Allosteric coupling from G protein to the agonist-binding pocket in GPCRs.

G-protein-coupled receptors (GPCRs) remain the primary conduit by which cells detect environmental stimuli and communicate with each other. Upon activation by extracellular agonists, these seven-transmembrane-domain-containing receptors interact with heterotrimeric G proteins to regulate downstream second messenger and/or protein kinase cascades. Crystallographic evidence from a prototypic GPCR, the ß2-adrenergic receptor (ß2AR), in complex with its cognate G protein, Gs, has provided a model for how agonist binding promotes conformational changes that propagate through the GPCR and into the nucleotide-binding pocket of the G protein α-subunit to catalyse GDP release, the key step required for GTP binding and activation of G proteins. The structure also offers hints about how G-protein binding may, in turn, allosterically influence ligand binding. Here we provide functional evidence that G-protein coupling to the ß2AR stabilizes a 'closed' receptor conformation characterized by restricted access to and egress from the hormone-binding site. Surprisingly, the effects of G protein on the hormone-binding site can be observed in the absence of a bound agonist, where G-protein coupling driven by basal receptor activity impedes the association of agonists, partial agonists, antagonists and inverse agonists. The ability of bound ligands to dissociate from the receptor is also hindered, providing a structural explanation for the G-protein-mediated enhancement of agonist affinity, which has been observed for many GPCR­G-protein pairs. Our data also indicate that, in contrast to agonist binding alone, coupling of a G protein in the absence of an agonist stabilizes large structural changes in a GPCR. The effects of nucleotide-free G protein on ligand-binding kinetics are shared by other members of the superfamily of GPCRs, suggesting that a common mechanism may underlie G-protein-mediated enhancement of agonist affinity.

Oxygen depletion speeds and simplifies diffusion in HeLa cells.

Many cell types undergo a hypoxic response in the presence of low oxygen, which can lead to transcriptional, metabolic, and structural changes within the cell. Many biophysical studies to probe the localization and dynamics of single fluorescently labeled molecules in live cells either require or benefit from low-oxygen conditions. In this study, we examine how low-oxygen conditions alter the mobility of a series of plasma membrane proteins with a range of anchoring motifs in HeLa cells at 37°C. Under high-oxygen conditions, diffusion of all proteins is heterogeneous and confined. When oxygen is reduced with an enzymatic oxygen-scavenging system for ≥ 15 min, diffusion rates increase by > 2-fold, motion becomes unconfined on the timescales and distance scales investigated, and distributions of diffusion coefficients are remarkably consistent with those expected from Brownian motion. More subtle changes in protein mobility are observed in several other laboratory cell lines examined under both high- and low-oxygen conditions. Morphological changes and actin remodeling are observed in HeLa cells placed in a low-oxygen environment for 30 min, but changes are less apparent in the other cell types investigated. This suggests that changes in actin structure are responsible for increased diffusion in hypoxic HeLa cells, although superresolution localization measurements in chemically fixed cells indicate that membrane proteins do not colocalize with F-actin under either experimental condition. These studies emphasize the importance of controls in single-molecule imaging measurements, and indicate that acute response to low oxygen in HeLa cells leads to dramatic changes in plasma membrane structure. It is possible that these changes are either a cause or consequence of phenotypic changes in solid tumor cells associated with increased drug resistance and malignancy.

A multicenter, randomized, open-label, 2-arm parallel study to compare the pharmacokinetics, safety and tolerability of AVT02 administered subcutaneously via prefilled syringe or autoinjector in healthy adults.

BACKGROUND: AVT02 is an adalimumab biosimilar, with bioequivalence previously established along with clinical similarity. This study assessed the pharmacokinetic (PK) similarity of a single dose of 100 mg/mL AVT02 administered via prefilled syringe (PFS) or autoinjector (AI). RESEARCH DESIGN AND METHODS: In this open-label, 2-arm, parallel-group study, healthy adults were randomized 1:1 to receive one 40 mg (100 mg/mL) dose of AVT02 subcutaneously via PFS (N = 102) or AI (N = 105). Primary PK parameters (Cmax, AUC0-t and AUC0-inf) were evaluated up to Day 64 of the study. Secondary PK parameters, safety, tolerability and immunogenicity were also assessed. RESULTS: The 90% CIs for the ratio of geometric least squares means were contained within the pre-specified 80-125% equivalence margins for the primary PK parameters, demonstrating bioequivalence of AVT02 when administered by PFS or AI. The incidence of treatment-emergent adverse events was comparable between the two groups, with a low frequency of injection site reactions observed. Immunogenicity profiles were also similar between the two groups. CONCLUSION: Bioequivalence was demonstrated for a single dose of AVT02 administered via PFS or AI. These results will help to increase availability of devices for patients, enabling treatment choice and flexibility.

