Diverse evolutionary pathways challenge the use of collateral sensitivity as a strategy to suppress resistance

Abstract
Data availability
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Abstract

Drug resistance remains a major obstacle to malaria control and eradication efforts, necessitating the development of novel therapeutic strategies to treat this disease. Drug combinations based on collateral sensitivity, wherein resistance to one drug causes increased sensitivity to the partner drug, have been proposed as an evolutionary strategy to suppress the emergence of resistance in pathogen populations. In this study, we explore collateral sensitivity between compounds targeting the Plasmodium dihydroorotate dehydrogenase (DHODH). We profiled the cross-resistance and collateral sensitivity phenotypes of several DHODH mutant lines to a diverse panel of DHODH inhibitors. We focus on one compound, TCMDC-125334, which was active against all mutant lines tested, including the DHODH C276Y line, which arose in selections with the clinical candidate DSM265. In six selections with TCMDC-125334, the most common mechanism of resistance to this compound was copy number variation of the dhodh locus, although we did identify one mutation, DHODH I263S, which conferred resistance to TCMDC-125334 but not DSM265. We found that selection of the DHODH C276Y mutant with TCMDC-125334 yielded additional genetic changes in the dhodh locus. These double mutant parasites exhibited decreased sensitivity to TCMDC-125334 and were highly resistant to DSM265. Finally, we tested whether collateral sensitivity could be exploited to suppress the emergence of resistance in the context of combination treatment by exposing wildtype parasites to both DSM265 and TCMDC-125334 simultaneously. This selected for parasites with a DHODH V532A mutation which were cross-resistant to both compounds and were as fit as the wildtype parent in vitro. The emergence of these cross-resistant, evolutionarily fit parasites highlights the mutational flexibility of the DHODH enzyme.

Data availability

The raw whole-genome sequencing data generated in this study have been submitted to the NCBI Sequence Read Archive database (https://www.ncbi.nlm.nih.gov/sra/) under accession number PRJNA689594. Sanger sequencing of the PCR amplified dhodh locus have been submitted to GenBank (NCBI) under accession numbers MZ571149-MZ571158.

The following data sets were generated

1. Mandt REK
2. Luth MR
3. Tye MA
4. Mazitschek R
5. Ottilie S
6. Winzeler EA
7. Lafuente-Monasterio M J
8. Gamo FJ
9. Wirth DF
10. Lukens AK
(2022) Plasmodium falciparum strain 3D7 A10 clone DT-F1-C1 dihydroorotate dehydrogenase (dhodh) gene, complete cds
NCBI, MZ571149.

https://www.ncbi.nlm.nih.gov/nuccore/MZ571149
1. Mandt REK
2. Luth MR
3. Tye MA
4. Mazitschek R
5. Ottilie S
6. Winzeler EA
7. Lafuente-Monasterio M J
8. Gamo FJ
9. Wirth DF
10. Lukens AK
(2022) Plasmodium falciparum strain 3D7 A10 clone DT-F1-C2 dihydroorotate dehydrogenase (dhodh) gene, complete cds
NCBI, MZ571150.

https://www.ncbi.nlm.nih.gov/nuccore/MZ571150
1. Mandt REK
2. Luth MR
3. Tye MA
4. Mazitschek R
5. Ottilie S
6. Winzeler EA
7. Lafuente-Monasterio M J
8. Gamo FJ
9. Wirth DF
10. Lukens AK
(2022) Plasmodium falciparum strain 3D7 A10 clone DT-F1-C3 dihydroorotate dehydrogenase (dhodh) gene, complete cds
NCBI, MZ571151.

https://www.ncbi.nlm.nih.gov/nuccore/MZ571151
1. Mandt REK
2. Luth MR
3. Tye MA
4. Mazitschek R
5. Ottilie S
6. Winzeler EA
7. Lafuente-Monasterio M J
8. Gamo FJ
9. Wirth DF
10. Lukens AK
(2022) Plasmodium falciparum strain 3D7 A10 clone DT-F1-C4 dihydroorotate dehydrogenase (dhodh) gene, complete cds
NCBI, MZ571152.

https://www.ncbi.nlm.nih.gov/nuccore/MZ571152
1. Mandt REK
2. Luth MR
3. Tye MA
4. Mazitschek R
5. Ottilie S
6. Winzeler EA
7. Lafuente-Monasterio M J
8. Gamo FJ
9. Wirth DF
10. Lukens AK
(2022) Plasmodium falciparum strain 3D7 A10 clone DT-F2-C1 dihydroorotate dehydrogenase (dhodh) gene, complete cds
NCBI, MZ571153.

