Miniaturized screening and performance prediction of tailored subcutaneous extended-release formulations for preclinical in vivo studies.

Microencapsulation of active pharmaceutical ingredients (APIs) for preparation of long acting injectable (LAI) formulations is an auspicious technique to enable preclinical characterization of a broad variety of APIs, ideally independent of their physicochemical and pharmacokinetic (PK) characteristics. During early API discovery, tunable LAI formulations may enable pharmacological proof-of-concept for the given variety of candidates by tailoring the level of plasma exposure over the duration of various timespans. Although numerous reports on small scale preparation methods for LAIs utilizing copolymers of lactic and glycolic acid (PLGA) and polymers of lactic acid (PLA) highlight their potential, application in formulation screening and use in preclinical in vivo studies is yet very limited. Transfer from downscale formulation preparation to in vivo experiments is hampered in early preclinical API screening by the large number of API candidates with simultaneously very limited available amount in the lower sub-gram scale, lack of formulation stability and deficient tunability of sustained release. We hereby present a novel comprehensive platform tool for tailored extended-release formulations, aiming to support a variety of preclinical in vivo experiments with ranging required plasma exposure levels and timespans. A novel small-scale spray drying process was successfully implemented by using an air brush based instrument for preparation of PLGA and PLA based formulations. Using Design of Experiments (DoE), required API amount of 250 mg was demonstrated to suffice for identification of dominant polymer characteristics with largest impact on sustained release capability for an individual API. BI-3231, a hydrophilic and weakly acidic small compound with good water solubility and permeability, but low metabolic stability, was used as an exemplary model for one of the many candidates during API discovery. Furthermore, an in vitro to in vivo correlation (IVIVC) of API release rate was established in mice, which enabled the prediction of in vivo plasma concentration plateaus after single subcutaneous injection, using only in vitro dissolution profiles of screened formulations. By tailoring LAI formulations and their doses for acute and sub-chronic preclinical experiments, we exemplary demonstrate the practical use for BI-3231. Pharmacological proof-of-concept could be enabled whilst circumventing the need of multiple administration as result of extensive hepatic metabolism and simultaneously superseding numerous in vivo experiments for formulation tailoring.

Label-free high-throughput screening via acoustic ejection mass spectrometry put into practice.

Acoustic droplet ejection-open port interface-mass spectrometry (ADE-OPI-MS) is a novel label-free analytical technique, promising to become a versatile readout for high-throughput screening (HTS) applications. The recent introduction of ADE-OPI-MS devices to the laboratory equipment market, paired with their compatibility with laboratory automation platforms, should facilitate the adoption of this technology by a broader community. Towards this goal, instrument robustness in the context of HTS campaigns - where up to millions of samples in complex matrices are tested in a short time frame - represents a major challenge, which explains the absence of detailed literature reports on this subject. Here, we present the results of our first fully automated HTS campaign, based on the ADE-OPI-MS technology, aiming to identify inhibitors of a metabolic enzyme in a >1 million compound library. The report encompasses the assay development and validation steps, as well as the adaptation for HTS requirements, where refinement of the capillary cleaning concept was crucial for final success. Altogether, our study unequivocally demonstrates the applicability of the ADE-OPI-MS technology for HTS-based drug discovery.

Differential analyte derivatization enables unbiased MALDI-TOF-based high-throughput screening: A proof-of-concept study for the discovery of catechol-o-methyltransferase inhibitors.

Recent advances in label-free high-throughput screening via matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) offer unprecedented opportunities for the identification of novel chemical starting points in target-based drug discovery. A clear advantage of the technology is the possibility for label-free, direct quantification of analytes with high precision and robustness. Here we have expanded the range of analytes and biology that can be addressed via MALDI-TOF HTS, by developing a method based on post-reaction pyrylium-based derivatization to detect 3-methoxytyramine, the physiological enzyme product of the catechol-O-methyltransferase (COMT) enzyme. The introduction of pyrylium-type reagents as universal derivatization strategy under aqueous conditions for molecules containing primary amines represents a valuable addition to the toolbox of MALDI-TOF assay development. Characterization of COMT's enzymatic activity and inhibition by reference inhibitors, and comparison of the results obtained in our assay with data from previous mechanistic studies validated the performance of this new method. To address the problem of isobaric interference, a source of false results in MALDI-TOF assays measuring low molecular weight analytes, we devised a differential derivatization workflow which can potentially replace other counter- or orthogonal assays in future screening campaigns. Finally, we report on the first label-free HTS campaign for the identification of COMT inhibitors performed in miniaturized 1536-well microtiter plate format via MALDI-TOF MS analysis.

