Project description:The inwardly rectifying potassium Kir4.2 channel plays a crucial role in regulating membrane potentials and maintaining potassium homeostasis. Kir4.2 has been implicated in various physiological processes, including insulin secretion, gastric acid regulation, and the pathogenesis of central nervous system diseases. Despite its significance, the number of identified ligands for Kir4.2 remains limited. In this study, we established a method to directly observe ligands avoiding a requirement to observe the high-mass ligand-membrane protein-detergent complexes. This method used collision-induced affinity selection mass spectrometry (CIAS-MS) to identify ligands dissociated from the Kir4.2 channel-detergent complex. The CIAS-MS approach integrated all stages of affinity selection within the mass spectrometer, offering advantages in terms of time efficiency and cost-effectiveness. Additionally, we explored the effect of collisional voltage ramps on the dissociation behavior of the ligand and the ligand at different concentrations, demonstrating dose dependency.
Project description:We report an enantioselective protein affinity selection mass spectrometry screening approach (EAS-MS) that enables the detection of weak binders, informs about selectivity, and generates orthogonal confirmation of binding. After method development with control proteins, we screened 31 human proteins against a designed library of 8,210 chiral compounds. 16 binders to 12 targets, including many proteins predicted to be "challenging to ligand", were discovered and confirmed in orthogonal assays. 7 binders to 6 targets bound in an enantioselective manner, with K Ds ranging from 3 to 20 μM. Binders for four targets (DDB1, WDR91, WDR55, and HAT1) were selected for in-depth characterization using X-ray crystallography. In all four cases, the mechanism for enantioselective selectivity was readily explained. EAS-MS can be used to identify and characterize selective and weakly-binding ligands for novel protein targets with unprecedented throughput and sensitivity.
Project description:Despite their potential, the preparation of large synthetic macrocyclic libraries for ligand discovery and development has been limited. Here, we produce 100-million-membered macrocyclic libraries containing natural and nonnatural amino acids. Near-quantitative intramolecular disulfide formation is facilitated by rapid oxidation with iodine in solution. After use in affinity selection, treatment with dithiothreitol enables near-quantitative reduction, rendering linear peptide analogs for standard tandem mass spectrometry. We use these libraries to discover macrocyclic binders to cadherin-2 and anti-hemagglutinin antibody clone 12ca5. Structure-activity relationship studies of an initial cadherin-binding peptide [CBP; apparent dissociation constant (Kd) = 53 nanomolar] reveal residues responsible for driving affinity (hotspots) and mutation-tolerant residues (coldspots). Two original macrocyclic libraries are prepared in which these hotspots and coldspots are derivatized with nonnatural amino acids. Following discovery and validation, high-affinity ligands are discovered from the coldspot library, with NCBP-4 demonstrating improved affinity (Kd = 29 nanomolar). Overall, we expect that this work will improve the use of macrocyclic libraries in therapeutic peptide development.
Project description:To overcome limiting factors in mass spectrometry-based screening methods such as automation while still facilitating the screening of complex mixtures such as botanical extracts, magnetic microbead affinity selection screening (MagMASS) was developed. The screening process involves immobilization of a target protein on a magnetic microbead using a variety of possible chemistries, incubation with mixtures of molecules containing possible ligands, a washing step that removes non-bound compounds while a magnetic field retains the beads in the microtiter well, and an organic solvent release step followed by LC-MS analysis. Using retinoid X receptor-α (RXRα) as an example, which is a nuclear receptor and target for anti-inflammation therapy as well as cancer treatment and prevention, a MagMASS assay was developed and compared with an existing screening assay, pulsed ultrafiltration (PUF)-MS. Optimization of MagMASS involved evaluation of multiple protein constructs and several magnetic bead immobilization chemistries. The full-length RXRα construct immobilized with amylose beads provided optimum results. Additional enhancements of MagMASS were the application of 96-well plates to enable automation, use of UHPLC instead of HPLC for faster MS analyses, and application of metabolomics software for faster, automated data analysis. Performance of MagMASS was demonstrated using mixtures of synthetic compounds and known ligands spiked into botanical extracts. Graphical Abstract ᅟ.
Project description:Two scan modes of the triple quadrupole tandem mass spectrometer, namely Collision Induced Dissociation Precursor Ion scan and Neutral Loss scan, allow selectively pinpointing, in a complex mixture, compounds that feature specific chemical groups, which yield characteristic fragment ions or are lost as distinctive neutral fragments. This feature of the triple quadrupole tandem mass spectrometer allows the non-target screening of mixtures for classes of components. The effective (center-of-mass) energy to achieve specific fragmentation depends on the inter-quadrupole voltage (laboratory-frame collision energy) and on the masses of the precursor molecular ion and of the collision gas, through a non-linear relationship. Thus, in a class of homologous compounds, precursor ions activated at the same laboratory-frame collision energy face different center-of-mass collision energy, and therefore the same fragmentation channel operates with different degrees of efficiency. This article reports a linear equation to calculate the laboratory-frame collision energy necessary to operate Collision-Induced Dissociation at the same center-of-mass energy on closely related compounds with different molecular mass. A routine triple quadrupole tandem mass spectrometer can operate this novel feature (iso-energetic collision-induced dissociation scan; i-CID) to analyze mixtures of endogenous metabolites by Precursor Ion and Neutral Loss scans. The latter experiment also entails the hitherto unprecedented synchronized scanning of all three quadrupoles of the triple quadrupole tandem mass spectrometer. To exemplify the application of this technique, this article shows two proof-of-principle approaches to the determination of biological mixtures, one by Precursor Ion analysis on alpha amino acid derivatized with a popular chromophore, and the other on modified nucleosides with a Neutral Fragment Loss scan.
