A new, sensitive ecto-5′-nucleotidase assay for compound screening

Freundlieb, Marianne; Zimmermann, Herbert; Müller, Christian

doi:10.1016/j.ab.2013.10.012

Cited by 37 publications

(45 citation statements)

References 45 publications

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“…The results in Fig 5 show that an IC 50 of 0.157 μM and 0.30 μM for the enzyme using 5 and 10 μM AMP as substrate, respectively, indicating a competitive inhibition mode of action for the inhibitor with respect to the substrate AMP, and it is similar to what was reported by others, IC 50 =0.236 μM (25).…”

Section: Resultssupporting

confidence: 87%

Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

Goueli

Hsiao

2019

Preprint

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14The success of immunotherapy treatment in oncology ushered a new modality for 15 treating a wide variety of cancers. However, lack of effect in some patients made it 16 imperative to identify other pathways that are exploited by cancer cells to 17 circumvent immune surveillance, and possibly synergize immune checkpoint 18 treatment in those cases. It has been recently recognized that adenosine levels 19 increased significantly in the tumor microenvironment and that 2 20 adenosine/adenosine receptors play a powerful role as immunosuppressant and 21 attenuating several effector T cell functions. The two main enzymes responsible for 22 generating adenosine in the microenvironment are the ectonucleotidases CD39 and 23 CD73, the former utilizes both ATP and ADP and producing AMP while the latter 24 utilizes AMP and generates adenosine. Thus, these two enzymes combined are the 25 major source for the bulk of adenosine produced in the microenvironment. They 26 were shown to be validated targets in oncology leading to several clinical trials that 27 include small molecules as well as antibodies, showing positive and encouraging 28 results in the preclinical arena. Towards the development of novel drugs to target 29 these enzymes, we have developed a platform that can be utilized to monitor the 30 activities of both enzymes in vitro (biochemical) as well as in cells (cell based) 31 assays. We have developed very sensitive and homogenous assays that enabled us 32 to monitor the activity of both enzymes and demonstrate selectivity of known 33 inhibitors as well as monoclonal antibodies. This should speed up screening for 34 novel inhibitors leading to more effective cancer therapy 35 Introduction: 36 Although immune-checkpoint blockers drugs showed tremendous success in 37 the clinic, many patients failed to respond to such treatments. Thus, identifying 38 other pathways exploited by cancer cells to circumvent immune surveillance, novel 39 pharmacological agents that will be synergistic with immune checkpoint inhibitors 3 40 are needed (1). The insightful observation that a significant increase in adenosine 41 levels was seen in the cancer microenvironment triggered interest in the role 42 played by this nucleoside in tumor onset and progression (2). Elevated adenosine 43 concentrations within neoplastic milieu, and activation of adenosine receptors were 44 shown to have powerful immunosuppressant activity and attenuation of several 45 effector T cell functions. Adenosine accumulation in solid tumors at high 46 concentrations stimulated tumor growth and angiogenesis, inhibited cytokine 47 synthesis, functions of T cells, macrophages, and natural killer cells (3). 48Overexpression of an ecto-5'-nucleotidase (CD73), the enzyme that 49 dephosphorylates extracellular adenosine monophosphate (AMP) to adenosine, 50 within the cancer microenvironment, was recognized as the leading candidate to 51 the generation of a strong immunosuppressive and pro-angiogenic adenosine halo, 52 that facilitates cancer onset and pr...

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Section: Resultssupporting

confidence: 87%

Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

Goueli

Hsiao

2019

Preprint

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“…These libraries have been used successfully for screening purposes for other targets (GPCRs and enzymes) to find hit compounds suitable as lead compounds for further optimization or as pharmacologic tools. 22,34–36 …”

Section: Resultsmentioning

confidence: 99%

Identification of Novel G Protein–Coupled Receptor 143 Ligands as Pharmacologic Tools for Investigating X-Linked Ocular Albinism

Filippo

Manga

Schiedel

2017

Invest. Ophthalmol. Vis. Sci.

