Protective efficacy of phosphodiesterase-1 inhibition against alpha-synuclein toxicity revealed by compound screening in LUHMES cells.

α-synuclein-induced neurotoxicity is a core pathogenic event in neurodegenerative synucleinopathies such as Parkinson's disease, dementia with Lewy bodies, or multiple system atrophy. There is currently no disease-modifying therapy available for these diseases. We screened 1,600 FDA-approved drugs for their efficacy to protect LUHMES cells from degeneration induced by wild-type α-synuclein and identified dipyridamole, a non-selective phosphodiesterase inhibitor, as top hit. Systematic analysis of other phosphodiesterase inhibitors identified a specific phosphodiesterase 1 inhibitor as most potent to rescue from α-synuclein toxicity. Protection was mediated by an increase of cGMP and associated with the reduction of a specific α-synuclein oligomeric species. RNA interference experiments confirmed PDE1A and to a smaller extent PDE1C as molecular targets accounting for the protective efficacy. PDE1 inhibition also rescued dopaminergic neurons from wild-type α-synuclein induced degeneration in the substantia nigra of mice. In conclusion, this work identifies inhibition of PDE1A in particular as promising target for neuroprotective treatment of synucleinopathies.

BK channel regulation by phosphodiesterase type 1: a novel signaling pathway controlling human detrusor smooth muscle function.

Large-conductance Ca(2+)-activated K(+) (BK) channels are critical regulators of detrusor smooth muscle (DSM) function. We aimed to investigate phosphodiesterase type 1 (PDE1) interactions with BK channels in human DSM to determine the mechanism by which PDE1 regulates human urinary bladder physiology. A combined electrophysiological, functional, and pharmacological approach was applied using human DSM specimens obtained from open bladder surgeries. The perforated whole cell patch-clamp technique was used to record transient BK currents (TBKCs) and the cell membrane potential in freshly isolated human DSM cells in combination with the selective PDE1 inhibitor, 8-methoxymethyl-3-isobutyl-1-methylxanthine (8MM-IBMX). Isometric DSM tension recordings were used to measure spontaneous phasic and electrical field stimulation-induced contractions in human DSM isolated strips. Selective pharmacological inhibition of PDE1 with 8MM-IBMX (10 µM) increased TBKC activity in human DSM cells, which was abolished by subsequent inhibition of protein kinase A (PKA) with H-89 (10 µM). The stimulatory effect of 8MM-IBMX on TBKCs was reversed upon activation of muscarinic acetylcholine receptors with carbachol (1 µM). 8MM-IBMX (10 µM) hyperpolarized the DSM cell membrane potential, an effect blocked by PKA inhibition. 8MM-IBMX significantly decreased spontaneous phasic and nerve-evoked contractions of human DSM isolated strips. The results reveal a novel mechanism that pharmacological inhibition of PDE1 attenuates human DSM excitability and contractility by activating BK channels via a PKA-dependent mechanism. The data also suggest interactions between PDE1 and muscarinic signaling pathways in human DSM. Inhibition of PDE1 can be a novel therapeutic approach for the treatment of overactive bladder associated with detrusor overactivity.

Synthesis of diethyl 4-substituted-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylates as a new series of inhibitors against yeast α-glucosidase.

1,4-Dihydropyridine-3,5-dicarboxylate derivatives (1-25) were synthesized in high yields via Hantzsch reaction and evaluated for their α-glucosidase inhibitory activity. Compounds 1, 2, 6-8, 11, 13-15, and 23-25 showed a potent inhibitory activity against yeast α-glucosidase with IC50 values in the range of 35.0-273.7 µM, when compared with the standard drug acarbose (IC50 = 937 ± 1.60 µM). Their structures were characterized by different spectroscopic techniques. The kinetics, selectivity, and toxicity studies on these compounds were also carried out. The kinetic studies on most active compounds 14 and 25 determined their modes of inhibition and dissociation constants Ki. Compound 14 was found to be a non-competitive inhibitor with Ki = 25.0 ± 0.06, while compound 25 was identified as a competitive inhibitor with Ki = 66.0 ± 0.07 µM.