Project description: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.
Project description:The identification of proteins by tandem mass spectrometry relies on knowledge of the products produced by collision-induced dissociation of peptide ions. Most previous work has focused on fragmentation statistics for ion trap systems. We analyzed fragmentation in MALDI TOF/TOF mass spectrometry, collecting statistics using a curated set of 2459 MS/MS spectra and applying bootstrap resampling to assess confidence intervals. We calculated the frequency of 18 product ion types, the correlation between both mass and intensity with ion type, the dependence of amide bond breakage on the residues surrounding the cleavage site, and the dependence of product ion detection on residues not adjacent to the cleavage site. The most frequently observed were internal ions, followed by y ions. A strong correlation between ion type and the mass and intensity of its peak was observed, with b and y ions producing the most intense and highest mass peaks. The amino acids P, W, D, and R had a strong effect on amide bond cleavage when situated next to the breakage site, whereas residues including I, K, and H had a strong effect on product ion observation when located in the peptide but not adjacent to the cleavage site, a novel observation.
Project description:In recent years, the incidence of diseases associated with hepatic injury has increased in prevalence. Targeting the mitochondria to protect liver function has gained momentum due to their central role in energy production, apoptotic cell death, oxidative stress, calcium homeostasis, and lipid metabolism. In this study, we employed a hepatic mitochondria-based centrifugal ultrafiltration/liquid chromatography/mass spectrometry method (CM-HMC) to identify hepatic mitochondria ligands from medicinal herbs (MHs) including Notopterygii Rhizoma et Radix (NRR) that possess hepatic-protective effects. A total of 4 newly identified mitochondrial ligands were successfully identified by CM-HMC. The mitochondria-regulating activities of 3 of the 4 hits were confirmed using isolated mitochondria. The hepatic-protective effects of one of these hits were validated in carbon tetrachloride-damaged human liver L02 cell models. We have thus identified new natural hepatic-protectants that enhance our understanding of the hepatic-protective mechanisms of MHs. CM-HMC was proven to efficiently screen for mitochondrial ligands from MHs.
Project description:Inducible T cell co-stimulator (ICOS) is a positive immune checkpoint receptor expressed on the surface of activated T cells, which could promote cell function after being stimulated with ICOS ligand (ICOS-L). Although clinical benefits have been reported in the ICOS modulation-based treatment for cancer and autoimmune disease, current modulators are restricted in biologics, whereas ICOS-targeted small molecules are lacking. To fill this gap, we performed an affinity selection mass spectrometry (ASMS) screening for ICOS binding using a library of 15,600 molecules. To the best of our knowledge, this is the first study that utilizes ASMS screening to discover small molecules targeting immune checkpoints. Compound 9 with a promising ICOS/ICOS-L inhibitory profile (IC50 = 29.38 ± 3.41 μM) was selected as the template for the modification. Following preliminary structure-activity relationship (SAR) study and molecular dynamic (MD) simulation revealed the critical role of the ortho-hydroxy group on compound 9 in the ICOS binding, as it could stabilize the interaction via the hydrogen bond formation with residuals on the glycan, and the depletion could lead to an activity lost. This work validates a promising inhibitor for the ICOS/ICOS-L interaction, and we anticipate future modifications could provide more potent modulators for this interaction.
Project description:Type 2 diabetes mellitus (T2DM) is recognized as a serious public health concern with increasing incidence. The dipeptidyl peptidase-4 (DPP-4) inhibitor sitagliptin has been used for the treatment of T2DM worldwide. Although sitagliptin has excellent therapeutic outcome, adverse effects are observed. In addition, previous studies have suggested that sitagliptin may have pleiotropic effects other than treating T2DM. These pieces of evidence point to the importance of further investigation of the molecular mechanisms of sitagliptin, starting from the identification of sitagliptin-binding proteins. In this study, by combining affinity purification mass spectrometry (AP-MS) and stable isotope labeling by amino acids in cell culture (SILAC), we discover seven high-confidence targets that can interact with sitagliptin. Surface plasmon resonance (SPR) assay confirms the binding of sitagliptin to three proteins, i. e., LYPLAL1, TCP1, and CCAR2, with binding affinities (K D) ranging from 50.1 μM to 1490 μM. Molecular docking followed by molecular dynamic (MD) simulation reveals hydrogen binding between sitagliptin and the catalytic triad of LYPLAL1, and also between sitagliptin and the P-loop of ATP-binding pocket of TCP1. Molecular mechanics Poisson-Boltzmann Surface Area (MMPBSA) analysis indicates that sitagliptin can stably bind to LYPLAL1 and TCP1 in active sites, which may have an impact on the functions of these proteins. SPR analysis validates the binding affinity of sitagliptin to TCP1 mutant D88A is ~10 times lower than that to the wild-type TCP1. Our findings provide insights into the sitagliptin-targets interplay and demonstrate the potential of sitagliptin in regulating gluconeogenesis and in anti-tumor drug development.