Enolase Inhibitor Research Articles

Abstract A39: Pomhex, a cell-permeable high potency enolase inhibitor with utility for collateral lethality treatment of cancer

Abstract Glycolysis inhibition is an active area of investigation for the treatment of cancer. However, few compounds have progressed beyond the cell culture stage. We have recently demonstrated that genomic passenger deletion of the glycolytic enzyme Enolase 1 (ENO1) leaves gliomas harboring such deletions solely reliant on ENO2, rendering them exquisitely sensitive to enolase inhibitors Collateral Lethality. However, the tool compound that we employed for these in vitro studies, Phosphonoacetohydroxamate (PhAH), has very poor pharmacological properties and was ineffective in vivo. We recently reported that a structural analogue of PhAH, the natural phosphonate antibiotic SF2312, is a high potency inhibitor of Enolase. While more potent than PhAH, SF2312 remains poorly cell permeable. Here, we generated a Pivaloyloxymethyl (POM) ester pro-drug derivative of SF2312, termed POMSF, which increased the potency in cell based systems by ~50-fold. POMSF is selectively active against ENO1-deleted glioma cells in culture at ~19 nM, versus μM for SF2312. However, POMSF displayed poor aqueous stability. A derivative of POMSF, termed POMHEX, showed greater stability and its active form, HEX, showed 4-fold preference for ENO1 over ENO2. Labeled 13C-glucose tracing shows that POMHEX inhibits glycolysis at the Enolase step in all cell lines tested, but with ~100-fold greater potency in ENO1-deleted lines. POMHEX selectively killed ENO1-deleted glioma cells with an IC50 <30nM, whilst non-deleted cells could readily tolerate μM levels of inhibitor. As such, POMHEX was selected for in vivo experiments. Using an orthotopic intracranial xenografted model where tumor growth and response to therapy are monitored by MRI, we show that POMHEX is capable of eradicating intracranial ENO1-deleted tumors, with mice remaining recurrence-free even after treatment discontinuation. Taken together, these results reinforce that glycolysis is a viable target and provide in vivo proof-of-principal for the concept of using passenger deletions as targetable vulnerabilities in personalized cancer therapy. Citation Format: Yu-Hsi Lin, Nikunj Satani, Naima Hammoudi, Federica Pisaneschi, Paul Leonard, David Maxwell, Zhenghong Peng, Todd Link, Lee IV R. Gilbert, Ananth Bosajou, Duoli Sun, Joe Marszalek, Yuting Sun, John S. McMurray, Pijus K. Mandal, Maria E. Di Francesco, Barbara Czako, Alan Wang, William Bornmann, Ronald A. DePinho, Florian Muller. Pomhex, a cell-permeable high potency enolase inhibitor with utility for collateral lethality treatment of cancer [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A39.

ENOblock Does Not Inhibit the Activity of the Glycolytic Enzyme Enolase.

Inhibition of glycolysis is of great potential for the treatment of cancer. However, inhibitors of glycolytic enzymes with favorable pharmacological profiles have not been forthcoming. Due to the nature of their active sites, most high-affinity transition-state analogue inhibitors of glycolysis enzymes are highly polar with poor cell permeability. A recent publication reported a novel, non-active site inhibitor of the glycolytic enzyme Enolase, termed ENOblock (N-[2-[2-2-aminoethoxy)ethoxy]ethyl]4-4-cyclohexylmethyl)amino]6-4-fluorophenyl)methyl]amino]1,3,5-triazin-2-yl]amino]benzeneacetamide). This would present a major advance, as this is heterocyclic and fully cell permeable molecule. Here, we present evidence that ENOblock does not inhibit Enolase enzymatic activity in vitro as measured by three different assays, including a novel 31P NMR based method which avoids complications associated with optical interferences in the UV range. Indeed, we note that due to strong UV absorbance, ENOblock interferes with the direct spectrophotometric detection of the product of Enolase, phosphoenolpyruvate. Unlike established Enolase inhibitors, ENOblock does not show selective toxicity to ENO1-deleted glioma cells in culture. While our data do not dispute the biological effects previously attributed to ENOblock, they indicate that such effects must be caused by mechanisms other than direct inhibition of Enolase enzymatic activity.

