Synthesis and Biological Evaluation of Novel MB327 Analogs as Resensitizers for Desensitized Nicotinic Acetylcholine Receptors after Intoxication with Nerve Agents.

Poisoning with organophosphorus compounds, which can lead to a cholinergic crisis due to the inhibition of acetylcholinesterase and the subsequent accumulation of acetylcholine (ACh) in the synaptic cleft, is a serious problem for which treatment options are currently insufficient. Our approach to broadening the therapeutic spectrum is to use agents that interact directly with desensitized nicotinic acetylcholine receptors (nAChRs) in order to induce functional recovery after ACh overstimulation. Although MB327, one of the most prominent compounds investigated in this context, has already shown positive properties in terms of muscle force recovery, this compound is not suitable for use as a therapeutic agent due to its insufficient potency. By means of in silico studies based on our recently presented allosteric binding pocket at the nAChR, i.e. the MB327-PAM-1 binding site, three promising MB327 analogs with a 4-aminopyridinium ion partial structure (PTM0056, PTM0062 and PTM0063) were identified. In this study, we present the synthesis and biological evaluation of a series of new analogs of the aforementioned compounds with a 4-aminopyridinium ion partial structure (PTM0064-PTM0072), as well as hydroxy-substituted analogs of MB327 (PTMD90-0012 and PTMD90-0015) designed to substitute entropically unfavorable water clusters identified during molecular dynamics simulations. The compounds were characterized in terms of their binding affinity towards the aforementioned binding site by applying the UNC0642 MS Binding Assays and in terms of their muscle force reactivation in rat diaphragm myography. More potent compounds were identified compared to MB327, as some of them showed a higher affinity towards MB327-PAM-1 and also a higher recovery of neuromuscular transmission at lower compound concentrations. To improve the treatment of organophosphate poisoning, direct targeting of nAChRs with appropriate compounds is a key step, and this study is an important contribution to this research.

The use of bispyridinium non-oxime analogues for the restoration of nerve agent impaired neuromuscular transmission in rat hemidiaphragms - Structure optimization.

Organophosphate pesticide poisoning challenges health care systems worldwide. Furthermore, nerve agents remain a continuous threat. The treatment options for organophosphate poisoning have virtually been unchanged for decades, relying on symptomatic treatment and the use of oximes to indirectly restore neuromuscular function. Hence, compounds targeting directly nicotinic acetylcholine receptors (nAChRs) might substantially improve treatment options. The current study investigated a series of bispyridinium analogues with a trimethylene or 2,2'-diethyloxy linker in a rat hemidiaphragm model, using indirect field stimulation. Methyl- and ethyl-substituted bispyridinium analogues restored neuromuscular function up to 37 ± 17% (MB419, a 3-methyl analogue) at a stimulation frequency of 20 Hz. The bispyridinium analogues with a 2- or 3-methyl group, or a 2- or 3-ethyl group, tended towards a higher restoration of neuromuscular function than those with a 4-methyl or 4-ethyl group, respectively. The current data can be used for future studies to optimize structure-based molecular modeling of compounds targeting the nAChR.

Identification of ligands binding to MB327-PAM-1, a binding pocket relevant for resensitization of nAChRs.

Desensitization of nicotinic acetylcholine receptors (nAChRs) can be induced by overstimulation with acetylcholine (ACh) caused by an insufficient degradation of ACh after poisoning with organophosphorus compounds (OPCs). Currently, there is no generally applicable treatment for OPC poisoning that directly targets the desensitized nAChR. The bispyridinium compound MB327, an allosteric modulator of nAChR, has been shown to act as a resensitizer of nAChRs, indicating that drugs binding directly to nAChRs can have beneficial effects after OPC poisoning. However, MB327 also acts as an inhibitor of nAChRs at higher concentrations and can thus not be used for OPC poisoning treatment. Consequently, novel, more potent resensitizers are required. To successfully design novel ligands, the knowledge of the binding site is of utmost importance. Recently, we performed in silico studies to identify a new potential binding site of MB327, MB327-PAM-1, for which a more affine ligand, UNC0646, has been described. In this work, we performed ligand-based screening approaches to identify novel analogs of UNC0646 to help further understand the structure-affinity relationship of this compound class. Furthermore, we used structure-based screenings and identified compounds representing four new chemotypes binding to MB327-PAM-1. One of these compounds, cycloguanil, is the active metabolite of the antimalaria drug proguanil and shows a higher affinity towards MB327-PAM-1 than MB327. Furthermore, cycloguanil can reestablish the muscle force in soman-inhibited rat muscles. These results can act as a starting point to develop more potent resensitizers of nAChR and to close the gap in the treatment after OPC poisoning.