Synthesis and Biological Potential Assessment of 2-Substituted Quinazolin-4(3H)-ones as Inhibitors of Phosphodiesterase-I and Carbonic Anhydrase-II.

A library of twenty-five derivatives of 2-substituted quinazolin-4(3H)-ones 1-25 was synthesized and evaluated against phosphodiesterase-I (PDE) and carbonic anhydrase-II (CA). Compounds 17 (IC50 = 210.7 ± 2.62 µM), 16 (IC50 = 301.6 ± 1.18 µM), and 13 (IC50 = 458.13 ± 3.60 µM), selectively exhibited PDE inhibition while compounds 22 (IC50 = 61.33 ± 2.38 µM), 1 (IC50 = 108.30 ± 0.93 µM), and 21 (IC50 = 191.93 ± 2.72 µM), discriminatingly exhibited CA inhibition as compared to standards EDTA (IC50 = 277.69 ± 2.52 µM) and acetazolamide (IC50 = 0.12 ± 0.03 µM), for PDE and CA inhibitions, respectively. However, compound 15 was found to be active against both enzymes with the IC50 values 344.33 ± 4.32 µM and 20.94 ± 0.58 µM, for PDE and CA inhibitions, respectively. Remaining compounds were found to be inactive against both the enzymes. Structure-activity relationship studies are discussed herein.

Oxadiazoles and thiadiazoles: novel α-glucosidase inhibitors.

Oxadiazoles and thiadiazoles 1-37 were synthesized and evaluated for the first time for their α-glucosidase inhibitory activities. As a result, fifteen of them 1, 4, 5, 7, 8, 13, 17, 23, 25, 30, 32, 33, 35, 36 and 37 were identified as potent inhibitors of the enzyme. Kinetic studies of the most active compounds (oxadiazoles 1, 23 and 25, and thiadiazoles 35 and 37) were carried out to determine their mode of inhibition and dissociation constants Ki. The most potent compound of the oxadiazole series (compound 23) was found to be a non-competitive inhibitor (Ki=4.36±0.017 µM), while most potent thiadiazole 35 was identified as a competitive inhibitor (Ki=6.0±0.059 µM). The selectivity and toxicity of these compounds were also studied by evaluating their potential against other enzymes, such as carbonic anhydrase-II and phosphodiesterase-I. Cytotoxicity was evaluated against rat fibroblast 3T3 cell line. Interestingly, these compounds were found to be inactive against other enzymes, exhibiting their selectivity towards α-glucosidase. Inhibition of α-glucosidase is an effective strategy for controlling post-prandial hyperglycemia in diabetic patients. α-Glucosidase inhibitors can also be used as anti-obesity and anti-viral drugs. Our study identifies two novel series of potent α-glucosidase inhibitors for further investigation.

Protective effects of phosphodiesterase-1 (PDE1) and ATP sensitive potassium (KATP) channel modulators against 3-nitropropionic acid induced behavioral and biochemical toxicities in experimental Huntington׳s disease.

Huntington׳s disease (HD), a devastating neurodegenerative disorder, is characterized by weight loss, impairment of motor function, cognitive dysfunction, neuropsychiatric disturbances and striatal damage. Phosphodiesterase-1 (PDE1) has been implicated in various neurological diseases. Mitochondrial potassium channels in the brain take part in neuroprotection. This study has been structured to investigate the role of vinpocetine, a selective PDE1 inhibitor as well as nicorandil, selective ATP sensitive potassium (KATP) channel opener in 3-nitropropionic acid (3-NP) induced HD symptoms in rats. Systemic administration of 3-NP significantly, reduced body weight, impaired locomotion, grip strength and impaired cognition. 3-NP elicited marked oxidative stress in the brain (enhanced malondialdehyde-MDA, reduced glutathione-GSH content, superoxide dismutase-SOD and catalase-CAT), elevated brain acetylcholinesterase activity and inflammation (myeloperoxidase-MPO), with marked nitrosative stress (nitrite/nitrate) in the brain. 3-NP has also induced mitochondrial dysfunction (impaired mitochondrial NADH dehydrogenase-complex I, succinate dehydrogenase-complex II and cytochrome oxidase-complex IV) activities in the striatum of the rat. Tetrabenazine was used as a positive control. Treatment with vinpocetine, nicorandil and tetrabenazine ameliorated 3-NP induced reduction in body weight, impaired locomotion, grip strength and impaired cognition. Treatment with these drugs reduced brain striatum oxidative (MDA, GSH, SOD and CAT) and nitrosative (nitrite/nitrate) stress, acetylcholinesterase activity, inflammation and mitochondrial dysfunctions. These results indicate that vinpocetine, a selective PDE1 inhibitor and nicorandil, a KATP channel opener have attenuated 3-NP induced experimental HD. Hence, pharmacological modulation of PDE1 as well as KATP channels may be considered as potential research targets for mitigation of HD.