375 - POMHEX, a cell-permeable enolase inhibitor for collateral lethality targeting of ENO1-deleted glioblastoma

375 - POMHEX, a cell-permeable enolase inhibitor for collateral lethality targeting of ENO1-deleted glioblastoma

Abstract 3837: Passenger deletion of ENO1 as a collateral lethality target in cancer

Abstract Large scale genomic characterization efforts such as TCGA have painted an unprecedentedly detailed picture of the genetic alterations that underlie tumorigenesis. Yet, the majority of genetic alterations are passenger rather than driver events and are considered unactionable. We have previously proposed that passenger or collateral deletions could serve as pharmacologically targetable vulnerabilities Collateral Lethality, in case passenger genes are homozygously deleted and member of a paralogous gene family carrying out an essential housekeeping function. We have presented proof-of-principal, whereby passenger deletion of the glycolytic gene Enolase 1 (ENO1) as part of the 1p36-tumor suppressor locus, renders glioma cells harboring such deletions highly sensitive to ablation of its redundant paralogue, ENO2. While our original analysis identified ENO1-homozygous deletions in Glioblastoma (GBM), recent bioinformatics analyzes, backed by immunohistochemistry, show that ENO1-homozygous deletions also occur in Hepatocellular Carcinoma and Cholangiocarcinoma. In GBM, multisector analysis of primary and recurrent tumors, indicate that ENO1 deletion is an early event which is homogenously distributed through the primary tumor and persist during recurrence. To pharmacologically exploit ENO1-deletion, we have pursued two approaches. First, we have synthesized cell-permeable prodrug derivatives of the natural Enolase inhibitor SF2312. The lead compound, POMHEX, shows potent killing of ENO1-deleted glioma cells in the low nM range while ENO1-restored isogenic or normal cells can tolerate μM doses. POMHEX has a short half-life yet can eradicate intracranial xenografted ENO1-deleted tumors, provided extensive breakdown of the blood-brain barrier. Our second approach to targeting ENO1-deletion consisted of chemical biology screening of drug libraries for the ability to kill ENO1-deleted but not isogenic rescued cells. We find that ENO1-deleted cells show a dramatic sensitization to inhibitors of the mitochondrial electron transport chain. These include tool compounds such as rotenone as well as compounds not previously associated with mitochondria, such as Mubritinib and an experimental anti-neoplastic agent previously described as a HIF1-inhibitor, now known to inhibit mitochondrial Complex I. The latter agent shows potent activity against ENO1-deleted intracranial xenografts. The likely cause for this sensitivity is the inability of ENO1-deleted cells to compensatory upregulate glycolysis in response mitochondrial inhibition, the typical response of ENO1-intact glioma cells and normal cells. Together, these data indicate that passenger deletion of ENO1 is an encouraging drug-target and provide support for collateral lethality as a viable therapeutic strategy, which, given the large number of passenger deletions in the cancer genome, may be broadly applicable. Citation Format: Yu-Hsi Lin, Nikunj Satani, Naima Hammoudi, Joe Marszalek, Yuting Sun, Marina Protopopova, Maria E. Di Francesco, Barbara Czako, Alan Wang, Ronald A. DePinho, Florian L. Muller. Passenger deletion of ENO1 as a collateral lethality target in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3837.

Abstract C183: Pomhex: a cell-permeable high potency Enolase inhibitor with in vivo anti-neoplastic activity

