Diverse evolutionary pathways challenge the use of collateral sensitivity as a strategy to suppress resistance
Curation statements for this article:-
Curated by eLife
eLife assessment
This study addresses an important question in the field of antimicrobial chemotherapy: whether combinations of enzyme inhibitors that select for mutations that confer resistance to one inhibitor and at the same time increased sensitization to the other inhibitor can provide a path towards mitigating resistance risks. The authors here investigated one such combination of inhibitors of Plasmodium falciparum DHODH (dihydroorotate dehydrogenase), finding that despite "collateral sensitivity", it was still possible to select a mutation that mediated resistance to both inhibitors without any change in parasite fitness. Additional cross-susceptibility and structural modeling strengthen this study, which is performed to a high technical standard and presents a convincing body of data.
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Microbiology and Infectious Disease (eLife)
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.
Article activity feed
-
-
Author response
Reviewer #1 (Public Review):
The usual strategy to combat antimicrobial drug resistance is to administer a combination of two drugs with distinct mechanisms. An alternative, however, would be to use two drugs that attack the same target, if resistance to one is incompatible with resistance to the other. The authors previously studied parasites resistant to the dihydroorotate dehydrogenase (DHODH) inhibitor DSM265 through an E182D mutation and found that resistance to another inhibitor, IDI-6273, resulted in a reversion to wild-type. Here, they screened various other inhibitors and found that TCMDC-125334 is more active on DSM265-resistant parasites than the wild-type. In this case, however, it was possible for the parasites to become resistant to both inhibitors, either by increasing the copy number of …
Author response
Reviewer #1 (Public Review):
The usual strategy to combat antimicrobial drug resistance is to administer a combination of two drugs with distinct mechanisms. An alternative, however, would be to use two drugs that attack the same target, if resistance to one is incompatible with resistance to the other. The authors previously studied parasites resistant to the dihydroorotate dehydrogenase (DHODH) inhibitor DSM265 through an E182D mutation and found that resistance to another inhibitor, IDI-6273, resulted in a reversion to wild-type. Here, they screened various other inhibitors and found that TCMDC-125334 is more active on DSM265-resistant parasites than the wild-type. In this case, however, it was possible for the parasites to become resistant to both inhibitors, either by increasing the copy number of DSM-265-resistant DHODH genes (with a C276Y mutation) or by the emergence of a different mutation. The selection of wild-type parasites with both compounds resulted in resistance but this took considerably longer than for either compound alone. (The actual frequency of double resistance emergence was not measured.)
Overall the results suggest that for DHODH, when pre-existing resistant parasites are selected with another inhibitor, the results will depend on both the initial mutation and the new inhibitor. The data are solid and convincing and suggest that DHODH has considerable scope for resistance development. The observations do have relevance for other inhibitors and/or enzyme drug targets. However from the data so far, the sweeping statements that the authors make concerning double resistance, in general, are not supported.
The formatting of the Figures requires some improvement and in some cases, more details of the statistical analyses are needed.
We thank Reviewer 1 for their kind and helpful comments. We have answered their specific concerns below. In particular, we have improved the formatting of the figures based on their recommendations. We have also edited the discussion based on reviewer 1’s comments.
Reviewer #2 (Public Review):
This article focuses on drug resistance acquired by Plasmodium falciparum malaria parasites that have been pressured with different inhibitors of the essential enzyme DHODH (dihydroorotate dehydrogenase). The study focuses on collateral sensitivity between DSM265, which has been evaluated in a human clinical trial and found to select for resistance via the point mutation C276Y (C276F and G181S were also implicated; PMID 29909069), and the GSK compound TMCDC-125334, against which a panel of DHODH mutant parasites (including C276Y) were found to have increased sensitivity. The authors herein explore this case of "collateral sensitivity" by examining whether these two inhibitors, when used simultaneously, might preclude the selection of resistant parasites. The answer, in this case, is no; collateral sensitivity did not prevent parasites from acquiring a novel mutation (V532A) that mediated resistance to both. Culture competition assays provide evidence that this mutant retains normal fitness. The authors conclude that for this target the idea of combining these inhibitors is not a viable therapeutic strategy. The authors also illustrate how TMCDC-125334 can select for resistance via a separate mutation (I263S) or amplification of a chromosomal segment containing dhodh. They also present modeling data to examine binding poses and how mutations could impact drug binding, which is allosteric to the enzyme's substrates (orotate and FMN). The data are thorough and provide convincing evidence that in this case collateral sensitization by distinct chemotypes does not translate into a viable strategy to inhibit DHODH in a way that can preclude mutations that confer cross-resistance.
We thank the reviewer for their kind comments and helpful recommendations.
