Pharmacological probes are important tools for exploring disease biology and discovering new therapies. Often molecules of insufficient quality are used instead, leading to spurious and misleading results. The Boehringer Ingelheim open innovation portal opnMe.com addresses this deficiency by sharing extensively validated pharmacological probes with the scientific community.

Rationale: Low speed and flexibility of most LC-MS/MS approaches in early drug discoverydelays sample analysis from routine in vivo studies within the same day of measurements. A highthroughput platform for the rapid quantification of drug compounds in various in vivo assays wasdeveloped and established in routine bioanalysis.Methods: Automated selection of an efficient and adequate LC method was realized byautonomous sample qualification for ultrafast batch gradients (9 s/sample) or for fast lineargradients (45 s/sample) if samples require chromatography. The hardware and softwarecomponents of our Rapid and Integrated Analysis System (RIAS) were streamlined for increasedanalytical throughput via state-of-the-art automation while keeping high analytical quality.Results: Online decision-making was based on a quick assay suitability test (AST) based on asmall and dedicated sample set evaluated by two different strategies. 84% of the acquired datapoints were within ±30% accuracy and 93% of the deviations between the lower limit ofquantitation (LLOQ) values were ≤2-fold compared to standard LC-MS/MS systems while speed,flexibility and overall automation was significantly improved.Conclusions: The developed platform provided an analysis time of only 10 min (batch-mode) and50 min (gradient-mode) per standard pharmacokinetic (PK) study (62 injections). Automation,data evaluation and results handling were optimized to pave the way for machine learning baseddecision-making regarding the evaluation strategy of the AST

Acoustic droplet ejection (ADE)–open port interface (OPI)–mass spectrometry (MS) has recently been introduced as a versatile analytical method that combines fast and contactless acoustic sampling with sensitive and accurate electrospray ionization (ESI)–MS-based analyte detection. The potential of the technology to provide label-free measurements in subsecond analytical cycle times makes it an attractive option for high-throughput screening (HTS). Here, we report the first implementation of ADE-OPI-MS in a fully automated HTS environment, based on the example of a biochemical assay aiming at the identification of small-molecule inhibitors of the cyclic guanosine monophosphate–adenosine monophosphate (GMP-AMP) synthase (cGAS). First, we describe the optimization of the method to enable sensitive and accurate determination of enzyme activity and inhibition in miniaturized 1536-well microtiter plate format. Then we show both results from a validation single-concentration screen using a test set of 5500 compounds, and the subsequent concentration–response testing of selected hits in direct comparison with a previously established matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) readout. Finally, we present the development of an in-line OPI cleaning procedure aiming to match the instrument robustness required for large-scale HTS campaigns. Overall, this work points to critical method development parameters and provides guidance for the establishment of integrated ADE-OPI-MS as HTS-compatible technology for early drug discovery.

RationaleThe low speed and low flexibility of most liquid chromatography/tandem mass spectrometry (LC/MS/MS) approaches in early drug discovery delay sample analysis from routine in vivo studies within the same day. A high‐throughput platform for the rapid quantification of drug compounds in various in vivo assays was developed and established in routine bioanalysis.MethodsAutomated selection of an efficient and adequate LC method was realized by autonomous sample qualification for ultrafast batch gradients (9 s/sample) or for fast linear gradients (45 s/sample) if samples required chromatography. The hardware and software components of our Rapid and Integrated Analysis System (RIAS) were streamlined for increased analytical throughput via state‐of‐the‐art automation while maintaining high analytical quality.ResultsOnline decision‐making was based on a quick assay suitability test (AST), based on a small and dedicated sample set evaluated by two different strategies. 84% of the acquired data points were within ±30% accuracy and 93% of the deviations between the lower limit of quantitation (LLOQ) values were ≤2‐fold compared with standard LC/MS/MS systems. Speed, flexibility and overall automation significantly improved.ConclusionsThe developed platform provided an analysis time of only 10 min (batch‐mode) and 47 min (gradient‐mode) per standard pharmacokinetic (PK) study (62 injections). Automation, data evaluation and results handling were optimized to pave the way for machine learning based on decision‐making regarding the evaluation strategy of the AST.

In order to overcome the challenges associated with a limited number of airway epithelial cells that can be obtained from clinical sampling and their restrained capacity to divide ex vivo, miniaturization of respiratory drug discovery assays is of pivotal importance. Thus, a 96-well microplate system was developed where primary human small airway epithelial (hSAE) cells were cultured at an air–liquid interface (ALI). After four weeks of ALI culture, a pseudostratified epithelium containing basal, club, goblet and ciliated cells was produced. The 96-well ALI cultures displayed a cellular composition, ciliary beating frequency, and intercellular tight junctions similar to 24-well conditions. A novel custom-made device for 96-parallelized transepithelial electric resistance (TEER) measurements, together with dextran permeability measurements, confirmed that the 96-well culture developed a tight barrier function during ALI differentiation. 96-well hSAE cultures were responsive to transforming growth factor β1 (TGF-β1) and tumor necrosis factor α (TNF-α) in a concentration dependent manner. Thus, the miniaturized cellular model system enables the recapitulation of a physiologically responsive, differentiated small airway epithelium, and a robotic integration provides a medium throughput approach towards pharmaceutical drug discovery, for instance, in respect of fibrotic distal airway/lung diseases.