Abstract Glycolysis inhibition is an active area of investigation in cancer. However, few compounds have progressed beyond the cell culture stage. We have recently demonstrated that genomic passenger deletion of the glycolytic enzyme Enolase 1 (ENO1) leaves gliomas harboring such deletions with less than 10% of normal enzymatic activity, rendering them exquisitely sensitive to enolase inhibitors. However, the tool compound that we employed for these in vitro studies, Phosphonoacetohydroxamate (PhAH), has very poor pharmacological properties and was ineffective in vivo. We performed a SAR studies to increase inhibitor specificity towards ENO2 as well as pro-druging to increase cell permeability. The lead compound generated by these efforts, termed POMHEX, is selectively active against ENO1-deleted glioma cells in culture at ∼35nM (versus μM for PhAH). Using an orthotopic intracranial xenografted model where tumor growth and response to therapy are monitored by MRI, we show that POMHEX is capable of eradicating intracranial ENO1-deleted tumors, with mice remaining recurrence-free even after treatment discontinuation. Taken together, these results reinforce that glycolysis is a viable target and provide in vivo proof-of-principal for the concept of using passenger deletions as targetable vulnerabilities in cancer therapy. Citation Format: Yu-Hsi Lin, Joe Marszalek, Yuting Sun, Naima Hammoudi, Paul Leonard, David Maxwell, Nikunj Satani, Peng Zhang, Todd Link, Gilbert Lee, Maria E. Di Francesco, Barbara Czako, Alan Y. Want, Ronald A. DePinho, Florian L. Muller. Pomhex: a cell-permeable high potency Enolase inhibitor with in vivo anti-neoplastic activity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C183.

Inhibition of Glycolysis for Glucose Estimation in Plasma: Recent Guidelines and their Implications.

Inhibition of Glycolysis for Glucose Estimation in Plasma: Recent Guidelines and their Implications.

A Unique Small Molecule Inhibitor of Enolase Clarifies Its Role in Fundamental Biological Processes

Enolase is a component of the glycolysis pathway and a "moonlighting" protein, with important roles in diverse cellular processes that are not related to its function in glycolysis. However, small molecule tools to probe enolase function have been restricted to crystallography or enzymology. In this study, we report the discovery of the small molecule "ENOblock", which is the first, nonsubstrate analogue that directly binds to enolase and inhibits its activity. ENOblock was isolated by small molecule screening in a cancer cell assay to detect cytotoxic agents that function in hypoxic conditions, which has previously been shown to induce drug resistance. Further analysis revealed that ENOblock can inhibit cancer cell metastasis in vivo. Moreover, an unexpected role for enolase in glucose homeostasis was revealed by in vivo analysis. Thus, ENOblock is the first reported enolase inhibitor that is suitable for biological assays. This new chemical tool may also be suitable for further study as a cancer and diabetes drug candidate.

Passenger deletions generate therapeutic vulnerabilities in cancer

Inactivation of tumor suppressor genes via homozygous deletion is a prototypic event in the cancer genome, yet such deletions often encompass neighboring genes. We hypothesized that homozygous deletions in such passenger genes can expose cancer-specific therapeutic vulnerabilities in the case where the collaterally deleted gene is a member of a functionally redundant family of genes exercising an essential function. The glycolytic gene Enolase 1 (ENO1) in the 1p36 locus is deleted in Glioblastoma (GBM), which is tolerated by expression of ENO2. We demonstrate that shRNA-mediated extinction of ENO2 selectively inhibits growth, survival, and tumorigenic potential of ENO1-deleted GBM cells and that the enolase inhibitor phosphonoacetohydroxamate (PhAH) is selectively toxic to ENO1-deleted GBM cells relative to ENO1-intact GBM cells or normal astrocytes. The principle of collateral vulnerability should be applicable to other passenger deleted genes encoding functionally-redundant essential activities and provide an effective treatment strategy for cancers harboring such genomic events.

Exploiting collateral damage

Some mutations in tumour cells play no part in causing cancer, but they generate cellular weak spots that may allow tumour cells to be selectively killed by drugs. See Article p.337 This Article introduces the concept of 'collateral damage' in cancer genomes as a possible basis for therapeutic strategies. Ronald DePinho and colleagues examine pairs of functionally redundant 'passenger' genes with 'housekeeping' roles, for example in cellular metabolism. They hypothesize that genetic deletions in cancer that encompass one such gene (as collateral damage caused by proximity to tumour-suppressor genes) may expose a selective vulnerability of cancer cells, but not normal cells, to pharmacological inhibition of the protein encoded by the second gene. They demonstrate this concept for the glycolytic enzymes ENO1 and ENO2. There is often homozygous deletion of the ENO1 gene on chromosome 1p36 in glioblastomas, which is shown here to render glioma cells sensitive to knockdown of ENO2 or to a small-molecule enolase inhibitor. The authors further analyse existing cancer genomics data sets for other examples of pairs of redundant housekeeping genes, one of which resides close to frequently deleted tumour-suppressor genes. They suggest that this concept may be generally applicable and could offer new therapeutic opportunities.

