Mass Spectrometry Binding Assays Research Articles

α-Synuclein Aggregation Inhibitory Prunolides and a Dibrominated β-Carboline Sulfamate from the Ascidian Synoicum prunum.

Seven new polyaromatic bis-spiroketal-containing butenolides, the prunolides D-I (4-9) and cis-prunolide C (10), a new dibrominated β-carboline sulfamate named pityriacitrin C (11), alongside the known prunolides A-C (1-3) were isolated from the Australian colonial ascidian Synoicum prunum. The prunolides D-G (4-7) represent the first asymmetrically brominated prunolides, while cis-prunolide C (10) is the first reported with a cis-configuration about the prunolide's bis-spiroketal core. The prunolides displayed binding activities with the Parkinson's disease-implicated amyloid protein α-synuclein in a mass spectrometry binding assay, while the prunolides (1-5 and 10) were found to significantly inhibit the aggregation (>89.0%) of α-synuclein in a ThT amyloid dye assay. The prunolides A-C (1-3) were also tested for inhibition of pSyn aggregate formation in a primary embryonic mouse midbrain dopamine neuron model with prunolide B (2) displaying statistically significant inhibitory activity at 0.5 μM. The antiplasmodial and antibacterial activities of the isolates were also examined with prunolide C (3) displaying only weak activity against the 3D7 parasite strain of Plasmodium falciparum. Our findings reported herein suggest that the prunolides could provide a novel scaffold for the exploration of future therapeutics aimed at inhibiting amyloid protein aggregation and the treatment of numerous neurodegenerative diseases.

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.

Development and validation of a mass spectrometry binding assay for mGlu5 receptor.

Mass spectrometry (MS) binding assays are a label-free alternative to radioligand or fluorescence binding assays, so the readout is based on direct mass spectrometric detection of the test ligand. The study presented here describes the development and validation of a highly sensitive, rapid, and robust MS binding assay for the quantification of the binding of the metabotropic glutamate 5 (mGlu5) negative allosteric modulator (NAM), MPEP (2-methyl-6-phenylethynylpyridine) at the mGlu5 allosteric binding site. The LC-ESI-MS/MS (liquid chromatography-electrospray ionization-tandem mass spectrometric) analytical method was established and validated with a deuterated analogue of MPEP as an internal standard. The developed MS binding assay described here allowed for the determination of MS binding affinity estimates that were in agreement with affinity estimates obtained from a tritiated MPEP radioligand saturation binding assay, indicating the suitability of this methodology for determining affinity estimates for compounds that target mGlu5 allosteric binding sites. Graphical abstract.

Insight into the Selectivity of Kir3.2 toward Phosphatidylinositides.

Activation of G-protein-gated inwardly rectifying potassium channels (Kir3.x) requires the direct binding of phosphorylated phosphatidylinositides (PIPs). Previous studies have established that PIP isoforms activate Kir channels to varying degrees and the binding affinity between PIPs and Kir3.2 appears to be correlated with the level of activation. However, how individual residues contribute to the selectivity of Kir channels toward PIP isoforms is poorly understood. Here, we employ native mass spectrometry (MS) and fluorescent lipid binding assays to gain insight into the contribution of specific Kir3.2 residues binding to phospholipids. For the wild-type channel, we demonstrate the importance of membrane protein samples devoid of co-purified contaminants for protein-lipid binding studies and show that PIP(4,5)P2 cooperatively binds Kir3.2 with a Hill coefficient of 2.7. We also find lipid binding profiles determined from native MS and solution binding assays are in direct agreement. Point mutations of Kir3.2 residues that interact with PIPs distinctly alter selective lipid binding. The K64Q mutation results in altered binding profiles with the highest binding affinity for PIP(4,5)P2 with specific acyl chains. Mutation of R92 to Pro, a residue found in Kir6.2, results in promiscuous binding of PIP isoforms. Kir3.2 with the K194A mutation results in a distinct binding preference for PIP(3,4,5)P3 over other PIP isoforms. Taken together, our results underscore the utmost importance of protein quality for protein-lipid binding studies and show that a single mutation in Kir3.2 can alter the selectivity toward PIPs.

