{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/381309"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/381309","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Investigating K-Ras inhibition by α-helical peptides","abstract":"Mutations in the KRAS gene are common drivers of human cancer and inhibition of KRas is a key goal in drug discovery. Recently, the development of covalent K-Ras(G12C) inhibitors has presented a breakthrough in the field and challenged the belief that K-Ras was \"undruggable\". Despite this, there are currently no approved inhibitors directly targeting other K-Ras variants and long-term efficacy of K-Ras(G12C) inhibitors is compromised by resistance. This necessitates investigation into alternative methods to target K-Ras. α-helical peptides present an effective method to inhibit protein-protein interactions such as those between K-Ras and its effectors. So far, K-Ras-targeting α-helical peptides have been resticted to SOS1 mimetics. This work aimed to investigate inhibition of K-Ras by α-helical peptides discovered by de novo screening or from a Ral GTPase effector protein domain (RLIP76 RBD) template. A single hit from a selection of an α-helical peptide library against K-Ras(G12D) was found to bind K-Ras(G12D) with low micromolar affinity. Further characterisation revealed that this peptide (P39) displayed selectivity for the GTP-bound state of K-Ras(G12D) and that binding was competitive with the K-Ras effector Raf. The P39 binding site on K-Ras(G12D) was mapped using a combination of NMR titrations and molecular dynamics simulations, revealing a novel binding pose between switch I and II of K-Ras(G12D). In addition to de novo screening, design of a K-Ras-binding α-helical peptide from RLIP76 RBD was attempted. RLIP76 RBD variants previously selected to bind K-Ras were validated biochemically, however their dimerisation prevented K-Ras binding. A point mutation successfully reversed dimerisation, although K-Ras binding of the resulting RBD could not be verified. Rational design of RLIP76 RBD point mutations enabled K-Ras binding in a single case, suggesting K-Ras affinity can be achieved by design. Finally, cellular studies revealed that when co-expressed in HEK-293T cells, the P39- K-Ras interaction could be seen. Surprisingly, treatment of a KRAS(G12D)-mutant colon cancer cell line with octa-Arg-appended P39 resulted in an increase in K-Ras activity and downstream MAPK and PI3K signalling. P39-induced hyperactivation of Ras signalling led to apoptosis in these cells. P39 therefore represents a useful starting point for further development.","abstract_html":"Mutations in the KRAS gene are common drivers of human cancer and inhibition of KRas is a key goal in drug discovery. Recently, the development of covalent K-Ras(G12C) inhibitors has presented a breakthrough in the field and challenged the belief that K-Ras was "undruggable". Despite this, there are currently no approved inhibitors directly targeting other K-Ras variants and long-term efficacy of K-Ras(G12C) inhibitors is compromised by resistance. This necessitates investigation into alternative methods to target K-Ras. α-helical peptides present an effective method to inhibit protein-protein interactions such as those between K-Ras and its effectors. So far, K-Ras-targeting α-helical peptides have been resticted to SOS1 mimetics. This work aimed to investigate inhibition of K-Ras by α-helical peptides discovered by de novo screening or from a Ral GTPase effector protein domain (RLIP76 RBD) template. A single hit from a selection of an α-helical peptide library against K-Ras(G12D) was found to bind K-Ras(G12D) with low micromolar affinity. Further characterisation revealed that this peptide (P39) displayed selectivity for the GTP-bound state of K-Ras(G12D) and that binding was competitive with the K-Ras effector Raf. The P39 binding site on K-Ras(G12D) was mapped using a combination of NMR titrations and molecular dynamics simulations, revealing a novel binding pose between switch I and II of K-Ras(G12D). In addition to de novo screening, design of a K-Ras-binding α-helical peptide from RLIP76 RBD was attempted. RLIP76 RBD variants previously selected to bind K-Ras were validated biochemically, however their dimerisation prevented K-Ras binding. A point mutation successfully reversed dimerisation, although K-Ras binding of the resulting RBD could not be verified. Rational design of RLIP76 RBD point mutations enabled K-Ras binding in a single case, suggesting K-Ras affinity can be achieved by design. Finally, cellular studies revealed that when co-expressed in HEK-293T cells, the P39- K-Ras interaction could be seen. Surprisingly, treatment of a KRAS(G12D)-mutant colon cancer cell line with octa-Arg-appended P39 resulted in an increase in K-Ras activity and downstream MAPK and PI3K signalling. P39-induced hyperactivation of Ras signalling led to apoptosis in these cells. P39 therefore represents a useful starting point for further development.","abstract_has_math":false,"creators":["Comfort, Hannah Megan