Coupling of surface plasmon resonance and mass spectrometry for molecular interaction studies in drug discovery.

Various analytical technologies have been developed for the study of target-ligand interactions. The combination of these technologies gives pivotal information on the binding mechanism, kinetics, affinity, residence time, and changes in molecular structures. Mass spectrometry (MS) offers structural information, enabling the identification and quantification of target-ligand interactions. Surface plasmon resonance (SPR) provides kinetic information on target-ligand interaction in real time. The coupling of MS and SPR complements each other in the studies of target-ligand interactions. Over the last two decades, the capabilities and added values of SPR-MS have been reported. This review summarizes and highlights the benefits, applications, and potential for further research of the SPR-MS approach.

Shining light on tryptamine-derived isocyanides: access to constrained spirocylic scaffolds.

Dearomatization of indoles through a charge transfer complex constitutes a powerful tool for synthesizing three-dimensional constrained structures. However, the implementation of this strategy for the dearomatization of tryptamine-derived isocyanides to generate spirocyclic scaffolds remains underdeveloped. In this work, we have demonstrated the ability of tryptamine-derived isocyanides to form aggregates at higher concentration, enabling a single electron transfer step to generate carbon-based-radical intermediates. Optical, HRMS and computational studies have elucidated key aspects associated with the photophysical properties of tryptamine-derived isocyanides. The developed protocol is operationally simple, robust and demonstrates a novel approach to generate conformationally constrained spirocyclic scaffolds, compounds with high demand in various fields, including drug discovery.

DNA Nanoparticle Based 2D Biointerface to Study the Effect of Dynamic RGD Presentation on Stem Cell Adhesion and Migration.

The native extracellular matrix (ECM) undergoes constant remodeling, where adhesive ligand presentation changes over time and in space to control stem cell function. As such, it is of interest to develop 2D biointerfaces able to study these complex ligand stem-cell interactions. In this study, a novel dynamic bio interface based on DNA hybridization is developed, which can be employed to control ligand display kinetics and used to study dynamic cell-ligand interaction. In this approach, mesoporous silica nanoparticles (MSN) are functionalized with single-strand DNA (MSN-ssDNA) and spin-coated on a glass substrate to create the 2D bio interface. Cell adhesive tripeptide RGD is conjugated to complementary DNA strands (csDNA) of 9, 11, or 20 nucleotides in length, to form csDNA-RGD. The resulting 3 csDNA-RGD conjugates can hybridize with the ssDNA on the MSN surface, presenting RGD with increased ligand dissociation rates as DNA length is shortened. Slow RGD dissociation rates led to enhanced stem cell adhesion and spreading, resulting in elongated cell morphology. Cells on surfaces with slow RGD dissociation rates also exhibited higher motility, migrating in multiple directions compared to cells on surfaces with fast RGD dissociation rates. This study contributes to the existing body of knowledge on dynamic ligand-stem cell interactions.

Matrix-assisted laser desorption/ionization mass spectrometry imaging for quorum sensing.

Quorum sensing (QS) is a complex communication system in bacteria, directing their response to the environment. QS is also one of the main regulators of bacterial biofilms' formation, maturation and dispersion. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) is a molecular imaging technique that allows the mapping of QS molecules in bacterial biofilms. Here, we highlight the latest advances in MALDI-MSI in recent years and how this technology can improve QS understanding at the molecular level.

Technological advances for analyzing the content of organ-on-a-chip by mass spectrometry.

