Binding kinetics drive G protein subtype selectivity at the ß1-adrenergic receptor.

G protein-coupled receptors (GPCRs) bind to different G protein α-subtypes with varying degrees of selectivity. The mechanism by which GPCRs achieve this selectivity is still unclear. Using 13C methyl methionine and 19F NMR, we investigate the agonist-bound active state of ß1AR and its ternary complexes with different G proteins in solution. We find the receptor in the ternary complexes adopts very similar conformations. In contrast, the full agonist-bound receptor active state assumes a conformation differing from previously characterised activation intermediates or from ß1AR in ternary complexes. Assessing the kinetics of binding for the agonist-bound receptor with different G proteins, we find the increased affinity of ß1AR for Gs results from its much faster association with the receptor. Consequently, we suggest a kinetic-driven selectivity gate between canonical and secondary coupling which arises from differential favourability of G protein binding to the agonist-bound receptor active state.

MEPIRAPIM-derived synthetic cannabinoids inhibit T-type calcium channels with divergent effects on seizures in rodent models of epilepsy.

Background: T-type Ca2+ channels (Cav3) represent emerging therapeutic targets for a range of neurological disorders, including epilepsy and pain. To aid the development and optimisation of new therapeutics, there is a need to identify novel chemical entities which act at these ion channels. A number of synthetic cannabinoid receptor agonists (SCRAs) have been found to exhibit activity at T-type channels, suggesting that cannabinoids may provide convenient chemical scaffolds on which to design novel Cav3 inhibitors. However, activity at cannabinoid type 1 (CB1) receptors can be problematic because of central and peripheral toxicities associated with potent SCRAs. The putative SCRA MEPIRAPIM and its analogues were recently identified as Cav3 inhibitors with only minimal activity at CB1 receptors, opening the possibility that this scaffold may be exploited to develop novel, selective Cav3 inhibitors. Here we present the pharmacological characterisation of SB2193 and SB2193F, two novel Cav3 inhibitors derived from MEPIRAPIM. Methods: The potency of SB2193 and SB2193F was evaluated in vitro using a fluorometric Ca2+ flux assay and confirmed using whole-cell patch-clamp electrophysiology. In silico docking to the cryo-EM structure of Cav3.1 was also performed to elucidate structural insights into T-type channel inhibition. Next, in vivo pharmacokinetic parameters in mouse brain and plasma were determined using liquid chromatography-mass spectroscopy. Finally, anticonvulsant activity was assayed in established genetic and electrically-induced rodent seizure models. Results: Both MEPIRAPIM derivatives produced potent inhibition of Cav3 channels and were brain penetrant, with SB2193 exhibiting a brain/plasma ratio of 2.7. SB2193 was further examined in mouse seizure models where it acutely protected against 6 Hz-induced seizures. However, SB2193 did not reduce spontaneous seizures in the Scn1a +/- mouse model of Dravet syndrome, nor absence seizures in the Genetic Absence Epilepsy Rat from Strasbourg (GAERS). Surprisingly, SB2193 appeared to increase the incidence and duration of spike-and-wave discharges in GAERS animals over a 4 h recording period. Conclusion: These results show that MEPIRAPIM analogues provide novel chemical scaffolds to advance Cav3 inhibitors against certain seizure types.

Evaluation of fluoropyruvate as nucleophile in reactions catalysed by N-acetyl neuraminic acid lyase variants: scope, limitations and stereoselectivity.

The catalysis of reactions involving fluoropyruvate as donor by N-acetyl neuraminic acid lyase (NAL) variants was investigated. Under kinetic control, the wild-type enzyme catalysed the reaction between fluoropyruvate and N-acetyl mannosamine to give a 90 : 10 ratio of the (3R,4R)- and (3S,4R)-configured products; after extended reaction times, equilibration occurred to give a 30 : 70 mixture of these products. The efficiency and stereoselectivity of reactions of a range of substrates catalysed by the E192N, E192N/T167V/S208V and E192N/T167G NAL variants were also studied. Using fluoropyruvate and (2R,3S)- or (2S,3R)-2,3-dihydroxy-4-oxo-N,N-dipropylbutanamide as substrates, it was possible to obtain three of the four possible diastereomeric products; for each product, the ratio of anomeric and pyranose/furanose forms was determined. The crystal structure of S. aureus NAL in complex with fluoropyruvate was determined, assisting rationalisation of the stereochemical outcome of C-C bond formation.

Structural insights into substrate specificity in variants of N-acetylneuraminic Acid lyase produced by directed evolution.

The substrate specificity of Escherichia coli N-acetylneuraminic acid lyase was previously switched from the natural condensation of pyruvate with N-acetylmannosamine, yielding N-acetylneuraminic acid, to the aldol condensation generating N-alkylcarboxamide analogues of N-acetylneuraminic acid. This was achieved by a single mutation of Glu192 to Asn. In order to analyze the structural changes involved and to more fully understand the basis of this switch in specificity, we have isolated all 20 variants of the enzyme at position 192 and determined the activities with a range of substrates. We have also determined five high-resolution crystal structures: the structures of wild-type E. coli N-acetylneuraminic acid lyase in the presence and in the absence of pyruvate, the structures of the E192N variant in the presence and in the absence of pyruvate, and the structure of the E192N variant in the presence of pyruvate and a competitive inhibitor (2R,3R)-2,3,4-trihydroxy-N,N-dipropylbutanamide. All structures were solved in space group P2(1) at resolutions ranging from 1.65 Å to 2.2 Å. A comparison of these structures, in combination with the specificity profiles of the variants, reveals subtle differences that explain the details of the specificity changes. This work demonstrates the subtleties of enzyme-substrate interactions and the importance of determining the structures of enzymes produced by directed evolution, where the specificity determinants may change from one substrate to another.

Atmospheric formaldehyde monitoring in the Greater Houston area in 2002.

A laser spectrometer based on difference frequency generation (DFG) was deployed for real-time long-term monitoring of HCHO concentrations at an environmental monitoring site located at Deer Park, Texas, in the Greater Houston area. Three HCHO concentration measurements were made during the periods of July 20-31 (period I), August 2-14 (period II), and August 24-September 25 (period III), 2002. In periods I and II, differences in HCHO concentrations are apparent between day and night measurements, with elevated concentrations during daylight hours. Most of the HCHO peak values are less than 20 ppbV except for two intense peaks on August 02 (approximately 25 ppbV) and August 04 (approximately 30 ppbV). The formaldehyde concentration levels in ambient air at the measurement site are produced mainly by the photochemical oxidation of volatile organic compounds (VOCs) caused by intense sunlight during periods I and II. This observation was made based on a comparison with the ozone concentration, solar radiation, temperature, relative humidity, and wind speed data obtained from the Texas Commission on Environmental Quality (TCEQ). During period III, data collected by a time-integrating wet-chemical technique are compared to the data collected by the spectroscopic instrument.