Development of a High-Throughput Screening Assay to Identify Inhibitors of the Major M17-Leucyl Aminopeptidase from Trypanosoma cruzi Using RapidFire Mass Spectrometry

Maikel Izquierdo; De Lin; Sandra O'Neill; Martin Zoltner; Lauren Webster; Anthony Hope; David W. Gray; Mark C. Field; Jorge González-Bacerio

doi:10.1177/2472555220923367

Development of a High-Throughput Screening Assay to Identify Inhibitors of the Major M17-Leucyl Aminopeptidase from Trypanosoma cruzi Using RapidFire Mass Spectrometry

Maikel Izquierdo, De Lin, Sandra O'Neill, Martin Zoltner, Lauren Webster, Anthony Hope, David W. Gray, Mark C. Field, Jorge González-Bacerio (Lead / Corresponding author)

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5 Citations (Scopus)

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Abstract

Leucyl aminopeptidases (LAPs) are involved in multiple cellular functions, which, in the case of infectious diseases, includes participation in the pathogen-host cell interface and pathogenesis. Thus, LAPs are considered good candidate drug targets, and the major M17-LAP from Trypanosoma cruzi (LAPTc) in particular is a promising target for Chagas disease. To exploit LAPTc as a potential target, it is essential to develop potent and selective inhibitors. To achieve this, we report a high-throughput screening method for LAPTc. Two methods were developed and optimized: a Leu-7-amido-4-methylcoumarin-based fluorogenic assay and a RapidFire mass spectrometry (RapidFire MS)-based assay using the LSTVIVR peptide as substrate. Compared with a fluorescence assay, the major advantages of the RapidFire MS assay are a greater signal-to-noise ratio as well as decreased consumption of enzyme. RapidFire MS was validated with the broad-spectrum LAP inhibitors bestatin (IC50 = 0.35 μM) and arphamenine A (IC50 = 15.75 μM). We suggest that RapidFire MS is highly suitable for screening for specific LAPTc inhibitors.

Original language	English
Pages (from-to)	1064-1071
Number of pages	8
Journal	SLAS Discovery
Volume	25
Issue number	9
Early online date	13 May 2020
DOIs	https://doi.org/10.1177/2472555220923367
Publication status	Published - 1 Oct 2020

Keywords

arphamenine
bestatin
high-throughput screening
M17-leucyl aminopeptidase
RapidFire mass spectrometry

ASJC Scopus subject areas

Analytical Chemistry
Molecular Medicine
Biochemistry
Biotechnology

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

Access to Document

10.1177/2472555220923367Licence: CC BY-NC-ND

Final published versionFinal published version, 380 KBLicence: CC BY-NC-ND

Cite this

Izquierdo, M., Lin, D., O'Neill, S., Zoltner, M., Webster, L., Hope, A., Gray, D. W., Field, M. C., & González-Bacerio, J. (2020). Development of a High-Throughput Screening Assay to Identify Inhibitors of the Major M17-Leucyl Aminopeptidase from Trypanosoma cruzi Using RapidFire Mass Spectrometry. SLAS Discovery, 25(9), 1064-1071. https://doi.org/10.1177/2472555220923367

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title = "Development of a High-Throughput Screening Assay to Identify Inhibitors of the Major M17-Leucyl Aminopeptidase from Trypanosoma cruzi Using RapidFire Mass Spectrometry",

abstract = "Leucyl aminopeptidases (LAPs) are involved in multiple cellular functions, which, in the case of infectious diseases, includes participation in the pathogen-host cell interface and pathogenesis. Thus, LAPs are considered good candidate drug targets, and the major M17-LAP from Trypanosoma cruzi (LAPTc) in particular is a promising target for Chagas disease. To exploit LAPTc as a potential target, it is essential to develop potent and selective inhibitors. To achieve this, we report a high-throughput screening method for LAPTc. Two methods were developed and optimized: a Leu-7-amido-4-methylcoumarin-based fluorogenic assay and a RapidFire mass spectrometry (RapidFire MS)-based assay using the LSTVIVR peptide as substrate. Compared with a fluorescence assay, the major advantages of the RapidFire MS assay are a greater signal-to-noise ratio as well as decreased consumption of enzyme. RapidFire MS was validated with the broad-spectrum LAP inhibitors bestatin (IC50 = 0.35 μM) and arphamenine A (IC50 = 15.75 μM). We suggest that RapidFire MS is highly suitable for screening for specific LAPTc inhibitors.",

keywords = "arphamenine, bestatin, high-throughput screening, M17-leucyl aminopeptidase, RapidFire mass spectrometry",

author = "Maikel Izquierdo and De Lin and Sandra O'Neill and Martin Zoltner and Lauren Webster and Anthony Hope and Gray, {David W.} and Field, {Mark C.} and Jorge Gonz{\'a}lez-Bacerio",

note = "The authors received no financial support for the research, authorship, and/or publication of this article: This work was supported by Wellcome Trust Centre awards (203134/Z/16/Z, 204697/Z/16/), the second to M.C.F.; the International Foundation for Sciences (grant F/4730-2) to J.G.-B.; and the project assigned to J.G.B. and associated with the Cuban National Program of Basic Science.",

