High-Throughput Compound Quality Assessment with High-Mass-Resolution Acoustic Ejection Mass Spectrometry: An Automatic Data Processing Toolkit.

Pharmacological screening heavily relies on the reliability of compound libraries. To ensure the accuracy of screening results, fast and reliable quality control (QC) of these libraries is essential. While liquid chromatography (LC) with ultraviolet (UV) or mass spectrometry (MS) detection has been employed for molecule QC on small sample sets, the analytical throughput becomes a bottleneck when dealing with large libraries. Acoustic ejection mass spectrometry (AEMS) is a high-throughput analytical platform that covers a broad range of chemical structural space. In this study, we present the utilization of an AEMS system equipped with a high-resolution MS analyzer for high-throughput compound QC. To facilitate efficient data processing, which is a key challenge for such a high-throughput application, we introduce an automatic data processing toolkit that allows for the high-throughput assessment of the sample standards' quantitative and qualitative characteristics, including purity calculation with the background processing option. Moreover, the toolkit includes a module for quantitatively comparing spectral similarity with the reference library. Integrating the described high-resolution AEMS system with the data processing toolkit effectively eliminates the analytical bottleneck, enabling a rapid and reliable compound quality assessment of large-scale compound libraries.

Busting Myths in Compound Handling Practices for Assay Developers.

Since the advent of modern-day screening collections in the early 2000s, various aspects of our knowledge of good handling practices have continued to evolve. Some early practices, however, continue to prevail due to the absence of defining data that would bust the myths of tradition. The lack of defining data leads to a gap between plate-based screeners, on the one hand, and compound sample handling groups, on the other, with the latter being the default party to blame when an assay goes awry.In this paper, we highlight recommended practices that ensure sample integrity and present myth busting data that can help determine the root cause of an assay gone bad. We show how a strong and collaborative relationship between screening and sample handling groups is the better state that leads to the accomplishment of the common goal of finding breakthrough medicines.

Rapid Compound Integrity Assessment for High-Throughput Screening Hit Triaging.

Hits from high-throughput screening (HTS) assays are typically evaluated using cheminformatics and/or empirical approaches before a decision for follow-up (activity confirmation and/or sample resynthesis) is made. However, the compound integrity (i.e., identity and purity) of these hits often remains largely unknown at this stage, since many compounds in the screening collection could undergo various changes such as degradation, polymerization, and precipitation during storage over time. When compound integrity is actually assessed for HTS hits postassay to address this issue, the process often increases the overall cycle time by weeks due to the reacquisition of the samples and the lengthy liquid chromatography-ultraviolet/mass spectrometric analysis time. Here we present a novel approach where compound integrity data are collected concurrently with the concentration-response curve (CRC) stage of HTS, with both assays occurring either in parallel on two distributions from the same liquid sample or serially using the original source liquid sample. The rapid generation of compound integrity data has been enabled by a high-speed ultra-high-pressure liquid chromatography-ultraviolet/mass spectrometric platform capable of analyzing ~2000 samples per instrument per week. From this parallel approach, both compound integrity and CRC potency results for screening hits become available to medicinal chemists at the same time, which has greatly enhanced the decision-making process for hit follow-up and progression. In addition, the compound integrity results from recent hits provide a real-time and representative "snapshot" of the sample integrity of the entire compound collection, and the data can be used for in-depth analyses of the screening collection.

Discovery of Selective Cannabinoid CB2 Receptor Agonists by High-Throughput Screening.

The endocannabinoid system (ECS) plays a diverse role in human physiology ranging from the regulation of mood and appetite to immune modulation and the response to pain. Drug development that targets the cannabinoid receptors (CB1 and CB2) has been explored; however, success in the clinic has been limited by the psychoactive side effects associated with modulation of the neuronally expressed CB1 that are enriched in the CNS. CB2, however, are expressed in peripheral tissues, primarily in immune cells, and thus development of CB2-selective drugs holds the potential to modulate pain among other indications without eliciting anxiety and other undesirable side effects associated with CB1 activation. As part of a collaborative effort among industry and academic laboratories, we performed a high-throughput screen designed to discover selective agonists or positive allosteric modulators (PAMs) of CB2. Although no CB2 PAMs were identified, 167 CB2 agonists were discovered here, and further characterization of four select compounds revealed two with high selectivity for CB2 versus CB1. These results broaden drug discovery efforts aimed at the ECS and may lead to the development of novel therapies for immune modulation and pain management with improved side effect profiles.

Acoustic Sample Deposition MALDI-MS (ASD-MALDI-MS): A Novel Process Flow for Quality Control Screening of Compound Libraries.

In the early stages of drug discovery, high-throughput screening (HTS) of compound libraries against pharmaceutical targets is a common method to identify potential lead molecules. For these HTS campaigns to be efficient and successful, continuous quality control of the compound collection is necessary and crucial. However, the large number of compound samples and the limited sample amount pose unique challenges. Presented here is a proof-of-concept study for a novel process flow for the quality control screening of small-molecule compound libraries that consumes only minimal amounts of samples and affords compound-specific molecular data. This process employs an acoustic sample deposition (ASD) technique for the offline sample preparation by depositing nanoliter volumes in an array format onto microscope glass slides followed by matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis. An initial study of a 384-compound array employing the ASD-MALDI-MS workflow resulted in a 75% first-pass positive identification rate with an analysis time of <1 s per sample.

Magic Angle Spinning NMR for Reaction Monitoring and Structure Determination of Molecules Attached to Multipin Crowns.

The first example of magic angle spinning NMR on crowns has been demonstrated. The ability to monitor a reaction on a crown and to confirm the structure of the reaction product directly on the crown without resorting to chemical cleavage should greatly enhance the utility of this convenient format for combinatorial chemistry.