Combining Label‐free and Flourometric Functional Assays with Biophysical Binding Assays in Drug Discovery: A Comparison of Positives from an HTS Campaign

Many human diseases are the result of abnormal expression or activation of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Not surprisingly, more than 30 tyrosine kinase inhibitors (TKIs) are currently in clinical use and provide unique treatment options for many patients. PTPs on the other hand have long been regarded as “undruggable” and only recently have gained increased attention in drug discovery. Striatal-enriched tyrosine phosphatase (STEP) is a neuron-specific PTP that is overactive in Alzheimer’s disease (AD) and other neurodegenerative and neuropsychiatric disorders, including Parkinson’s disease, schizophrenia, and fragile X syndrome. An emergent model suggests that the increase in STEP activity interferes with synaptic function and contributes to the characteristic cognitive and behavioral deficits present in these diseases. Prior efforts to generate STEP inhibitors with properties that warrant clinical development have largely failed. To identify novel STEP inhibitor scaffolds, we developed a biophysical, label-free high-throughput screening (HTS) platform based on the protein thermal shift (PTS) technology. In contrast to conventional HTS using STEP enzymatic assays, we found the PTS platform highly robust and capable of identifying true hits with confirmed STEP inhibitory activity and selectivity. This new platform promises to greatly advance STEP drug discovery and should be applicable to other PTP targets.

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Development of a Novel Label-Free and High-Throughput Arginase-1 Assay Using Self-Assembled Monolayer Desorption Ionization Mass Spectrometry

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/24725552211000677 ◽

2021 ◽

pp. 247255522110006

Author(s):

Michael D. Scholle ◽

Zachary A. Gurard-Levin

Keyword(s):

Mass Spectrometry ◽

Drug Discovery ◽

High Throughput ◽

Ionization Mass Spectrometry ◽

Ionization Mass ◽

Label Free ◽

Desorption Ionization ◽

Arginase 1 ◽

Self Assembled ◽

High Throughput Assay

Arginase-1, an enzyme that catalyzes the reaction of L-arginine to L-ornithine, is implicated in the tumor immune response and represents an interesting therapeutic target in immuno-oncology. Initiating arginase drug discovery efforts remains a challenge due to a lack of suitable high-throughput assay methodologies. This report describes the combination of self-assembled monolayers and matrix-assisted laser desorption ionization mass spectrometry to enable the first label-free and high-throughput assay for arginase activity. The assay was optimized for kinetically balanced conditions and miniaturized, while achieving a robust assay (Z-factor > 0.8) and a significant assay window [signal-to-background ratio > 20] relative to fluorescent approaches. To validate the assay, the inhibition of the reference compound nor-NOHA (Nω-hydroxy-nor-L-arginine) was evaluated, and the IC50 measured to be in line with reported results (IC50 = 180 nM). The assay was then used to complete a screen of 175,000 compounds, demonstrating the high-throughput capacity of the approach. The label-free format also eliminates opportunities for false-positive results due to interference from library compounds and optical readouts. The assay methodology described here enables new opportunities for drug discovery for arginase and, due to the assay flexibility, can be more broadly applicable for measuring other amino acid–metabolizing enzymes.

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Cellular Dielectric Spectroscopy: A Label-Free Technology for Drug Discovery

JALA Journal of the Association for Laboratory Automation ◽

10.1016/j.jala.2005.06.002 ◽

2005 ◽

Vol 10 (4) ◽

pp. 258-269 ◽

Cited By ~ 19

Author(s):

G LEUNG ◽

H TANG ◽

R MCGUINNESS ◽

E VERDONK ◽

J MICHELOTTI ◽

...

Keyword(s):

Drug Discovery ◽

Dielectric Spectroscopy ◽

Label Free

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Integration of an In Situ MALDI-Based High-Throughput Screening Process: A Case Study with Receptor Tyrosine Kinase c-MET

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555217727701 ◽

2017 ◽

Vol 22 (10) ◽

pp. 1203-1210 ◽

Cited By ~ 5

Author(s):

Katrin Beeman ◽

Jens Baumgärtner ◽

Manuel Laubenheimer ◽

Karlheinz Hergesell ◽

Martin Hoffmann ◽

...

