Affinity Selection Mass Spectrometry Research Articles

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.

Affinity selection-mass spectrometry for the discovery of pharmacologically active compounds from combinatorial libraries and natural products.

Invented to address the high-throughput screening (HTS) demands of combinatorial chemistry, affinity selection-mass spectrometry (AS-MS) utilizes binding interactions between ligands and receptors to isolate pharmacologically active compounds from mixtures of small molecules and then relies on the selectivity, sensitivity, and speed of mass spectrometry to identify them. No radiolabels, fluorophores, or chromophores are required. Although many variations of AS-MS have been devised, three approaches have emerged as the most flexible, productive, and popular, and they differ primarily in how ligand-receptor complexes are separated from nonbinding compounds in the mixture. These are pulsed ultrafiltration (PUF) AS-MS, size exclusion chromatography (SEC) AS-MS, and magnetic microbead affinity selection screening (MagMASS). PUF and SEC AS-MS are solution-phase screening approaches, and MagMASS uses receptors immobilized on magnetic microbeads. Because pools of compounds are screened using AS-MS, each containing hundreds to thousands of potential ligands, hundreds of thousands of compounds can be screened per day. AS-MS is also compatible with complex mixtures of chemically diverse natural products in extracts of botanicals and fungi and microbial cultures, which often contain fluorophores and chromophores that can interfere with convention HTS. Unlike conventional HTS, AS-MS may be used to discover ligands binding to allosteric as well as orthosteric receptor sites, and AS-MS has been useful for discovering ligands to targets that are not easily incorporated into conventional HTS such as membrane-bound receptors.

Solving the Competitive Binding Equilibria between Many Ligands: Application to High-Throughput Screening and Affinity Optimization.

Modern small-molecule drug discovery relies on the selective targeting of biological macromolecules by low-molecular weight compounds. Therefore, the binding affinities of candidate drugs to their targets are key for pharmacological activity and clinical use. For drug discovery methods where multiple drug candidates can simultaneously bind to the same target, a competition is established, and the resulting equilibrium depends on the dissociation constants and concentration of all the species present. Such coupling between all equilibrium-governing parameters complicates analysis and development of improved mixture-based, high-throughput drug discovery techniques. In this work, we present an iterative computational algorithm to solve coupled equilibria between an arbitrary number of ligands and a biomolecular target that is efficient and robust. The algorithm does not require the estimation of initial values to rapidly converge to the solution of interest. We explored binding equilibria under ligand/receptor conditions used in mixture-based library screening by affinity selection-mass spectrometry (AS-MS). Our studies support a facile method for affinity-ranking hits. The ranking method involves varying the receptor-to-ligand concentration ratio in a pool of candidate ligands in two sequential AS-MS analyses. The ranking is based on the relative change in bound ligand concentration. The method proposed does not require a known reference ligand and produces a ranking that is insensitive to variations in the concentration of individual compounds, thereby enabling the use of unpurified compounds generated by mixture-based combinatorial synthesis techniques.

Combination of MS Binding Assays and affinity selection mass spectrometry for screening of structurally homogenous libraries as exemplified for a focused oxime library addressing the neuronal GABA transporter 1.

This study presents an efficient screening approach based on combination of mass spectrometry (MS) based binding assays (MS Binding Assays) and affinity selection mass spectrometry (ASMS) customized for screening of structurally homogeneous libraries sharing a common mass spectrometric fragmentation pattern. After reaction of a nipecotic acid derivative possessing a hydroxylamine functionality with aldehydes, the resulting oxime library was screened accordingly toward the GABA transporter subtype 1 (GAT1), a drug target for several neurological disorders. After assessing sublibraries' activities for inhibition of reporter ligand binding, hits in active ones were directly identified. This could be achieved by recording mass transitions for the reporter ligand as well as those predicted for the library components in a single LC-MS/MS run with a triple quadrupole mass spectrometer in the multiple reaction monitoring mode. Identification of hits with a predefined affinity could be reliably accomplished by calculation of IC50-values from specific binding concentrations of library constituents and reporter ligand. Application of this strategy revealed six hits, from which two of them were resynthesized for further biological evaluation. Thereby, the best one displayed a pKi of 7.38 in MS Binding Assays and a pIC50 of 6.82 in [3H]GABA uptake assays for GAT1.

Revolution of Small Molecule Drug Discovery by Affinity Selection-Mass Spectrometry Technology.