Emission Spectroscopy-Based Sensor System to Correlate the In-Cylinder Combustion Temperature of a Diesel Engine to NOx Emissions.

Due to a rising importance of the reduction of pollutant, produced by conventional energy technologies, the knowledge of pollutant forming processes during a combustion is of great interest. In this study the in-cylinder temperature, of a near series diesel engine, is examined with a minimal invasive emission spectroscopy sensor. The soot, nearly a black body radiator, emits light, which is spectrally detected and evaluated with a modified function of Planck's law. The results show a good correlation between the determined temperatures and the NOx concentration, measured in the exhaust gas of the engine, during a variety of engine operating points. A standard deviation between 25 K and 49 K was obtained for the in-cylinder temperature measurements.

Restoration of nerve agent impaired neuromuscular transmission in rat diaphragm by bispyridinium non-oximes - Structure-activity relationships.

Organophosphate (OP) poisoning is currently treated with atropine, oximes and benzodiazepines. The nicotinic signs, i.e., respiratory impairment, can only be targeted indirectly via the use of oximes as reactivators of OP-inhibited acetylcholinesterase. Hence, compounds selectively targeting nicotinic acetylcholine receptors (nAChRs) might fundamentally improve current treatment options. The bispyridinium compound MB327 has previously shown some therapeutic effect against nerve agents in vitro and in vivo. Nevertheless, compound optimization was deemed necessary, due to limitations (e.g., toxicity and efficacy). The current study investigated a series of 4-tert-butyl bispyridinium compounds and of corresponding bispyridinium compounds without substituents in a rat diaphragm model using an indirect field stimulation technique. The length of the respective linker influenced the ability of the bispyridinium compounds to restore muscle function in rat hemidiaphragms. The current data show structure-activity relationships for a series of bispyridinium compounds and provide insight for future structure-based molecular modeling.

Screening for new ligands of the MB327-PAM-1 binding site of the nicotinic acetylcholine receptor.

Intoxications with organophosphorus compounds (OPCs) effect a severe impairment of cholinergic neurotransmission that, as a result of overstimulation may lead to desensitization of nicotinic acetylcholine receptors (nAChRs) and finally to death due to respiratory paralysis. So far, therapeutics, that are capable to address and revert desensitized neuromuscular nAChRs into their resting, i.e. functional state are still missing. Still, among a class of compounds termed bispyridinium salts, which are characterized by the presence of two pyridinium subunits, constituents have been identified, that can counteract organophosphate poisoning by resensitizing desensitized nAChRs. According to comprehensive modeling studies this effect is mediated by an allosteric binding site at the nAChR termed MB327-PAM-1 site. For MB327, the most prominent representative of the bispyridinium salts and all other analogues studied so far, the affinity for the aforementioned binding site and the intrinsic activity measured in ex vivo and in in vivo experiments are distinctly too low, to meet the criteria to be fulfilled for therapeutic use. Hence, in order to identify new compounds with higher affinities for the MB327-PAM-1 binding site, as a basic requirement for an enhanced potency, two compound libraries, the ChemDiv library with 60 constituents and the Tocriscreen Plus library with 1280 members have been screened for hit compounds addressing the MB327-PAM-1 binding site, utilizing the [2H6]MB327 MS Binding Assay recently developed by us. This led to the identification of a set of 10 chemically diverse compounds, all of which exhibit an IC50 value of ≤ 10 µM (in the [2H6]MB327 MS Binding Assay), which had been defined as selection criteria. The three most affine ligands, which besides a quinazoline scaffold share similarities with regard to the substitution pattern and the nature of the substituents, are UNC0638, UNC0642 and UNC0646. With binding affinities expressed as pKi values of 6.01 ± 0.10, 5.97 ± 0.05 and 6.23 ± 0.02, respectively, these compounds exceed the binding affinity of MB327 by more than one log unit. This renders them promising starting points for the development of drugs for the treatment of organophosphorus poisoning by addressing the MB327-PAM-1 binding site of the nAChR.

