{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/385276"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/385276","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Novel Modulators of CD39 and CD73","abstract":"CD39 and CD73 are crucial in purinergic signalling, converting triphosphate and diphosphate nucleotides such as ATP and ADP into AMP, which CD73 further degrades into adenosine. CD39 and CD73 dysregulation is linked to various diseases. In cancer, their upregulation is associated with immune suppression, while downregulation in autoimmune disorders leads to immune hyperactivation. The study hypothesises that drugs targeting purinergic binding sites in other proteins might modulate CD39 and CD73 activity. It aims to screen P2Y12 antagonists and ATP-competitive kinase inhibitors to identify novel modulators of CD39 and CD73, explore their mechanisms of action, investigate their binding sites via in-silico docking, and evaluate the ex-vivo activity of specific CD39 modulators. The malachite green assay was employed for in-vitro screening of modulators using recombinant soluble human CD39 and CD73. The protocol was validated using the reported inhibitors POM-1 for CD39 and AB680 for CD73. The IC50s of Identified leads were determined, and their mechanisms of action were analysed by examining the effects on Vmax and Km through non-linear regression. Potential modulator binding sites were identified using blind docking with CB-Dock2. This protocol was validated by successfully docking ADP and AMP into the active sites of CD39 and CD73, respectively. An ex-vivo assay was conducted to assess the effects of specific CD39 modulators on platelet aggregation, with validation performed using the POM-1. During validation experiments, the novel phenomenon of substrate inhibition was observed for CD39 with substrates ADP, ATP, GTP, CDP, CTP, and UTP and for CD73 with GMP. The new substrates of CD39, 2-MeSADP and 2-MeSATP were identified during the validation. To avoid complications from substrate inhibition, UDP and AMP were chosen as substrates for CD39 and CD73, respectively, in the subsequent screens to investigate potential modulators. The pIC50 value is defined as the negative logarithm to the base 10 of the IC50 value. Novel competitive inhibitors included KN-62 (pIC50 of 4.7, IC50 of 19.9 µM) and R406 (pIC50 of 4.4, IC50 of 39.8 µM) for CD39, and AG-183 (pIC50 of 5.1, IC50 of 7.9 µM) for CD73. AR-C 66096 was identified as a new inhibitor class by acting as a poor substrate inhibitor of CD39. New mixed inhibitors included prasugrel (pIC50 of 4, IC50 of 100 µM), PSB-0739 (pIC50 of 5.7, IC50 of 2 µM) and elinogrel (pIC50 of 4.1, IC50 of 79.4 µM) for CD39, and AR-C 66096 (pIC50 of 4.3, IC50 of 50.1 µM ), PSB-0739 (pIC50 of 4.6, IC50 of 25.1 µM) for CD73. Elinogrel (pIC50 of 4.1, IC50 of 79.4 µM) was identified as a novel mixed inhibitor of CD39. Novel mixed inhibitors included ABT-869 (pIC50 of 4.4, IC50 of 39.8 µM),and CAY10578 (pIC50 of 5.3, IC50 of 5 µM ) for CD73. Novel uncompetitive inhibitors of CD73 include KN-62 (pIC50 of 4.7, IC50 of 19.9 µM), R406 (pIC50 of 4.5, IC50 of 31.6 µM), and AG-494 (pIC50 of 4.9, IC50 of 12.6 µM). Ticagrelor M5 metabolite was determined to be a new competitive inhibitor of CD39 (pIC50 of 4, IC50 of 100 µM), displaying non-competitive inhibition kinetic patterns based on its interactions with certain residues suspected to be in the active site in the in-silico docking study. Adenosine was determined to be a new competitive inhibitor of CD39 based on its interactions with suspected active site residues in CD39. However, kinetic studies could not be performed to confirm this mechanism due to its weak inhibition. Prasugrel was also identified as a new ex-vivo modulator of CD39. Unexpectedly, this study also revealed novel positive allosteric modulators such as ticagrelor, GNF-5, and phthalazinone pyrazole for CD39 and ticagrelor, AG-1478, and GNF-5 for CD73. These are the first reported small molecule positive allosteric modulators of these enzymes, to the best of our knowledge, and provide