Affinity selection (AS)-MS is a label-free binding assay technology for the analysis of interactions between targets and small drug molecules, which does not require modification of targets or compounds. AS-MS technology has been used in drug discovery research for more than 10 years, and is currently one of the most important affinity-based screening techniques. As such, it may be the driving force for novel small molecule drug discovery. This review introduces the principles of AS-MS technology and its use in high-throughput screening (HTS), then discusses strategies for its use in drug discovery and its application in target identification.

A Library Screening Strategy Combining the Concepts of MS Binding Assays and Affinity Selection Mass Spectrometry

The primary objective of early drug development is to identify hits and leads for a target of interest. To achieve this aim, rapid, and reliable screening techniques for a huge number of compounds are needed. Mass spectrometry based binding assays (MS Binding Assays) represent a well-established technique for library screening based on competitive binding experiments revealing active sublibraries due to reduced binding of a reporter ligand and following hit identification for active libraries by deconvolution in further competitive binding experiments. In the present study, we combined the concepts of MS Binding Assays and affinity selection mass spectrometry (ASMS) to improve the efficiency of the hit identification step. In that case, only a single competitive binding experiment is performed that is in the first step analyzed for reduced binding of the reporter ligand and—only if a sublibrary is active—additionally for specific binding of individual library components. Subsequently, affinities of identified hits as well as activities of reduced sublibraries (i.e., all sublibrary components without hit) are assessed in additional competitive binding experiments. We exemplified this screening concept for the identification of ligands addressing the most widespread GABA transporter subtype in the brain (GAT1) studying in the beginning a library composed of 128 and further on a library of 1,280 well-characterized GAT1 inhibitors, drug substances, and pharmacological tool compounds. Determination of sublibraries' activities was done by quantification of bound NO711 as reporter ligand and hit identification for the active ones achieved in a further LC-ESI-MS/MS run in the multiple reaction monitoring mode enabling detection of all sublibrary components followed by hit verification and investigation of reduced sublibraries in further competitive binding experiments. In this way, we could demonstrate that all GAT1 inhibitors reducing reporter ligand binding below 50% at a concentration of 1 μM are detected reliably without generation of false positive or false negative hits. As the described strategy is apart from its reliability also highly efficient, it can be assumed to become a valuable tool in early drug research, especially for membrane integrated drug targets that are often posing problems in established screening techniques.

RNA-ALIS: Methodology for screening soluble RNAs as small molecule targets using ALIS affinity-selection mass spectrometry.

Recent advances resulting from the completion of the human genome have shown that RNA has the promise to be a target for small molecule drugs, and therefore represents a previously unexploited class of targets for novel human therapeutics. We recently reported the adaptation of an affinity selection mass spectrometry screening technique, termed ALIS (Automatic Ligand Identification System), to screen and characterize a variety of RNA species from both prokaryotic and eukaryotic sources. We demonstrated that the ALIS technique, which had previously been used for protein targets, was also compatible for screening, ranking and characterizing small molecule ligands for RNA targets. We present here a detailed description of the use of ALIS for screening and characterizing ligands for RNA and discuss issues of validating and testing RNA for use in the ALIS system. We have also further elaborated on issues of RNA stability and testing in the ALIS system and demonstrate that the affinity-selection screening system has the potential to be a general solution for label-free screening and characterization of small molecule drug candidates for RNA targets.

Identification and characterization of small molecule inhibitors of the ubiquitin ligases Siah1/2 in melanoma and prostate cancer cells

Inhibition of ubiquitin ligases with small molecule remains a very challenging task, given the lack of catalytic activity of the target and the requirement of disruption of its interactions with other proteins. Siah1/2, which are E3 ubiquitin ligases, are implicated in melanoma and prostate cancer and represent high-value drug targets. We utilized three independent screening approaches in our efforts to identify small-molecule Siah1/2 inhibitors: Affinity Selection-Mass Spectrometry, a protein thermal shift-based assay and an in silico based screen. Inhibitors were assessed for their effect on viability of melanoma and prostate cancer cultures, colony formation, prolyl-hydroxylase-HIF1α signaling, expression of selected Siah2-related transcripts, and Siah2 ubiquitin ligase activity. Several analogs were further characterized, demonstrating improved efficacy. Combination of the top hits identified in the different assays demonstrated an additive effect, pointing to complementing mechanisms that underlie each of these Siah1/2 inhibitors.