Phosphodiesterase I-inhibiting Diels-Alder adducts from the leaves of Morus mesozygia.

A new 2-arylbenzofuran derivative, (+)-dimethylsmoracin O (1), and three new Diels-Alder type adducts, mesozygins A­C (2­4), in addition to eight known compounds, artonin I (5), chalcomaracin (6), norartocarpetin (7), moracin L (8), mulberrofuran F (9), moracin M (10), moracin C (11), and morachalcone A (12),were isolated from the leaves of Morus mesozygia Stapf. Structures were elucidated by spectroscopic data analyses. Compounds 2-7 displayed a potent phosphodiesterase I inhibitory activity.

Kinetic analysis of autotaxin reveals substrate-specific catalytic pathways and a mechanism for lysophosphatidic acid distribution.

Autotaxin (ATX) is a secreted lysophospholipase D that hydrolyzes lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), initiating signaling cascades leading to cancer metastasis, wound healing, and angiogenesis. Knowledge of the pathway and kinetics of LPA synthesis by ATX is critical for developing quantitative physiological models of LPA signaling. We measured the individual rate constants and pathway of the LPA synthase cycle of ATX using the fluorescent lipid substrates FS-3 and 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-LPC. FS-3 binds rapidly (k(1) ≥500 µm(-1) s(-1)) and is hydrolyzed slowly (k(2) = 0.024 s(-1)). Release of the first hydrolysis product is random and rapid (≥1 s(-1)), whereas release of the second is slow and rate-limiting (0.005-0.007 s(-1)). Substrate binding and hydrolysis are slow and rate-limiting with LPC. Product release is sequential with choline preceding LPA. The catalytic pathway and kinetics depend strongly on the substrate, suggesting that ATX kinetics could vary for the various in vivo substrates. Slow catalysis with LPC reveals the potential for LPA signaling to spread to cells distal to the site of LPC substrate binding by ATX. An ATX mutant in which catalytic threonine at position 210 is replaced with alanine binds substrate weakly, favoring a role for Thr-210 in binding as well as catalysis. FTY720P, the bioactive form of a drug currently used to treat multiple sclerosis, inhibits ATX in an uncompetitive manner and slows the hydrolysis reaction, suggesting that ATX inhibition plays a significant role in lymphocyte immobilization in FTY720P-based therapeutics.

Efficient synthesis of 3-O-thia-cPA and preliminary analysis of its biological activity toward autotaxin.

The efficient synthesis of 3-O-thia-cPAs (4a-d), sulfur analogues of cyclic phosphatidic acid (cPA), has been achieved. The key step of the synthesis is an intramolecular Arbuzov reaction to construct the cyclic thiophosphate moiety. The present synthetic route enables the synthesis of 4a-d in only four steps from the commercially available glycidol. Preliminary biological experiments showed that 4a-d exhibited a similar inhibitory effect on autotaxin (ATX) as original cPA.

Purification and characterization of phosphodiesterase i from Walterinnesia aegyptia venom.