Reviewer #3 (Public Review):
'Collateral sensitivity' occurs when drug-resistance mutations render a drug target more sensitive to inhibition by another drug, which has been previously described in some detail for malaria parasite dihydroorotate dehydrogenase (DHODH - see refs 36, 46, and 47, for example). Although it has been suggested that combinations of such drugs could potentially suppress the emergence of resistance, cross-resistance-associated mutation (or copy-number variation, CNV) could render such combination strategies ineffective. In the current study, the authors assess a new pairing of DHODH-targeting drugs. Cross-resistant parasites with DHODH mutation or CNV arise following either sequential or combined drug selection, suggesting that the drug combination described would likely fail to effectively suppress the emergence of resistance.
The strength of the study is that it describes, for a particular drug combination, different mutations associated either with collateral sensitivity or with cross-resistance, and the authors conclude that "combination treatment with DSM265 and TCMDC-125334 failed to suppress resistance". They go on to say that this "brings into question the usefulness of pursuing further DHODH inhibitors." More specific interpretations and implications of the study are as follows:
a. Other combinations may also fail but there may be combinations that can effectively suppress resistance. A more exhaustive analysis of mutational space will likely be required to determine which combinations if any, would be predicted to succeed in a clinical setting.
b. It was previously reported that "a combination of [DHODH] wild-type and mutant-type selective inhibitors led to resistance far less often than either drug alone. ... Comparative growth assays demonstrated that two mutant parasites grew less robustly than their wild-type parent, and the purified protein of those mutants showed a decrease in catalytic efficiency, thereby suggesting a reason for the diminished growth rate" (Ref 46). Also, "selection with a combination of Genz-669178, a wild-type PfDHODH inhibitor, and IDI-6273, a mutant-selective PfDHODH inhibitor, did not yield resistant parasites" (Ref 36). It is possible that these previously tested combinations would also yield cross-resistant mutants if selected further.
c. Although increased DHODH copy number "confers only moderately reduced susceptibility" to the drug used for selection and although these clones were not assessed here for cross-resistance, it seems likely that CNV may represent a general mechanism that could undermine other collateral resistance strategies.
We thank the reviewer for their kind and helpful comments.
-
eLife assessment
This study addresses an important question in the field of antimicrobial chemotherapy: whether combinations of enzyme inhibitors that select for mutations that confer resistance to one inhibitor and at the same time increased sensitization to the other inhibitor can provide a path towards mitigating resistance risks. The authors here investigated one such combination of inhibitors of Plasmodium falciparum DHODH (dihydroorotate dehydrogenase), finding that despite "collateral sensitivity", it was still possible to select a mutation that mediated resistance to both inhibitors without any change in parasite fitness. Additional cross-susceptibility and structural modeling strengthen this study, which is performed to a high technical standard and presents a convincing body of data.
-
Reviewer #1 (Public Review):
The usual strategy to combat antimicrobial drug resistance is to administer a combination of two drugs with distinct mechanisms. An alternative, however, would be to use two drugs that attack the same target, if resistance to one is incompatible with resistance to the other. The authors previously studied parasites resistant to the dihydroorotate dehydrogenase (DHODH) inhibitor DSM265 through an E182D mutation and found that resistance to another inhibitor, IDI-6273, resulted in a reversion to wild-type. Here, they screened various other inhibitors and found that TCMDC-125334 is more active on DSM265-resistant parasites than the wild-type. In this case, however, it was possible for the parasites to become resistant to both inhibitors, either by increasing the copy number of DSM-265-resistant DHODH genes …
Reviewer #1 (Public Review):
The usual strategy to combat antimicrobial drug resistance is to administer a combination of two drugs with distinct mechanisms. An alternative, however, would be to use two drugs that attack the same target, if resistance to one is incompatible with resistance to the other. The authors previously studied parasites resistant to the dihydroorotate dehydrogenase (DHODH) inhibitor DSM265 through an E182D mutation and found that resistance to another inhibitor, IDI-6273, resulted in a reversion to wild-type. Here, they screened various other inhibitors and found that TCMDC-125334 is more active on DSM265-resistant parasites than the wild-type. In this case, however, it was possible for the parasites to become resistant to both inhibitors, either by increasing the copy number of DSM-265-resistant DHODH genes (with a C276Y mutation) or by the emergence of a different mutation. The selection of wild-type parasites with both compounds resulted in resistance but this took considerably longer than for either compound alone. (The actual frequency of double resistance emergence was not measured.)
Overall the results suggest that for DHODH, when pre-existing resistant parasites are selected with another inhibitor, the results will depend on both the initial mutation and the new inhibitor. The data are solid and convincing and suggest that DHODH has considerable scope for resistance development. The observations do have relevance for other inhibitors and/or enzyme drug targets. However from the data so far, the sweeping statements that the authors make concerning double resistance, in general, are not supported.
The formatting of the Figures requires some improvement and in some cases, more details of the statistical analyses are needed.