Acoustic Ejection Mass Spectrometry: A Fully Automatable Technology for High-Throughput Screening in Drug Discovery.

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.

MALDI-TOF-Based Affinity Selection Mass Spectrometry for Automated Screening of Protein-Ligand Interactions at High Throughput.

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.

Ultrahigh-Throughput ESI-MS: Sampling Pushed to Six Samples per Second by Acoustic Ejection Mass Spectrometry.

We present an acoustic ejection mass spectrometry (AEMS) setup for contactless electrospray ionization mass spectrometry (ESI-MS)-based sample injection at a sampling rate faster than current ESI and matrix-assisted laser desorption ionization (MALDI) techniques. For the direct transfer of samples out of 384-well plates into a modified ESI source, an open port interface (OPI) was combined with a modified acoustic droplet ejection (ADE) system. AEMS has the potential to eliminate bottlenecks known from classical MS approaches, such as speed, reproducibility, carryover, ion suppression, as well as sample preparation and consumption. This setup provided a drastically reduced transfer distance between OPI and ESI electrode for optimum throughput performance and broadens the scope of applications for this emerging technique. To simulate label-free applications of drug metabolism and pharmacokinetics (DMPK) analysis and high-throughput screening (HTS) campaigns, two stress tests were performed regarding ion suppression and system endurance in combination with minor sample preparation. The maximum sampling rate was 6 Hz for dextromethorphan and d3-dextrorphan (each 100 nM) for 1152 injections in 63 s at full width at half-maximum (FWHM) of 105 ms and a relative standard deviation (%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). An HTS endurance buffer was used for >25 000 injections with minor OPI pollution and constant signals (%RSD = 8.5%, FWHM of 177 ms ± 8.5%, n = 10 557). The optimized hardware and method setup resulted in high-throughput performance and enables further implementation in a fully automated platform for ESI-MS-based high-throughput screening.

MALDI-TOF Mass Spectrometry-Based High-Throughput Screening for Inhibitors of the Cytosolic DNA Sensor cGAS.

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.

Lifespan-increasing drug nordihydroguaiaretic acid inhibits p300 and activates autophagy.

Aging is characterized by the progressive loss of physiological function in all organisms. Remarkably, the aging process can be modulated by environmental modifications, including diet and small molecules. The natural compound nordihydroguaiaretic acid (NDGA) robustly increases lifespan in flies and mice, but its mechanism of action remains unclear. Here, we report that NDGA is an inhibitor of the epigenetic regulator p300. We find that NDGA inhibits p300 acetyltransferase activity in vitro and suppresses acetylation of a key p300 target in histones (i.e., H3K27) in cells. We use the cellular thermal shift assay to uniquely demonstrate NDGA binding to p300 in cells. Finally, in agreement with recent findings indicating that p300 is a potent blocker of autophagy, we show that NDGA treatment induces autophagy. These findings identify p300 as a target of NDGA and provide mechanistic insight into its role in longevity.

Cofactor Analogues as Active Site Probes in Lysine Acetyltransferases.

Lysine acetyltransferases (KATs, also termed histone acetyltransferases, HATs) catalyze the acetylation of substrate lysine residues by employing the cofactor acetyl-coenzyme A (AcCoA), thereby providing a dynamic control mechanism of protein function. Because of their major involvement in cell development and homeostasis, small-molecule modulators of KAT activity are urgently needed to assess their therapeutic potential and for probing their underlying biology. Recent advances in the field suggest that targeting the cofactor binding site represents a promising strategy for identifying potent and selective ligands. Here, we present the synthesis of two functional cofactor-based chemical probes and their usage as mechanistic tools in a broadly applicable assay platform. A fluorescence polarization (FP)-based binding assay was combined with biolayer interferometry competition analysis and a FP competition activity immunoassay to enable easy, reliable, and profound evaluation of ligands that target the KAT cofactor binding site.

KATching-Up on Small Molecule Modulators of Lysine Acetyltransferases.

The reversible acetylation of lysines is one of the best characterized epigenetic modifications. Its involvement in many key physiological and pathological processes has been documented in numerous studies. Lysine deacetylases (KDACs) and acetyltransferases (KATs) maintain the acetylation equilibrium at histones but also many other proteins. Besides acetylation, also other acyl groups are reversibly installed at the side chain of lysines in proteins. Because of their involvement in disease, KDACs and KATs were proposed to be promising drug targets, and for KDACs, indeed, five inhibitors are now approved for human use. While there is a similar level of evidence for the potential of KATs as drug targets, no inhibitor is in clinical trials. Here, we review the evidence for the diverse roles of KATs in disease pathology, provide an overview of structural features and the available modulators, including those targeting the bromodomains of KATs, and present an outlook.