A phosphodiesterase I (EC 3.1.4.1; PDE-I) was purified from Walterinnesia aegyptia venom by preparative native polyacrylamide gel electrophoresis (PAGE). A single protein band was observed in analytical native PAGE and sodium dodecyl sulfate (SDS)-PAGE. PDE-I was a single-chain glycoprotein with an estimated molecular mass of 158 kD (SDS-PAGE). The enzyme was free of 5'-nucleotidase and alkaline phosphatase activities. The optimum pH and temperature were 9.0 and 60°C, respectively. The energy of activation (Ea) was 96.4, the V(max) and K(m) were 1.14 µM/min/mg and 1.9 × 10(-3) M, respectively, and the K(cat) and K(sp) were 7 s(-1) and 60 M(-1) min(-1) respectively. Cysteine was a noncompetitive inhibitor, with K(i) = 6.2 × 10(-3) M and an IC(50) of 2.6 mM, whereas adenosine diphosphate was a competitive inhibitor, with K(i) = 0.8 × 10(-3) M and an IC(50) of 8.3 mM. Glutathione, o-phenanthroline, zinc, and ethylenediamine tetraacetic acid (EDTA) inhibited PDE-I activity whereas Mg(2+) slightly potentiated the activity. PDE-I hydrolyzed thymidine-5'-monophosphate p-nitrophenyl ester most readily, whereas cyclic 3'-5'-AMP was least susceptible to hydrolysis. PDE-I was not lethal to mice at a dose of 4.0 mg/kg, ip, but had an anticoagulant effect on human plasma. These findings indicate that W. aegyptia PDE-I shares various characteristics with this enzyme from other snake venoms.

Benzyl and naphthalene methylphosphonic acid inhibitors of autotaxin with anti-invasive and anti-metastatic activity.

Autotaxin (ATX, NPP2) is a member of the nucleotide pyrophosphate phosphodiesterase enzyme family. ATX catalyzes the hydrolytic cleavage of lysophosphatidylcholine (LPC) by lysophospholipase D activity, which leads to generation of the growth-factor-like lipid mediator lysophosphatidic acid (LPA). ATX is highly upregulated in metastatic and chemotherapy-resistant carcinomas and represents a potential target to mediate cancer invasion and metastasis. Herein we report the synthesis and pharmacological characterization of ATX inhibitors based on the 4-tetradecanoylaminobenzylphosphonic acid scaffold, which was previously found to lack sufficient stability in cellular systems. The new 4-substituted benzylphosphonic acid and 6-substituted naphthalen-2-ylmethylphosphonic acid analogues block ATX activity with K(i) values in the low micromolar to nanomolar range against FS3, LPC, and nucleotide substrates through a mixed-mode inhibition mechanism. None of the compounds tested inhibit the activity of related enzymes (NPP6 and NPP7). In addition, the compounds were evaluated as agonists or antagonists of seven LPA receptor (LPAR) subtypes. Analogues 22 and 30 b, the two most potent ATX inhibitors, inhibit the invasion of MM1 hepatoma cells across murine mesothelial and human vascular endothelial monolayers in vitro in a dose-dependent manner. The average terminal half-life for compound 22 is 10±5.4 h and it causes a long-lasting decrease in plasma LPA levels. Compounds 22 and 30 b significantly decrease lung metastasis of B16-F10 syngeneic mouse melanoma in a post-inoculation treatment paradigm. The 4-substituted benzylphosphonic acids and 6-substituted naphthalen-2-ylmethylphosphonic acids described herein represent new lead compounds that effectively inhibit the ATX-LPA-LPAR axis both in vitro and in vivo.

Synthesis of enantiopure 2-carba-cyclic phosphatidic acid and effects of its chirality on biological functions.

Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator, which has a quite unique cyclic phosphate ring at sn-2 and sn-3 positions of the glycerol backbone. We have designed and chemically synthesized several metabolically stabilized derivatives of cPA. 2-Carba-cPA (2ccPA) is one of the synthesized compounds in which the phosphate oxygen was replaced with a methylene group at the sn-2 position, and it showed much more potent biological activities than natural cPA. Here, we developed a new method of 2ccPA enantiomeric synthesis. And we examined the effects of 2ccPA enantiomers on autotaxin (ATX) activity, cancer cell invasion and nociceptive reflex. As well as racemic-2ccPA, both enantiomers showed inhibitory effects on ATX activity, cancer cell invasion and nociceptive reflex. As their effects were not significantly different from each other, the chirality of 2ccPA may not be critical for these biological functions of 2ccPA.

