CRISPR-based oligo recombineering prioritizes apicomplexan cysteines for drug discovery

H. J. Benns; M. Storch; J. A. Falco; F. R. Fisher; Fabio Tamaki; E. Alves; C. J. Wincott; Rachel Milne; Natalie Wiedemar; G. Craven; Beatriz Baragaña; Susan Wyllie; Jake Baum; G. S. Baldwin; Eranthie  Weerapana; Edward W. Tate; M. A. Child

doi:10.1038/s41564-022-01249-y

CRISPR-based oligo recombineering prioritizes apicomplexan cysteines for drug discovery

H. J. Benns, M. Storch, J. A. Falco, F. R. Fisher, Fabio Tamaki, E. Alves, C. J. Wincott, Rachel Milne, Natalie Wiedemar, G. Craven, Beatriz Baragaña, Susan Wyllie, Jake Baum, G. S. Baldwin, Eranthie Weerapana, Edward W. Tate, M. A. Child

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Abstract

Nucleophilic amino acids are important in covalent drug development yet underutilized as anti-microbial targets. Chemoproteomic technologies have been developed to mine chemically accessible residues via their intrinsic reactivity towards electrophilic probes but cannot discern which chemically reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based oligo recombineering (CORe) platform to support the rapid identification, functional prioritization and rational targeting of chemically reactive sites in haploid systems. Our approach couples protein sequence and function with biological fitness of live cells. Here we profile the electrophile sensitivity of proteinogenic cysteines in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. Electrophile-sensitive cysteines decorating the ribosome were found to be critical for parasite growth, with target-based screening identifying a parasite-selective anti-malarial lead molecule and validating the apicomplexan translation machinery as a target for ongoing covalent ligand development.

Original language	English
Pages (from-to)	1891-1905
Number of pages	30
Journal	Nature Microbiology
Volume	7
Issue number	11
DOIs	https://doi.org/10.1038/s41564-022-01249-y
Publication status	Published - 20 Oct 2022

Keywords

Mutagenesis
Parasitology
Proteomics
Target identification

ASJC Scopus subject areas

Applied Microbiology and Biotechnology
Microbiology (medical)
Genetics
Cell Biology
Microbiology
Immunology

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10.1038/s41564-022-01249-yLicence: CC BY

Final Published VersionFinal published version, 12.8 MBLicence: CC BY

Cite this

Benns, H. J., Storch, M., Falco, J. A., Fisher, F. R., Tamaki, F., Alves, E., Wincott, C. J., Milne, R., Wiedemar, N., Craven, G., Baragaña, B., Wyllie, S., Baum, J., Baldwin, G. S., Weerapana, E., Tate, E. W., & Child, M. A. (2022). CRISPR-based oligo recombineering prioritizes apicomplexan cysteines for drug discovery. Nature Microbiology, 7(11), 1891-1905. https://doi.org/10.1038/s41564-022-01249-y

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title = "CRISPR-based oligo recombineering prioritizes apicomplexan cysteines for drug discovery",

abstract = "Nucleophilic amino acids are important in covalent drug development yet underutilized as anti-microbial targets. Chemoproteomic technologies have been developed to mine chemically accessible residues via their intrinsic reactivity towards electrophilic probes but cannot discern which chemically reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based oligo recombineering (CORe) platform to support the rapid identification, functional prioritization and rational targeting of chemically reactive sites in haploid systems. Our approach couples protein sequence and function with biological fitness of live cells. Here we profile the electrophile sensitivity of proteinogenic cysteines in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. Electrophile-sensitive cysteines decorating the ribosome were found to be critical for parasite growth, with target-based screening identifying a parasite-selective anti-malarial lead molecule and validating the apicomplexan translation machinery as a target for ongoing covalent ligand development.",

keywords = "Mutagenesis, Parasitology, Proteomics, Target identification",

author = "Benns, {H. J.} and M. Storch and Falco, {J. A.} and Fisher, {F. R.} and Fabio Tamaki and E. Alves and Wincott, {C. J.} and Rachel Milne and Natalie Wiedemar and G. Craven and Beatriz Baraga{\~n}a and Susan Wyllie and Jake Baum and Baldwin, {G. S.} and Eranthie Weerapana and Tate, {Edward W.} and Child, {M. A.}",

note = "Funding Information: This work was supported by grants BB/M011178/1 from the BBSRC (to H.J.B., E.W.T. and M.A.C.), 202553/Z/16/Z from the Wellcome Trust & Royal Society (to M.A.C.), NIH grant R35GM134964 (to E.W.) and 100993/Z/13/Z from the Wellcome Trust (to J.B.). Studies in Dundee were supported by funding from Medicines for Malaria Venture (RD-09-0043) and also from the Wellcome Trust (203134/Z/16/Z), which supports The Wellcome Centre for Anti-Infectives Research. This funding covers all Dundee participants. We acknowledge and thank I. Andrew and L. Game at the UKRI London Institute of Medical Sciences Genomics Laboratory for providing resources and support that contributed to the research results reported within this paper. Copyright: {\textcopyright} 2022, The Author(s).",