MS Binding Assays with UNC0642 as reporter ligand for the MB327 binding site of the nicotinic acetylcholine receptor.

Intoxications with organophosphorus compounds (OPCs) based chemical warfare agents and insecticides may result in a detrimental overstimulation of muscarinic and nicotinic acetylcholine receptors evolving into a cholinergic crisis leading to death due to respiratory failure. In the case of the nicotinic acetylcholine receptor (nAChR), overstimulation leads to a desensitization of the receptor, which cannot be pharmacologically treated so far. Still, compounds interacting with the MB327 binding site of the nAChR like the bispyridinium salt MB327 have been found to re-establish the functional activity of the desensitized receptor. Only recently, a series of quinazoline derivatives with UNC0642 as one of the most prominent representatives has been identified to address the MB327 binding site of the nAChR, as well. In this study, UNC0642 has been utilized as a reporter ligand to establish new Binding Assays for this target. These assays follow the concept of MS Binding Assays for which by assessing the amount of bound reporter ligand by mass spectrometry no radiolabeled material is required. According to the results of the performed MS Binding Assays comprising saturation and competition experiments it can be concluded, that UNC0642 used as a reporter ligand addresses the MB327 binding site of the Torpedo-nAChR. This is further supported by the outcome of ex vivo studies carried out with poisoned rat diaphragm muscles as well as by in silico studies predicting the binding mode of UNC0646, an analog of UNC0642 with the highest binding affinity, in the recently proposed binding site of MB327 (MB327-PAM-1). With UNC0642 addressing the MB327 binding site of the Torpedo-nAChR, this and related quinazoline derivatives represent a promising starting point for the development of novel ligands of the nAChR as antidotes for the treatment of intoxications with organophosphorus compounds. Further, the new MS Binding Assays are a potent alternative to established assays and of particular value, as they do not require the use of radiolabeled material and are based on a commercially available compound as reporter ligand, UNC0642, exhibiting one of the highest binding affinities for the MB327 binding site known so far.

Two-color four-wave mixing contribution to an electrostrictive laser-induced grating signal in CO2-N2 mixtures and gas diagnostics: publisher's note.

This publisher's note corrects content in Appl. Opt.62, 8115 (2023)APOPAI0003-693510.1364/AO.497467.

Two-color four-wave mixing contribution to an electrostrictive laser-induced grating signal in CO2-N2 mixtures and gas diagnostics.

Multiparameter determination in the gas phase using the versatile laser-induced grating (LIG) technique is a challenging task due to interdependence of observables on multiple thermodynamic parameters. In C O 2-N 2 mixtures, simultaneous determination of species concentration and gas temperature can be achieved by using an additional C O 2 concentration-dependent contribution to the LIG signal, which appears if 1064 nm pump pulses are employed. This contribution can be attributed to a direct, quasi-resonant two-color four-wave mixing (TCFWM) of the pump and probe radiations in C O 2. A detailed study of the laser power and beam polarization, as well as mixture composition, pressure, and temperature dependencies of the TCFWM intensity relative to that of the LIG signal, allowed for the formulation of analytical relations enabling simultaneous mixture composition and temperature determination.

Recombinant cellular model system for human muscle-type nicotinic acetylcholine receptor α12ß1δε.

