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7. Supramolecular self-associating amphiphiles (SSAs) as nanoscale enhancers of cisplatin anticancer activity

Author

Dora, Nova O., primary, Blackburn, Edith, additional, Boles, Jessica E., additional, Williams, George T., additional, White, Lisa J., additional, Turner, Scarlett E. G., additional, Hothersall, J. Daniel, additional, Askwith, Trevor, additional, Doolan, Jack A., additional, Mulvihill, Daniel P., additional, Garrett, Michelle D., additional, and Hiscock, Jennifer R., additional

Published
2021
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8. Prolonged inhibition of 5-HT3 receptors by palonosetron results from surface receptor inhibition rather than inducing receptor internalization

Author

Hothersall, J Daniel, Moffat, Christopher, and Connolly, Christopher N

Subjects
irreversible antagonism, Nystatin, Quinuclidines, Down-Regulation, Granisetron, Radioligand Assay, Allosteric Regulation, Chlorocebus aethiops, endocytosis, Animals, Humans, Serotonin 5-HT3 Receptor Antagonists, palonosetron, allosterism, Cell Membrane, Hydrazones, emesis, receptor trafficking, Isoquinolines, Research Papers, Recombinant Proteins, Kinetics, Protein Transport, 5-HT3 receptor, COS Cells, cancer therapy, Antiemetics, Receptors, Serotonin, 5-HT3
Abstract

Background and Purpose The 5-HT3 receptor antagonist palonosetron is an important treatment for emesis and nausea during cancer therapy. Its clinical efficacy may result from its unique binding and clearance characteristics and receptor down-regulation mechanisms. We investigated the mechanisms by which palonosetron exerts its long-term inhibition of 5-HT3 receptors for a better understanding of its clinical efficacy. Experimental Approach Cell surface receptors (recombinantly expressed 5HT3A or 5HT3AB in COS-7 cells) were monitored using [3H]granisetron binding and ELISA after exposure to palonosetron. Receptor endocytosis was investigated using immunofluorescence microscopy. Key Results Chronic exposure to palonosetron reduced the number of available cell surface [3H]granisetron binding sites. This down-regulation was not sensitive to either low temperature or pharmacological inhibitors of endocytosis (dynasore or nystatin) suggesting that internalization did not play a role. This was corroborated by our observation that there was no change in cell surface 5-HT3 receptor levels or increase in endocytic rate. Palonosetron exhibited slow dissociation from the receptor over many hours, with a significant proportion of binding sites being occupied for at least 4 days. Furthermore, our observations suggest that chronic receptor down-regulation involved interactions with an allosteric binding site. Conclusions and Implications Palonosetron acts as a pseudo-irreversible antagonist causing prolonged inhibition of 5-HT3 receptors due to its very slow dissociation. In addition, an irreversible binding mode persists for at least 4 days. Allosteric receptor interactions appear to play a role in this phenomenon.

Published
2013

9. Structure-Activity Relationships of the Sustained Effects of Adenosine A2A Receptor Agonists Driven by Slow Dissociation Kinetics

Author

Hothersall, J. Daniel, primary, Guo, Dong, additional, Sarda, Sunil, additional, Sheppard, Robert J., additional, Chen, Hongming, additional, Keur, Wesley, additional, Waring, Michael J., additional, IJzerman, Adriaan P., additional, Hill, Stephen J., additional, Dale, Ian L., additional, and Rawlins, Philip B., additional

Published
2016
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11. The design, synthesis and pharmacological characterization of novel β2-adrenoceptor antagonists

Author

Hothersall, J Daniel, Black, James, Caddick, Stephen, Vinter, Jeremy G, Tinker, Andrew, and Baker, James R

Subjects
Male, Models, Molecular, Guinea Pigs, Stereoisomerism, Research Papers, Propanolamines, Trachea, Structure-Activity Relationship, Adrenergic beta-2 Receptor Antagonists, Drug Design, Cyclic AMP, Animals, Combinatorial Chemistry Techniques, Female, Receptors, Adrenergic, beta-2, Protein Binding, Signal Transduction
Abstract