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Benns, HJ, Storch, M, Falco, JA, Fisher, FR, Tamaki, F, Alves, E, Wincott, CJ, Milne, R, Wiedemar, N, Craven, G, Baragaña, B , Wyllie, S, Baum, J, Baldwin, GS, Weerapana, E, Tate, EW & Child, MA 2022, 'CRISPR-based oligo recombineering prioritizes apicomplexan cysteines for drug discovery', Nature Microbiology, vol. 7, no. 11, pp. 1891-1905. https://doi.org/10.1038/s41564-022-01249-y

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T1 - CRISPR-based oligo recombineering prioritizes apicomplexan cysteines for drug discovery

AU - Benns, H. J.

AU - Storch, M.

AU - Falco, J. A.

AU - Fisher, F. R.

AU - Tamaki, Fabio

AU - Alves, E.

AU - Wincott, C. J.

AU - Milne, Rachel

AU - Wiedemar, Natalie

AU - Craven, G.

AU - Baragaña, Beatriz

AU - Wyllie, Susan

AU - Baum, Jake

AU - Baldwin, G. S.

AU - Weerapana, Eranthie

AU - Tate, Edward W.

AU - Child, M. A.

N1 - Funding Information: This work was supported by grants BB/M011178/1 from the BBSRC (to H.J.B., E.W.T. and M.A.C.), 202553/Z/16/Z from the Wellcome Trust & Royal Society (to M.A.C.), NIH grant R35GM134964 (to E.W.) and 100993/Z/13/Z from the Wellcome Trust (to J.B.). Studies in Dundee were supported by funding from Medicines for Malaria Venture (RD-09-0043) and also from the Wellcome Trust (203134/Z/16/Z), which supports The Wellcome Centre for Anti-Infectives Research. This funding covers all Dundee participants. We acknowledge and thank I. Andrew and L. Game at the UKRI London Institute of Medical Sciences Genomics Laboratory for providing resources and support that contributed to the research results reported within this paper. Copyright: © 2022, The Author(s).

PY - 2022/10/20

Y1 - 2022/10/20

N2 - Nucleophilic amino acids are important in covalent drug development yet underutilized as anti-microbial targets. Chemoproteomic technologies have been developed to mine chemically accessible residues via their intrinsic reactivity towards electrophilic probes but cannot discern which chemically reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based oligo recombineering (CORe) platform to support the rapid identification, functional prioritization and rational targeting of chemically reactive sites in haploid systems. Our approach couples protein sequence and function with biological fitness of live cells. Here we profile the electrophile sensitivity of proteinogenic cysteines in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. Electrophile-sensitive cysteines decorating the ribosome were found to be critical for parasite growth, with target-based screening identifying a parasite-selective anti-malarial lead molecule and validating the apicomplexan translation machinery as a target for ongoing covalent ligand development.

AB - Nucleophilic amino acids are important in covalent drug development yet underutilized as anti-microbial targets. Chemoproteomic technologies have been developed to mine chemically accessible residues via their intrinsic reactivity towards electrophilic probes but cannot discern which chemically reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based oligo recombineering (CORe) platform to support the rapid identification, functional prioritization and rational targeting of chemically reactive sites in haploid systems. Our approach couples protein sequence and function with biological fitness of live cells. Here we profile the electrophile sensitivity of proteinogenic cysteines in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. Electrophile-sensitive cysteines decorating the ribosome were found to be critical for parasite growth, with target-based screening identifying a parasite-selective anti-malarial lead molecule and validating the apicomplexan translation machinery as a target for ongoing covalent ligand development.

KW - Mutagenesis

KW - Parasitology

KW - Proteomics

KW - Target identification

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U2 - 10.1038/s41564-022-01249-y

DO - 10.1038/s41564-022-01249-y

M3 - Article

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EP - 1905

JO - Nature Microbiology

JF - Nature Microbiology

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ER -

CRISPR-based oligo recombineering prioritizes apicomplexan cysteines for drug discovery

Abstract

Keywords

ASJC Scopus subject areas

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Wellcome Centre for Anti-Infectives Research

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CRISPR-based oligo recombineering prioritizes apicomplexan cysteines for drug discovery

Abstract

Keywords

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

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Wellcome Centre for Anti-Infectives Research

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