Application of the concept of oxime library screening by mass spectrometry (MS) binding assays to pyrrolidine-3-carboxylic acid derivatives as potential inhibitors of γ-aminobutyric acid transporter 1 (GAT1).

In the present study, the concept of oxime library screening by MS Binding Assays was successfully extended to N-substituted lipophilic pyrrolidine-3-carboxylic acid derivatives in the pursuit of varying the amino acid motif in order to identify new inhibitors for GAT1 and to broaden structure-activity-relationships for this target, the most abundant GABA transporter in the central nervous system. For the screening, 28 different oxime sub-libraries were employed that were generated by simple condensation reaction of an excess of pyrrolidine-3-carboxylic acid derivatives carrying a hydroxylamine functionality with various sub-libraries each assembled of eight aldehydes with broadly varying chemical structures and functionalities. The compounds responsible for the activity of an oxime sub-library were identified by deconvolution experiments performed by employing single oximes. Binding affinities of the oxime hits were confirmed in full-scale competitive MS Binding Assays. Thereby, oxime derivatives with a 1,1'-biphenyl moiety were found as the first inhibitors of mGAT1 comprising a pyrrolidine-3-carboxylic acid motif with affinities in the submicromolar range.

MS binding assays for GlyT1 based on Org24598 as nonlabelled reporter ligand

In this study an alternative to radioligand binding assays addressing the glycine transporter 1 (GlyT1) based on quantification of a nonlabelled reporter ligand by means of mass spectrometry (MS) is presented. The established MS Binding Assays employ the GlyT1 inhibitor Org24598 as reporter ligand for which a highly sensitive LC-ESI-MS/MS (liquid chromatography electrospray ionization tandem mass spectrometry) method was developed. A validation of this LC-ESI-MS/MS method with respect to selectivity, linearity, accuracy and precision according to the FDA guidance demonstrated its reliability for quantification of Org24598 in binding experiments. For the implementation of GlyT1 binding experiments conditions in accordance to known GlyT1 radioligand binding assays and already known filtration based MS Binding Assays were chosen. In saturation experiments the affinity of Org24598 towards GlyT1 could be characterized with an equilibrium dissociation constant (Kd) of 16.8 ± 2.2 nM that is well in agreement with the affinity determined in radioligand binding assays. Finally, several known GlyT ligands were studied in competition experiments and the determined inhibition constants (Ki) compared with results from radioligand binding and uptake assays. The almost perfect correlation of the affinities obtained in the MS based binding experiments with results from literature clearly indicates that the established GlyT1 MS Binding Assays are a powerful substitute for the GlyT1 radioligand binding assays so far used for affinity profiling and screening.This article is part of the issue entitled ‘Special Issue on Neurotransmitter Transporters’.

Screening oxime libraries by means of mass spectrometry (MS) binding assays: Identification of new highly potent inhibitors to optimized inhibitors γ-aminobutyric acid transporter 1.

Generation and screening of oxime libraries by competitive MS Binding Assays represents a powerful tool for the identification of new compounds, with affinity to mGAT1, the most abundant plasma membrane bound GABA transporter in the CNS. By screening a guvacine derived oxime library, new potent inhibitors of mGAT1 had been revealed. In the present study, oxime libraries generated by reaction of a large excess of a rac-nipecotic acid derivative displaying a hydroxylamine functionality in which various aldehydes under suitable conditions, were examined for new potent inhibitors of mGAT1. The pKi values obtained of the best hits were compared with those of related compounds displaying a guvacine instead of a nipecotic acid subunit as hydrophilic moiety. Amongst the new compounds one of the most affine ligands of mGAT1 known so far (pKi = 8.55 ± 0.04) was found.

Mass spectrometry-based ligand binding assays on adenosine A1 and A2A receptors.