Three-dimensional (3D) cell cultures, including organ-on-a-chip (OOC) devices, offer the possibility to mimic human physiology conditions better than 2D models. The organ-on-a-chip devices have a wide range of applications, including mechanical studies, functional validation, and toxicology investigations. Despite many advances in this field, the major challenge with the use of organ-on-a-chips relies on the lack of online analysis methods preventing the real-time observation of cultured cells. Mass spectrometry is a promising analytical technique for real-time analysis of cell excretes from organ-on-a-chip models. This is due to its high sensitivity, selectivity, and ability to tentatively identify a large variety of unknown compounds, ranging from metabolites, lipids, and peptides to proteins. However, the hyphenation of organ-on-a-chip with MS is largely hampered by the nature of the media used, and the presence of nonvolatile buffers. This in turn stalls the straightforward and online connection of organ-on-a-chip outlet to MS. To overcome this challenge, multiple advances have been made to pre-treat samples right after organ-on-a-chip and just before MS. In this review, we summarised these technological advances and exhaustively evaluated their benefits and shortcomings for successful hyphenation of organ-on-a-chip with MS.

Ionic mononuclear [Fe] and heterodinuclear [Fe,Ru] bis(diphenylphosphino)alkane complexes: Synthesis, spectroscopy, DFT structures, cytotoxicity, and biomolecular interactions.

Iron(II) and Ru(II) half-sandwich compounds encompass some promising pre-clinical anticancer agents whose efficacy may be tuned by structural modification of the coordinated ligands. Here, we combine two such bioactive metal centres in cationic bis(diphenylphosphino)alkane-bridged heterodinuclear [Fe2+, Ru2+] complexes to delineate how ligand structural variations modulate compound cytotoxicity. Specifically, Fe(II) complexes of the type [(η5-C5H5)Fe(CO)2(κ1-PPh2(CH2)nPPh2)]{PF6} (n = 1-5), compounds 1-5, and heterodinuclear [Fe2+, Ru2+] complexes, [(η5-C5H5)Fe(CO)2(µ-PPh2(CH2)nPPh2))(η6-p-cymene)RuCl2]{PF6} (n = 2-5) (compounds 7-10), were synthesized and characterised. The mononuclear complexes were moderately cytotoxic against two ovarian cancer cell lines (A2780 and cisplatin resistant A2780cis) with IC50 values ranging from 2.3 ± 0.5 µM to 9.0 ± 1.4 µM. For 7-10, the cytotoxicity increased with increasing Fe⋅⋅⋅Ru distance, consistent with their DNA affinity. UV-visible spectroscopy suggested the chloride ligands in heterodinuclear 8-10 undergo stepwise substitution by water on the timescale of the DNA interaction experiments, probably affording the species [RuCl(OH2)(η6-p-cymene)(PRPh2)]2+ and [Ru(OH)(OH2)(η6-p-cymene)(PRPh2)]2+ (where PRPh2 has R = [-(CH2)5PPh2-Fe(C5H5)(CO)2]+). One interpretation of the combined DNA-interaction and kinetic data is that the mono(aqua) complex may interact with dsDNA through nucleobase coordination. Heterodinuclear 10 reacts with glutathione (GSH) to form stable mono- and bis(thiolate) adducts, 10-SG and 10-SG2, with no evidence of metal ion reduction (k1 = 1.07 ± 0.17 × 10-1 min-1 and k2 = 6.04 ± 0.59 × 10-3 min-1 at 37 °C). This work highlights the synergistic effect of the Fe2+/Ru2+ centres on both the cytotoxicity and biomolecular interactions of the present heterodinuclear complexes.

High-resolution ion mobility spectrometry-mass spectrometry for isomeric separation of prostanoids after Girard's reagent T derivatization.

