Exploration and characterization of the antimalarial activity of cyclopropyl carboxamides that target the mitochondrial protein, cytochrome b

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dc.contributor.authorAwalt, Jon Kyleen
dc.contributor.authorSu, Wenyinen
dc.contributor.authorNguyen, Williamen
dc.contributor.authorLoi, Katieen
dc.contributor.authorJarman, Kate E.en
dc.contributor.authorPenington, Jocelyn S.en
dc.contributor.authorRamesh, Saishyamen
dc.contributor.authorFairhurst, Kate J.en
dc.contributor.authorYeo, Tomasen
dc.contributor.authorPark, Heekuken
dc.contributor.authorUhlemann, Anne Catrinen
dc.contributor.authorChandra Maity, Bikashen
dc.contributor.authorDe, Nirupamen
dc.contributor.authorMukherjee, Parthaen
dc.contributor.authorChakraborty, Arnishen
dc.contributor.authorChurchyard, Alisjeen
dc.contributor.authorFamodimu, Mufuliat T.en
dc.contributor.authorDelves, Michael J.en
dc.contributor.authorBaum, Jakeen
dc.contributor.authorMittal, Nimishaen
dc.contributor.authorWinzeler, Elizabeth A.en
dc.contributor.authorPapenfuss, Anthony T.en
dc.contributor.authorChowdury, Mrittikaen
dc.contributor.authorde Koning-Ward, Tania F.en
dc.contributor.authorMaier, Alexander G.en
dc.contributor.authorvan Dooren, Giel G.en
dc.contributor.authorBaud, Delphineen
dc.contributor.authorBrand, Stephenen
dc.contributor.authorFidock, David A.en
dc.contributor.authorJackson, Paul F.en
dc.contributor.authorCowman, Alan F.en
dc.contributor.authorDans, Madeline G.en
dc.contributor.authorSleebs, Brad E.en
dc.date.accessioned2025-05-23T16:25:29Z
dc.date.available2025-05-23T16:25:29Z
dc.date.issued2024-12-15en
dc.description.abstractDrug resistance against antimalarials is rendering them increasingly ineffective and so there is a need for the development of new antimalarials. To discover new antimalarial chemotypes a phenotypic screen of the Janssen Jumpstarter library against the P. falciparum asexual stage was undertaken, uncovering the cyclopropyl carboxamide structural hit class. Structure-activity analysis revealed that each structural moiety was largely resistant to change, although small changes led to the frontrunner compound, WJM280, which has potent asexual stage activity (EC50 40 nM) and no human cell cytotoxicity. Forward genetics uncovered that cyclopropyl carboxamide resistant parasites have mutations and an amplification in the cytochrome b gene. Cytochrome b was then verified as the target with profiling against cytochrome b drug-resistant parasites and a mitochondrial oxygen consumption assay. Accordingly, the cyclopropyl carboxamide class was shown to have slow-acting asexual stage activity and activity against male gametes and exoerythrocytic forms. Enhancing metabolic stability to attain efficacy in malaria mouse models remains a challenge in the future development of this antimalarial chemotype.en
dc.description.sponsorshipIt is not entirely clear whether there is a direct link between the mutations found in the W466-resistant Dd2-Pol\u03B4 F3_A6 and F3_H4 clones and the mutations found in cyt b. 4-Hydroxybenzoate polyprenyltransferase is an enzyme putatively involved in the biosynthesis of ubiquinone [35] required for the function of the mitochondrial electron transport chain. 4-Hydroxy-3-methylbut-2-en-1-yl diphosphate synthase is an essential component of the isoprenoid biosynthesis pathway which is necessary for supplying precursors for ubiquinone biosynthesis [36]. The mutations in these proteins may be a compensatory mechanism for the inhibition of cyt b by W466 (1). The role of these mutations on the function of these enzymes is unknown and requires reverse genetics to delineate. The role or the survival advantage of the mutations found in the other proteins of W466-resistant Dd2-Pol\u03B4 F3_A6 and F3_H4 clones on the function cyt b or the mitochondrial electron transport chain is unclear and therefore further investigation is required. Overall, the whole genome sequencing on the W466 recrudescence parasites from the MIR study supports cyt b as the target of the cyclopropyl carboxamide antimalarial series.To provide further evidence to support the cyclopropyl carboxamide series targeting cyt b, an oxygen consumption rate (OCR) assay was performed using a Seahorse XFe96 Flux Analyzer [17]. The primary role of the mitochondria