VoHV1 formed a separate branch, which may indicate a new gammaherpesvirus genus. The genomes contained 60 predicted open reading frames (ORFs) homologous to those in eutherian herpesviruses and 20 ORFs not yet found in any other herpesvirus. Seven of these ORFs were shared by the two viruses, indicating that they were probably acquired prespeciation, approximately 30 to 40 mya. One of these shared genes encodes a putative nucleoside triphosphate diphosphohydrolase (NTPDase). NTPDases are usually found in mammals and higher-order eukaryotes, with a very small number being found in bacteria. This is the first time that an NTPDase has been identified in any viral genome. Interrogation of public transcriptomic data sets from two koalas identified PhaHV1-specific transcripts in multiple host tissues, including transcripts for the novel NTPDase. PhaHV1 ATPase activity was also demonstrated in vitro, suggesting that the encoded NTPDase is functional during viral infection. In mammals, NTPDases are important in downregulation of the inflammatory and immune responses, but the role of the PhaHV1 NTPDase during viral infection remains to be determined.IMPORTANCE The genome sequences of the koala and wombat gammaherpesviruses show that the viruses form a distinct branch, indicative of a novel genus within the Gammaherpesvirinae Their genomes contain several new ORFs, including ORFs encoding a ß-galactoside α-2,6-sialyltransferase that is phylogenetically closest to poxvirus and insect homologs and the first reported viral NTPDase. NTPDases are ubiquitously expressed in mammals and are also present in several parasitic, fungal, and bacterial pathogens. In mammals, these cell surface-localized NTPDases play essential roles in thromboregulation, inflammation, and immune suppression. In this study, we demonstrate that the virus-encoded NTPDase is enzymatically active and is transcribed during natural infection of the host. Understanding how these enzymes benefit viruses can help to inform how they may cause disease or evade host immune defenses.

Recombinant expression of ecto-nucleotide pyrophosphatase/phosphodiesterase 4 (NPP4) and development of a luminescence-based assay to identify inhibitors.

Nucleotide pyrophosphatase/phosphodiesterase 4 (NPP4) is a membrane-bound enzyme that hydrolyzes extracellular diadenosine polyphosphates such as diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) yielding mononucleotides. NPP4 on the surface of endothelial cells was reported to promote platelet aggregation by hydrolyzing Ap3A to ADP, which activates pro-thrombotic G protein-coupled P2Y1 and P2Y12 receptors. Thus, NPP4 inhibitors have potential as novel antithrombotic drugs. In the present study we expressed soluble human NPP4 in Sf9 insect cells and established an enzyme assay using diadenosine tetraphosphate (Ap4A) as a substrate. The reaction product ATP was quantified by luciferin-luciferase reaction in a 96-well plate format. The sensitive method displayed a limit of detection (LOD) of 14.6 nM, and a Z'-factor of 0.68 indicating its suitability for high-throughput screening. The new assay was applied for studying enzyme kinetics and led to the identification of the first NPP4 inhibitors.

Development of a nitinol-actuated surgical instrument for laparoscopic renal denervation: feasibility test in a swine survival model.

Purpose: In this study, we developed a novel nitinol-actuated surgical instrument to conduct laparoscopic renal denervation for the treatment of resistant hypertension. We investigated whether shape and temperature settings of nitinol specimens fit well into the design goals. Furthermore, we conducted a pilot study to validate the mechanical and physiological performance of nerve ablation without damaging the renal artery.Method: Tensile tests were performed to observe temperature-dependent thermomechanical properties and the original shape of nitinol specimens was set considering our design goal. We performed strain gage experiments to measure bending strain. We developed surgical instrument and operated laparoscopic renal denervation in a swine model. Subsequent impedance spectroscopy experiments were conducted to measure changes in impedance magnitudes during the operation and histological analyses were performed to visualize thermogenic damage to arteries and nerves.Results: Tensile testing showed that the shape memory effect begins above 37 °C. Measured strains on nitinol surfaces were 2.10% ± 0.769%, below the strain limit of 8%. Impedance spectroscopy experiments showed decreases in magnitude in all six trials. After operation of laparoscopic renal denervation following the protocol, renal arteries and nerves were harvested and thermogenic damage was observed in nerves but not arteries.Conclusion: We developed a novel nitinol-actuated surgical instrument with which to perform laparoscopic renal denervation. The feasibility of our device was verified using thermomechanical analyses of nitinol, and assessments of mechanical and physiological performance. Our device could be used in other laparoscopic procedures that require large degrees of freedom while restricting to trocar size.

The effects of visual feedback balance training on the pain and physical function of patients with chronic degenerative knee arthritis.

