An Integrated Approach toward NanoBRET Tracers for Analysis of GPCR Ligand Engagement

Gaining insight into the pharmacology of ligand engagement with G-protein coupled receptors (GPCRs) under biologically relevant conditions is vital to both drug discovery and basic research. NanoLuc-based bioluminescence resonance energy transfer (NanoBRET) monitoring competitive binding between fluorescent tracers and unmodified test compounds has emerged as a robust and sensitive method to quantify ligand engagement with specific GPCRs genetically fused to NanoLuc luciferase or the luminogenic HiBiT peptide. However, development of fluorescent tracers is often challenging and remains the principal bottleneck for this approach. One way to alleviate the burden of developing a specific tracer for each receptor is using promiscuous tracers, which is made possible by the intrinsic specificity of BRET. Here, we devised an integrated tracer discovery workflow that couples machine learning-guided in silico screening for scaffolds displaying promiscuous binding to GPCRs with a blend of synthetic strategies to rapidly generate multiple tracer candidates. Subsequently, these candidates were evaluated for binding in a NanoBRET ligand-engagement screen across a library of HiBiT-tagged GPCRs. Employing this workflow, we generated several promiscuous fluorescent tracers that can effectively engage multiple GPCRs, demonstrating the efficiency of this approach. We believe that this workflow has the potential to accelerate discovery of NanoBRET fluorescent tracers for GPCRs and other target classes.     

Dechlorination of chloroethylenes by cob(I)alamin and cobalamin model complexes

Cobalt-mediated dehalogenation reactions, specifically those that employ cobalamin, have attracted particular attention because these complexes rapidly degrade tetrachloroethylene (PCE) and trichloroethylene (TCE), which are common groundwater contaminants. Although questions remain about the relative importance of several pathways, both radicals and organometallic intermediates, specifically chlorovinyl complexes, play an important role in these processes. This Perspective highlights recent studies focused on elucidating the mechanism of chloroethylene degradation, including experimental studies on PCE and TCE dechlorination, computational studies, preparation of model complexes, and the study of model catalytic systems.     

An Experimental Method to Measure Gaseous Diffusivity in Tight and Partially Saturated Porous Media via Continuously Monitored Mass Spectrometry

Transport in Porous Media -     

The Spin‐Forbidden Reaction of Ground‐State Sulfur Atoms with Ethylene

Ground‐state S‐atoms (³P_J) were generated by pulsed laser photolysis of carbonyl sulfide (OCS) precursor and monitored by time‐resolved resonance fluorescence. The kinetics were studied over the temperature range of 291–1052 K. Below 900 K, the effective bimolecular rate constant k₁ was found to be independent of pressure and also to be in good accord with prior measurements made at 442 K and below. At higher temperatures, fall‐off curves were characterized. These demonstrate that the reaction is dominated by addition. The high‐pressure limit is summarized as k_∞ = 1.5 × 10^?11 exp(?8.4 kJ mol^?1/RT) cm³ molecule^?1 s^?1. The low‐pressure limiting rate constant is also obtained. The observation of formation of one or more adducts even at ～1000 K constrains their thermochemistry, and comparison with the computed reaction enthalpies for various candidates in the literature shows the addition products can only be accessed via intersystem crossing. Estimated Rice–Ramsperger–Kassel–Marcus (RRKM) addition rate constants at the low‐pressure limit for formation of vinylthiol and ethanethial are in accord with the observed kinetics assuming that the collisional efficiency near 1000 K is about 0.1.     