Identification of G-Quadruplex-Binding Inhibitors of Myc Expression through Affinity Selection–Mass Spectrometry

The Myc oncogene is overexpressed in many cancers, yet targeting it for cancer therapy has remained elusive. One strategy for inhibition of Myc expression is through stabilization of the G-quadruplex (G4), a G-rich DNA secondary structure found within the Myc promoter; stabilization of G4s has been shown to halt transcription of downstream gene products. Here we used the Automated Ligand Identification System (ALIS), an affinity selection–mass spectrometry method, to identify compounds that bind to the Myc G4 out of a pool of compounds that had previously been shown to inhibit Myc expression in a reporter screen. Using an ALIS-based screen, we identified hits that bound to the Myc G4, a small subset of which bound preferentially relative to G4s from the promoters of five other genes. To determine functionality and specificity of the Myc G4-binding compounds in cell-based assays, we compared inhibition of Myc expression in cells with and without Myc G4 regulation. Several compounds inhibited Myc expression only in the Myc G4-containing line, and one compound was verified to function through Myc G4 binding. Our study demonstrates that ALIS can be used to identify selective nucleic acid-binding compounds from phenotypic screen hits, increasing the pool of drug targets beyond proteins.

Streamlining bioactive molecular discovery through integration and automation

The discovery of bioactive small molecules is generally driven via iterative design–make–purify–test cycles. Automation is routinely harnessed at individual stages of these cycles to increase the productivity of drug discovery. Here, we describe recent progress to automate and integrate two or more adjacent stages within discovery workflows. Examples of such technologies include microfluidics, liquid-handling robotics and affinity-selection mass spectrometry. The value of integrated technologies is illustrated in the context of specific case studies in which modulators of targets, such as protein kinases, nuclear hormone receptors and protein–protein interactions, were discovered. We note that to maximize impact on the productivity of discovery, each of the integrated stages would need to have both high and matched throughput. We also consider the longer-term goal of realizing the fully autonomous discovery of bioactive small molecules through the integration and automation of all stages of discovery. The discovery of bioactive small molecules is generally driven via iterative design–make–purify–test cycles. Progress has been made towards the automation and integration of adjacent stages within such discovery workflows, which can increase the efficiency and effectiveness of bioactive small-molecule discovery.

Nanoscale synthesis and affinity ranking

Most drugs are developed through iterative rounds of chemical synthesis and biochemical testing to optimize the affinity of a particular compound for a protein target of therapeutic interest. This process is challenging because candidate molecules must be selected from a chemical space of more than 1060 drug-like possibilities 1 , and a single reaction used to synthesize each molecule has more than 107 plausible permutations of catalysts, ligands, additives and other parameters 2 . The merger of a method for high-throughput chemical synthesis with a biochemical assay would facilitate the exploration of this enormous search space and streamline the hunt for new drugs and chemical probes. Miniaturized high-throughput chemical synthesis3-7 has enabled rapid evaluation of reaction space, but so far the merger of such syntheses with bioassays has been achieved with only low-density reaction arrays, which analyse only a handful of analogues prepared under a single reaction condition8-13. High-density chemical synthesis approaches that have been coupled to bioassays, including on-bead 14 , on-surface 15 , on-DNA 16 and mass-encoding technologies 17 , greatly reduce material requirements, but they require the covalent linkage of substrates to a potentially reactive support, must be performed under high dilution and must operate in a mixture format. These reaction attributes limit the application of transition-metal catalysts, which are easily poisoned by the many functional groups present in a complex mixture, and of transformations for which thekinetics require a high concentration of reactant. Here we couple high-throughput nanomole-scale synthesis with a label-free affinity-selection mass spectrometry bioassay. Each reaction is performed at a 0.1-molar concentration in a discrete well toenable transition-metal catalysis while consuming less than 0.05 milligrams of substrate per reaction. The affinity-selection mass spectrometry bioassay is then used to rank the affinity of the reaction products to target proteins, removing the need for time-intensive reaction purification. This method enables the primary synthesis and testing steps that are critical to the invention of protein inhibitors to be performed rapidly and with minimal consumption of starting materials.

Discovery of Selective RNA-Binding Small Molecules by Affinity-Selection Mass Spectrometry.