RATIONALE: Isomeric separation of prostanoids is often a challenge and requires chromatography and time-consuming sample preparation. Multiple prostanoid isomers have distinct in vivo functions crucial for understanding the inflammation process, including prostaglandins E2 (PGE2 ) and D2 (PGD2 ). High-resolution ion mobility spectrometry (IMS) based on linear ion transport in low-to-moderate electric fields and nonlinear ion transport in strong electric fields emerges as a broad approach for rapid separations prior to mass spectrometry. METHODS: Derivatization with Girard's reagent T (GT) was used to overcome inefficient ionization of prostanoids in negative ionization mode due to poor deprotonation of the carboxylic acid group. Three high-resolution IMS techniques, namely linear cyclic IMS, linear trapped IMS, and nonlinear high-field asymmetric waveform IMS, were compared for the isomeric separation and endogenous detection of prostanoids present in intestinal tissue. RESULTS: Direct infusion of GT-derivatized prostanoids proved to increase the ionization efficiency in positive ionization mode by a factor of >10, which enabled detection of these molecules in endogenous concentration levels. The high-resolution IMS comparison revealed its potential for rapid isomeric analysis of biologically relevant prostanoids. Strengths and weaknesses of both linear and nonlinear IMS are discussed. Endogenous prostanoid detection in intestinal tissue extracts demonstrated the applicability of our approach in biomedical research. CONCLUSIONS: The applied derivatization strategy offers high sensitivity and improved stereoisomeric separation for screening of complex biological systems. The high-resolution IMS comparison indicated that the best sensitivity and resolution are achieved by linear and nonlinear IMS, respectively.

Uncovering the behaviour of ions in the gas-phase to predict the ion mobility separation of isomeric steroid compounds.

Bile acids are steroid compounds involved in biological mechanisms of neurodegenerative diseases making them potential biomarkers for diagnosis or treatment. These compounds exist as structural and conformational isomers, which hinder distinguishing them in physiological processes. We aimed to develop tandem mass spectrometry-ion mobility spectrometry (MS/MS-IMS) methodologies to explore and understand the behaviour of isomeric steroids in the gas-phase and rapidly separate them. Unlike previously published ion mobility data, various isomers were investigated in mixtures to better mimic complex (pre-) clinical samples. The experimental collision cross sections (CCS)s were compared to the theoretical CCS values for an in-depth analysis of isomeric ions' behaviour in the gas-phase. Based on density-functional theory, we identified the impact of adduct positioning on the 3D conformation of enantiomers, diastereomers and structural isomers. The curling of the large side chains hedged the small differences among the isomers and lowered the CCS values. On the other hand, fragmenting off the identical side branches as well as imposing the bending of the steroid ring resulted in ion mobility differentiation. Careful data evaluation revealed the tendency of isomers to form homo-cluster in the mixture solutions and assist the separation. Our fundamental and experimental findings enable the ion mobility separation of isomeric steroids to be predicted. The introduced rapid and optimal MS/MS-IMS analytical methodology can be applied to distinguish isomeric bile acids both in a solution and potentially in patients' tissue samples, and consequently, reveal their molecular pathways.

Cholecalciferol as Bioactive Plasticizer of High Molecular Weight Poly(D,L-Lactic Acid) Scaffolds for Bone Regeneration.

Synthetic thermoplastic polymers are a widespread choice as material candidates for scaffolds for tissue engineering (TE), thanks to their ease of processing and tunable properties with respect to biological polymers. These features made them largely employed in melt-extrusion-based additive manufacturing, with particular application in hard-TE. In this field, high molecular weight (Mw) polymers ensuring entanglement network strength are often favorable candidates as scaffold materials because of their enhanced mechanical properties compared with lower Mw grades. However, this is accompanied by high viscosities once processed in molten conditions, which requires driving forces not always accessible technically or compatible with often chemically nonstabilized biomedical grades. When possible, this is circumvented by increasing the operating temperature, which often results in polymer chain scission and consequent degradation of properties. In addition, synthetic polymers are mostly considered bioinert compared with biological materials, and additional processing steps are often required to make them favorable for tissue regeneration. In this study, we report the plasticization of a common thermoplastic polymer with cholecalciferol, the metabolically inactive form of vitamin D3 (VD3). Plasticization of the polymer allowed us to reduce its melt viscosity, and therefore the energy requirements (mechanical [torque] and heat [temperature]) for extrusion, limiting ultimately polymer degradation. In addition, we evaluated the effect of cholecalciferol, which is more easily available than its active counterpart, on the osteogenic differentiation of human mesenchymal stromal cells (hMSCs). Results indicated that cholecalciferol supported osteogenic differentiation more than the osteogenic culture medium, suggesting that hMSCs possess the enzymatic toolbox for VD3 metabolism. Impact statement Limitations in mechanical and biological performances of scaffolds manufactured through melt deposition may result from material thermal degradation during processing and inherent bioinertness of synthetic polymers. Current approaches involve the incorporation of chemical additives to reduce the extent of thermal degradation, which are often nonbiocompatible or may lead to uncontrolled modifications to the polymer structure. Lack of polymer bioactivity is tackled by postfunctionalization methods that often involve extra processes extending scaffold production time. Therefore, new methods to improve scaffolds performances should consider preserving the integrity of the molecular structure and improving biological responsiveness of the material while keeping the process as straightforward as possible.