Autotaxin inhibitors: a perspective on initial medicinal chemistry efforts.

The lysophospholipase D enzyme, autotaxin (ATX), has been linked to numerous human diseases including cancer, neurophatic pain, obesity and Alzheimer's disease. Although the ATX protein was initially purified and characterized in 1992, a link to bioactive lipid metabolism was not made until 2002. In the past decade, metal chelators, lysophospholipid product analogs, and more recently, small non-lipid inhibitors of the enzyme were successfully identified. The majority of these inhibitors have been characterized using recombinant purified ATX in vitro, with very few examples studied in more complex systems. Translation of ATX inhibitors from the hands of medicinal chemists to clinical use will require substantially expanded characterization of ATX inhibitors in vivo.

Synthesis and pharmacological evaluation of the stereoisomers of 3-carba cyclic-phosphatidic acid.

Cyclic phosphatidic acid (CPA) is a naturally occurring analog of lysophosphatidic acid (LPA) in which the sn-2 hydroxy group forms a five-membered ring with the sn-3 phosphate. Here, we describe the synthesis of R-3-CCPA and S-3-CCPA along with their pharmacological properties as inhibitors of lysophospholipase D/autotaxin, agonists of the LPA(5) GPCR, and blockers of lung metastasis of B16-F10 melanoma cells in a C57BL/6 mouse model. S-3CCPA was significantly more efficacious in the activation of LPA(5) compared to the R-stereoisomer. In contrast, no stereoselective differences were found between the two isomers toward the inhibition of autotaxin or lung metastasis of B16-F10 melanoma cells in vivo. These results extend the potential utility of these compounds as potential lead compounds warranting evaluation as cancer therapeutics.

Development of an activity-based probe for autotaxin.

Autotaxin (ATX), or ecto-nucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), is a secreted lysophospholipase D that hydrolyses lysophosphatidylcholine into the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX has been implicated in tumour progression and inflammation, and might serve as a biomarker. Here we describe the development of a fluorescent activity-based probe that covalently binds to the active site of ATX. The probe consists of a lysophospholipid-based backbone linked to a trapping moiety that becomes reactive after phosphate ester hydrolysis, and a Cy5 fluorescent dye to allow visualisation of active ATX. The probe reacts specifically with the three known isoforms of ATX, it competes with small-molecule inhibitors for binding to ATX and allows ATX activity in plasma to be determined. Our activity-based reporter will be useful for monitoring ATX activity in biological fluids and for inhibitor screening.

Synthesis and structure-activity relationships of tyrosine-based inhibitors of autotaxin (ATX).

Autotaxin (ATX) is a secreted soluble enzyme that generates lysophosphatidic acid (LPA) through its lysophospholipase D activity. Because of LPA's role in neoplastic diseases, ATX is an attractive therapeutic target due to its involvement in LPA biosynthesis. Here we describe the SAR of ATX inhibitor, VPC8a202, and apply this SAR knowledge towards developing a high potency inhibitor. We found that electron density in the pyridine region greatly influences activity of our inhibitors at ATX.

Discovery and optimization of boronic acid based inhibitors of autotaxin.

Autotaxin (ATX) is an extracellular enzyme that hydrolyzes lysophosphatidylcholine (LPC) to produce the lipid mediator lysophosphatidic acid (LPA). The ATX-LPA signaling axis has been implicated in diverse physiological and pathological processes, including vascular development, inflammation, fibrotic disease, and tumor progression. Therefore, targeting ATX with small molecule inhibitors is an attractive therapeutic strategy. We recently reported that 2,4-thiazolidinediones inhibit ATX activity in the micromolar range. Interestingly, inhibitory potency was dramatically increased by introduction of a boronic acid moiety, designed to target the active site threonine in ATX. Here we report on the discovery and further optimization of boronic acid based ATX inhibitors. The most potent of these compounds inhibits ATX-mediated LPC hydrolysis in the nanomolar range (IC(50) = 6 nM). The finding that ATX can be targeted by boronic acids may aid the development of ATX inhibitors for therapeutic use.

Targeting melanoma growth and viability reveals dualistic functionality of the phosphonothionate analogue of carba cyclic phosphatidic acid.

