Endothelial cells cope with hypoxia-induced depletion of ATP via activation of cellular purine turnover and phosphotransfer networks

Losenkova, Karolina; Zuccarini, Mariachiara; Helenius, Mikko; Jacquemet, Guillaume; Gerasimovskaya, Evgenia; Tallgren, Camilla; Jalkanen, Sirpa; Yegutkin, Gennady G.

doi:10.1016/j.bbadis.2018.03.001

Cited by 23 publications

(19 citation statements)

References 53 publications

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“…Due to residual phosphate in the POM-1 formulation, POM-1 was desalted using Zeba spin columns (Thermo Scientific) prior to use in inhibition assays. The following antibodies were used in this study: a mouse anti-CD39 (clone A1, eBioscience, Cat: 14–0399-82), guinea pig anti-human CD39 (hN1-1 C, provided by Prof. Jean Sevigny; http.//ectonucleotidases-ab.com), mouse anti-CD73 (clone 4G4, generated in-house), 63 and polyclonal rabbit anti-ALPL (TNAP) antibody (MyBioSource, Cat: MBS003198). Secondary Alexa Fluor® 488-conjugated goat anti-mouse (Cat: A-11001) and Alexa Fluor® 633-conjugated goat anti-rabbit (Cat: A-21070) antibodies, and ProLong® Gold Antifade reagent with 4,6-diamidino-2-phenylindole (DAPI) were from Invitrogen™ (ThermoFisher Life Technologies).…”

Section: Methodsmentioning

confidence: 99%

“…Aliquots of the mixture were applied onto Alugram® SIL G/UV 254 TLC sheets (Machery-Nagel), separated using appropriate solvent systems and then either developed by autoradiography using Kodak BioMax MS films (Carestream) or quantified by scintillation β-counting, as described elsewhere. 63 , 71 …”

Section: Methodsmentioning

confidence: 99%

“…The adherent fixed cells were processed for staining of membrane-bound nucleotidases, as described earlier. 63 The coverslips were mounted with ProLong® medium with DAPI and examined using a 3i spinning disk confocal microscope CSU-W1 with Photometrics Evolve EM-CCD camera, and Slidebook 6.0 software (Intelligent Imaging Innovations, Inc.). Z-stacks of the images were captured using C-Apochromat 40×/1.1 objective (Carl Zeiss) at identical exposure times for each antibody.…”

Section: Methodsmentioning

confidence: 99%

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Fully human anti-CD39 antibody potently inhibits ATPase activity in cancer cells via uncompetitive allosteric mechanism

Spatola¹,

Lerner

Wong³

et al. 2020

mAbs

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The extracellular ATP/adenosine axis in the tumor microenvironment (TME) has emerged as an important immune-regulatory pathway. Nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), otherwise known as CD39, is highly expressed in the TME, both on infiltrating immune cells and tumor cells across a broad set of cancer indications. CD39 processes pro-inflammatory extracellular ATP to ADP and AMP, which is then processed by Ecto-5ʹ-nucleotidase/CD73 to immunosuppressive adenosine. Directly inhibiting the enzymatic function of CD39 via an antibody has the potential to unleash an immune-mediated anti-tumor response via two mechanisms: 1) increasing the availability of immunostimulatory extracellular ATP released by damaged and/or dying cells, and 2) reducing the generation and accumulation of suppressive adenosine within the TME. Tizona Therapeutics has engineered a novel first-in-class fully human anti-CD39 antibody, TTX-030, that directly inhibits CD39 ATPase enzymatic function with sub-nanomolar potency. Further characterization of the mechanism of inhibition by TTX-030 using CD39 + human melanoma cell line SK-MEL-28 revealed an uncompetitive allosteric mechanism (α < 1). The uncompetitive mechanism of action enables TTX-030 to inhibit CD39 at the elevated ATP concentrations reported in the TME. Maximal inhibition of cellular CD39 ATPase velocity was 85%, which compares favorably to results reported for antibody inhibitors to other enzyme targets. The allosteric mechanism of TTX-030 was confirmed via mapping the epitope to a region of CD39 distant from its active site, which suggests possible models for how potent inhibition is achieved. In summary, TTX-030 is a potent allosteric inhibitor of CD39 ATPase activity that is currently being evaluated in clinical trials for cancer therapy.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Fully human anti-CD39 antibody potently inhibits ATPase activity in cancer cells via uncompetitive allosteric mechanism

Spatola¹,

Lerner

Wong³

et al. 2020

mAbs

Self Cite

View full text Add to dashboard Cite

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“…Therefore, the oxygen concentration required for hypoxia is also different between tissues. Blood vessels release nitric oxide as an acute repose to hypoxia leading to dilation of the vessels to fulfill the oxygen demand 45 . During more prolonged states of hypoxia, there are certain pathways which are activated, the most well studied involving the transcription factor hypoxia inducible factor (HIF1) 46 ( Figure 3).…”