promising leads for potential therapeutic applications such as autoimmune disease. Future studies will need to investigate if these identified modulators exert an effect on CD39 and CD73 in vivo. Likewise, to confirm the binding sites of modulators that were identified through in-silico docking as well as gain a greater understanding of their mechanism of action, it may be necessary to perform both molecular dynamic simulations and empirical studies such as X-ray crystallography.","abstract_html":"CD39 and CD73 are crucial in purinergic signalling, converting triphosphate and diphosphate nucleotides such as ATP and ADP into AMP, which CD73 further degrades into adenosine. CD39 and CD73 dysregulation is linked to various diseases. In cancer, their upregulation is associated with immune suppression, while downregulation in autoimmune disorders leads to immune hyperactivation. The study hypothesises that drugs targeting purinergic binding sites in other proteins might modulate CD39 and CD73 activity. It aims to screen P2Y12 antagonists and ATP-competitive kinase inhibitors to identify novel modulators of CD39 and CD73, explore their mechanisms of action, investigate their binding sites via in-silico docking, and evaluate the ex-vivo activity of specific CD39 modulators. The malachite green assay was employed for in-vitro screening of modulators using recombinant soluble human CD39 and CD73. The protocol was validated using the reported inhibitors POM-1 for CD39 and AB680 for CD73. The IC50s of Identified leads were determined, and their mechanisms of action were analysed by examining the effects on Vmax and Km through non-linear regression. Potential modulator binding sites were identified using blind docking with CB-Dock2. This protocol was validated by successfully docking ADP and AMP into the active sites of CD39 and CD73, respectively. An ex-vivo assay was conducted to assess the effects of specific CD39 modulators on platelet aggregation, with validation performed using the POM-1. During validation experiments, the novel phenomenon of substrate inhibition was observed for CD39 with substrates ADP, ATP, GTP, CDP, CTP, and UTP and for CD73 with GMP. The new substrates of CD39, 2-MeSADP and 2-MeSATP were identified during the validation. To avoid complications from substrate inhibition, UDP and AMP were chosen as substrates for CD39 and CD73, respectively, in the subsequent screens to investigate potential modulators. The pIC50 value is defined as the negative logarithm to the base 10 of the IC50 value. Novel competitive inhibitors included KN-62 (pIC50 of 4.7, IC50 of 19.9 µM) and R406 (pIC50 of 4.4, IC50 of 39.8 µM) for CD39, and AG-183 (pIC50 of 5.1, IC50 of 7.9 µM) for CD73. AR-C 66096 was identified as a new inhibitor class by acting as a poor substrate inhibitor of CD39. New mixed inhibitors included prasugrel (pIC50 of 4, IC50 of 100 µM), PSB-0739 (pIC50 of 5.7, IC50 of 2 µM) and elinogrel (pIC50 of 4.1, IC50 of 79.4 µM) for CD39, and AR-C 66096 (pIC50 of 4.3, IC50 of 50.1 µM ), PSB-0739 (pIC50 of 4.6, IC50 of 25.1 µM) for CD73. Elinogrel (pIC50 of 4.1, IC50 of 79.4 µM) was identified as a novel mixed inhibitor of CD39. Novel mixed inhibitors included ABT-869 (pIC50 of 4.4, IC50 of 39.8 µM),and CAY10578 (pIC50 of 5.3, IC50 of 5 µM ) for CD73. Novel uncompetitive inhibitors of CD73 include KN-62 (pIC50 of 4.7, IC50 of 19.9 µM), R406 (pIC50 of 4.5, IC50 of 31.6 µM), and AG-494 (pIC50 of 4.9, IC50 of 12.6 µM). Ticagrelor M5 metabolite was determined to be a new competitive inhibitor of CD39 (pIC50 of 4, IC50 of 100 µM), displaying non-competitive inhibition kinetic patterns based on its interactions with certain residues suspected to be in the active site in the in-silico docking study. Adenosine was determined to be a new competitive inhibitor of CD39 based on its interactions with suspected active site residues in CD39. However, kinetic studies could not be performed to confirm this mechanism due to its weak inhibition. Prasugrel was also identified as a new ex-vivo modulator of CD39. Unexpectedly, this study also revealed novel