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PurposeGPR143 regulates melanosome biogenesis and organelle size in pigment cells. The mechanisms underlying receptor function remain unclear. G protein–coupled receptors (GPCRs) are excellent pharmacologic targets; thus, we developed and applied a screening approach to identify potential GPR143 ligands and chemical modulators.MethodsGPR143 interacts with β-arrestin; we therefore established a β-arrestin recruitment assay to screen for compounds that modulate activity. Because GPR143 is localized intracellularly, screening with the wild-type receptor would be restricted to agents absorbed by the cell. For the screen we used a mutant receptor, which shows similar basal activity as the wild type but traffics to the plasma membrane. We tested two compound libraries and investigated validated hits for their effects on melanocyte pigmentation.ResultsGPR143, which showed high constitutive activity in the β-arrestin assay, was inhibited by several compounds. The three validated inhibitors (pimozide, niclosamide, and ethacridine lactate) were assessed for impact on melanocytes. Pigmentation and expression of tyrosinase, a key melanogenic enzyme, were reduced by all compounds. Because GPR143 appears to be constitutively active, these compounds may turn off its activity.ConclusionsX-linked ocular albinism type I, characterized by developmental eye defects, results from GPR143 mutations. Identifying pharmacologic agents that modulate GPR143 activity will contribute significantly to our understanding of its function and provide novel tools with which to study GPCRs in melanocytes and retinal pigment epithelium. Pimozide, one of three GPR143 inhibitors identified in this study, maybe be a good lead structure for development of more potent compounds and provide a platform for design of novel therapeutic agents.

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“…22 Recombinant rat eN expressed in Sf9 insect cells was employed. 47 For all compounds full concentration-response curves were determined in at least three separate experiments using 10 different concentrations.…”

Section: Pharmacological Evaluationmentioning

confidence: 99%

“…20 analog of ADP, is one of the most potent competitive inhibitors of eN known to date. 22 In addition to this nucleotide analog, only anthraquinones, 24 sulfonamides, 25 various polyphenols 26 and some polyoxometalates (POMs) 27 are currently known to potently inhibit eN. Among them the most potent inhibitor is the anthraquinone derivative IV (Figure 1).…”

Section: Introductionmentioning

confidence: 99%

α,β-Methylene-ADP (AOPCP) Derivatives and Analogues: Development of Potent and Selective ecto-5′-Nucleotidase (CD73) Inhibitors

et al. 2015

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ecto-5'-Nucleotidase (eN, CD73) catalyzes the hydrolysis of extracellular AMP to adenosine. eN inhibitors have potential for use as cancer therapeutics. The eN inhibitor α,β-methylene-ADP (AOPCP, adenosine-5'-O-[(phosphonomethyl)phosphonic acid]) was used as a lead structure, and derivatives modified in various positions were prepared. Products were tested at rat recombinant eN. 6-(Ar)alkylamino substitution led to the largest improvement in potency. N(6)-Monosubstitution was superior to symmetrical N(6),N(6)-disubstitution. The most potent inhibitors were N(6)-(4-chlorobenzyl)- (10l, PSB-12441, Ki 7.23 nM), N(6)-phenylethyl- (10h, PSB-12425, Ki 8.04 nM), and N(6)-benzyl-adenosine-5'-O-[(phosphonomethyl)phosphonic acid] (10g, PSB-12379, Ki 9.03 nM). Replacement of the 6-NH group in 10g by O (10q, PSB-12431) or S (10r, PSB-12553) yielded equally potent inhibitors (10q, 9.20 nM; 10r, 9.50 nM). Selected compounds investigated at the human enzyme did not show species differences; they displayed high selectivity versus other ecto-nucleotidases and ADP-activated P2Y receptors. Moreover, high metabolic stability was observed. These compounds represent the most potent eN inhibitors described to date.

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A new, sensitive ecto-5′-nucleotidase assay for compound screening

Cited by 37 publications

References 45 publications

Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

Identification of Novel G Protein–Coupled Receptor 143 Ligands as Pharmacologic Tools for Investigating X-Linked Ocular Albinism

α,β-Methylene-ADP (AOPCP) Derivatives and Analogues: Development of Potent and Selective ecto-5′-Nucleotidase (CD73) Inhibitors

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