Molecular modelling, docking and interaction studies of human-plasmogen and salmonella enolase with enolase inhibitors.

Salmonella enteric serovar Typhi Ty2 is a human specific pathogen and an etiological agent for typhoid fever. Most of Salmonella serotypes produce glycogen which has a comparatively minor role in virulence and colonization, but has a more significant role in survival. Enzymes present in glycolytic pathway of bacteria help bacteria to survive by activating other factors inside host. Numerous pathogenic bacteria species intervene with the plasminogen system, and this plasminogen–enolase association may play a critical role in the virulence of S. Typhi by causing direct damage to the host cell extracellular matrix, possibly by enzymic degradation of extracellular matrix proteins or other protein constituents. In this study, molecular modelling of enolase of Salmonella has been accomplished in silico by comparative modelling; we have then analyzed Human alpha enolase which is a homodimer and serves on epithelial cells with our model. Both Structures were docked by D-tartronate semialdehyde phosphate (TSP) and 3-aminoenolpyruvate phosphate (AEP) enolase inhibitors. Our study shows that salmonella enolase and human enolase have different active sites in their structure. This will help in development of new ligands, more suitable for inhibiting bacterial survival inside host as vaccines for typhoid fever are not fully protective. The study also confirmed that enolase Salmonella and Human Plasminogen suggested direct physical interaction between both of them as the activation loop of plasminogen residues showed conformational changes similar to the tissue type plasminogen activator. Various computational biology tools were used for our present study such as Modeller, Molegro Virtual Docker, Grommacs.

Mefloquine interferes with glycolysis in schistosomula of Schistosoma mansoni via inhibition of enolase

The antimalarial drug mefloquine has promising antischistosomal properties killing haematophagous adult schistosomes as well as schistosomula. The mode of action and involved drug targets of mefloquine in Schistosoma mansoni schistosomula are unknown. In order to identify mefloquine-binding proteins and thus potential drug targets, mefloquine affinity chromatography with S. mansoni schistosomula crude extracts was performed. We found one specific mefloquine-binding protein that was identified by mass spectrometry as the glycolytic enzyme enolase (Q27877). Enolase activity assays were performed on schistosomula crude extracts and on the recombinant enolase Q27877 expressed in Escherichia coli. In schistosomula crude extracts enolase activity was inhibited by mefloquine and by the enolase inhibitor sodium fluoride, while activity of the recombinant enolase was not affected. In contrast to enolase from crude extracts, recombinant Q27877 did not bind to mefloquine-agarose. Using isothermal microcalorimetry, we next investigated the metabolic inhibition of mefloquine and 3 known glycolytic inhibitors in Schistosoma spp., namely sodium fluoride, 3-bromopyruvate and menadione on schistosomula in the presence or absence of glucose. We found that in the presence of glucose, schistosomula were less affected by mefloquine, sodium fluoride and 3-bromopyruvate, whereas glucose had no protective effect when schistosomula had been exposed to menadione. These results suggest a potential role of mefloquine as an inhibitor of glycolysis, at least in stages where other targets like haem degradation are not relevant.

Role of glycolysis and antioxidant enzymes in the toxicity of amyloid beta peptide Aβ25–35 to erythrocytes

The role of glycolysis and antioxidant enzymes in amyloid beta peptide Abeta(25-35) toxicity to human and rat erythrocytes was studied. The erythrotoxicity of Abeta(25-35) was shown to increase two- to fourfold both in the absence of glucose in the incubation medium and upon the addition of sodium fluoride, an enolase inhibitor. Potassium cyanide, a Cu,Zn-superoxide dismutase inhibitor, abolishes the toxic effect of Abeta(25-35) to erythrocytes, whereas mercaptosuccinate, a glutathione peroxidase inhibitor, and ouabain, a Na+,K+-ATPase inhibitor, promote it. Sodium azide, a catalase inhibitor, did not affect the cell lysis under the action of Abeta(25-35) . The results support the hypothesis that H2O2, Cu,Zn superoxide dismutase, and glutathione peroxidase are involved in the toxicity mechanism rather than superoxide radical. Glycolysis and Na+,K+-ATPase play a substantial protective role. Fullerene C(60) nanoparticles are toxic to erythrocytes of both types; their toxicity is not related to enhanced oxidative stress and the mechanism of toxicity differs from that of Abeta(25-35) .