-
Reviewer #2 (Public Review):
This article focuses on drug resistance acquired by Plasmodium falciparum malaria parasites that have been pressured with different inhibitors of the essential enzyme DHODH (dihydroorotate dehydrogenase). The study focuses on collateral sensitivity between DSM265, which has been evaluated in a human clinical trial and found to select for resistance via the point mutation C276Y (C276F and G181S were also implicated; PMID 29909069), and the GSK compound TMCDC-125334, against which a panel of DHODH mutant parasites (including C276Y) were found to have increased sensitivity. The authors herein explore this case of "collateral sensitivity" by examining whether these two inhibitors, when used simultaneously, might preclude the selection of resistant parasites. The answer, in this case, is no; collateral …
Reviewer #2 (Public Review):
This article focuses on drug resistance acquired by Plasmodium falciparum malaria parasites that have been pressured with different inhibitors of the essential enzyme DHODH (dihydroorotate dehydrogenase). The study focuses on collateral sensitivity between DSM265, which has been evaluated in a human clinical trial and found to select for resistance via the point mutation C276Y (C276F and G181S were also implicated; PMID 29909069), and the GSK compound TMCDC-125334, against which a panel of DHODH mutant parasites (including C276Y) were found to have increased sensitivity. The authors herein explore this case of "collateral sensitivity" by examining whether these two inhibitors, when used simultaneously, might preclude the selection of resistant parasites. The answer, in this case, is no; collateral sensitivity did not prevent parasites from acquiring a novel mutation (V532A) that mediated resistance to both. Culture competition assays provide evidence that this mutant retains normal fitness. The authors conclude that for this target the idea of combining these inhibitors is not a viable therapeutic strategy. The authors also illustrate how TMCDC-125334 can select for resistance via a separate mutation (I263S) or amplification of a chromosomal segment containing dhodh. They also present modeling data to examine binding poses and how mutations could impact drug binding, which is allosteric to the enzyme's substrates (orotate and FMN). The data are thorough and provide convincing evidence that in this case collateral sensitization by distinct chemotypes does not translate into a viable strategy to inhibit DHODH in a way that can preclude mutations that confer cross-resistance.
-
Reviewer #3 (Public Review):
'Collateral sensitivity' occurs when drug-resistance mutations render a drug target more sensitive to inhibition by another drug, which has been previously described in some detail for malaria parasite dihydroorotate dehydrogenase (DHODH - see refs 36, 46, and 47, for example). Although it has been suggested that combinations of such drugs could potentially suppress the emergence of resistance, cross-resistance-associated mutation (or copy-number variation, CNV) could render such combination strategies ineffective. In the current study, the authors assess a new pairing of DHODH-targeting drugs. Cross-resistant parasites with DHODH mutation or CNV arise following either sequential or combined drug selection, suggesting that the drug combination described would likely fail to effectively suppress the emergence …
Reviewer #3 (Public Review):
'Collateral sensitivity' occurs when drug-resistance mutations render a drug target more sensitive to inhibition by another drug, which has been previously described in some detail for malaria parasite dihydroorotate dehydrogenase (DHODH - see refs 36, 46, and 47, for example). Although it has been suggested that combinations of such drugs could potentially suppress the emergence of resistance, cross-resistance-associated mutation (or copy-number variation, CNV) could render such combination strategies ineffective. In the current study, the authors assess a new pairing of DHODH-targeting drugs. Cross-resistant parasites with DHODH mutation or CNV arise following either sequential or combined drug selection, suggesting that the drug combination described would likely fail to effectively suppress the emergence of resistance.
The strength of the study is that it describes, for a particular drug combination, different mutations associated either with collateral sensitivity or with cross-resistance, and the authors conclude that "combination treatment with DSM265 and TCMDC-125334 failed to suppress resistance". They go on to say that this "brings into question the usefulness of pursuing further DHODH inhibitors." More specific interpretations and implications of the study are as follows:
a. Other combinations may also fail but there may be combinations that can effectively suppress resistance. A more exhaustive analysis of mutational space will likely be required to determine which combinations if any, would be predicted to succeed in a clinical setting.
b. It was previously reported that "a combination of [DHODH] wild-type and mutant-type selective inhibitors led to resistance far less often than either drug alone. ... Comparative growth assays demonstrated that two mutant parasites grew less robustly than their wild-type parent, and the purified protein of those mutants showed a decrease in catalytic efficiency, thereby suggesting a reason for the diminished growth rate" (Ref 46). Also, "selection with a combination of Genz-669178, a wild-type PfDHODH inhibitor, and IDI-6273, a mutant-selective PfDHODH inhibitor, did not yield resistant parasites" (Ref 36). It is possible that these previously tested combinations would also yield cross-resistant mutants if selected further.
c. Although increased DHODH copy number "confers only moderately reduced susceptibility" to the drug used for selection and although these clones were not assessed here for cross-resistance, it seems likely that CNV may represent a general mechanism that could undermine other collateral resistance strategies. -