positive allosteric modulators such as ticagrelor, GNF-5, and phthalazinone pyrazole for CD39 and ticagrelor, AG-1478, and GNF-5 for CD73. These are the first reported small molecule positive allosteric modulators of these enzymes, to the best of our knowledge, and provide promising leads for potential therapeutic applications such as autoimmune disease. Future studies will need to investigate if these identified modulators exert an effect on CD39 and CD73 in vivo. Likewise, to confirm the binding sites of modulators that were identified through in-silico docking as well as gain a greater understanding of their mechanism of action, it may be necessary to perform both molecular dynamic simulations and empirical studies such as X-ray crystallography.","abstract_has_math":false,"creators":["Vadlamani, Venkat"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Harper, Matthew"],"committee_chairs":[],"committee_members":[],"year":2024,"date_issued":"2024-09-16","date_published":"2024-09-16","updated_at":"2026-07-24T01:33:33Z","subjects":["CD39","CD73","Immune Checkpoint Modulation","Cancer","Autoimmune","Ectonucleotidase"],"languages":[],"rights":[],"rights_urls":["https://www.repository.cam.ac.uk/bitstreams/d86b1ad1-0c52-4009-b1dc-48183ed06437/download","http://purl.org/NET/rdflicense/allrightsreserved"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.118957","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Harper, Matthew"]},{"key":"dc:creator","label":"Author","values":["Vadlamani, Venkat"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2024-09-16"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/385276"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["CD39","CD73","Immune Checkpoint Modulation","Cancer","Autoimmune","Ectonucleotidase"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:rights","label":"Dc Rights","values":["https://www.repository.cam.ac.uk/bitstreams/d86b1ad1-0c52-4009-b1dc-48183ed06437/download","http://purl.org/NET/rdflicense/allrightsreserved"]},{"key":"dc:rights.embargodate","label":"Dc Rights Embargodate","values":["2026-06-11"]},{"key":"dc:rights.embargotype","label":"Dc Rights Embargotype","values":["embargo"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.118957"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://www.repository.cam.ac.uk/bitstreams/1d18680a-bc1c-4fb1-b966-cf8c5243f499/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["CD39 and CD73 are crucial in purinergic signalling, converting triphosphate and diphosphate nucleotides such as ATP and ADP into AMP, which CD73 further degrades into adenosine. CD39 and CD73 dysregulation is linked to various diseases. In cancer, their upregulation is associated with immune suppression, while downregulation in autoimmune disorders leads to immune hyperactivation. The study hypothesises that drugs targeting purinergic binding sites in other proteins might modulate CD39 and CD73 activity. It aims to screen P2Y12 antagonists and ATP-competitive kinase inhibitors to identify novel modulators of CD39 and CD73, explore their mechanisms of action, investigate their binding sites via in-silico docking, and evaluate the ex-vivo activity of specific CD39 modulators. The malachite green assay was employed for in-vitro screening of modulators using recombinant soluble human CD39 and CD73. The protocol was validated using the reported inhibitors POM-1 for CD39 and AB680 for CD73. The IC50s of Identified leads were determined, and their mechanisms of action were analysed by examining the effects on Vmax and Km through non-linear regression. Potential modulator binding sites were identified using blind docking with CB-Dock2. This protocol was validated by successfully docking ADP and AMP into the active sites of CD39 and CD73, respectively. An ex-vivo assay was conducted to assess the effects of specific CD39 modulators on platelet aggregation, with validation performed using the POM-1. During validation experiments, the novel phenomenon of substrate inhibition was observed for CD39 with substrates ADP, ATP, GTP, CDP, CTP, and UTP and for