PURIFICATION AND CHARACTERIZATION OF A HOMODIMERIC ENOLASE FROM SYNECHOCOCCUS PCC 6301 (CYANOPHYCEAE)1

Enolase from Synechococcus PCC 6301 was purified 1450‐fold to electrophoretic homogeneity and a final specific activity of 68 μmol of phosphoenolpyruvate produced·min−1·mg protein−1. Analytical gel filtration and nondenaturing and SDS‐gel electrophoresis demonstrated that this enolase exists as a 118‐kDa homodimer composed of 56‐kDa subunits. The purified enzyme displayed 1) a broad pH‐activity profile with maximal activity occurring at pH 8.0 and 7.5 for the forward and reverse reactions, respectively, 2) a forward‐to‐reverse maximal activity ratio of about 1.6, 3) a Km (2‐phosphoglycerate) of 0.28 mM, and 4) an absolute requirement for a divalent metal cation cofactor that was best satisfied by Mg2+ (Km=0.62 mM). Enolase activity increased by about 200% after the first purification step (60° C heat treatment), whereas addition of increasing amounts of a clarified extract led to a progressive 70% inhibition in the activity of the purified enzyme. This was reflected by a reduction in enolase's Vmax from 73 to 22 U·mg−1 and forward‐to‐reverse activity ratio from 1.6 to 1.3. This inhibition was negated when the clarified extract was either preincubated with trypsin or warmed to approximately 40° for 5 min. Results are indicative of a heat‐labile enolase inhibitor protein in Synechococcus PCC 6301. By contrast, the purified enolase lost no activity when incubated at 70° C for up to 5 min. This study represents the first purification of enolase from the Cyanophyceae. Characterization of the purified enzyme's physical and kinetic features has provided insights into the structural and functional properties of cyanobacterial enolase.

The Crystal Structure of Trypanosoma brucei Enolase: Visualisation of the Inhibitory Metal Binding Site III and Potential as Target for Selective, Irreversible Inhibition

The glycolytic enzymes of the trypanosomatids, that cause a variety of medically and agriculturally important diseases, are validated targets for drug design. Design of species-specific inhibitors is facilitated by the availability of structural data. Irreversible inhibitors, that bound covalently to the parasite enzyme alone, would be potentially particularly effective. Here we determine the crystal structure of enolase from Trypanosoma brucei and show that two cysteine residues, located in a water-filled cavity near the active-site, are modified by iodoacetamide leading to loss of catalytic activity. Since these residues are specific to the Trypanosomatidae lineage, this finding opens the way for the development of parasite-specific, irreversibly binding enolase inhibitors. In the present structure, the catalytic site is partially occupied by sulphate and two zinc ions. Surprisingly, one of these zinc ions illustrates the existence of a novel enolase-binding site for divalent metals. Evidence suggests that this is the first direct visualization of the elusive inhibitory metal site, whose existence has hitherto only been inferred from kinetic data.

Effect of fructose on the biochemical toxicity of hydrazine in isolated rat hepatocytes