Comprehensive and unbiased detection methods are a prerequisite for high-throughput screening (HTS) campaigns within drug discovery research. Label-free matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has been introduced as an HTS-compatible readout for biochemical test systems to support the drug discovery process. So far, reported HTS applications were based on surface-modified systems or proof-of-concept studies. We present the utilization of a MALDI-TOF-based screening platform to identify inhibitors of human cyclic GMP-AMP synthase (cGAS), a mediator of innate immune response whose aberration has been causally correlated to a number of inflammatory disorders. In this context, the development and validation of a MALDI-TOF-based activity assay is reported to demonstrate fast, robust, and accurate detection of chemical cGAS inhibition by direct quantification of the physiological reaction product cyclic GMP-ATP (cGAMP). Results from a screen of a diverse library of more than 1 million small molecules in 1536-well format against the catalytic cGAS activity are presented with excellent assay performance and data quality. Identified hits were qualified in dose-response experiments and confirmed by RapidFire-MS measurements. Conclusively, the presented data provide the first proof of applicability of direct automated MALDI-TOF MS as a readout strategy for large-scale drug discovery HTS campaigns.

We present an acoustic ejection mass spectrometry (AEMS) setup for ESI-MS based sample injection at a sampling rate faster than current ESI and MALDI techniques. A modified acoustic droplet ejection system was combined with an open port interface and a modified ESI source. To simulate applications of drug metabolism and pharmacokinetics analysis and high-throughput screening campaigns, two stress tests were performed regarding ion suppression and system endurance in combination with minor sample preparation. Maximum sampling rate was 6 Hz for dextromethorphan and d3-dextrorphan (each 100 nM) for 1152 injections in 63 s at FWHM of 105 ms and %RSD of 7.7%/7.5% without internal standard correction. Enzyme assay buffer and crude dog plasma caused signal suppression of 51%/73% at %RSD of 5.7%/6.7% (n = 120) and stable OPI performance during 1100 injections. An endurance buffer revealed minor OPI pollution and constant signals for >25.000 injections (%RSD = 8.5%, n = 10,557).

Demonstration of in vitro target engagement for small-molecule ligands by measuring binding to a molecular target is an established approach in early drug discovery and a pivotal step in high-throughput screening (HTS)-based compound triaging. We describe the setup, evaluation, and application of a ligand binding assay platform combining automated affinity selection (AS)-based sample preparation and label-free matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. The platform enables mass spectrometry (MS)-based HTS for small-molecule target interactions from single-compound incubation mixtures and is embedded into a regular assay automation environment. Efficient separation of target–ligand complexes is achieved by in-plate size exclusion chromatography (SEC), and small-molecule ligands are subsequently identified by MALDI-TOF analysis. In contrast to alternative HTS-capable binding assay formats, MALDI-TOF AS-MS is capable of identifying orthosteric and allosteric ligands, as shown for the model system protein tyrosine phosphatase 1B (PTP1B), irrespective of protein function. Furthermore, determining relative binding affinities (RBAs) enabled ligand ranking in accordance with functional inhibition and reference data for PTP1B and a number of diverse protein targets. Finally, we present a validation screen of more than 23,000 compounds within 24 h, demonstrating the general applicability of the platform for the HTS-compatible assessment of protein–ligand interactions.

Label-free in vitro potency assays are an emerging field in drug discovery to enable more physiological conditions, to improve the readout quality, and to save time. For this approach mass spectrometry (MS) is a powerful technology to directly follow physiological processes. The speed of this methodology, however, was for a long time not compatible with chemiluminescence- or fluorescence-based assays. Recent advances in matrix-assisted laser desorption/ionization (MALDI) instrumentation paved the way for high-throughput MS analysis of label-free assays for large compound libraries, whereas electrospray ionization (ESI)-based mass spectrometers equipped with RapidFire autosamplers were limited to medium throughput. Here we present a technological advancement of the RapidFire device to enable cycle times of 2.5 s per sample. This newly developed BLAZE-mode substantially boosted the ESI-MS analysis speed, providing an alternative technology for label-free high-throughput screening.