The impact of deep versus standard neuromuscular block on intraoperative safety during laparoscopic surgery: an international multicenter randomized controlled double-blind strategy trial - EURO-RELAX TRIAL.

BACKGROUND: Muscle relaxants are routinely used during anesthesia to facilitate endotracheal intubation and to optimize surgical conditions. However, controversy remains about the required depth of neuromuscular block (NMB) needed for optimal surgical working conditions and how this relates to other outcomes. For instance, a deep neuromuscular block yields superior surgical working conditions compared to a standard NMB in laparoscopic surgery, however, a robust association to other (safety) outcomes has not yet been established. METHODS: Trial design: an international multicenter randomized controlled double-blind strategy trial. Trial population: 922 patients planned for elective, laparoscopic or robotic, abdominal surgery. INTERVENTION: Patients will be randomized to a deep NMB (post-tetanic count 1-2 twitches) or standard care (single-dose muscle relaxant administration at induction and repeated only if warranted by surgical team). Main trial endpoints: Primary endpoint is the difference in incidence of intraoperative adverse events during laparoscopic surgery graded according to ClassIntra® classification (i.e., ClassIntra® grade ≥ 2) between both groups. Secondary endpoints include the surgical working conditions, 30-day postoperative complications, and patients' quality of recovery. DISCUSSION: This trial was designed to analyze the effect of deep neuromuscular block compared to standard neuromuscular block on intra- and postoperative adverse events in patients undergoing laparoscopic surgery. TRIAL REGISTRATION: ClinicalTrials.gov NCT04124757 (EURO-RELAX); registration URL: https://clinicaltrials.gov/ct2/show/NCT04124757 , registered on October 11th, 2019.

Cholinergic Chemotransmission and Anesthetic Drug Effects at the Carotid Bodies.

General anesthesia is obtained by administration of potent hypnotics, analgesics and muscle relaxants. Apart from their intended effects (loss of consciousness, pain relief and muscle relaxation), these agents profoundly affect the control of breathing, in part by an effect within the peripheral chemoreflex loop that originates at the carotid bodies. This review assesses the role of cholinergic chemotransmission in the peripheral chemoreflex loop and the mechanisms through which muscle relaxants and hypnotics interfere with peripheral chemosensitivity. Additionally, consequences for clinical practice are discussed.

Reduced postoperative pain using Nociception Level-guided fentanyl dosing during sevoflurane anaesthesia: a randomised controlled trial.