Rat hepatocyte suspensions were incubated with various concentrations of hydrazine (0, 8, 12, 16, 20 mM) for 1, 2 and 3 h. In some experiments fructose (10 mM) was added either during the preincubation period, or 1 h after the start of hydrazine treatment. In certain experiments in which fructose was added, the glycolytic inhibitor sodium fluoride (3 mM) was also added during the preincubation period. Hepatocytes incubated with hydrazine alone demonstrated both a concentration- and time-dependent loss of cell viability as measured by increased Trypan blue uptake and lactate dehydrogenase (LDH) leakage. These parameters were reduced and delayed by fructose when added either before or 1 h after hydrazine treatment. There was also both a concentration- and time-dependent loss of ATP and reduced glutathione (GSH) content with hydrazine treatment. Moreover, fructose caused an initial rapid depletion of ATP but thereafter ATP levels were increased in control hepatocytes. Fructose reduced both the depletion of ATP and GSH in hydrazine- treated hepatocytes. Urea synthesis was inhibited by all concentrations of hydrazine studied but fructose treatment after 1 h did not alter this. This study also demonstrated that fluoride, an enolase inhibitor, abolished the protection against depletion of ATP levels provided by fructose, without affecting cell viability or GSH levels. These findings suggest that the cytotoxicity of hydrazine and its effects on urea synthesis and GSH levels are not a direct result of ATP depletion. The protective effects of fructose against the cytotoxicity may be due to a direct interaction with hydrazine.

Predominance of gluconate formation from glucose during germination of Bacillus megaterium QM B1551 spores.

Metabolic pathways of glucose during germination of Bacillus megaterium QM B1551 spores were studied by using specifically labeled glucose and gluconate. The Embden-Meyerhof pathway, the pentose cycle, and the direct oxidation route of glucose to gluconate (the gluconate pathway) were all operative at this stage; among those, gluconate accumulation was most predominant, especially in the early stage. Potassium fluoride, an enolase inhibitor, abolished the catabolism by the Embden-Meyerhof pathway totally without affecting gluconate accumulation. Under these conditions glucose was exclusively oxidized to gluconate. Gluconate thus accumulated could be metabolized further via phosphorylation by gluconate kinase. Remarkable gluconate accumulation was also demonstrated in several other spores requiring alanine as an effective germinant. NADH formed by the direct glucose oxidation may serve as a initial ATP source to phosphorylate glucose in germinating spores.

29] Active site-specific reagents and transition-state analogs for enolase

Publisher Summary This chapter describes the active site-specific reagents and transition-state analogs for enolase. There is only one active site-specific reagent known to form a covalent derivative with the enzyme— namely, glycidol phosphate (1,2- epoxipropanol 3-phosphate). In addition, there are pairs of analogs, D-tartronate semialdehyde 2-phosphate and 3-amino enolpyruvate phosphate, which form very tight noncovalent complexes with the active site of enolases. Because of the strong UV absorbance of the complexes, these compounds have been useful in establishing the number of active sites in the enzyme, and as likely transition state analogs, their interaction with the enzyme has shed some light on the mechanism of catalysis. Glycidol phosphate is readily prepared by phosphorylation of commercially available glycidol with POC1 3 , and it satisfies the major requirements for an active site-specific reagent for rabbit muscle enolase. The chapter describes dissociation constants and stoichiometry of enolase-inhibitor complexes, the spectral properties of the enolase inhibitors tartronate semialdehyde phosphate (Ttal 2P) and 3-amino enolpyruvate, and the synthesis of Ttal 2P from commercially available D-gluconolactone. A representation of the complexes of Ttal 2P and Am- e PrvP with the active site of enolase is given as a basis for the comparison with the proposed transition state in the reversible dehydration of glycerate 2-phosphate to enolpyruvate phosphate. It is shown that the enolase-analog complexes form only in the presence of Mg 2+ and that both compounds compete with substrate for the active site support the proposition that the two compounds behave as true substrate (transition state) analogs.

Studies on two high-affinity enolase inhibitors. Reaction with enolases.

Studies on two high-affinity enolase inhibitors. Reaction with enolases.

Studies on two high-affinity enolase inhibitors. Chemical characterization.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTTwo high-affinity enolase inhibitors. Chemical characterizationThomas G. Spring and Finn WoldCite this: Biochemistry 1971, 10, 25, 4649–4654Publication Date (Print):December 7, 1971Publication History Published online1 May 2002Published inissue 7 December 1971https://doi.org/10.1021/bi00801a009RIGHTS & PERMISSIONSArticle Views188Altmetric-Citations24LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (633 KB) Get e-Alerts Get e-Alerts