Assessment of real-life patient handling experience of AVT02 administered subcutaneously via autoinjector in patients with moderate to severe active rheumatoid arthritis: an open-label, single-arm clinical trial, then an extension phase of AVT02 administered with a prefilled syringe.

BACKGROUND: This study investigated the ability of patients, naïve to adalimumab treatment and self-injection with an autoinjector (AI), to successfully self-administer AVT02, an adalimumab biosimilar, using a custom, ergonomic AI (Alvotech hf., Reykjavik, Iceland). RESEARCH DESIGN AND METHODS: This was a single-arm, open-label study, consisting of an 8-week active period and 48-week extension phase. Patients with moderate to severe rheumatoid arthritis (RA) self-administered 40 mg AVT02 subcutaneously via AI in the active period, followed by prefilled syringe in the extension phase. The primary endpoint was the percentage of successful self-injections up to Week 8. Usability and robustness of the AI were evaluated in the active period; safety, efficacy, pharmacokinetic and immunogenicity data were assessed throughout the study. RESULTS: The AI success rate was 100%. No handling events were noted up to Week 8. Both Ctrough measurements and immunogenicity profile were in line with expectations from previous studies, with no unexpected safety signals. CONCLUSIONS: This study demonstrated that AVT02-AI can be successfully and reliably used for repeated self-injections of AVT02 by moderate to severe RA patients, despite no previous experience of adalimumab self-administration. The extension phase provides long-term efficacy and safety data for AVT02 in RA. STUDY IDENTIFIER: NCT04224194.

Subunit stabilization and polyethylene glycolation of cocaine esterase improves in vivo residence time.

No small-molecule therapeutic is available to treat cocaine addiction, but enzyme-based therapy to accelerate cocaine hydrolysis in serum has gained momentum. Bacterial cocaine esterase (CocE) is the fastest known native enzyme that hydrolyzes cocaine. However, its lability at 37°C has limited its therapeutic potential. Cross-linking subunits through disulfide bridging is commonly used to stabilize multimeric enzymes. Herein we use structural methods to guide the introduction of two cysteine residues within dimer interface of CocE to facilitate intermolecular disulfide bond formation. The disulfide-crosslinked enzyme displays improved thermostability, particularly when combined with previously described mutations that enhance stability (T172R-G173Q). The newly modified enzyme yielded an extremely stable form of CocE (CCRQ-CocE) that retained greater than 90% of its activity after 41 days at 37°C, representing an improvement of more than 4700-fold over the wild-type enzyme. CCRQ-CocE could also be modified by polyethylene glycol (PEG) polymers, which improved its in vivo residence time from 24 to 72 h, as measured by a cocaine lethality assay, by self-administration in rodents, and by measurement of inhibition of cocaine-induced cardiovascular effects in rhesus monkeys. PEG-CCRQ elicited negligible immune response in rodents. Subunit stabilization and PEGylation has thus produced a potential protein therapeutic with markedly higher stability both in vitro and in vivo.

Interactions of amyloid-ß peptides on lipid bilayer studied by single molecule imaging and tracking.

The amyloid-ß peptides (Aß40 and Aß42) feature prominently in the synaptic dysfunction and neuronal loss associated with Alzheimer's disease (AD). This has been proposed to be due either to interactions between Aß and cell surface receptors affecting cell signaling, or to the formation of calcium-permeable channels in the membrane that disrupt calcium homeostasis. In both mechanisms the cell membrane is the primary cellular structure with which Aß interacts. Aß concentrations in human bodily fluids are very low (pM-nM) rendering studies of the size, composition, cellular binding sites and mechanism of action of the oligomers formed in vivo very challenging. Most studies, therefore, have utilized Aß oligomers prepared at micromolar peptide concentrations, where Aß forms oligomeric species which possess easily observable cell toxicity. Such toxicity has not been observed when nM concentrations of peptide are used in the experiment highlighting the importance of employing physiologically relevant peptide concentrations for the results to be of biological significance. In this paper single-molecule microscopy was used to monitor Aß oligomer formation and diffusion on a supported lipid bilayer at nanomolar peptide concentrations. Aß monomers, the dominant species in solution, tightly associate with the membrane and are highly mobile whereas trimers and higher-order oligomers are largely immobile. Aß dimers exist in a mixture of mobile and immobile states. Oligomer growth on the membrane is more rapid for Aß40 than for the more amyloidogenic Aß42 but is largely inhibited for a 1:1 Aß40:Aß42 mixture. The mechanism underlying these Aß40-Aß42 interactions may feature in Alzheimer's pathology.