BACKGROUND: The majority of postoperative patients report moderate to severe pain, possibly related to opioid underdosing or overdosing during surgery. Objective guidance of opioid dosing using the Nociception Level (NOL) index, a multiparameter artificial intelligence-driven index designed to monitor nociception during surgery, may lead to a more appropriate analgesic regimen, with effects beyond surgery. We tested whether NOL-guided opioid dosing during general anaesthesia results in less postoperative pain. METHODS: In this two-centre RCT, 50 patients undergoing abdominal surgery under fentanyl/sevoflurane anaesthesia were randomised to NOL-guided fentanyl dosing or standard care in which fentanyl dosing was based on haemodynamics. The primary endpoint of the study was postoperative pain assessed in the PACU. RESULTS: Median postoperative pain scores were 3.2 (inter-quartile range 1.3-4.3) and 4.8 (3.0-5.3) in NOL-guided and standard care groups, respectively (P=0.006). Postoperative morphine consumption (standard deviation) was 0.06 (0.07) mg kg-1 (NOL-guided group) and 0.09 (0.09) mg kg-1 (control group; P=0.204). During surgery, fentanyl dosing was not different between groups (NOL-guided group: 6.4 [4.2] µg kg-1vs standard care: 6.0 [2.2] µg kg-1, P=0.749), although the variation between patients was greater in the NOL-guided group (% coefficient of variation 66% in the NOL-guided group vs 37% in the standard care group). CONCLUSIONS: Despite absence of differences in fentanyl and morphine consumption during and after surgery, a 1.6-point improvement in postoperative pain scores was observed in the NOL-guided group. We attribute this to NOL-driven rather than BP- and HR-driven fentanyl dosing during anaesthesia. CLINICAL TRIAL REGISTRATION: www.trialregister.nl under identifier NL7845.

Probing Protein Denaturation during Size-Exclusion Chromatography Using Native Mass Spectrometry.

Size-exclusion chromatography employing aqueous mobile phases with volatile salts at neutral pH combined with electrospray-ionization mass spectrometry (SEC-ESI-MS) is a useful tool to study proteins in their native state. However, whether the applied eluent conditions actually prevent protein-stationary phase interactions, and/or protein denaturation, often is not assessed. In this study, the effects of volatile mobile phase additives on SEC retention and ESI of proteins were thoroughly investigated. Myoglobin was used as the main model protein, and eluents of varying ionic strength and pH were applied. The degree of interaction between protein and stationary phase was evaluated by calculating the SEC distribution coefficient. Protein-ion charge state distributions obtained during offline and online native ESI-MS were used to monitor alterations in protein structure. Interestingly, most of the supposedly mild eluent compositions induced nonideal SEC behavior and/or protein unfolding. SEC experiments revealed that the nature, ionic strength, and pH of the eluent affected protein retention. Protein-stationary phase interactions were effectively avoided using ammonium acetate at ionic strengths above 0.1 M. Direct-infusion ESI-MS showed that the tested volatile eluent salts seem to follow the Hofmeister series: no denaturation was induced using ammonium acetate (kosmotropic), whereas ammonium formate and bicarbonate (both chaotropic) caused structural changes. Using a mobile phase of 0.2 M ammonium acetate (pH 6.9), several proteins (i.e., myoglobin, carbonic anhydrase, and cytochrome c) could be analyzed by SEC-ESI-MS using different column chemistries without compromising their native state. Overall, with SEC-ESI-MS, the effect of nonspecific interactions between protein and stationary phase on the protein structure can be studied, even revealing gradual structural differences along a peak.

TMTHSI, a superior 7-membered ring alkyne containing reagent for strain-promoted azide-alkyne cycloaddition reactions.

We describe the development of TMTH-SulfoxImine (TMTHSI) as a superior click reagent. This reagent combines a great reactivity, with small size and low hydrophobicity and compares outstandingly with existing click reagents. TMTHSI can be conveniently functionalized with a variety of linkers allowing attachment of a diversity of small molecules and (peptide, nucleic acid) biologics.

The Crystalline Sponge Method in Water.

The crystalline sponge method entails the elucidation of the (absolute) structure of molecules from a solution phase using single-crystal X-ray diffraction and eliminates the need for crystals of the target compound. An important limitation for the application of the crystalline sponge method is the instability of the available crystalline sponges that can act as host crystals. The host crystal that is most often used decomposes in protic or nucleophilic solvents, or when guest molecules with Lewis basic substituents are introduced. Here a new class of (water) stable host crystals based on f-block metals is disclosed. It can be shown that these hosts not only increase the scope of the crystalline sponge method to a wider array of solvents and guests, but that they can even be applied to aqueous solutions containing hydrophilic guest molecules, thereby extending the crystalline sponge method to the important field of water-based chemistry.