ETC is to produce ATP oxidative phosphorylation, and thus the oxygen consumption directly quantifies mitochondrial ETC activity. In this assay, malate, which is a substrate of malate quinone dehydrogenase (MQD), was added to provide a base line of oxygen consumption at approximately 50 pmol/min (Fig. 4). After adding atovaquone (ATQ) which inhibits cyt b (complex III), an OCR reduction was detected while the DHODH inhibitor, DSM265 didn't change the basal OCR. Both W466 (1) and W499 (2) reduced the OCR to the same level as ATQ. Tetramethylphenylendiamin (TMPD), an electron donor was then added which recovered the OCR, indicating none of the compounds inhibit cyt c. Finally, NaN3 was added to inhibit complex IV to bring the OCR back to basal levels. The same OCR pattern between ATQ and both W466 (1) and W499 (2) supports cyt b as the target of cyclopropyl carboxamide series. This data also suggests that the amplification of DHODH is a compensatory mechanism of W466- and W499-resistant populations and is not the molecular target of the cyclopropyl carboxamide class.The Qo site of cyt b was confirmed as the binding site and molecular target of the cyclopropyl carboxamide class evaluating the cyclopropyl carboxamide class against ATQ and ELQ300 resistant populations. This data showed that W466 (1), W499 (2), and WJM280 (108) showed decreased sensitivity to ATQ resistant populations with Qo site mutations but not to an ELQ300 resistant population with a Qi site mutation. Curiously, ATQ showed the same level of sensitivity to W466-resistant populations with a Qo site A122T and F264L mutation to wildtype 3D7 parasites, suggesting structural divergence and a different binding mode between ATQ and the cyclopropyl carboxamide class. Expectedly, ATQ, ELQ300, and DSM265 showed decreased sensitivity to W499-resistant populations with an amplification of DHODH. These findings were further supported by the specific inhibition of complex III in the mitochondrial ETC by W466 (1) and W499 (2) using an oxygen consumption reduction assay.This work was funded by the National Health and Medical Research Council of Australia (Development Grant 1135421 to B.E.S. and A.F.C.; Ideas Grant 1182369 to G.G.v.D and A.G.M. and Synergy Grant 1185354 to T.F.dK-W), the National Institutes of Health (1R01AI152533 to E.A.W.) the Victorian State Government Operational Infrastructure Support and the Australian Government NHMRC IRIISS. J.B. acknowledges support from Wellcome (100993/Z/13/Z) and J.B. D.A.F. and B.E.S. acknowledge the support of Medicines for Malaria Venture (RD-08-2800, RD-08-0015, and RD-08-0003, respectively). We thank and acknowledge the Australian Red Cross Lifeblood & UK NHS Blood and Transplant for the provision of fresh red blood cells. We thank Assoc Prof Paul Gilson from the Burnet Institute and Assoc Prof Darren Creek from Monash University for the provision of the Dd2 ScDHODH parasite strain and SB1-A6 parasite strain, respectively. We thank Dr Keith Watson from the Walter and Eliza Hall Institute for the helpful advice. We thank the University of Melbourne for provision of a Research Scholarship to W.S. A.F.C. is a Howard Hughes International Scholar and an Australia Fellow of the NHMRC. B.E.S. is a Corin Centenary Fellow. This work was funded by the National Health and Medical Research Council of Australia (Development Grant 1135421 to B.E.S. and A.F.C.; Ideas Grant 1182369 to G.G.v.D and A.G.M., and Synergy Grant 1185354 to T.F.dK-W), the National Institutes of Health (1R01AI152533 to E.A.W.) the Victorian State Government Operational Infrastructure Support and the Australian Government NHMRC IRIISS. J.B. acknowledges support from Wellcome (100993/Z/13/Z) and J.B., D.A.F. and B.E.S. acknowledge the support of Medicines for Malaria Venture (RD-08-2800, RD-08-0015, and RD-08-0003, respectively). We thank and acknowledge the Australian Red Cross Lifeblood & UK NHS Blood and Transplant for the provision of fresh red blood cells. We thank Assoc Prof Paul Gilson from the Burnet Institute and Assoc Prof Darren Creek from Monash University for the provision of the Dd2 ScDHODH parasite strain and SB1-A6 parasite strain, respectively. We thank Dr Keith Watson from the Walter and Eliza Hall Institute for the helpful advice. We thank the University of Melbourne for provision of a Research Scholarship to W.S. A.F.C. is a Howard Hughes International Scholar and an Australia Fellow of the NHMRC. B.E.S. is a Corin Centenary Fellow.en