Microbial-dependent trimethylamine (TMA) generation from dietary precursors such as choline was recently linked to cardiovascular diseases (CVDs) as well as chronic kidney disease (CKD). Inhibition of TMA-generating enzymes in gut bacteria would be an innovative approach to treat these diseases. The potential to accurately quantify secreted TMA levels highlights the capacity of mass spectrometry (MS) for tracking microbial TMA-lyase activity. However, high-throughput screening (HTS) by conventional MS instrumentation is hampered by limited sample throughput. Recent advancement in liquid handling and instrumentation of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS provides an HTS-compatible MS technology. The deciphering of enzymatic reactions using this label-free readout has been successfully applied but has thus far been limited to peptide/protein-centric activity assays. Here, we demonstrate the versatile applicability of MALDI-TOF by tracking a small molecule within a highly complex sample background. The key to success for this concept was chemical derivatization of the target molecule enabling quantitative assessment of microbial TMA formation. Further, its potential was demonstrated in a side-by-side comparison to RapidFire-MS in a primary screen and subsequent dose-response experiments. Overall, the established assay enables the screening for microbial TMA-lyase inhibitors and serves as a proof of concept for the applicability of MALDI-TOF for demanding assay concepts per se.

In drug discovery, there is an increasing demand for more physiological in vitro models that recapitulate the disease situation in patients. Human induced pluripotent stem (hiPS) cell-derived model cells could serve this purpose. To date, several directed differentiation approaches have been described to generate definitive endoderm (DE) from hiPS cells, but protocols suitable for drug development and high-throughput screening (HTS) have not been reported yet. In this work, a large-scale expansion of hiPS cells for high-throughput adaption is presented and an optimized stepwise differentiation of hiPS cells into DE cells is described. The produced DE cells were demonstrated to express classical DE markers on the gene expression and protein level. The here described DE cells are multipotent progenitors and act as starting points for a broad spectrum of endodermal model cells in HTS and other areas of drug discovery.

Label-free, mass spectrometric (MS) deciphering of enzymatic reactions by direct analysis of substrate-to-product conversion provides the next step toward more physiological relevant assays within drug discovery campaigns. Reduced risk of suffering from compound interference combined with diminished necessity for tailored signal mediators emphasizes the valuable role of label-free readouts. However, MS-based detection has not hitherto met high-throughput screening (HTS) requirements because of the lack of HTS-compatible sample introduction. In the present study, we report on a fully automated liquid-handling concept built in-house to concatenate biochemical assays with matrix-assisted laser desorption/ionization time-of-flight closing this technological gap. The integrated reformatting from 384- to 1536-well format enables cycle times of 0.6 s/sample for automated spotting and 0.4 s/sample for MS analysis, matching the requirements of HTS compatibility. In-depth examination of spotting quality, quantification accuracy, and instrument robustness together with the implementation of a protein tyrosine phosphatase 1B (PTP1B) inhibitor screening (4896 compounds) demonstrate the potential of the heavily inquired HTS integration of the label-free MS readout. Overall, the presented data demonstrate that the introduced automation concept makes label-free MS-based readouts accessible for HTS within drug discovery campaigns but also in other research areas requiring ultrafast MS-based detection.

A fierce dispute has arisen between the supporters of phenotypic and target-focused screening regarding which path grants the higher probability of successful drug development. A chance to reconcile these two approaches lies in successful target deconvolution (TD) after phenotypic screens. But, despite the panoply of available in vitro TD methods, the task of matching a phenotypically active compound with a biomolecular target remains challenging. Consequently, this review details the latest developments of in silico techniques that expedite TD. Ultimately, the deconvoluted target allows us to reap the benefits of the phenotypic and target-focused approaches.

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.

Deficient functional expression of drug transporters incapacitates most hepatic cell lines as a reliable tool for evaluating transporter-mediated drug-drug interactions. Recently, genetically modified cells (referred to as upcyte hepatocytes) have emerged as an expandable, noncancerous source of human hepatic cells. Herein, we quantified mRNA and protein levels of key hepatobiliary transporters and we assessed associated uptake activity in short- and long-term cultures of upcyte human hepatocytes (UHH) in comparison to cryopreserved primary human hepatocytes (cPHH). Expression of canalicular efflux pumps, such as MRD1/ABCB1, MATE1/SLC47A1, and MRP2/ABCC2, was relatively well preserved in UHH. By contrast, long-term cultivation of UHH in a two-dimensional sandwich configuration [sandwich-cultured upcyte human hepatocytes (SCUHH)] was required to upregulate organic anion-transporting polypeptide OATP1B1/SLCO1B1, OATP2B1/SLCO2B1, NTCP/SLC10A1, and OCT1/SLC22A1 mRNA expression, which correlated well with respective protein abundances. However, mRNA and protein levels of sinusoidal solute carrier transporters, except for NTCP and OATP2B1, remained low in SCUHH compared to sandwich-cultured cPHH. OCT1- and NTCP-mediated uptake of N-methyl-4-phenylpyridinium acetate and taurocholate was demonstrated in both hepatic models, whereas active uptake of OATP1B1/1B3-selective marker substrates, paralleled by markedly reduced SLCO1B1/1B3 expression, were not detectable in SCUHH. Uptake studies under Na+-depletion and excess of taurocholate confirmed the presence of functional NTCP protein and indicated that NTCP, apart from OATP2B1, contributed substantially to the overall hepatic uptake of rosuvastatin in SCUHH. In conclusion, our data suggest that SCUHH, despite their limitation for evaluating OATP1B1/1B3-mediated transport processes, retain NTCP, OATP2B1, and OCT1 transport activities and thus may be considered as a tool for elucidating compensatory uptake pathways for OATP1B1/1B3 substrates.