Adduct ion formation as a tool for the molecular structure assessment of ten isomers in traveling wave and trapped ion mobility spectrometry.

RATIONALE: The separation of isomeric compounds with major differences in their physiochemical and pharmacokinetic properties is of particular importance in pharmaceutical R&D. However, the structural assessment and separation of these compounds with current analytical techniques and methods are still a challenge. In this study, we describe strategies to separate the various structural and stereo-isomers. METHODS: The separation of ten structural and stereo-isomers was investigated using Trapped and Travelling Wave ion mobility spectrometry (TIMS and TWIMS). Different strategies including adduct ion formation with Na, Li, Ag and Cs as well as fragmentation before and after the ion mobility cell were applied to separate the isomeric compounds. RESULTS: All the counter ions (in particular Na) strongly coordinated with the test analytes in all the IMS systems. The highest resolving power was achieved for the sodium and lithium adducts using TIMS-time-of-flight (TOF). However, some separation was attained on a Synapt HDMS system with its unique potential to monitor the ion mobility of the product ions. The elution order of the adduct ions was the same in all instruments, in which, unexpectedly, the para-substituted isomer of the [M + Na]+ species had the lowest collision cross section followed by the meta- and ortho-isomers. CONCLUSIONS: The formation of adduct ions could facilitate the separation of structural and even stereo-isomers by generating different molecular conformations. In addition, fragmenting isomers before or after the ion mobility cell is a valuable strategy to separate and also to assess the structures of adducts and different conformers.

Racemic and Enantiopure Camphene and Pinene Studied by the Crystalline Sponge Method.

The use of an achiral metal-organic framework for structure determination of chiral compounds is demonstrated for camphene and pinene. The structure of enantiopure ß-pinene can be resolved using the crystalline sponge method. However, α-pinene cannot be resolved using enantiopure material alone because no ordering of guest molecules takes place in that case. Interestingly, enantiomeric pairs order inside the channels of the host framework when impure (+)-camphene is offered to the host, which is also the case when a racemic mixture of α-pinene is used. A mixture of (+)-α-pinene and (-)-ß-pinene also leads to ordered incorporation in the host, showing the influence of the presence of an inversion center in the host framework. We further show that powder X-ray diffraction provides a direct view on incorporation of ordered guest molecules. This technique, therefore, provides a way to determine the optimal and/or minimal soaking time. In contrast, color change of the crystal only demonstrates guest uptake, not ordering. Moreover, we show that color change can also be caused by guest-induced host degradation.

Nanofractionation Platform with Parallel Mass Spectrometry for Identification of CYP1A2 Inhibitors in Metabolic Mixtures.

With early assessment of inhibitory properties of drug candidates and their circulating metabolites toward cytochrome P450 enzymes, drug attrition, especially later in the drug development process, can be decreased. Here we describe the development and validation of an at-line nanofractionation platform, which was applied for screening of CYP1A2 inhibitors in Phase I metabolic mixtures. With this platform, a metabolic mixture is separated by liquid chromatography (LC), followed by parallel nanofractionation on a microtiter well plate and mass spectrometry (MS) analysis. After solvent evaporation, all metabolites present in the nanofractionated mixture are assayed utilizing a fluorescence CYP1A2 inhibition bioassay performed on the plate. Next, a bioactivity chromatogram is constructed from the bioassay results. By peak shape and retention time correlation of the bioactivity peaks with the obtained MS data, CYP1A2-bioactive inhibiting metabolites can be identified. The method correctly evaluated the potency of five CYP1A2 inhibitors. Mixtures comprising potent inhibitors of CYP1A2 or in vitro-generated metabolites of ellipticine were evaluated for their inhibitory bioactivities. In both cases, good LC separation of all compounds was achieved and bioactivity data could be accurately correlated with the parallel recorded MS data. Generation and evaluation of Phase II metabolites of hydroxylated ellipticine was also pursued.