dc.description.statusPeer-revieweden
dc.identifier.issn0223-5234en
dc.identifier.otherPubMed:39388903en
dc.identifier.otherORCID:/0000-0003-2455-9821/work/184104384en
dc.identifier.scopus85205932032en
dc.identifier.urihttp://www.scopus.com/inward/record.url?scp=85205932032&partnerID=8YFLogxKen
dc.identifier.urihttps://hdl.handle.net/1885/733752703
dc.language.isoenen
dc.rightsPublisher Copyright: © 2024 The Author(s)en
dc.sourceEuropean Journal of Medicinal Chemistryen
dc.subjectAntimalarialen
dc.subjectCytochrome ben
dc.subjectMalariaen
dc.subjectMitochondriaen
dc.subjectPlasmodiumen
dc.titleExploration and characterization of the antimalarial activity of cyclopropyl carboxamides that target the mitochondrial protein, cytochrome ben
dc.typeJournal articleen
dspace.entity.typePublicationen
local.contributor.affiliationAwalt, Jon Kyle; Walter and Eliza Hall Institute of Medical Researchen
local.contributor.affiliationSu, Wenyin; Walter and Eliza Hall Institute of Medical Researchen
local.contributor.affiliationNguyen, William; Walter and Eliza Hall Institute of Medical Researchen
local.contributor.affiliationLoi, Katie; Walter and Eliza Hall Institute of Medical Researchen
local.contributor.affiliationJarman, Kate E.; Walter and Eliza Hall Institute of Medical Researchen
local.contributor.affiliationPenington, Jocelyn S.; Walter and Eliza Hall Institute of Medical Researchen
local.contributor.affiliationRamesh, Saishyam; ANU College of Science and Medicine, The Australian National Universityen
local.contributor.affiliationFairhurst, Kate J.; Columbia Universityen
local.contributor.affiliationYeo, Tomas; Columbia Universityen
local.contributor.affiliationPark, Heekuk; Columbia Universityen
local.contributor.affiliationUhlemann, Anne Catrin; Columbia Universityen
local.contributor.affiliationChandra Maity, Bikash; TCG Lifesciences Pvt. Ltd.en
local.contributor.affiliationDe, Nirupam; TCG Lifesciences Pvt. Ltd.en
local.contributor.affiliationMukherjee, Partha; TCG Lifesciences Pvt. Ltd.en
local.contributor.affiliationChakraborty, Arnish; TCG Lifesciences Pvt. Ltd.en
local.contributor.affiliationChurchyard, Alisje; Imperial College Londonen
local.contributor.affiliationFamodimu, Mufuliat T.; London School of Hygiene and Tropical Medicineen
local.contributor.affiliationDelves, Michael J.; London School of Hygiene and Tropical Medicineen
local.contributor.affiliationBaum, Jake; Imperial College Londonen
local.contributor.affiliationMittal, Nimisha; University of California at San Diegoen
local.contributor.affiliationWinzeler, Elizabeth A.; University of California at San Diegoen
local.contributor.affiliationPapenfuss, Anthony T.; Walter and Eliza Hall Institute of Medical Researchen
local.contributor.affiliationChowdury, Mrittika; Deakin Universityen
local.contributor.affiliationde Koning-Ward, Tania F.; Deakin Universityen
local.contributor.affiliationMaier, Alexander G.; Division of Biomedical Science & Biochemistry, Research School of Biology, ANU College of Science and Medicine, The Australian National Universityen
local.contributor.affiliationvan Dooren, Giel G.; Division of Biomedical Science & Biochemistry, Research School of Biology, ANU College of Science and Medicine, The Australian National Universityen
local.contributor.affiliationBaud, Delphine; Medicines for Malaria Ventureen
local.contributor.affiliationBrand, Stephen; Medicines for Malaria Ventureen
local.contributor.affiliationFidock, David A.; Columbia Universityen
local.contributor.affiliationJackson, Paul F.; Johnson & Johnsonen
local.contributor.affiliationCowman, Alan F.; Walter and Eliza Hall Institute of Medical Researchen
local.contributor.affiliationDans, Madeline G.; Walter and Eliza Hall Institute of Medical Researchen
local.contributor.affiliationSleebs, Brad E.; Walter and Eliza Hall Institute of Medical Researchen
local.identifier.citationvolume280en
local.identifier.doi10.1016/j.ejmech.2024.116921en
local.identifier.pure98083aaa-e453-4ba6-adb3-7685fb6b1449en
local.identifier.urlhttps://www.scopus.com/pages/publications/85205932032en
local.type.statusPublisheden

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