The human muscle-type nicotinic acetylcholine receptor α12ß1δε (nAChR) is a complex transmembrane receptor needed for drug screening for disorders like congenital myasthenic syndromes and multiple pterygium syndrome. Until today, most models are still using the nAChR from Torpedo californica electric ray. A simple reproducible cellular system expressing functional human muscle-type nAChR is still missing. This study addressed this issue and further tested the hypothesis that different chaperones, both biological and chemical, and posttranslational modification supporting substances as well as hypothermic incubation are able to increase the nAChR yield. Therefore, Gibson cloning was used to generate transfer plasmids carrying the sequence of nAChR or chosen biological chaperones to support the nAChR folding in the cellular host. Viral transduction was used for stable integration of these transgenes in Chinese hamster ovary cells (CHO). Proteins were detected with Western blot, in-cell and on-cell Western, and the function of the receptor with voltage clamp analysis. We show that the internalization of nAChR into plasma membranes was sufficient for detection and function. Additional transgenic overexpression of biological chaperones did result in a reduced nAChR expression. Chemical chaperones, posttranslational modification supporting substances, and hypothermic conditions are well-suited supporting applications to increase the protein levels of different subunits. This study presents a stable and functional cell line that expresses human muscle-type nAChR and yields can be further increased using the chemical chaperone nicotine without affecting cell viability. The simplified access to this model system should enable numerous applications beyond drug development. Graphical abstract created with http://biorender.com.

Laser Applications to Chemical, Security, and Environmental Analysis: introduction to the feature issue.

The eighteenth topical meeting on Laser Applications to Chemical, Security, and Environmental Analysis (LACSEA) was held in Vancouver, Canada from 11-15 July 2022, as part of the Optica Optical Sensors and Sensing Congress in a hybrid format allowing on-site and online attendance. The meeting featured a broad range of distinguished papers focusing on recent advances in laser and optical spectroscopy. A total of 52 contributed and invited papers were presented during the meeting, including topics such as photo-acoustic spectroscopy, imaging, non-linear technologies, frequency combs, remote sensing, environmental monitoring, aerosols, combustion diagnostics, hypersonic flow diagnostics, nuclear diagnostics, fs/ps applications, and machine learning and computational sensing.

A novel binding site in the nicotinic acetylcholine receptor for MB327 can explain its allosteric modulation relevant for organophosphorus-poisoning treatment.

Organophosphorus compounds (OPCs) are highly toxic compounds that can block acetylcholine esterase (AChE) and thereby indirectly lead to an overstimulation of muscarinic and nicotinic acetylcholine receptors (nAChRs). The current treatment with atropine and AChE reactivators (oximes) is insufficient to prevent toxic effects, such as respiratory paralysis, after poisonings with various OPCs. Thus, alternative treatment options are required to increase treatment efficacy. Novel therapeutics, such as the bispyridinium non-oxime MB327, have been found to reestablish neuromuscular transmission by interacting directly with nAChR, probably via allosteric mechanisms. To rationally design new, more potent drugs addressing nAChR, knowledge of the binding mode of MB327 is fundamental. However, the binding pocket of MB327 has remained elusive. Here, we identify a new potential allosteric binding pocket (MB327-PAM-1) of MB327 located at the transition of the extracellular to the transmembrane region using blind docking experiments and molecular dynamics simulations. MB327 forms striking interactions with the receptor at this site. The interacting amino acids are highly conserved among different subunits and different species. Correspondingly, MB327 can interact with several nAChR subtypes from different species. We predict by rigidity analysis that MB327 exerts an allosteric effect on the orthosteric binding pocket and the transmembrane domain after binding to MB327-PAM-1. Furthermore, free ligand diffusion MD simulations reveal that MB327 also has an affinity to the orthosteric binding pocket, which agrees with recently published results that related bispyridinium compounds show inhibitory effects via the orthosteric binding site. The newly identified binding site allowed us to predict structural modifications of MB327, resulting in the more potent resensitizers PTM0062 and PTM0063.

Pilot Testing of a Nurse-Led Basic Symptom Self-management Support for Patients Receiving First-Line Systemic Outpatient Anticancer Treatment: A Cluster-Randomized Study (Symptom Navi Pilot Study).