Recent advances in understanding the relevance of noncoding RNA (ncRNA) to disease have increased interest in drugging ncRNA with small molecules. The recent discovery of ribocil, a structurally distinct synthetic mimic of the natural ligand of the flavin mononucleotide (FMN) riboswitch, has revealed the potential chemical diversity of small molecules that target ncRNA. Affinity-selection mass spectrometry (AS-MS) is theoretically applicable to high-throughput screening (HTS) of small molecules binding to ncRNA. Here, we report the first application of the Automated Ligand Detection System (ALIS), an indirect AS-MS technique, for the selective detection of small molecule-ncRNA interactions, high-throughput screening against large unbiased small-molecule libraries, and identification and characterization of novel compounds (structurally distinct from both FMN and ribocil) that target the FMN riboswitch. Crystal structures reveal that different compounds induce various conformations of the FMN riboswitch, leading to different activity profiles. Our findings validate the ALIS platform for HTS screening for RNA-binding small molecules and further demonstrate that ncRNA can be broadly targeted by chemically diverse yet selective small molecules as therapeutics.

Discovery and characterization of selective human sphingomyelin synthase 2 inhibitors

Sphingomyelin synthase (SMS) is a membrane enzyme that catalyzes the synthesis of sphingomyelin, is required for the maintenance of plasma membrane microdomain fluidity, and has two isoforms: SMS1 and SMS2. Although these isoforms exhibit the same SMS activity, they are different enzymes with distinguishable subcellular localizations. It was reported that SMS2 KO mice displayed lower inflammatory responses and anti-atherosclerotic effects, suggesting that inhibition of SMS2 would be a potential therapeutic approach for controlling inflammatory responses and atherosclerosis.This study aimed to discover a novel small-molecule compound that selectively inhibits SMS2 enzymatic activity. We developed a human SMS2 enzyme assay with a high-throughput mass spectrometry-based screening system. We characterized the enzymatic properties of SMS2 and established a high-throughput screening-compatible assay condition. To identify human SMS2 inhibitors, we conducted compound screening using the enzyme assay. We identified a 2-quinolone derivative as a SMS2 selective inhibitor with an IC50 of 950 nM and >100-fold selectivity for SMS2 over SMS1. The 2-quinolone exhibited efficacy in a cell-based engagement assay. We demonstrated that a more potent derivative directly bound to SMS2-expressing membrane fractions in an affinity selection mass spectrometry assay. Mutational analyses revealed that the interaction of the inhibitor with SMS2 required the presence of the amino acids S227 and H229, which are located in the catalytic domain of SMS2.In conclusion, we discovered novel SMS2-selective inhibitors. 2-Quinolone SMS2 inhibitors are considered applicable for leading optimization studies. Further investigations using these SMS2 inhibitors would provide validation tools for SMS2-relevant pathways in vitro and in vivo.

Affinity Selection-Mass Spectrometry Identifies a Novel Antibacterial RNA Polymerase Inhibitor.

The growing prevalence of drug resistant bacteria is a significant global threat to human health. The antibacterial drug rifampin, which functions by inhibiting bacterial RNA polymerase (RNAP), is an important part of the antibacterial armamentarium. Here, in order to identify novel inhibitors of bacterial RNAP, we used affinity-selection mass spectrometry to screen a chemical library for compounds that bind to Escherichia coli RNAP. We identified a novel small molecule, MRL-436, that binds to RNAP, inhibits RNAP, and exhibits antibacterial activity. MRL-436 binds to RNAP through a binding site that differs from the rifampin binding site, inhibits rifampin-resistant RNAP derivatives, and exhibits antibacterial activity against rifampin-resistant strains. Isolation of mutants resistant to the antibacterial activity of MRL-436 yields a missense mutation in codon 622 of the rpoC gene encoding the RNAP β' subunit or a null mutation in the rpoZ gene encoding the RNAP ω subunit, confirming that RNAP is the functional cellular target for the antibacterial activity of MRL-436, and indicating that RNAP β' subunit residue 622 and the RNAP ω subunit are required for the antibacterial activity of MRL-436. Similarity between the resistance determinant for MRL-436 and the resistance determinant for the cellular alarmone ppGpp suggests a possible similarity in binding site and/or induced conformational state for MRL-436 and ppGpp.