[Purpose] This study aimed to investigate the effects of visual feedback balance training on the pain and dysfunction of patients with chronic degenerative knee arthritis. [Participants and Methods] Twenty-six patients with chronic degenerative knee arthritis participated in this study; the control group (n=13) performed muscle strength training and the experimental group (n=13) performed visual feedback balance training. General physical therapy was applied to both groups three times a week for eight weeks. The visual analog scale was used to measure the patient's pain scale, and the K-WOMAC (Korean Western Ontario and McMaster Universities Osteoarthritis Index) was used as a tool to evaluate their physical function. [Results] In the intra-group comparisons, significant decreases in the visual analog scale and the K-WOMAC were observed for the control group and the experimental group. No significant difference was found in the inter-group comparisons after treatment. [Conclusion] Visual feedback balance training is considered to be an effective intervention method for improving pain and dysfunction in patients with chronic degenerative knee arthritis.

Effects of resistance exercise using the elastic band on the pain and function of patients with degenerative knee arthritis.

[Purpose] This study examined the effects of resistance exercise using the elastic band on the pain and function of patients with degenerative knee arthritis. [Participants and Methods] Thirty patients with degenerative knee arthritis were classified into an experimental group of 15 patients on whom resistance exercise using the elastic band was applied and a control group of 15 patients on whom conservative physical therapy was applied. Both groups received treatments three times a week for four weeks. Pain was measured by the visual analogue scale and function was evaluated by the Korean Western Ontario and McMaster Universities Osteoarthritis Index (K-WOMAC). [Results] The intragroup comparison showed significant decreases in the visual analogue scale and the K-WOMAC in both the experimental and control groups. In the intergroup comparison after treatment, the experimental group showed significantly lower visual analogue scale and K-WOMAC values than the control group. [Conclusion] The results suggest that resistance exercise using the elastic band is an effective intervention for the pain and function of patients with degenerative knee arthritis.

The effects of manual manipulation therapy on pain and dysfunction in patients with lumbar spinal stenosis.

[Purpose] This study investigated the effects of manual manipulation therapy on the pain and dysfunction of patients with lumbar spinal stenosis. [Participants and Methods] In this study, 30 patients with chronic back pain were evenly divided into an experimental group, who received manual traction therapy, and a control group, who received intermittent traction therapy. Both groups received therapy three times a week for eight weeks. A visual analogue scale was used to measure participants' back pain, and the Oswestry disability index (ODI) was used to check the functional impediment they experienced as a result. [Results] The intragroup comparison showed that the visual analog scale and the ODI significantly decreased in the control group and the experimental group, respectively. The intergroup comparison after treatment showed that the visual analog scale and the ODI of the experimental group were significantly lower than in the control group. [Conclusion] The results of this study suggest that manual manipulation therapy is an effective intervention for treating pain and dysfunction in patients with lumbar spinal stenosis.

Correction to: Identification of adenine-N9-(methoxy)ethyl-ß-bisphosphonate as NPP1 inhibitor attenuates NPPase activity in human osteoarthritic chondrocytes.

The original version of the article unfortunately contained an error.

Identification of adenine-N9-(methoxy)ethyl-ß-bisphosphonate as NPP1 inhibitor attenuates NPPase activity in human osteoarthritic chondrocytes.

Overproduction of extracellular diphosphate due to hydrolysis of ATP by NPP1 leads to pathological calcium diphosphate (pyrophosphate) dihydrate deposition (CPPD) in cartilage, resulting in a degenerative joint disease that today lacks a cure. Here, we targeted the identification of novel NPP1 inhibitors as potential therapeutic agents for CPPD deposition disease. Specifically, we synthesized novel analogs of AMP (NPP1 reaction product) and ADP (NPP1 inhibitor). These derivatives incorporate several chemical modifications of the natural nucleotides including (1) a methylene group replacing the Pα,ß-bridging oxygen atom to provide metabolic resistance, (2) sulfonate group(s) replacing phosphonate(s) to improve binding to NPP1's catalytic zinc ions, (3) an acyclic nucleotide analog to allow flexible binding in the NPP1 catalytic site, and (4) a benzimidazole base replacing adenine. Among the investigated compounds, adenine-N9-(methoxy)ethyl-ß-bisphosphonate, 10, was identified as an NPP1 inhibitor (Ki 16.3 µM vs. the artificial substrate p-nitrophenyl thymidine-5'-monophosphate (p-Nph-5'-TMP), and 9.60 µM vs. the natural substrate, ATP). Compound 10 was selective for NPP1 vs. human NPP3, human CD39, and tissue non-specific alkaline phosphatase (TNAP), but also inhibited human CD73 (Ki 12.6 µM). Thus, 10 is a dual NPP1/CD73 inhibitor, which could not only be of interest for treating CPPD deposition disease and calcific aortic valve disease but may also be considered for the immunotherapy of cancer. Compound 10 proved to be a promising inhibitor, which almost completely reduces NPPase activity in human osteoarthritic chondrocytes at a concentration of 100 µM.