BACKGROUND: The Symptom Navi Program (SNP) is a nurse-led intervention supporting basic symptom self-management for patients with any cancer diagnosis. The SNP has been accepted by patients and healthcare professionals alike. OBJECTIVE: The aims of this study were to pilot the SNP and evaluate patient-reported symptom outcomes, nursing support for symptom management, and patient safety. METHODS: Using a cluster-randomized design, we randomized centers to the intervention (SNP) or control group (usual care). Adult patients starting first-line systemic cancer treatment were included. The primary outcome was the change in symptom interference with daily functions from treatment onset to 16 weeks. Secondary outcomes included changes in symptom severity, symptom burden, self-efficacy, and perceived symptom management support and patient safety. We used linear and logistic mixed-effects models to pilot-test differences in mean changes between groups. The trial was registered with ClinicalTrials.gov (NCT03649984). RESULTS: Changes in symptom interference with daily functions did not differ (mean difference at 16 weeks: -0.50; 95% confidence interval, -1.38 to 0.38; P = 0.25) between SNP (3 centers, 49 patients) and control (5 centers, 85 patients) as for all other outcomes. No adverse events were reported. CONCLUSIONS: Our preliminary findings did not indicate an effect of the SNP on patient-reported symptom outcomes, self-efficacy, or symptom management support. Inadequate power and SNP components (eg, insufficient training, low number of follow-up consultations) may be attributed to the lack of an observed effect. IMPLICATIONS FOR PRACTICE: The SNP training content and intervention procedures merit reconsideration.

Improvement of the coherent model function for S-branch Raman linewidth determination in oxygen.

Determination of S-branch Raman linewidths of oxygen from picosecond time-domain pure rotational coherent anti-Stokes Raman spectroscopy (RCARS) measurements requires consideration of coherence beating. We present an optimization of the established model for fitting the coherence decay in oxygen, which leads to an improvement in Raman linewidth data quality, especially for the challenging small signal intensity and decay constant regime, enabling the application for low oxygen concentrations. Two modifications to the fitting procedure are discussed, which aim at reliably fitting the second coherence beat properly. These are evaluated statistically against simulated decay traces, and weighing the data by the inverse of the data magnitude gives the best agreement. The presented temperature dependent ${{\rm O}_2} {-} {{\rm O}_2}$ S-branch Raman linewidth from the modified model shows an improved data quality over the original model function for all studied temperatures. ${{\rm O}_2} {-} {{\rm N}_2}$ linewidths of oxygen in air for the temperature range from 295 K to 1900 K demonstrate applicability to small concentrations. Use of the determined RCARS ${{\rm O}_2} {-} {{\rm O}_2}$ S-branch linewidth instead of regularly used Q-branch derived linewidths leads to a lowering in evaluated RCARS temperature by about 21 K, thereby, a much better agreement with thermocouple measurements.

Temperature dependent determination of the S-branch Raman linewidths of oxygen and carbon dioxide in an oxyfuel relevant mixture.

The temperature dependence of the ${\rm O}_2$ and ${\rm CO}_2$ S-branch linewidths in a 30/70% ${\rm O}_2 - {\rm CO}_2$ mixture between 295 K and 1900 K has been studied by a picosecond time-resolved pure rotational coherent anti-Stokes Raman scattering (RCARS) approach. The S-branch Raman linewidths are required for diagnostics of thermodynamic properties in oxyfuel combustion processes by RCARS, where this mixture is of special interest, because it is regularly used to replace air when transiting from air-fed to oxyfuel combustion. The obtained linewidths for oxygen and carbon dioxide show a strong deviation from pure self-broadened linewidths and previously used Q-branch linewidths, respectively. A discussion on the expected impact on RCARS thermometry and concentration evaluations as well as a description of specific properties of oxygen and carbon dioxide and their effect on the dephasing behavior of the Raman coherences and, thereby the Raman linewidths, is included, along tabulated linewidths data of both molecules.

Laser applications to chemical, security, and environmental analysis: introduction to the feature issue.

This Applied Optics feature issue on laser applications to chemical, security, and environmental analysis (LACSEA) highlights papers presented at the LACSEA 2020 Seventeenth Topical Meeting sponsored by The Optical Society (OSA).

CH and NO planar laser-induced fluorescence and Rayleigh-scattering in turbulent flames using a multimode optical parametric oscillator.