Electrospray ionization-voltage sweep-Ion mobility spectrometry for biomolecules and complex samples

Voltage Sweep Ion Mobility Spectrometry (VSIMS) has been applied to complex samples using electrospray ionization (ESI). The usable range of VSIMS has been extended from that obtained in previous studies where only volatile compounds were investigated. Using ESI, VSIMS was evaluated with compounds with reduced mobility values as low as 0.3 V²cm^?1 s^?1. The primary advantage of VSIMS is to enable a drift time ion mobility spectrometer (DTIMS) to detect both fast and slow moving ions at optimal resolving power, thus improving the peak capacity. In this work ESI-VSIMS was applied to a series of small peptides and drugs spanning a large range of reduced mobility values in order to demonstrate ESI-VSIMS to separation. To demonstrate improved peak capacity of IMS with voltage scan operation, oligomers of silicone oil provided a series of evenly-spaced peaks, ranging in reduced mobility values from 0.85 to 0.3 V²cm^?1 s^?1. The peak capacity of 61 for a standard IMS was improved to 102 when voltage sweep operation was employed. In addition, VSIMS increased the average resolving power of the DTIMS from 66 to 106 for silicone oil.     

Rigidity for Nonnegatively Curved Metrics on S 2 × R3

We address the question: how large is the family of complete metricswith nonnegative sectional curvature on S ² × R³? We classify theconnection metrics, and give several examples of nonconnection metrics.We provide evidence that the family is small by proving some rigidityresults for metrics more general than connection metrics.

     

MALDI ionization mechanisms investigated by comparison of isomers of dihydroxybenzoic acid

Matrix‐assisted laser desorption/ionization (MALDI) ion formation mechanisms were investigated by comparison of isomers of dihydroxybenzoic acid (DHB). These exhibit substantially different MALDI performance, the basis for which was not previously understood. Luminescence decay curves are used here to estimate excited electronic state properties relevant for the coupled chemical and physical dynamics (CPCD) model. With these estimates, the CPCD predictions for relative total ion and analyte ion yields are in good agreement with the data for the DHB isomers. Predictions of a thermal equilibrium model were also compared and found to be incompatible with the data. Copyright © 2015 John Wiley & Sons, Ltd.     

E/Z Oxime Isomerism in PhC(NOH)CN

The reaction of nitric oxide with benzyl cyanide in the presence of potassium methoxide at low temperature gave the dipotassium salt of a bis‐diazeniumdiolate 2 in excellent yield. Two new stereospecific syntheses of E or Z 2‐(hydroxyimino)‐2‐phenylacetonitrile from 2 have been found. The thermodynamics of the E/Z isomerization has been investigated spectroscopically in solution, in the solid state by differential scanning calorimetry (DSC), and theoretically in the gas phase. Evidence of catalysis by NO of E/Z oxime isomerization has been observed.     

The complex relationship between guest-free polymorphic products and desolvation of p-tert-butylcalix[4]arene inclusion compounds

Depending on the details of the technique employed, desolvation of tBC inclusion compounds leads to either a dense, guest-free polymorph, or a guest-free low density polymorph, the latter also having distinct high and low temperature forms.     

Thin-film solid-state proton NMR measurements using a synthetic mica substrate: Polymer blends

Solid-state proton nuclear magnetic resonance (NMR) measurements are performed successfully on polymer blend thin films through the use of synthetic mica as a substrate. When used as a substrate, synthetic fluorophlogopite mica with its proton-free, diamagnetic character, allows for adequate measurement sensitivity while minimally perturbing the proton thin-film spectra, especially relative to more commonly available natural micas. Specifically, we use multiple-pulse techniques in the presence of magic-angle spinning to measure the degree of mixing in two different polymer blend thin films, polystyrene/poly(xylylene ether) and poly(1-methyladamantyl methacrylate) (PMAdMA)/triphenylsulfonium perfluorobutanesulfonate (TPS-PFBS), spin-coated onto mica substrates. Our earlier studies had focused on bulk systems where NMR signals are stronger, but may not be representative of thin films of the same systems that are relevant to many applications such as photoresist formulations in the electronics industry. The superiority of synthetic over natural paramagnetic mica is demonstrated by the maintenance of resolution and spinning sideband intensities (relative to bulk samples) for the synthetic mica samples. In contrast, degraded resolution and large spinning sidebands are shown to typify spectra of the natural mica samples. This approach can be applied to many other proton measurements of solid thin films, thereby greatly extending the types of systems to be investigated. Magnetic susceptibility measurements are also reported for all micas used.