Adenine-(methoxy)-ethoxy-Pα,α-dithio-triphosphate inhibits pathologic calcium pyrophosphate deposition in osteoarthritic human chondrocytes.

Nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) inhibitors have been suggested as a potential treatment for calcium pyrophosphate dihydrate (CPPD) deposition disease. Here, we targeted the development of improved NPP1 inhibitors based on acyclic mimics of Pα,α-phosphorodithioate-substituted adenine nucleotides, 7-10. The latter were obtained in a facile two-step synthesis from adenine-(methoxy)ethanol. Among analogs 7-10, adenine-(methoxy)ethoxy-Pα,α-dithio-triphosphate, 8, was the most potent NPP1 inhibitor both with purified enzyme (IC50 0.645 µM) and in osteoarthritic human chondrocytes (IC50 0.033 µM). Furthermore, it efficaciously (10-fold vs. control) inhibited ATP-induced CPPD in human articular chondrocytes. Importantly, 8 was a highly selective NPP1 inhibitor which showed only minor inhibition of NPP3, CD39 and CD73, and did not inhibit TNAP (tissue nonspecific alkaline phosphatase) activity in human chondrocytes. Furthermore, 8 did not activate P2Y1,2,6 receptors. Analog 8 was not toxic to cultured chondrocytes at 100 µM. Therefore, 8 may be suitable for further development as a drug candidate for the treatment of CPPD arthritis and other NPP1-related diseases.

The effect of leg angle during push-up plus exercise on shoulder stabilization muscle activity.

[Purpose] This study investigated the effect of different leg angles during push-up plus exercise on shoulder stabilization muscle activity. [Participants and Methods] Fifteen healthy adult males participated in this study. The smart phone application Clinometer was used to measure leg angles of 70°, 90°, and 110° during push-up plus exercise. The muscle activities of the serratus anterior, upper trapezius fibers, and pectoralis major muscles involved in shoulder stabilization were analyzed using surface electromyogram. [Results] Leg angle significantly affected serratus anterior muscle activity, but it did not affect activities of the upper trapezius fibers or pectoralis major muscles. Post-hoc analysis revealed that serratus anterior muscle activity at the leg angle of 110° was significantly higher than at leg angles of 70° and 90°. [Conclusion] A higher leg angle during push-up plus exercise is a more effective intervention for the serratus anterior muscle activity.

Effects of the flexion-distraction technique and drop technique on straight leg raising angle and intervertebral disc height of patients with an intervertebral disc herniation.

[Purpose] The purpose of this study was to determine the effects of the flexion-distraction technique and drop technique on the straight leg raising angle and intervertebral disc height of patients with an intervertebral disc herniation. [Participants and Methods] Thirty patients with herniated intervertebral discs were divided into either an experimental group (n=15), who underwent flexion-distraction and drop techniques, or a control group (n=15), who obtained spinal decompression therapy. Both groups were treated three times per week for eight weeks. [Results] An intragroup comparison showed that the straight leg raising angle and the intervertebral disc height significantly increased in both groups. But it was not significantly difference in the intergroup comparison. [Conclusion] The flexion-distraction technique and the drop technique may serve as effective interventions for the straight leg raising angle and intervertebral disc height in patients with intervertebral disc herniations.

Development of a selective and highly sensitive fluorescence assay for nucleoside triphosphate diphosphohydrolase1 (NTPDase1, CD39).