An optical parametric oscillator (OPO) is developed and characterized for the simultaneous generation of ultraviolet (UV) and near-UV nanosecond laser pulses for the single-shot Rayleigh scattering and planar laser-induced-fluorescence (PLIF) imaging of methylidyne (CH) and nitric oxide (NO) in turbulent flames. The OPO is pumped by a multichannel, 8-pulse Nd:YAG laser cluster that produces up to 225 mJ/pulse at 355 nm with pulse spacing of 100 µs. The pulsed OPO has a conversion efficiency of 9.6% to the signal wavelength of ∼430nm when pumped by the multimode laser. Second harmonic conversion of the signal, with 3.8% efficiency, is used for the electronic excitation of the A-X (1,0) band of NO at ∼215nm, while the residual signal at 430 nm is used for direct excitation of the A-X (0,0) band of the CH radical and elastic Rayleigh scattering. The section of the OPO signal wavelength for simultaneous CH and NO PLIF imaging is performed with consideration of the pulse energy, interference from the reactant and product species, and the fluorescence signal intensity. The excitation wavelengths of 430.7 nm and 215.35 nm are studied in a laminar, premixed CH4-H2-NH3-air flame. Single-shot CH and NO PLIF and Rayleigh scatter imaging is demonstrated in a turbulent CH4-H2-NH3 diffusion flame using a high-speed intensified CMOS camera. Analysis of the complementary Rayleigh scattering and CH and NO PLIF enables identification and quantification of the high-temperature flame layers, the combustion product zones, and the fuel-jet core. Considerations for extension to simultaneous, 10-kHz-rate acquisition are discussed.

Diagnostics and treatment of nerve agent poisoning-current status and future developments.

Although 193 states have committed to the Chemical Weapons Convention and 98% of the declared chemical weapons stockpiles have been destroyed so far, nerve agent poisoning remains a lingering threat. The recent dissemination of sarin in Syria, the assassination of Kim Jong-Nam in Malaysia, and the assault on Sergei Skripal in the United Kingdom underline the need for effective treatment. The current therapeutic options of a muscarinic receptor antagonist, an oxime, and an anticonvulsant have been unchanged for decades. Therefore, new therapeutic strategies, for example, bioscavengers and receptor-active substances, are promising concepts that have to be examined for their benefits and limitations. In order to facilitate rapid diagnosis in challenging clinical situations, point-of-care diagnostics and detection are of importance. Therapeutic guidance concerning the duration and success of the current oxime therapy via determination of the cholinesterase status can contribute to an optimal use of resources. In summary, the challenges of current and future therapies for nerve agent poisoning and key diagnostic devices will be discussed.

Impact of soman and acetylcholine on the effects of propofol in cultured cortical networks.

Patients intoxicated with organophosphorous compounds may need general anaesthesia to enable mechanical ventilation or for control of epileptiform seizures. It is well known that cholinergic overstimulation attenuates the efficacy of general anaesthetics to reduce spontaneous network activity in the cortex. However, it is not clear how propofol, the most frequently used intravenous anaesthetic today, is affected. Here, we investigated the effects of cholinergic overstimulation induced by soman and acetylcholine on the ability of propofol to depress spontaneous action potential activity in organotypic cortical slices measured by extracellular voltage recordings. Cholinergic overstimulation by co-application of soman and acetylcholine (10 µM each) did not reduce the relative inhibition of propofol (1.0 µM; mean normalized action potential firing rate 0.49 ± 0.06 of control condition, p < 0.001, Wilcoxon signed rank test) but clearly reduced its efficacy. Co-application of atropine (10 nM) did not improve the efficacy. Propofol preserved its relative inhibitory potential but did not produce a degree of neuronal depression which can be expected to assure hypnosis in humans. Since a combination with atropine did not improve its efficacy, an increase in dosage will probably be necessary when propofol is used in victims suffering from organophosphorous intoxication.

Determination of N2-N2 and N2-O2 S-branch Raman linewidths using time-resolved picosecond pure rotational coherent anti-Stokes Raman scattering.

N2-N2 and N2-O2 S-branch Raman linewidths have been determined using picosecond dual-broadband pure rotational coherent anti-Stokes Raman scattering (CARS). Time-resolved rotational CARS measurements were performed in gas-phase N2 and air for temperatures up to 1900 K in order to determine the time constants of the coherence decay to subsequently calculate the S-branch Raman linewidths. Coherence decay time traces and the resulting S-branch Raman linewidths are presented for N2-N2 and N2-O2 collisions. Therewith, we reduce the gap of widely missing S-branch linewidth data in the temperature regime of many combustion processes. Further, we demonstrate that the standard monoexponential fitting of the coherence decay, as it is commonly done for nitrogen, is not applicable to oxygen.