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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13 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)

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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

KW - RapidFire mass spectrometry

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DO - 10.1177/2472555220923367

M3 - Article

C2 - 32400260

SN - 2472-5552

VL - 25

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

Wellcome Centre for Anti-Infectives Research

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

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

Wellcome Centre for Anti-Infectives Research

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

Cite this