Ecto-nucleoside triphosphate diphosphohydrolase1 (NTPDase1, CD39) is a major ectonucleotidase that hydrolyzes proinflammatory ATP via ADP to AMP, which is subsequently converted by ecto-5'-nucleotidase (CD73) to immunosuppressive adenosine. Activation of CD39 has potential for treating inflammatory diseases, while inhibition was suggested as a novel strategy for the immunotherapy of cancer. In the present study, we developed a selective and highly sensitive capillary electrophoresis (CE) assay using a novel fluorescent CD39 substrate, a fluorescein-labelled ATP (PSB-170621A) that is converted to its AMP derivative. To accelerate the assays, a two-directional (forward and reverse) CE system was implemented using 96-well plates, which is suitable for the screening of compound libraries (Z'-factor: 0.7). The detection limits for the forward and reverse operation were 11.7 and 2.00 pM, respectively, indicating a large enhancement in sensitivity as compared to previous methods (e.g. malachite-green assay: 1 000 000-fold, CE-UV assay: 500 000-fold, fluorescence polarization immunoassay: 12 500-fold). Enzyme kinetic studies at human CD39 revealed a Km value of 19.6 µM, and a kcat value of 119 × 10-3 s-1 for PSB-170621A, which shows similar substrate properties as ATP (11.4 µM and 82.5 × 10-3 s-1). The compound displayed similar properties at rat and mouse CD39. Subsequent docking studies into a homology model of human CD39 revealed a hydrophobic pocket that accommodates the fluorescein tag. PSB-170621A was found to be preferably hydrolyzed by CD39 as compared to other ectonucleotidases. The new assay was validated by performing inhibition assays with several standard CD39 inhibitors yielding results that were consonant with data using the natural substrates.

Effects of isometric contraction of the affected-side upper limb in the supine position on the opposite side muscle activity of the body trunk muscles of normal adults.

[Purpose] This study examines the effect of isometric contraction of the upper limb on the affected side in the supine position on the activity of body trunk muscles on the opposite side of normal adults. [Participants and Methods] The research participants included 10 normal adults. A handheld dynamometer device was used to measure the isometric contraction of the upper limb. The muscle activity of the body trunk was analyzed by electromyography. [Results] The muscle activities of rectus abdominis, internal oblique abdominis, erector spinae muscle, and multifidus muscle of the body trunk on the opposite side were significantly larger in the case of the adduction and flexion of the shoulder joint on the affected side than in the case of abduction and extension. [Conclusion] The isometric contraction induced by the adduction and flexion of the shoulder joint on the affected side altered the muscle activity of the body trunk on the opposite side.

The impact of manual spinal traction therapy on the pain and Oswestry disability index of patients with chronic back pain.

[Purpose] The purpose of this study was to examine the effect of manual spinal traction therapy on the pain and Oswestry disability index of patients with chronic back pain. [Participants and Methods] In this study, 30 patients with chronic back pain were evenly divided into an experimental group 1, who received manual traction therapy, and an experimental group 2, who received intermittent traction therapy. Both groups received therapy three times a week for eight weeks. A visual analogue scale was used to measure participants' back pain, and the Oswestry disability index was used to check the functional impediment they experienced as a result. [Results] In a within-group comparison, visual analogue scale and Oswestry disability index significantly decreased in both the experimental 1 and experimental 2. In a between-group comparison after treatment, there was a significantly greater decrease in visual analogue scale and Oswestry disability index in the experimental group 1 compared to the experimental 2. [Conclusion] The manual spinal traction therapy was an effective intervention scheme for the treatment of pain and disorder in patients with chronic back pain.

The effects of flexion-distraction and drop techniques on disorders and Ferguson's angle in female patients with lumbar intervertebral disc herniation.

[Purpose] This study examines the effects of the flexion-distraction technique and the drop technique on disorders and on Ferguson's angle in female patients with lumbar intervertebral disc herniation. [Subjects and Methods] Thirty female patients with lumbar intervertebral disc herniation were divided into an experimental group (n=15) treated with flexion-distraction and drop techniques and a control group (n=15) treated with spinal decompression therapy. Both groups were treated three times a week over an eight-week period. [Results] In the comparison of changes within each group after treatment, both groups showed statistically significant decreases in disorders and in Ferguson's angle. [Conclusion] Flexion-distraction and drop techniques may be an effective intervention to improve disorders and Ferguson's angle in female patients with lumbar intervertebral disc herniation.

Quantification of green fluorescent protein-(GFP-) tagged membrane proteins by capillary gel electrophoresis.

A fast and robust procedure for the quantification of GFP-tagged membrane proteins in cell homogenates was developed employing capillary gel electrophoresis coupled to laser-induced fluorescence detection (CGE-LIF). The new method was found to be highly sensitive and applicable to structurally diverse membrane proteins including synaptic vesicle protein 2A (SV2A), adenosine A2A receptor (A2AAR), and connexin 43 (Cx43). Quantification of SV2A and A2AAR using radioligand binding assays confirmed the results obtained with CGE-LIF. The CGE-LIF method showed significantly higher sensitivity as compared to fluorimetric measurement in a microplate. Importantly, CGE-LIF involves separation of the target proteins and their degradation products prior to quantification and thereby ensures specificity. We anticipate broad applicability of the method for any fluorophore-tagged protein.