Comprehensive mutagenesis on yeast cytosine deaminase yields improvements in 5‐fluorocytosine toxicity in HT1080 cells

Warren, Tiana D.; Patel, Krishna; Rivera, Jordan L.; Eshleman, James R.; Ostermeier, Marc

doi:10.1002/aic.16688

“…The lack of cytosine deaminase activity in humans has made the enzyme a promising candidate for pro-drug gene therapy, where it is targeted to cancer cells to induce tumor specific toxicity 17,18 . This has led to extensive engineering of Fcy1p to optimize its properties as a pro-drug gene therapeutic agent [19][20][21] . Because of this wealth of knowledge, .…”

Section: Introductionmentioning

confidence: 99%

Asymmetrical dose responses shape the evolutionary trade-off between antifungal resistance and nutrient use

Després

¹

,

Cisneros

²

,

Alexander

³

et al. 2022

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Antimicrobial resistance is an emerging threat for public health. The success of resistance mutations depends on the trade-off between the benefits and costs they incur. This trade-off is largely unknown and uncharacterized for antifungals. Here, we systematically catalog the effect of all amino acid substitutions in the yeast cytosine deaminase FCY1, the target of the antifungal 5-FC. We identify over 900 missense mutations granting resistance to 5-FC, a large fraction of which appear to act through destabilisation of the protein. The relationship between 5-FC resistance and growth sustained by cytosine deamination is characterized by a sharp trade-off, such that small gains in resistance universally lead to large losses in canonical enzyme function. We show that this steep relationship can be explained by differences in the dose-response function of 5-FC and cytosine. Our results provide a powerful resource and platform for interpreting drug target variants in fungal pathogens as well as unprecedented insights into resistance-function trade-offs..

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“…The lack of cytosine deaminase activity in humans has made the enzyme a promising candidate for pro-drug gene therapy, where it is targeted to cancer cells to induce tumor specific toxicity 17, 18 . This has led to extensive engineering of Fcy1p to optimize its properties as a pro-drug gene therapeutic agent 19–21 . Because of this wealth of knowledge, Fcy1p is a powerful model to study the evolution of resistance and the potentially associated trade-offs.…”

Section: Introductionmentioning

confidence: 99%

Asymmetrical dose-responses shape the evolutionary trade-off between antifungal resistance and nutrient use

Després

¹

,

Cisneros

²

,

Alexander

³

et al. 2021

Preprint

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Antimicrobial resistance is an emerging threat for public health. The success of resistance mutations depends on the trade-off between the benefits and costs they incur. This trade-off is largely unknown and uncharacterized for antifungals. Here, we systematically catalog the effect of all amino acid substitutions in the yeast cytosine deaminase FCY1, the target of the antifungal 5-FC. We identify over 900 missense mutations granting resistance to 5-FC, a large fraction of which appear to act through destabilisation of the protein. The relationship between 5-FC resistance and growth sustained by cytosine deamination is characterized by a sharp trade-off, such that small gains in resistance universally lead to large losses in canonical enzyme function. We show that this steep relationship can be explained by differences in the dose-response function of 5-FC and cytosine. Our results provide a powerful resource and platform for interpreting drug target variants in fungal pathogens as well as unprecedented insights into resistance-function trade-offs.

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“…The changes in the abundance of each mutant are then used to calculate the ratio of sequence counts before and after selection, which can be used to estimate the relative activities of mutant proteins. This approach has been applied to diverse proteins that function in cellular catalysis. − However, it has not yet been applied to multiheme cytochromes that mediate EET.…”

mentioning

confidence: 99%

Determinants of Multiheme Cytochrome Extracellular Electron Transfer Uncovered by Systematic Peptide Insertion

Campbell

¹

,

Atkinson

²

,

Carpenter

³

et al. 2022

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The multiheme cytochrome MtrA enables microbial respiration by transferring electrons across the outer membrane to extracellular electron acceptors. While structural studies have identified residues that mediate the binding of MtrA to hemes and to other cytochromes that facilitate extracellular electron transfer (EET), the relative importance of these interactions for EET is not known. To better understand EET, we evaluated how insertion of an octapeptide across all MtrA backbone locations affects Shewanella oneidensis MR-1 respiration on Fe(III). The EET efficiency was found to be inversely correlated with the proximity of the insertion to the heme prosthetic groups. Mutants with decreased EET efficiencies also arose from insertions in a subset of the regions that make residue–residue contacts with the porin MtrB, while all sites contacting the extracellular cytochrome MtrC presented high peptide insertion tolerance. MtrA variants having peptide insertions within the CXXCH motifs that coordinate heme cofactors retained some ability to support respiration on Fe(III), although these variants presented significantly decreased EET efficiencies. Furthermore, the fitness of cells expressing different MtrA variants under Fe(III) respiration conditions correlated with anode reduction. The peptide insertion profile, which represents the first comprehensive sequence–structure–function map for a multiheme cytochrome, implicates MtrA as a strategic protein engineering target for the regulation of EET.

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Comprehensive mutagenesis on yeast cytosine deaminase yields improvements in 5‐fluorocytosine toxicity in HT1080 cells

Cited by 8 publications

References 26 publications

Asymmetrical dose responses shape the evolutionary trade-off between antifungal resistance and nutrient use

Asymmetrical dose responses shape the evolutionary trade-off between antifungal resistance and nutrient use

Asymmetrical dose-responses shape the evolutionary trade-off between antifungal resistance and nutrient use

Determinants of Multiheme Cytochrome Extracellular Electron Transfer Uncovered by Systematic Peptide Insertion

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