https://www.ncbi.nlm.nih.gov/nuccore/MZ571153
1. Mandt REK
2. Luth MR
3. Tye MA
4. Mazitschek R
5. Ottilie S
6. Winzeler EA
7. Lafuente-Monasterio M J
8. Gamo FJ
9. Wirth DF
10. Lukens AK
(2022) Plasmodium falciparum strain 3D7 A10 clone DT-F2-C2 dihydroorotate dehydrogenase (dhodh) gene, complete cds
NCBI, MZ571154.

https://www.ncbi.nlm.nih.gov/nuccore/MZ571154
1. Mandt REK
2. Luth MR
3. Tye MA
4. Mazitschek R
5. Ottilie S
6. Winzeler EA
7. Lafuente-Monasterio M J
8. Gamo FJ
9. Wirth DF
10. Lukens AK
(2022) Plasmodium falciparum strain 3D7 A10 clone DT-F2-C3 dihydroorotate dehydrogenase (dhodh) gene, complete cds
NCBI, MZ571155.

https://www.ncbi.nlm.nih.gov/nuccore/MZ571155
1. Mandt REK
2. Luth MR
3. Tye MA
4. Mazitschek R
5. Ottilie S
6. Winzeler EA
7. Lafuente-Monasterio M J
8. Gamo FJ
9. Wirth DF
10. Lukens AK
(2022) Plasmodium falciparum strain 3D7 A10 clone DT-F2-C4 dihydroorotate dehydrogenase (dhodh) gene, complete cds
NCBI, MZ571156.

https://www.ncbi.nlm.nih.gov/nuccore/MZ571156
1. Mandt REK
2. Luth MR
3. Tye MA
4. Mazitschek R
5. Ottilie S
6. Winzeler EA
7. Lafuente-Monasterio M J
8. Gamo FJ
9. Wirth DF
10. Lukens AK
(2022) Plasmodium falciparum strain 3D7 A10 clone DT-F2-C5 dihydroorotate dehydrogenase (dhodh) gene, complete cds
NCBI, MZ571157.

https://www.ncbi.nlm.nih.gov/nuccore/MZ571157
1. Mandt REK
2. Luth MR
3. Tye MA
4. Mazitschek R
5. Ottilie S
6. Winzeler EA
7. Lafuente-Monasterio M J
8. Gamo FJ
9. Wirth DF
10. Lukens AK
(2022) Plasmodium falciparum strain 3D7 A10 clone DT-F2-C6 dihydroorotate dehydrogenase (dhodh) gene, complete cds
NCBI, MZ571158.

https://www.ncbi.nlm.nih.gov/nuccore/MZ571158
1. University of California
2. San Diego
(2021) WGS of Plasmodium falciparum selected with DHODH inhibitors
NCBI Bioproject, PRJNA689594.

https://www.ncbi.nlm.nih.gov/bioproject/PRJNA689594/

Article and author information

Author details

Rebecca EK Mandt

Harvard T H Chan School of Public Health, Boston, United States

For correspondence
rebeccamandt@gmail.com

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0001-7165-7876
Madeline R Luth

University of California, San Diego, San Diego, United States

Competing interests
No competing interests declared.
Mark A Tye

Massachusetts General Hospital, Boston, United States

Competing interests
No competing interests declared.
Ralph Mazitschek

Massachusetts General Hospital, Boston, United States

Competing interests
No competing interests declared.
Sabine Ottilie

University of California, San Diego, San Diego, United States

Competing interests
No competing interests declared.
Elizabeth A Winzeler

University of California, San Diego, San Diego, United States

Competing interests
Elizabeth A Winzeler, Sits on the advisory board of the Tres Cantos Open Lab Foundation.

"This ORCID iD identifies the author of this article:" 0000-0002-4049-2113
Maria Jose Lafuente-Monasterio

GlaxoSmithKline, Tres Cantos, Spain

Competing interests
Maria Jose Lafuente-Monasterio, GlaxoSmithKline employee.
Francisco Javier Gamo

GlaxoSmithKline, Tres Cantos, Spain

Competing interests
Francisco Javier Gamo, GlaxoSmithKline employee.
Dyann F Wirth

Harvard T H Chan School of Public Health, Boston, United States

Competing interests
Dyann F Wirth, Sits on the advisory board of Medicines for Malaria Venture.
Amanda K Lukens

Broad Institute, Cambridge, United States

For correspondence
alukens@broadinstitute.org

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0002-9560-7643

Funding

National Institutes of Health (R01 AI093716)

Rebecca EK Mandt
Dyann F Wirth
Amanda K Lukens

Bill and Melinda Gates Foundation (OPP1132451)

Rebecca EK Mandt
Maria Jose Lafuente-Monasterio
Francisco Javier Gamo
Dyann F Wirth
Amanda K Lukens

National Institutes of Health (T32 GM008666)

Madeline R Luth

ExxonMobil Foundation

Rebecca EK Mandt
Dyann F Wirth
Amanda K Lukens

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.