Selective and potent antagonists for the β(2) -adrenoceptor are potentially interesting as experimental and clinical tools, and we sought to identify novel ligands with this pharmacology.A range of pharmacological assays was used to assess potency, affinity, selectivity (β(2) -adrenoceptor vs. β(1) -adrenoceptor) and efficacy.Ten novel compounds were identified but none had as high affinity as the prototypical β(2) -adrenoceptor blocker ICI-118,551, although one of the novel compounds was more selective for β(2) -adrenoceptors. Most of the ligands were inverse agonists for β(2) -adrenoceptor-cAMP signalling, although one (5217377) was a partial agonist and another a neutral antagonist (7929193). None of the ligands were efficacious with regard to β(2) -adrenoceptor-β-arrestin signalling. The (2S,3S) enantiomers were identified as the most active, although unusually the racemates were the most selective for the β(2) -adrenoceptors. This was taken as evidence for some unusual enantiospecific behaviour.In terms of improving on the pharmacology of the ligand ICI-118,551, one of the compounds was more selective (racemic JB-175), while one was a neutral antagonist (7929193), although none had as high an affinity. The results substantiate the notion that β-blockers do more than simply inhibit receptor activation, and differences between the ligands could provide useful tools to investigate receptor biology.

Published
2011

17. Structure-Activity Relationships of the Sustained Effects of Adenosine A2A Receptor Agonists Driven by Slow Dissociation Kinetics

Author

Hothersall, J. Daniel, Guo, Dong, Sarda, Sunil, Sheppard, Robert J., Chen, Hongming, Keur, Wesley, Waring, Michael J., IJzerman, Adriaan P., Hill, Stephen J., Dale, Ian L., and Rawlins, Philip B.

Abstract

The duration of action of adenosine A2Areceptor (A2A) agonists is critical for their clinical efficacy, and we sought to better understand how this can be optimized. The in vitro temporal response profiles of a panel of A2A agonists were studied using cAMP assays in recombinantly (CHO) and endogenously (SH-SY5Y) expressing cells. Some agonists (e.g., 3cd; UK-432,097) but not others (e.g., 3ac; CGS-21680) demonstrated sustained wash-resistant agonism, where residual receptor activation continued after washout. The ability of an antagonist to reverse pre-established agonist responses was used as a surrogate read-out for agonist dissociation kinetics, and together with radioligand binding studies suggested a role for slow off-rate in driving sustained effects. One compound, 3ch, showed particularly marked sustained effects, with a reversal t1/2> 6 hours and close to maximal effects that remained for at least 5 hours after washing. Based on the structure-activity relationship of these compounds, we suggest that lipophilic N6 and bulky C2 substituents can promote stable and long-lived binding events leading to sustained agonist responses, although a high compound logD is not necessary. This provides new insight into the binding interactions of these ligands and we anticipate that this information could facilitate the rational design of novel long-acting A2A agonists with improved clinical efficacy.

Published
2017
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18. 5-Hydroxytryptamine (5-HT) Cellular Sequestration during Chronic Exposure Delays 5-HT3 Receptor Resensitization due to Its Subsequent Release.

Author

Hothersall, J. Daniel, Alexander, Amy, Samson, Andrew J., Moffat, Christopher, Bollan, Karen A., and Connolly, Christopher N.

Subjects
*SEROTONIN, *NEUROTRANSMITTERS, *TRYPTAMINE, *SEQUESTRATION (Chemistry), *MASKING (Chemistry)
Abstract