Conventional methods to measure ligand-receptor binding parameters typically require radiolabeled ligands as probes. Despite the robustness of radioligand binding assays, they carry inherent disadvantages in terms of safety precautions, expensive synthesis, special lab requirements, and waste disposal. Mass spectrometry (MS) is a method that can selectively detect ligands without the need of a label. The sensitivity of MS equipment increases progressively, and currently, it is possible to detect low ligand quantities that are usually found in ligand binding assays. We developed a label-free MS ligand binding (MS binding) assay on the adenosine A1 and A2A receptors (A1AR and A2AAR), which are well-characterized members of the class A G protein-coupled receptor (GPCR) family. Radioligand binding assays for both receptors are well established, and ample data is available to compare and evaluate the performance of an MS binding assay. 1,3-Dipropyl-8-cyclopentyl-xanthine (DPCPX) and 4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a]-[1,3,5]triazin-5-yl)amino)ethyl)phenol (ZM-241,385) are high-affinity ligands selective for the A1AR and A2AAR, respectively. To proof the feasibility of MS binding on the A1AR and A2AAR, we first developed an MS detection method for unlabeled DPCPX and ZM-241,385. To serve as internal standards, both compounds were also deuterium-labeled. Subsequently, we investigated whether the two unlabeled compounds could substitute for their radiolabeled counterparts as marker ligands in binding experiments, including saturation, displacement, dissociation, and competition association assays. Furthermore, we investigated the accuracy of these assays if the use of internal standards was excluded. The results demonstrate the feasibility of the MS binding assay, even in the absence of a deuterium-labeled internal standard, and provide great promise for the further development of label-free assays based on MS for other GPCRs.Electronic supplementary materialThe online version of this article (doi:10.1007/s11302-015-9477-0) contains supplementary material, which is available to authorized users.

Development and validation of an LC-ESI-MS/MS method for the triple reuptake inhibitor indatraline enabling its quantification in MS Binding Assays.

We herein present the first LC-MS/MS quantification method for indatraline, a highly potent nonselective inhibitor of the three monoamine transporters (for dopamine, DAT; norepinephrine, NET; serotonin, SERT), and its application to MS Binding Assays. For HPLC, an R18 column with a mobile phase composed of acetonitrile and ammonium bicarbonate buffer (5 mmol L(-1), pH 10.0) in a ratio of 90:10 (v/v) at a flow rate of 600 μL min(-1) was used. Recording indatraline at m/z 292.2/261.0 and ((2)H(7))-indatraline, employed as internal standard, at m/z 299.2/268.0 allowed reliable quantification from 5 pmol L(-1) (LLOQ) to 5 nmol L(-1) in biological matrices without additional sample preparation. Validation of the developed quantification method showed that selectivity, calibration standard curve, accuracy, as well as precision meet the criteria of the CDER guideline. Applying this method to mass spectrometry (MS) Binding Assays, a label-free MS-based alternative to conventional radioligand binding assays, binding of indatraline's eutomer, (1R,3S)-indatraline, towards NET could be characterized directly for the first time, revealing an equilibrium dissociation constant (K d) of 805 pmol L(-1). Additionally, it could be shown that the established MS Binding Assays enable characterization of test compounds in competition experiments. As the established setup is based on a 96-well format and an LC MS/MS method with a short chromatographic cycle time (1.5 min), the developed MS Binding Assays enable considerable throughput and are therefore well suited as substitute for corresponding radioligand binding assays.

Focused Pseudostatic Hydrazone Libraries Screened by Mass Spectrometry Binding Assay: Optimizing Affinities toward γ-Aminobutyric Acid Transporter 1

Mass spectrometric (MS) binding assays, a powerful tool to determine affinities of single drug candidates toward chosen targets, were recently demonstrated to be suitable for the screening of compound libraries generated with reactions of dynamic combinatorial chemistry when rendering libraries pseudostatic. Screening of small hydrazone libraries targeting γ-aminobutyric acid transporter 1 (GAT1), the most abundant γ-aminobutyric acid (GABA) transporter in the central nervous system, revealed two nipecotic acid derived binders with submicromolar affinities. Starting from the biphenyl carrying hit as lead structure, the objective of the present study was to discover novel high affinity GAT1 binders by screening of biphenyl focused pseudostatic hydrazone libraries formed from hydrazine 10 and 36 biphenylcarbaldehydes 11c-al. Hydrazone 12z that carried a 2',4'-dichlorobiphenyl residue was found to be the most potent binder with low nanomolar affinity (pK(i) = 8.094 ± 0.098). When stable carba analogues of representative hydrazones were synthesized and evaluated, the best binder 13z was again displaying the 2',4'-dichlorobiphenyl moiety (pK(i) = 6.930 ± 0.021).