Integration of Affinity Selection–Mass Spectrometry and Functional Cell-Based Assays to Rapidly Triage Druggable Target Space within the NF-κB Pathway

The primary objective of early drug discovery is to associate druggable target space with a desired phenotype. The inability to efficiently associate these often leads to failure early in the drug discovery process. In this proof-of-concept study, the most tractable starting points for drug discovery within the NF-κB pathway model system were identified by integrating affinity selection–mass spectrometry (AS-MS) with functional cellular assays. The AS-MS platform Automated Ligand Identification System (ALIS) was used to rapidly screen 15 NF-κB proteins in parallel against large-compound libraries. ALIS identified 382 target-selective compounds binding to 14 of the 15 proteins. Without any chemical optimization, 22 of the 382 target-selective compounds exhibited a cellular phenotype consistent with the respective target associated in ALIS. Further studies on structurally related compounds distinguished two chemical series that exhibited a preliminary structure-activity relationship and confirmed target-driven cellular activity to NF-κB1/p105 and TRAF5, respectively. These two series represent new drug discovery opportunities for chemical optimization. The results described herein demonstrate the power of combining ALIS with cell functional assays in a high-throughput, target-based approach to determine the most tractable drug discovery opportunities within a pathway.

Application of the ultrafiltration-based LC-MS approach for screening PTP1B inhibitors from Chinese red yeast rice

Ultrafiltration-based affinity selection mass spectrometry was utilized to rapidly screen potential PTP1B inhibitors in Chinese red yeast rice.

Biochemical characterization of smoothened receptor antagonists by binding kinetics against drug-resistant mutant

Hedgehog (Hh) signaling critical for development, differentiation, and cell growth is involved in several cancers, including medulloblastoma and basal cell carcinoma. Although antagonism of the smoothened receptor (SMO), which mediates Hh signaling, is an attractive therapeutic target, a drug-resistant mutation in SMO (SMO-D473H) was identified in a clinical trial of the approved drug vismodegib. TAK-441 potently inhibits SMO-D473H, unlike vismodegib and another SMO antagonist, cyclopamine, whereas the differences in binding modes between these antagonists remain unknown. Here we report the biochemical characterization of TAK-441, vismodegib, and cyclopamine by binding kinetics. The association (kon) and dissociation (koff) rates were determined by kinetic binding studies using [3H]TAK-441, and dissociation was confirmed by label-free affinity selection-mass spectrometry (AS-MS). In the [3H]TAK-441 competition assay, TAK-441 but not vismodegib and cyclopamine showed time-dependent inhibition. Quantitative kinetic binding analysis revealed that koff of TAK-441 was >10-fold smaller than those of vismodegib and cyclopamine. To further assess the binding mode of antagonists, kinetic binding analysis was performed against SMO-D473H. The D473H mutation affected koff of TAK-441 but not kon. In contrast, only kon was changed by the D473H mutation in the case of vismodegib and cyclopamine. These results suggest that the difference in antagonist efficacy against D473H is associated with the binding mode of antagonists. These findings provide a new insight into the drug action of SMO antagonists and help develop potential therapeutics for drug-resistant mutants.

Ultracentrifugation-based multi-target affinity selection mass spectrometry

A rate-zonal density gradient ultracentrifugation-based affinity selection mass spectrometry approach has been developed for simultaneous multi-target screening.

Immobilized magnetic beads-based multi-target affinity selection coupled with HPLC-MS for screening active compounds from traditional Chinese medicine and natural products.

Screening and identifying active compounds from traditional Chinese medicine (TCM) and other natural products plays an important role in drug discovery. Here, we describe a magnetic beads-based multi-target affinity selection-mass spectrometry approach for screening bioactive compounds from natural products. Key steps and parameters including activation of magnetic beads, enzyme/protein immobilization, characterization of functional magnetic beads, screening and identifying active compounds from a complex mixture by LC/MS, are illustrated. The proposed approach is rapid and efficient in screening and identification of bioactive compounds from complex natural products.

Development of a CCK1R-membrane nanoparticle as a fish-out tool for bioactive peptides

The cholecystokinin receptor type 1 (CCK1R) is a G protein-coupled receptor (GPCR) that is involved in several biological processes including the regulation of the secretion of digestive enzymes. The peptide hormone cholecystokinin (CCK) binds to CCK1R, which is an important pharmacological target for several diseases, including obesity. Interestingly, nutritional dietary peptides also appear to activate CCK1R, and may play a role in CCK1R signaling in the gut. In this study, a novel technique to screen for CCK1R ligands based on affinity-selection is described. Functional expressed CCK1R is reconstituted into membrane nanoparticles called NABBs (nanoscale apo-lipoprotein bound bilayers). NABBs are native-like bilayer membrane systems for incorporation of GPCRs. CCK1R-NABBs were characterized using a fluorescently labeled CCK analog and can be used as a cutting-edge technology to screen for CCK1R ligands using affinity-selection mass spectrometry.