BACKGROUND: Although the incidence of melanoma in the U.S. is rising faster than any other cancer, the FDA-approved chemotherapies lack efficacy for advanced disease, which results in poor overall survival. Lysophosphatidic acid (LPA), autotaxin (ATX), the enzyme that produces LPA, and the LPA receptors represent an emerging group of therapeutic targets in cancer, although it is not known which of these is most effective. RESULTS: Herein we demonstrate that thio-ccPA 18:1, a stabilized phosphonothionate analogue of carba cyclic phosphatidic acid, ATX inhibitor and LPA1/3 receptor antagonist, induced a marked reduction in the viability of B16F10 metastatic melanoma cells compared with PBS-treated control by 80-100%. Exogenous LPA 18:1 or D-sn-1-O-oleoyl-2-O-methylglyceryl-3-phosphothioate did not reverse the effect of thio-ccPA 18:1. The reduction in viability mediated by thio-ccPA 18:1 was also observed in A375 and MeWo melanoma cell lines, suggesting that the effects are generalizable. Interestingly, siRNA to LPA3 (siLPA3) but not other LPA receptors recapitulated the effects of thio-ccPA 18:1 on viability, suggesting that inhibition of the LPA3 receptor is an important dualistic function of the compound. In addition, siLPA3 reduced proliferation, plasma membrane integrity and altered morphology of A375 cells. Another experimental compound designed to antagonize the LPA1/3 receptors significantly reduced viability in MeWo cells, which predominantly express the LPA3 receptor. CONCLUSIONS: Thus the ability of thio-ccPA 18:1 to inhibit the LPA3 receptor and ATX are key to its molecular mechanism, particularly in melanoma cells that predominantly express the LPA3 receptor. These observations necessitate further exploration and exploitation of these targets in melanoma.

Autotaxin and lipid signaling pathways as anticancer targets.

Tumor progression and metastasis depend on signals in the tumor microenvironment acting on both the malignant cells and benign stroma to create an environment favorable to tumor expansion. A factor of emerging importance that acts in the tumor microenvironment is lysophosphatidic acid (LPA), a small signaling molecule that not only induces the transformation of benign cells into malignant invasive tumors, but also increases tumor growth, invasion, metastasis and angiogenesis. This review examines the LPA signaling pathway, its role in selected human malignancies, and the current state of development of inhibitors targeting molecules in this pathway.

A novel autotaxin inhibitor reduces lysophosphatidic acid levels in plasma and the site of inflammation.

Autotaxin is the enzyme responsible for the production of lysophosphatidic acid (LPA) from lysophosphatidyl choline (LPC), and it is up-regulated in many inflammatory conditions, including but not limited to cancer, arthritis, and multiple sclerosis. LPA signaling causes angiogenesis, mitosis, cell proliferation, and cytokine secretion. Inhibition of autotaxin may have anti-inflammatory properties in a variety of diseases; however, this hypothesis has not been tested pharmacologically because of the lack of potent inhibitors. Here, we report the development of a potent autotaxin inhibitor, PF-8380 [6-(3-(piperazin-1-yl)propanoyl)benzo[d]oxazol-2(3H)-one] with an IC(50) of 2.8 nM in isolated enzyme assay and 101 nM in human whole blood. PF-8380 has adequate oral bioavailability and exposures required for in vivo testing of autotaxin inhibition. Autotaxin's role in producing LPA in plasma and at the site of inflammation was tested in a rat air pouch model. The specific inhibitor PF-8380, dosed orally at 30 mg/kg, provided >95% reduction in both plasma and air pouch LPA within 3 h, indicating autotaxin is a major source of LPA during inflammation. At 30 mg/kg PF-8380 reduced inflammatory hyperalgesia with the same efficacy as 30 mg/kg naproxen. Inhibition of plasma autotaxin activity correlated with inhibition of autotaxin at the site of inflammation and in ex vivo whole blood. Furthermore, a close pharmacokinetic/pharmacodynamic relationship was observed, which suggests that LPA is rapidly formed and degraded in vivo. PF-8380 can serve as a tool compound for elucidating LPA's role in inflammation.