Library Screening by Means of Mass Spectrometry (MS) Binding Assays—Exemplarily Demonstrated for a Pseudostatic Library Addressing γ‐Aminobutyric Acid (GABA) Transporter 1 (GAT1)

In the present study, the application of mass spectrometry (MS) binding assays as a tool for library screening is reported. For library generation, dynamic combinatorial chemistry (DCC) was used. These libraries can be screened by means of MS binding assays when appropriate measures are taken to render the libraries pseudostatic. That way, the efficiency of MS binding assays to determine ligand binding in compound screening with the ease of library generation by DCC is combined. The feasibility of this approach is shown for γ-aminobutyric acid (GABA) transporter 1 (GAT1) as a target, representing the most important subtype of the GABA transporters. For the screening, hydrazone libraries were employed that were generated in the presence of the target by reacting various sets of aldehydes with a hydrazine derivative that is delineated from piperidine-3-carboxylic acid (nipecotic acid), a common fragment of known GAT1 inhibitors. To ensure that the library generated is pseudostatic, a large excess of the nipecotic acid derivative is employed. As the library is generated in a buffer system suitable for binding and the target is already present, the mixtures can be directly analyzed by MS binding assays-the process of library generation and screening thus becoming simple to perform. The binding affinities of the hits identified by deconvolution were confirmed in conventional competitive MS binding assays performed with single compounds obtained by separate synthesis. In this way, two nipecotic acid derivatives exhibiting a biaryl moiety, 1-{2-[2'-(1,1'-biphenyl-2-ylmethylidene)hydrazine]ethyl}piperidine-3-carboxylic acid and 1-(2-{2'-[1-(2-thiophenylphenyl)methylidene]hydrazine}ethyl)piperidine-3-carboxylic acid, were found to be potent GAT1 ligands exhibiting pK(i) values of 6.186 ± 0.028 and 6.229 ± 0.039, respectively. This method enables screening of libraries, whether generated by conventional chemistry or DCC, and is applicable to all kinds of targets including membrane-bound targets such as G protein coupled receptors (GPCRs), ion channels and transporters. As such, this strategy displays high potential in the drug discovery process.

Cover Picture: (S)‐ and (R)‐Fluoxetine as Native Markers in Mass Spectrometry (MS) Binding Assays Addressing the Serotonin Transporter (ChemMedChem 10/2011)

The cover picture shows a label-free screening technique to characterize target–ligand interactions. In the foreground, a triple quadrupole mass spectrometer positioned in a flowering meadow illustrates the environmentally friendly opportunity to realize binding assays based on mass spectrometric quantification of a nonlabeled marker compound for a desired target. By peeling back the illustration of this concept—referred to as MS binding assays—the cover picture visualizes the implementation of this approach to the serotonin transporter (SERT). The study describes the efforts to address binding of nonlabeled fluoxetine enantiomers to SERT, as well as the promising results generated by the application of this MS binding assay strategy. For more details, see the Full Paper by Klaus T. Wanner et al. on p. 1900 ff.