Autotaxin (ATX) is a promising drug target for the treatment of several diseases, such as cancer and fibrosis. ATX hydrolyzes lysophosphatidyl choline (LPC) into bioactive lysophosphatidic acid (LPA). The potency of ATX inhibitors can be readily determined by using fluorescence-based LPC derivatives. While such assays are ultra-high throughput, they are prone to false positives compared to assays based on natural LPC. Here we report the development of ultrafast mass spectrometry-based ATX assays enabling the measurement of data points within 13 s, which is 10 times faster than classic liquid chromatography-mass spectrometry. To this end, we set up a novel in vitro and whole-blood assay. We demonstrate that the potencies determined with these assays are in good agreement with the in vivo efficacy and that the whole-blood assay has the best predictive power. This high-throughput label-free approach paired with the translatable data quality is highly attractive for appropriate guidance of medicinal chemists for constructing strong structure-activity relationships.

In vitro models based on primary human hepatocytes (PHH) have been advanced for clearance (CL) prediction of metabolically stable compounds, representing state-of-the-art assay systems for drug discovery and development. Yet, limited cell availability and large interindividual variability of metabolic profiles remain shortcomings of PHH. Upcyte human hepatocytes (UHH) represent a novel hepatic cell system derived from PHH, exhibiting proliferative capacity for approximately 35 population doublings. UHH from three donors were evaluated during culture for up to 18 days, investigating relative mRNA expression and in situ enzyme activity of cytochrome P450s (P450s), UDP-glucuronosyltransferases, and sulfotransferases. Furthermore, UHH were used for predicting hepatic CL of 21 marketed low to intermediate CL drugs. In a typical experiment, expansion from 3.9 × 106 up to 8.5 × 107 cells was achieved during subculture. When maintained at confluence, transcripts of major P450s were expressed at donor-specific levels with sustained activities for the majority of isoforms, showing generally low CYP1A2 and high CYP2B6 activity levels. For donor 151-03, CL prediction based on depletion experiments resulted in an average fold error of 2.0, and 80% of compounds being predicted within twofold to in vivo CL for a subset of 10 low CL drugs. UHH showed sustained and consistent activity of drug-metabolizing enzymes (DME), resulting in highly reproducible CL prediction performance. In conclusion, UHH show promising potential as alternative to PHH for standardized in vitro applications in discovery research based on their stable, hepatocyte-like DME phenotype and virtually unlimited cell availability.

BI 1002494 [(R)-4-{(R)-1-[7-(3,4,5-trimethoxy-phenyl)-[1,6] napthyridin-5-yloxy]-ethyl} pyrrolidin-2-one] is a novel, potent, and selective spleen tyrosine kinase (SYK) inhibitor with sustained plasma exposure after oral administration in rats, which qualifies this molecule as a good in vitro and in vivo tool compound. BI 1002494 exhibits higher potency in inhibiting high-affinity IgE receptor-mediated mast cell and basophil degranulation (IC50 = 115 nM) compared with B-cell receptor-mediated activation of B cells (IC50 = 810 nM). This may be explained by lower kinase potency when the physiologic ligand B-cell linker was used, suggesting that SYK inhibitors may exhibit differential potency depending on the cell type and the respective signal transduction ligand. A 3-fold decrease in potency was observed in rat basophils (IC50 = 323 nM) compared with human basophils, but a similar species potency shift was not observed in B cells. The lower potency in rat basophils was confirmed in both ex vivo inhibition of bronchoconstriction in precision-cut rat lung slices and in reversal of anaphylaxis-driven airway resistance in rats. The different cellular potencies translated into different in vivo efficacy; full efficacy in a rat ovalbumin model (that contains an element of mast cell dependence) was achieved with a trough plasma concentration of 340 nM, whereas full efficacy in a rat collageninduced arthritis model (that contains an element of B-cell dependence) was achieved with a trough plasma concentration of 1400 nM. Taken together, these data provide a platform from which different estimates of human efficacious exposures can be made according to the relevant cell type for the indication intended to be treated.