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Izquierdo, M, Lin, D , O'Neill, S, Zoltner, M, Webster, L, Hope, A, Gray, DW , Field, MC & González-Bacerio, J 2020, 'Development of a High-Throughput Screening Assay to Identify Inhibitors of the Major M17-Leucyl Aminopeptidase from Trypanosoma cruzi Using RapidFire Mass Spectrometry', SLAS Discovery, vol. 25, no. 9, pp. 1064-1071. https://doi.org/10.1177/2472555220923367

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AU - Izquierdo, Maikel

AU - Lin, De

AU - O'Neill, Sandra

AU - Zoltner, Martin

AU - Webster, Lauren

AU - Hope, Anthony

AU - Gray, David W.

AU - Field, Mark C.

AU - González-Bacerio, Jorge

N1 - The authors received no financial support for the research, authorship, and/or publication of this article: This work was supported by Wellcome Trust Centre awards (203134/Z/16/Z, 204697/Z/16/), the second to M.C.F.; the International Foundation for Sciences (grant F/4730-2) to J.G.-B.; and the project assigned to J.G.B. and associated with the Cuban National Program of Basic Science.

PY - 2020/10/1

Y1 - 2020/10/1

N2 - Leucyl aminopeptidases (LAPs) are involved in multiple cellular functions, which, in the case of infectious diseases, includes participation in the pathogen-host cell interface and pathogenesis. Thus, LAPs are considered good candidate drug targets, and the major M17-LAP from Trypanosoma cruzi (LAPTc) in particular is a promising target for Chagas disease. To exploit LAPTc as a potential target, it is essential to develop potent and selective inhibitors. To achieve this, we report a high-throughput screening method for LAPTc. Two methods were developed and optimized: a Leu-7-amido-4-methylcoumarin-based fluorogenic assay and a RapidFire mass spectrometry (RapidFire MS)-based assay using the LSTVIVR peptide as substrate. Compared with a fluorescence assay, the major advantages of the RapidFire MS assay are a greater signal-to-noise ratio as well as decreased consumption of enzyme. RapidFire MS was validated with the broad-spectrum LAP inhibitors bestatin (IC50 = 0.35 μM) and arphamenine A (IC50 = 15.75 μM). We suggest that RapidFire MS is highly suitable for screening for specific LAPTc inhibitors.

AB - Leucyl aminopeptidases (LAPs) are involved in multiple cellular functions, which, in the case of infectious diseases, includes participation in the pathogen-host cell interface and pathogenesis. Thus, LAPs are considered good candidate drug targets, and the major M17-LAP from Trypanosoma cruzi (LAPTc) in particular is a promising target for Chagas disease. To exploit LAPTc as a potential target, it is essential to develop potent and selective inhibitors. To achieve this, we report a high-throughput screening method for LAPTc. Two methods were developed and optimized: a Leu-7-amido-4-methylcoumarin-based fluorogenic assay and a RapidFire mass spectrometry (RapidFire MS)-based assay using the LSTVIVR peptide as substrate. Compared with a fluorescence assay, the major advantages of the RapidFire MS assay are a greater signal-to-noise ratio as well as decreased consumption of enzyme. RapidFire MS was validated with the broad-spectrum LAP inhibitors bestatin (IC50 = 0.35 μM) and arphamenine A (IC50 = 15.75 μM). We suggest that RapidFire MS is highly suitable for screening for specific LAPTc inhibitors.

KW - arphamenine

KW - bestatin

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KW - M17-leucyl aminopeptidase

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DO - 10.1177/2472555220923367

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SN - 2472-5552

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SP - 1064

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ER -

Development of a High-Throughput Screening Assay to Identify Inhibitors of the Major M17-Leucyl Aminopeptidase from Trypanosoma cruzi Using RapidFire Mass Spectrometry

Abstract

Keywords

ASJC Scopus subject areas

UN SDGs

Access to Document

Other files and links

Fingerprint

A Systems Approach for Understanding Cell Surface Dynamics in Trypanosomes (Investigator Award)

Wellcome Centre for Anti-Infectives Research

Cite this

Development of a High-Throughput Screening Assay to Identify Inhibitors of the Major M17-Leucyl Aminopeptidase from Trypanosoma cruzi Using RapidFire Mass Spectrometry

Abstract

Keywords

ASJC Scopus subject areas

UN SDGs

Access to Document

Other files and links

Fingerprint

Projects

A Systems Approach for Understanding Cell Surface Dynamics in Trypanosomes (Investigator Award)

Wellcome Centre for Anti-Infectives Research

Cite this