(S)‐ and (R)‐Fluoxetine as Native Markers in Mass Spectrometry (MS) Binding Assays Addressing the Serotonin Transporter

A recently established and validated LC-ESI-MS/MS method for quantification of fluoxetine was used to implement MS binding assays for the human serotonin transporter (hSERT)-the primary target in the treatment of depression and emotional disorders. As a label-free screening technique, MS binding assays offer the opportunity to perform kinetic, saturation and competition assays using both (S)- and (R)-fluoxetine as native markers. In kinetic experiments, an association rate constant (k(+1) of 0.92±0.17×10(6) M(-1) s(-1) and a dissociation rate constant (k(-1)) of 0.0032±0.0002 s(-1) for (S)-fluoxetine binding to hSERT were determined. Saturation experiments provided K(d) values of 4.4±0.4 nM and 5.2±0.9 nM for (S)- and (R)-fluoxetine, respectively; statistical analysis revealed that the two enantiomers are equipotent. In competitive experiments with (S)-fluoxetine as a marker, K(i) values were obtained for various known inhibitors with a broad range of affinities for hSERT that correlate well with literature data obtained from radioligand binding experiments with [(3)H]imipramine. Additional competitive experiments using (R)-fluoxetine as a marker led to K(i) values for SERT inhibitors that deviate only marginally from those determined using the (S)-enantiomer. No changes in the rank order of affinities occurred, indicating that there is no difference in the binding characteristics of the two enantiomers.

Development and validation of a rapid LC-ESI-MS/MS method for quantification of fluoxetine and its application to MS binding assays

In the present study, a rapid and sensitive LC-ESI-MS/MS method for quantification of (S)-fluoxetine as a native marker in mass spectrometry (MS) binding assays addressing the human serotonin transporter (hSERT) was developed and validated. The concept of MS binding assays based on mass spectrometric quantification of a nonlabeled marker recently introduced by us represents a promising alternative to conventional radioligand binding without the drawbacks inherently connected with radioisotope labeling. For high-performance liquid chromatography (HPLC), a 20 × 2-mm RP-18 column with a mobile phase composed of acetonitrile and ammonium bicarbonate buffer (5 mmol L(-1), pH 9.5) at a ratio of 80:20 (v/v) and a flow rate of 800 μL min(-1) in an isocratic mode were used, resulting in a chromatographic cycle time of 60 s. Employing [(2)H(5)]fluoxetine as internal standard enabled ESI-MS/MS quantification of (S)-fluoxetine between 3 nmol L(-1) and 50 pmol L(-1) (LLOQ) in matrix obtained from binding experiments without the need of any sample preparation. Validation of the method showed that linearity, intra-, and inter-batch accuracy as well as precision meet the requirements of the FDA guidance for bioanalytical method validation. Considering sensitivity and speed, the established method is clearly superior to those published for biological matrices so far. Furthermore, the method was transferred to other RP-18 columns of different lengths and respective validation experiments demonstrated its versatility and chromatographic robustness. Finally, the newly developed method was successfully applied to MS binding assays for hSERT. The affinity determined for (S)-fluoxetine in saturation experiments was in good agreement with literature data obtained in respective radioligand binding assays.

Editing of the HLA-DR-peptide repertoire by HLA-DM.

Antigenic peptide loading of classical major histocompatibility complex (MHC) class II molecules requires the exchange of the endogenous invariant chain fragment CLIP (class II associated Ii peptide) for peptides derived from antigenic proteins. This process is facilitated by the non-classical MHC class II molecule HLA-DM (DM) which catalyzes the removal of CLIP. Up to now it has been unclear whether DM releases self-peptides other than CLIP and thereby modifies the peptide repertoire presented to T cells. Here we report that DM can release a variety of peptides from HLA-DR molecules. DR molecules isolated from lymphoblastoid cell lines were found to carry a sizeable fraction of self-peptides that are sensitive to the action of DM. The structural basis for this DM sensitivity was elucidated by high-performance size exclusion chromatography and a novel mass spectrometry binding assay. The results demonstrate that the overall kinetic stability of a peptide bound to DR determines its sensitivity to removal by DM. We show that DM removes preferentially those peptides that contain at least one suboptimal side chain at one of their anchor positions or those that are shorter than 11 residues. These findings provide a rationale for the previously described ligand motifs and the minimal length requirements of naturally processed DR-associated self-peptides. Thus, in endosomal compartments, where peptide loading takes place, DM can function as a versatile peptide editor that selects for high-stability MHC class II-peptide complexes by kinetic proofreading before these complexes are presented to T cells.