CD73 with GMP. The new substrates of CD39, 2-MeSADP and 2-MeSATP were identified during the validation. To avoid complications from substrate inhibition, UDP and AMP were chosen as substrates for CD39 and CD73, respectively, in the subsequent screens to investigate potential modulators. The pIC50 value is defined as the negative logarithm to the base 10 of the IC50 value. Novel competitive inhibitors included KN-62 (pIC50 of 4.7, IC50 of 19.9 µM) and R406 (pIC50 of 4.4, IC50 of 39.8 µM) for CD39, and AG-183 (pIC50 of 5.1, IC50 of 7.9 µM) for CD73. AR-C 66096 was identified as a new inhibitor class by acting as a poor substrate inhibitor of CD39. New mixed inhibitors included prasugrel (pIC50 of 4, IC50 of 100 µM), PSB-0739 (pIC50 of 5.7, IC50 of 2 µM) and elinogrel (pIC50 of 4.1, IC50 of 79.4 µM) for CD39, and AR-C 66096 (pIC50 of 4.3, IC50 of 50.1 µM ), PSB-0739 (pIC50 of 4.6, IC50 of 25.1 µM) for CD73. Elinogrel (pIC50 of 4.1, IC50 of 79.4 µM) was identified as a novel mixed inhibitor of CD39. Novel mixed inhibitors included ABT-869 (pIC50 of 4.4, IC50 of 39.8 µM),and CAY10578 (pIC50 of 5.3, IC50 of 5 µM ) for CD73. Novel uncompetitive inhibitors of CD73 include KN-62 (pIC50 of 4.7, IC50 of 19.9 µM), R406 (pIC50 of 4.5, IC50 of 31.6 µM), and AG-494 (pIC50 of 4.9, IC50 of 12.6 µM). Ticagrelor M5 metabolite was determined to be a new competitive inhibitor of CD39 (pIC50 of 4, IC50 of 100 µM), displaying non-competitive inhibition kinetic patterns based on its interactions with certain residues suspected to be in the active site in the in-silico docking study. Adenosine was determined to be a new competitive inhibitor of CD39 based on its interactions with suspected active site residues in CD39. However, kinetic studies could not be performed to confirm this mechanism due to its weak inhibition. Prasugrel was also identified as a new ex-vivo modulator of CD39. Unexpectedly, this study also revealed novel positive allosteric modulators such as ticagrelor, GNF-5, and phthalazinone pyrazole for CD39 and ticagrelor, AG-1478, and GNF-5 for CD73. These are the first reported small molecule positive allosteric modulators of these enzymes, to the best of our knowledge, and provide promising leads for potential therapeutic applications such as autoimmune disease. Future studies will need to investigate if these identified modulators exert an effect on CD39 and CD73 in vivo. Likewise, to confirm the binding sites of modulators that were identified through in-silico docking as well as gain a greater understanding of their mechanism of action, it may be necessary to perform both molecular dynamic simulations and empirical studies such as X-ray crystallography."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["7f25fb43cc66cf15a4c821da25b29c1a","87eda9de84448d1f82354d60eee3eb5f"]},{"key":"dc:title","label":"Title","values":["Novel Modulators of CD39 and CD73"]}]}],"canonical_facts":{"dc:contributor.advisor":["Harper, Matthew"],"dc:creator":["Vadlamani, Venkat"],"dc:date.issued":["2024-09-16"],"dc:description.abstract":["CD39 and CD73 are crucial in purinergic signalling, converting triphosphate and diphosphate nucleotides such as ATP and ADP into AMP, which CD73 further degrades into adenosine. CD39 and CD73 dysregulation is linked to various diseases. In cancer, their upregulation is associated with immune suppression, while downregulation in autoimmune disorders leads to immune hyperactivation. The study hypothesises that drugs targeting purinergic binding sites in other proteins might modulate CD39 and CD73 activity. It aims to screen P2Y12 antagonists and ATP-competitive kinase inhibitors to identify novel modulators of CD39 and CD73, explore their mechanisms of action, investigate their binding sites via in-silico docking, and evaluate the ex-vivo activity of specific CD39 modulators. The malachite green assay was employed for in-vitro screening of modulators using recombinant soluble human CD39 and CD73. The protocol was validated using the reported inhibitors POM-1 for CD39 and AB680 for CD73. The IC50s of Identified leads were