Section: Hypoxia Signalingmentioning

confidence: 99%

Investigating mechanisms of angiogenesis in health and disease using zebrafish models

Ali

2018

Linköping University Medical Dissertations

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Angiogenesis, the growth of blood vessels from an existing vasculature, can occur by sprouting from preexisting vessels or by vessel splitting (intussusception). Pathological angiogenesis drives choroidal neovascularization (CNV) in age related macular degeneration (AMD) which is commonly restricted under the retinal pigment epithelium (RPE), called occult CNV, but may also involve vessels penetrating through the RPE into the sub-retinal space. Pathological vessels are poorly developed, insufficiently perfused and highly leaky, phenotypes that are considered to drive disease progression and lead to poor prognosis. Currently, a number of anti-angiogenic drugs exists, the majority of which target vascular endothelial factor (VEGF), but although they often are highly beneficial for treating eye diseases in the short-term, they are generally of limited efficacy in other diseases such as cancer, and also have poorer efficacy when used for treatment of eye diseases in the long-term. A better understanding of the mechanisms underlying pathological angiogenesis can generate new targets for treatment leading to development of better drugs for cancer and retinopathies, but perhaps also other angiogenesis-dependent diseases, in the future. In this thesis mechanisms involved in developmental angiogenesis or pathological angiogenesis in the choroid, cornea or melanoma was identified. These findings highlight the need to further elaborate our knowledge related to angiogenesis in different tissues/conditions for a more targeted, and potentially effective treatment of diseases in the future.

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“…Several chromatographic procedures such as high‐performance liquid chromatography (HPLC) with ultraviolet detection (UV), thin‐layer chromatography (TLC), capillary electrochromatography (CEC), LC/mass spectrometry (MS), or LC/MS/MS have been adopted for the analysis of adenosine and its related impurities. However, these techniques are not suitable for structural determination of unknown compounds at trace levels.…”

Section: Introductionmentioning

confidence: 99%

Identification of new trace related impurities in adenosine products using ultra‐high‐performance liquid chromatography/electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Shen

Zhai

et al. 2019

Rapid Comm Mass Spectrometry

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Rationale: Adenosine can induce various physiopathological effects and has been adopted as a drug to treat certain forms of supraventricular tachycardia. Adenosine is predominantly produced via fermentation that may generate many other bioactive compounds with similar structures at trace levels. Thus, it is necessary to identify these trace structurally related impurities. A new method using ultra-highperformance liquid chromatography (UHPLC) coupled with quadrupole time-of-flight tandem mass spectrometry (QTOF-MS/MS) is proposed to separate complex components at trace amounts; to obtain accurate mass measurements providing the elemental composition of unknown compounds; and MS/MS spectra for elucidation of structures. Methods:A UHPLC/QTOF-MS method was developed to separate and detect trace related impurities with structures similar to adenosine in formation samples.MS and MS/MS spectra of the impurities detected in real samples were acquired and used to propose their structures. Available reference standards were used to confirm the identification of some of the impurities detected.Results: Nine trace impurities of adenosine were separated and characterized. Of these nine, five were confirmed as 5'-adenylic acid, hypoxanthine, inosine, adenine, and 2'-deoxyadenosine by comparison with their available reference standards. The remaining four were proposed to be ADP-ribose, two S-epimers of 5'-deoxy-5'methylsulfinyl adenosine, and 3'-α-glucosyl adenosine based on their MS and MS/ MS spectra, and available literature. Additionally, the MS/MS fingerprints of the monosaccharide glycosyl groups were discovered and discussed. Conclusions:A UHPLC/QTOF-MS method was established and used for the separation and characterization of nine trace related impurities of adenosine. Their structures were identified based on the MS and MS/MS spectra and retention times. In addition, the specific MS/MS fingerprints of the monosaccharide glycosyl moieties of the adenosine impurity analogs were summarized.

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Endothelial cells cope with hypoxia-induced depletion of ATP via activation of cellular purine turnover and phosphotransfer networks

Cited by 23 publications

References 53 publications

Fully human anti-CD39 antibody potently inhibits ATPase activity in cancer cells via uncompetitive allosteric mechanism

Fully human anti-CD39 antibody potently inhibits ATPase activity in cancer cells via uncompetitive allosteric mechanism

Investigating mechanisms of angiogenesis in health and disease using zebrafish models

Identification of new trace related impurities in adenosine products using ultra‐high‐performance liquid chromatography/electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

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