The serotonergic synapse is dynamically regulated by serotonin (5-hydroxytryptamine (5-HT)) with elevated levels leading to the down-regulation of the serotonin transporter and a variety of 5-HT receptors, including the 5-HT type-3 (5-HT3) receptors. We report that recombinantly expressed 5-HT3 receptor binding sites are reduced by chronic exposure to 5-HT (IC50 of 154.0 ± 45.7 μm, t1/2 = 28.6 min). This is confirmed for 5-HT3 receptor-induced contractions in the guinea pig ileum, which are down-regulated after chronic, but not acute, exposure to 5-HT. The loss of receptor function does not involve endocytosis, and surface receptor levels are unaltered. The rate and extent of down-regulation is potentiated by serotonin transporter function (IC50 of 2.3 ± 1.0 μm, t1/2 = 3.4 min). Interestingly, the level of 5-HT uptake correlates with the extent of down-regulation. Using TX-114 extraction, we find that accumulated 5-HT remains soluble and not membrane-bound. This cytoplasmically sequestered 5-HT is readily releasable from both COS-7 cells and the guinea pig ileum. Moreover, the 5-HT level released is sufficient to prevent recovery from receptor desensitization in the guinea pig ileum. Together, these findings suggest the existence of a novel mechanism of down-regulation where the chronic release of sequestered 5-HT prolongs receptor desensitization. [ABSTRACT FROM AUTHOR]

Published
2014
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19. Prolonged inhibition of 5- HT3 receptors by palonosetron results from surface receptor inhibition rather than inducing receptor internalization.

Author

Hothersall, J Daniel, Moffat, Christopher, and Connolly, Christopher N

Subjects
*SEROTONIN receptors, *SEROTONIN antagonists, *ISOQUINOLINE, *DRUG efficacy, *VOMITING treatment, *NAUSEA treatment, *CANCER treatment
Abstract

Background and Purpose The 5- HT3 receptor antagonist palonosetron is an important treatment for emesis and nausea during cancer therapy. Its clinical efficacy may result from its unique binding and clearance characteristics and receptor down-regulation mechanisms. We investigated the mechanisms by which palonosetron exerts its long-term inhibition of 5- HT3 receptors for a better understanding of its clinical efficacy. Experimental Approach Cell surface receptors (recombinantly expressed 5HT3A or 5HT3AB in COS-7 cells) were monitored using [3H]granisetron binding and ELISA after exposure to palonosetron. Receptor endocytosis was investigated using immunofluorescence microscopy. Key Results Chronic exposure to palonosetron reduced the number of available cell surface [3H]granisetron binding sites. This down-regulation was not sensitive to either low temperature or pharmacological inhibitors of endocytosis (dynasore or nystatin) suggesting that internalization did not play a role. This was corroborated by our observation that there was no change in cell surface 5- HT3 receptor levels or increase in endocytic rate. Palonosetron exhibited slow dissociation from the receptor over many hours, with a significant proportion of binding sites being occupied for at least 4 days. Furthermore, our observations suggest that chronic receptor down-regulation involved interactions with an allosteric binding site. Conclusions and Implications Palonosetron acts as a pseudo-irreversible antagonist causing prolonged inhibition of 5- HT3 receptors due to its very slow dissociation. In addition, an irreversible binding mode persists for at least 4 days. Allosteric receptor interactions appear to play a role in this phenomenon. [ABSTRACT FROM AUTHOR]

Published
2013
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20. Structure-activity relationships of the sustained effects of adenosine A2A receptor agonists driven by slow dissociation kinetics

Author

Hothersall, J. Daniel, Guo, Dong, Sarda, Sunil, Sheppard, Robert J., Chen, Hongming, Keur, Wesley, Waring, Michael J., IJzerman, Adriaan P., Hill, Stephen J., Dale, Ian L., Rawlins, Philip B., Hothersall, J. Daniel, Guo, Dong, Sarda, Sunil, Sheppard, Robert J., Chen, Hongming, Keur, Wesley, Waring, Michael J., IJzerman, Adriaan P., Hill, Stephen J., Dale, Ian L., and Rawlins, Philip B.

