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

Abstract

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 IC 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.

Multiscale modelling of drug transport and metabolism in liver spheroids

“…making certain that the permeability is either relatively high or relatively low to ensure delivery to the spheroid centre. Validation of these in silico investigations could involve emerging technologies such as MALDI (matrix-assisted laser desorption/ionization)-mass spectrometry imaging, which provide label-free mass spectrometric detection within tissue sections [41]. This detection methodology is rapidly being developed to provide a quantitative measure of drug penetration within a tissue/spheroid at different time-points that could potentially be compared with our model.…”

High-throughput matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry–based deubiquitylating enzyme assay for drug discovery

“…This allowed the miniaturization and scalability of MALDI-TOF MS-based assays to 384 and 1,536 well formats. To date, MALDI-TOF MS-based assays have been established for a large variety of enzymes-including E3 ligases 62 , kinases 63,64 , phosphatases 65,66 , β-secretases (BACE1) 67 , histone demethylases and acetylcholinesterases 68 and cyclic GMP-AMP synthases 69 -and have been applied to the analysis of N-glycans [70][71][72] . The success of MALDI-TOF MS within HTS campaigns relies on several factors.…”

Automated MALDI Target Preparation Concept: Providing Ultra-High-Throughput Mass Spectrometry–Based Screening for Drug Discovery

“…Label-based as well as label-free detection of PTP1B inhibition were conducted by AlphaScreen and MALDI-TOF, respectively, as previously published with minor modifications. 16 Briefly, for single-dose concentration screening, 100 nL DMSO containing 0.5 mg/mL compound was transferred into 384-well microplates (No. 781075, Greiner, Frickenhausen, Germany) using the CyBio Well vario (Analytik Jena, Jena, Germany) liquid-handling unit equipped with a capillary head.…”