Freeman"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Mott, Helen"],"committee_chairs":[],"committee_members":[],"year":2024,"date_issued":"2024-09-27","date_published":"2024-09-27","updated_at":"2026-07-22T22:24:20Z","subjects":["K-Ras","peptide","therapeutic"],"languages":["eng"],"rights":[],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/f63c473b-8fb2-4a5a-b190-5a9220231de7/download","http://purl.org/NET/rdflicense/allrightsreserved"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.116542","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Mott, Helen"]},{"key":"dc:creator","label":"Author","values":["Comfort, Hannah Megan Freeman"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2024-09-27"]},{"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/1810/381309"]},{"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":["K-Ras","peptide","therapeutic"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/f63c473b-8fb2-4a5a-b190-5a9220231de7/download","http://purl.org/NET/rdflicense/allrightsreserved"]},{"key":"dc:rights.embargodate","label":"Dc Rights Embargodate","values":["2026-03-11"]},{"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.116542"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/e033ea7a-f29b-4486-8fdf-cf3d87caf6b7/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Mutations in the KRAS gene are common drivers of human cancer and inhibition of KRas is a key goal in drug discovery. Recently, the development of covalent K-Ras(G12C) inhibitors has presented a breakthrough in the field and challenged the belief that K-Ras was \"undruggable\". Despite this, there are currently no approved inhibitors directly targeting other K-Ras variants and long-term efficacy of K-Ras(G12C) inhibitors is compromised by resistance. This necessitates investigation into alternative methods to target K-Ras. α-helical peptides present an effective method to inhibit protein-protein interactions such as those between K-Ras and its effectors. So far, K-Ras-targeting α-helical peptides have been resticted to SOS1 mimetics. This work aimed to investigate inhibition of K-Ras by α-helical peptides discovered by de novo screening or from a Ral GTPase effector protein domain (RLIP76 RBD) template. A single hit from a selection of an α-helical peptide library against K-Ras(G12D) was found to bind K-Ras(G12D) with low micromolar affinity. Further characterisation revealed that this peptide (P39) displayed selectivity for the GTP-bound state of K-Ras(G12D) and that binding was competitive with the K-Ras effector Raf. The P39 binding site on K-Ras(G12D) was mapped using a combination of NMR titrations and molecular dynamics simulations, revealing a novel binding pose between switch I and II of K-Ras(G12D). In addition to de novo screening, design of a K-Ras-binding α-helical peptide from RLIP76 RBD was attempted. RLIP76 RBD variants previously selected to bind K-Ras were validated biochemically, however their dimerisation prevented K-Ras binding. A point mutation successfully reversed dimerisation, although K-Ras binding of the resulting RBD could not be verified. Rational design of RLIP76 RBD point mutations enabled K-Ras binding in a single case, suggesting K-Ras affinity can be achieved by design. Finally, cellular studies revealed that when co-expressed in HEK-293T cells, the P39- K-Ras interaction could be seen. Surprisingly, treatment of a KRAS(G12D)-mutant colon cancer cell line with octa-Arg-appended P39 resulted in an increase in K-Ras activity and downstream MAPK and PI3K signalling. P39-induced hyperactivation of Ras signalling led to apoptosis in these cells. P39 therefore represents a useful starting point for further development."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["87eda9de84448d1f82354d60eee3eb5f","b2e5980cd19e6d8c2080b2dfc0829816"]},{"key":"dc:title","label":"Title","values":["Investigating K-Ras inhibition by α-helical peptides"]}]}],"canonical_facts":{"dc:contributor.advisor":["Mott, Helen"],"dc:creator":["Comfort, Hannah Megan Freeman"],"dc:date.issued":["2024-09-27"],"dc:description.abstract":["Mutations in the KRAS gene are common drivers of human cancer and inhibition of KRas is a key goal in drug discovery. 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Further characterisation revealed that this peptide (P39) displayed selectivity for the GTP-bound state of K-Ras(G12D) and that binding was competitive with the K-Ras effector Raf. The P39 binding site on K-Ras(G12D) was mapped using a combination of NMR titrations and molecular dynamics simulations, revealing a novel binding pose between switch I and II of K-Ras(G12D). In addition to de novo screening, design of a K-Ras-binding α-helical peptide from RLIP76 RBD was attempted. RLIP76 RBD variants previously selected to bind K-Ras were validated biochemically, however their dimerisation prevented K-Ras binding. A point mutation successfully reversed dimerisation, although K-Ras binding of the resulting RBD could not be verified. Rational design of RLIP76 RBD point mutations enabled K-Ras binding in a single case, suggesting K-Ras affinity can be achieved by design. Finally, cellular studies revealed that when co-expressed in HEK-293T cells, the P39- K-Ras interaction could be seen. Surprisingly, treatment of a KRAS(G12D)-mutant colon cancer cell line with octa-Arg-appended P39 resulted in an increase in K-Ras activity and downstream MAPK and PI3K signalling. P39-induced hyperactivation of Ras signalling led to apoptosis in these cells. 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