Abstract

The duration of action of adenosine A2A receptor (A2A) agonists is critical for their clinical efficacy, and we sought to better understand how this can be optimized. The in vitro temporal response profiles of a panel of A2A agonists were studied using cAMP assays in recombinantly (CHO) and endogenously (SH-SY5Y) expressing cells. Some agonists (e.g., 3cd; UK-432,097) but not others (e.g., 3ac; CGS-21680) demonstrated sustained wash-resistant agonism, where residual receptor activation continued after washout. The ability of an antagonist to reverse pre-established agonist responses was used as a surrogate read-out for agonist dissociation kinetics, and together with radioligand binding studies suggested a role for slow off-rate in driving sustained effects. One compound, 3ch, showed particularly marked sustained effects, with a reversal t1/2 > 6 hours and close to maximal effects that remained for at least 5 hours after washing. Based on the structure-activity relationship of these compounds, we suggest that lipophilic N6 and bulky C2 substituents can promote stable and long-lived binding events leading to sustained agonist responses, although a high compound logD is not necessary. This provides new insight into the binding interactions of these ligands and we anticipate that this information could facilitate the rational design of novel long-acting A2A agonists with improved clinical efficacy.

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21. Structure-activity relationships of the sustained effects of adenosine A2A receptor agonists driven by slow dissociation kinetics

Author

Hothersall, J. Daniel, Guo, Dong, Sarda, Sunil, Sheppard, Robert J., Chen, Hongming, Keur, Wesley, Waring, Michael J., IJzerman, Adriaan P., Hill, Stephen J., Dale, Ian L., Rawlins, Philip B., Hothersall, J. Daniel, Guo, Dong, Sarda, Sunil, Sheppard, Robert J., Chen, Hongming, Keur, Wesley, Waring, Michael J., IJzerman, Adriaan P., Hill, Stephen J., Dale, Ian L., and Rawlins, Philip B.

Abstract

The duration of action of adenosine A2A receptor (A2A) agonists is critical for their clinical efficacy, and we sought to better understand how this can be optimized. The in vitro temporal response profiles of a panel of A2A agonists were studied using cAMP assays in recombinantly (CHO) and endogenously (SH-SY5Y) expressing cells. Some agonists (e.g., 3cd; UK-432,097) but not others (e.g., 3ac; CGS-21680) demonstrated sustained wash-resistant agonism, where residual receptor activation continued after washout. The ability of an antagonist to reverse pre-established agonist responses was used as a surrogate read-out for agonist dissociation kinetics, and together with radioligand binding studies suggested a role for slow off-rate in driving sustained effects. One compound, 3ch, showed particularly marked sustained effects, with a reversal t1/2 > 6 hours and close to maximal effects that remained for at least 5 hours after washing. Based on the structure-activity relationship of these compounds, we suggest that lipophilic N6 and bulky C2 substituents can promote stable and long-lived binding events leading to sustained agonist responses, although a high compound logD is not necessary. This provides new insight into the binding interactions of these ligands and we anticipate that this information could facilitate the rational design of novel long-acting A2A agonists with improved clinical efficacy.

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22. Structure-activity relationships of the sustained effects of adenosine A2A receptor agonists driven by slow dissociation kinetics

Author

Hothersall, J. Daniel, Guo, Dong, Sarda, Sunil, Sheppard, Robert J., Chen, Hongming, Keur, Wesley, Waring, Michael J., IJzerman, Adriaan P., Hill, Stephen J., Dale, Ian L., Rawlins, Philip B., Hothersall, J. Daniel, Guo, Dong, Sarda, Sunil, Sheppard, Robert J., Chen, Hongming, Keur, Wesley, Waring, Michael J., IJzerman, Adriaan P., Hill, Stephen J., Dale, Ian L., and Rawlins, Philip B.

Abstract

The duration of action of adenosine A2A receptor (A2A) agonists is critical for their clinical efficacy, and we sought to better understand how this can be optimized. The in vitro temporal response profiles of a panel of A2A agonists were studied using cAMP assays in recombinantly (CHO) and endogenously (SH-SY5Y) expressing cells. Some agonists (e.g., 3cd; UK-432,097) but not others (e.g., 3ac; CGS-21680) demonstrated sustained wash-resistant agonism, where residual receptor activation continued after washout. The ability of an antagonist to reverse pre-established agonist responses was used as a surrogate read-out for agonist dissociation kinetics, and together with radioligand binding studies suggested a role for slow off-rate in driving sustained effects. One compound, 3ch, showed particularly marked sustained effects, with a reversal t1/2 > 6 hours and close to maximal effects that remained for at least 5 hours after washing. Based on the structure-activity relationship of these compounds, we suggest that lipophilic N6 and bulky C2 substituents can promote stable and long-lived binding events leading to sustained agonist responses, although a high compound logD is not necessary. This provides new insight into the binding interactions of these ligands and we anticipate that this information could facilitate the rational design of novel long-acting A2A agonists with improved clinical efficacy.