Keyword(s):

Drug Discovery ◽

High Throughput ◽

High Throughput Screening ◽

Maldi Ms ◽

Kinase Assay ◽

Label Free ◽

Screening Process ◽

Label Free Detection ◽

Ic50 Values

Mass spectrometry (MS) is known for its label-free detection of substrates and products from a variety of enzyme reactions. Recent hardware improvements have increased interest in the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for high-throughput drug discovery. Despite interest in this technology, several challenges remain and must be overcome before MALDI-MS can be integrated as an automated “in-line reader” for high-throughput drug discovery. Two such hurdles include in situ sample processing and deposition, as well as integration of MALDI-MS for enzymatic screening assays that usually contain high levels of MS-incompatible components. Here we adapt our c-MET kinase assay to optimize for MALDI-MS compatibility and test its feasibility for compound screening. The pros and cons of the Echo (Labcyte) as a transfer system for in situ MALDI-MS sample preparation are discussed. We demonstrate that this method generates robust data in a 1536-grid format. We use the MALDI-MS to directly measure the ratio of c-MET substrate and phosphorylated product to acquire IC50 curves and demonstrate that the pharmacology is unaffected. The resulting IC50 values correlate well between the common label-based capillary electrophoresis and the label-free MALDI-MS detection method. We predict that label-free MALDI-MS-based high-throughput screening will become increasingly important and more widely used for drug discovery.

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Self-referencing a single waveguide grating sensor in a micron-sized deep flow chamber for label-free biomolecular binding assays

Lab on a Chip ◽

10.1039/b501219h ◽

2005 ◽

Vol 5 (9) ◽

pp. 959 ◽

Cited By ~ 17

Author(s):

Po Ki Yuen ◽

Norman H. Fontaine ◽

Mark A. Quesada ◽

Prantik Mazumder ◽

Richard Bergman ◽

...

Keyword(s):

Flow Chamber ◽

Label Free ◽

Binding Assays ◽

Waveguide Grating ◽

Biomolecular Binding

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Label-free MS imaging from drug discovery to preclinical development

Bioanalysis ◽

10.4155/bio.14.202 ◽

2014 ◽

Vol 6 (20) ◽

pp. 2775-2788 ◽

Cited By ~ 9

Author(s):

Guillaume Hochart ◽

Gregory Hamm ◽

Jonathan Stauber

Keyword(s):

Drug Discovery ◽

Label Free ◽

Preclinical Development ◽

Ms Imaging

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Ligand-Induced Conformational Changes in HSP90 Monitored Time Resolved and Label Free-Towards a Conformational Activity Screening for Drug Discovery

Angewandte Chemie International Edition ◽

10.1002/anie.201802603 ◽

2018 ◽

Vol 57 (31) ◽

pp. 9955-9960 ◽

Cited By ~ 4

Author(s):

Jörn Güldenhaupt ◽

Marta Amaral ◽

Carsten Kötting ◽

Jonas Schartner ◽

Djordje Musil ◽

...

Keyword(s):

Drug Discovery ◽

Conformational Changes ◽

Label Free ◽

Time Resolved ◽

Activity Screening

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Establishing MALDI-TOF as Versatile Drug Discovery Readout to Dissect the PTP1B Enzymatic Reaction

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555218759267 ◽

2018 ◽

Vol 23 (6) ◽

pp. 561-573 ◽

Cited By ~ 7

Author(s):

Martin Winter ◽

Tom Bretschneider ◽

Carola Kleiner ◽

Robert Ries ◽

Jörg P. Hehn ◽

...

Keyword(s):

Drug Discovery ◽

High Throughput Screening ◽

Tyrosine Phosphatase ◽

Enzymatic Reaction ◽

Enzymatic Reactions ◽

Label Free ◽

Ionization Time ◽

Maldi Tof ◽

Ptp1b Inhibitors ◽

Promising Perspective

Label-free, mass spectrometric (MS) detection is an emerging technology in the field of drug discovery. Unbiased deciphering of enzymatic reactions is a proficient advantage over conventional label-based readouts suffering from compound interference and intricate generation of tailored signal mediators. Significant evolvements of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, as well as associated liquid handling instrumentation, triggered extensive efforts in the drug discovery community to integrate the comprehensive MS readout into the high-throughput screening (HTS) portfolio. Providing speed, sensitivity, and accuracy comparable to those of conventional, label-based readouts, combined with merits of MS-based technologies, such as label-free parallelized measurement of multiple physiological components, emphasizes the advantages of MALDI-TOF for HTS approaches. Here we describe the assay development for the identification of protein tyrosine phosphatase 1B (PTP1B) inhibitors. In the context of this precious drug target, MALDI-TOF was integrated into the HTS environment and cross-compared with the well-established AlphaScreen technology. We demonstrate robust and accurate IC50 determination with high accordance to data generated by AlphaScreen. Additionally, a tailored MALDI-TOF assay was developed to monitor compound-dependent, irreversible modification of the active cysteine of PTP1B. Overall, the presented data proves the promising perspective for the integration of MALDI-TOF into drug discovery campaigns.

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