determined, and their mechanisms of action were analysed by examining the effects on Vmax and Km through non-linear regression. Potential modulator binding sites were identified using blind docking with CB-Dock2. This protocol was validated by successfully docking ADP and AMP into the active sites of CD39 and CD73, respectively. An ex-vivo assay was conducted to assess the effects of specific CD39 modulators on platelet aggregation, with validation performed using the POM-1. During validation experiments, the novel phenomenon of substrate inhibition was observed for CD39 with substrates ADP, ATP, GTP, CDP, CTP, and UTP and for CD73 with GMP. The new substrates of CD39, 2-MeSADP and 2-MeSATP were identified during the validation. To avoid complications from substrate inhibition, UDP and AMP were chosen as substrates for CD39 and CD73, respectively, in the subsequent screens to investigate potential modulators. The pIC50 value is defined as the negative logarithm to the base 10 of the IC50 value. Novel competitive inhibitors included KN-62 (pIC50 of 4.7, IC50 of 19.9 µM) and R406 (pIC50 of 4.4, IC50 of 39.8 µM) for CD39, and AG-183 (pIC50 of 5.1, IC50 of 7.9 µM) for CD73. AR-C 66096 was identified as a new inhibitor class by acting as a poor substrate inhibitor of CD39. New mixed inhibitors included prasugrel (pIC50 of 4, IC50 of 100 µM), PSB-0739 (pIC50 of 5.7, IC50 of 2 µM) and elinogrel (pIC50 of 4.1, IC50 of 79.4 µM) for CD39, and AR-C 66096 (pIC50 of 4.3, IC50 of 50.1 µM ), PSB-0739 (pIC50 of 4.6, IC50 of 25.1 µM) for CD73. Elinogrel (pIC50 of 4.1, IC50 of 79.4 µM) was identified as a novel mixed inhibitor of CD39. Novel mixed inhibitors included ABT-869 (pIC50 of 4.4, IC50 of 39.8 µM),and CAY10578 (pIC50 of 5.3, IC50 of 5 µM ) for CD73. Novel uncompetitive inhibitors of CD73 include KN-62 (pIC50 of 4.7, IC50 of 19.9 µM), R406 (pIC50 of 4.5, IC50 of 31.6 µM), and AG-494 (pIC50 of 4.9, IC50 of 12.6 µM). Ticagrelor M5 metabolite was determined to be a new competitive inhibitor of CD39 (pIC50 of 4, IC50 of 100 µM), displaying non-competitive inhibition kinetic patterns based on its interactions with certain residues suspected to be in the active site in the in-silico docking study. Adenosine was determined to be a new competitive inhibitor of CD39 based on its interactions with suspected active site residues in CD39. However, kinetic studies could not be performed to confirm this mechanism due to its weak inhibition. Prasugrel was also identified as a new ex-vivo modulator of CD39. Unexpectedly, this study also revealed novel positive allosteric modulators such as ticagrelor, GNF-5, and phthalazinone pyrazole for CD39 and ticagrelor, AG-1478, and GNF-5 for CD73. These are the first reported small molecule positive allosteric modulators of these enzymes, to the best of our knowledge, and provide promising leads for potential therapeutic applications such as autoimmune disease. Future studies will need to investigate if these identified modulators exert an effect on CD39 and CD73 in vivo. Likewise, to confirm the binding sites of modulators that were identified through in-silico docking as well as gain a greater understanding of their mechanism of action, it may be necessary to perform both molecular dynamic simulations and empirical studies such as X-ray crystallography."],"dc:format.checksum.md5":["7f25fb43cc66cf15a4c821da25b29c1a","87eda9de84448d1f82354d60eee3eb5f"],"dc:identifier.doi":["https://doi.org/10.17863/CAM.118957"],"dc:identifier.uri":["https://www.repository.cam.ac.uk/bitstreams/1d18680a-bc1c-4fb1-b966-cf8c5243f499/download"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/385276"],"dc:rights":["https://www.repository.cam.ac.uk/bitstreams/d86b1ad1-0c52-4009-b1dc-48183ed06437/download","http://purl.org/NET/rdflicense/allrightsreserved"],"dc:rights.embargodate":["2026-06-11"],"dc:rights.embargotype":["embargo"],"dc:subject":["CD39","CD73","Immune Checkpoint Modulation","Cancer","Autoimmune","Ectonucleotidase"],"dc:title":["Novel Modulators of CD39 and CD73"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-24T01:33:33Z"}