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23. Structure-activity relationships of the sustained effects of adenosine A2A receptor agonists driven by slow dissociation kinetics

Author

Hothersall, J. Daniel, Guo, Dong, Sarda, Sunil, Sheppard, Robert J., Chen, Hongming, Keur, Wesley, Waring, Michael J., IJzerman, Adriaan P., Hill, Stephen J., Dale, Ian L., Rawlins, Philip B., Hothersall, J. Daniel, Guo, Dong, Sarda, Sunil, Sheppard, Robert J., Chen, Hongming, Keur, Wesley, Waring, Michael J., IJzerman, Adriaan P., Hill, Stephen J., Dale, Ian L., and Rawlins, Philip B.

Abstract

The duration of action of adenosine A2A receptor (A2A) agonists is critical for their clinical efficacy, and we sought to better understand how this can be optimized. The in vitro temporal response profiles of a panel of A2A agonists were studied using cAMP assays in recombinantly (CHO) and endogenously (SH-SY5Y) expressing cells. Some agonists (e.g., 3cd; UK-432,097) but not others (e.g., 3ac; CGS-21680) demonstrated sustained wash-resistant agonism, where residual receptor activation continued after washout. The ability of an antagonist to reverse pre-established agonist responses was used as a surrogate read-out for agonist dissociation kinetics, and together with radioligand binding studies suggested a role for slow off-rate in driving sustained effects. One compound, 3ch, showed particularly marked sustained effects, with a reversal t1/2 > 6 hours and close to maximal effects that remained for at least 5 hours after washing. Based on the structure-activity relationship of these compounds, we suggest that lipophilic N6 and bulky C2 substituents can promote stable and long-lived binding events leading to sustained agonist responses, although a high compound logD is not necessary. This provides new insight into the binding interactions of these ligands and we anticipate that this information could facilitate the rational design of novel long-acting A2A agonists with improved clinical efficacy.

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24. High-affinity fluorescent ligands for the 5-HT3 receptor

Author

Simonin, Jonathan, Vernekar, Sanjeev Kumar V., Thompson, Andrew J., Hothersall, J. Daniel, Connolly, Christopher N., Lummis, Sarah C.R., and Lochner, Martin

Subjects
*PROTEIN receptors, *CHEMICAL synthesis, *LIGANDS (Biochemistry), *QUANTUM biochemistry, *GREEN fluorescent protein, *STRUCTURE-activity relationship in pharmacology, *POLYETHYLENE glycol
Abstract

Abstract: The synthesis, photophysical and biological characterization of a small library of fluorescent 5-HT3 receptor ligands is described. Several of these novel granisetron conjugates have high quantum yields and show high affinity for the human 5-HT3AR. [Copyright &y& Elsevier]

Published
2012
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25. An efficient asymmetric synthesis of the potent beta-blocker ICI-118,551 allows the determination of enantiomer dependency on biological activity.

Author

Baker JR, Hothersall JD, Fitzmaurice RJ, Tucknott M, Vinter A, Tinker A, and Caddick S

Subjects
Adrenergic beta-1 Receptor Antagonists, Adrenergic beta-2 Receptor Antagonists, Adrenergic beta-Antagonists chemistry, Crystallography, X-Ray, Molecular Conformation, Propanolamines chemistry, Receptors, Adrenergic, beta-1 metabolism, Receptors, Adrenergic, beta-2 metabolism, Stereoisomerism, Adrenergic beta-Antagonists chemical synthesis, Propanolamines chemical synthesis
Abstract

A highly efficient, practical and flexible two-step asymmetric synthesis of the beta(2)-selective beta-blocker ICI 118,551 is reported, allowing an unambiguous determination of the dependency of biological activity with optical activity, revealing the S,S-enantiomer to be the most potent.

Published
2010
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