Covalent agonists for studying G protein-coupled receptor activation

Structural studies on G protein-coupled receptors (GPCRs) provide important insights into the architecture and function of these important drug targets. However, the crystallization of GPCRs in active states is particularly challenging, requiring the formation of stable and conformationally homogeneous ligand-receptor complexes. Native hormones, neurotransmitters, and synthetic agonists that bind with low affinity are ineffective at stabilizing an active state for crystallogenesis. To promote structural studies on the pharmacologically highly relevant class of aminergic GPCRs, we here present the development of covalently binding molecular tools activating G_s-, G_i-, and G_q-coupled receptors. The covalent agonists are derived from the monoamine neurotransmitters noradrenaline, dopamine, serotonin, and histamine, and they were accessed using a general and versatile synthetic strategy. We demonstrate that the tool compounds presented herein display an efficient covalent binding mode and that the respective covalent ligand-receptor complexes activate G proteins comparable to the natural neurotransmitters. A crystal structure of the β₂-adrenoreceptor in complex with a covalent noradrenaline analog and a conformationally selective antibody (nanobody) verified that these agonists can be used to facilitate crystallogenesis.One of the major obstacles to the investigation of the structural basis of G protein-coupled receptor (GPCR) activation is the flexibility of their seven-transmembrane core, particularly in the active state (1), and the resulting biochemical instability of the solubilized protein (2, 3). Protein crystallography, the most powerful tool for the study of GPCR structure, requires the formation of stable and conformationally homogeneous ligand-receptor complexes (4). High-affinity agonists with dissociation constants in the low to subnanomolar range and low off-rates facilitate stabilization of the protein throughout the process of expression, purification, and crystallogenesis (2); however, endogenous neurotransmitters usually show poor binding affinity. Low binding affinity with rapid association and dissociation rates leads to conformational heterogeneity that prevents the formation of diffraction-quality crystals. The rapid dissociation rate of agonists also makes it difficult to generate active-state stabilizing proteins, such as the camelid antibodies (nanobodies) that have been used to obtain active-state structures of the β₂-adrenergic receptor (β₂AR) (5) and M2 muscarinic receptor (6).To prevent ligand dissociation, irreversible ligation of electrophilic moieties like halomethylketones, isothiocyanates, Michael acceptors, or aziridinium groups of small-molecule ligands with a suitably positioned nucleophilic residue in the receptor has been used (7–16). However, irreversible ligands often suffer from incomplete cross-linking (15) and reduced receptor activation when covalent binding leads to loss of agonist efficacy (10, 16). Furthermore, their highly electrophilic nature and the abundance of nucleophilic groups in biological systems may lead to a low coupling selectivity (7, 8).Disulfide-based cross-linking approaches (17, 18) offer the advantage that the covalent binding of disulfide-containing compounds is chemoselective for cysteine and enforced by the affinity of the ligand-pharmacophore rather than by the electrophilicity of the cross-linking function (19). We refer to the described ligands as covalent rather than irreversible agonists because cleavage may be promoted by reducing agents and the disulfide transfer process is a reversible chemical reaction in general.Structural information on the target protein facilitates the development of covalent ligand-receptor pairs. Mutation of H93^2.64 in the β₂AR to cysteine introduced an anchor for the disulfide-based covalent agonist FAUC50, which does not perturb ligand binding or the activation of the receptor, and thus enabled, to our knowledge, the first agonist-bound GPCR structure (20). Taking advantage of the high structural homology among aminergic GPCRs, we reasoned that the introduction of cysteine into position X^2.64 should also result in a covalently binding receptor mutant for other aminergic GPCRs.We here report a methodology to generate disulfide-based covalent ligand-receptor pairs to promote structural and functional studies on GPCRs. We demonstrate that even the low-affinity endogenous agonists noradrenaline, dopamine, and serotonin can be converted into efficient covalently binding molecular tools for the β₂AR, the dopamine D₂ receptor (D₂R), and the 5-hydroxytryptamine 2A (5-HT_2A) serotonergic subtype representing G_s-, G_i-, and G_q-coupled GPCRs, respectively. Analogous studies were conducted starting from histamine and the receptor subtype H₁. We applied this strategy to obtain an active-state crystal structure of the β₂AR^H93C and a covalent (nor)adrenaline analog.     

Antigen affinity and antigen dose exert distinct influences on CD4 T-cell differentiation

Cumulative T-cell receptor signal strength and ensuing T-cell responses are affected by both antigen affinity and antigen dose. Here we examined the distinct contributions of these parameters to CD4 T-cell differentiation during infection. We found that high antigen affinity positively correlates with T helper (Th)1 differentiation at both high and low doses of antigen. In contrast, follicular helper T cell (T_FH) effectors are generated after priming with high, intermediate, and low affinity ligand. Unexpectedly, memory T cells generated after priming with very low affinity antigen remain impaired in their ability to generate secondary Th1 effectors, despite being recalled with high affinity antigen. These data challenge the view that only strongly stimulated CD4 T cells are capable of differentiating into the T_FH and memory T-cell compartments and reveal that differential strength of stimulation during primary T-cell activation imprints unique and long lasting T-cell differentiation programs.Following infection, T-cell receptor (TCR) interactions with foreign peptide/MHC (pMHC) drive the rapid clonal expansion and differentiation of T cells into distinct effector subsets specialized against different classes of microbes. An early bifurcation in CD4 T-cell responses results in the generation of T helper (Th)1 effectors, which regulate innate cell microbicidal function and follicular helper T (T_FH) cells, which migrate to B-cell follicles to regulate germinal center (GC) responses and antimicrobial antibody production (1). After pathogen is cleared, T cells undergo a contraction phase during which the majority of effectors die by apoptosis, leaving behind a population of long-lived memory cells to provide protection upon subsequent reinfection. The decision to differentiate into Th1 and T_FH lineages appears to occur very early in the immune response (2, 3). Initial T-cell priming by dendritic cells (DCs) is sufficient to induce fate-committed Th1 and T_FH cells as early as 3 d after infection, whereas maintenance and further expansion of the T_FH compartment depends on T-cell interactions with B cells (2). Similarly, memory T-cell differentiation occurs very early after infection and is critically dependent on B-cell interactions for optimal priming (4, 5). Importantly, CD4 T-cell differentiation is coupled to division, and unlike CD8 T-cell differentiation, requires constant antigen recognition (6, 7).Although the strength of TCR–pMHC interactions has been shown to directly modulate T-cell expansion and clonal dominance within the Th cell compartment (8, 9), how this influences CD4 T-cell fate is not well understood. Cumulative TCR signaling can be influenced by both antigen affinity and antigen dose (10). In terms of proliferation, higher antigen dose can compensate for lower antigen affinity to some extent, but several reports have shown independent effects on T-cell responses both in vitro and in vivo (10–12). These data indicate that antigen affinity and antigen dose may promote qualitatively distinct TCR signals. Recently, modulation of the overall TCR signal by varying either TCR affinity or antigen dose was shown to influence the pattern of effector T-cell differentiation, with higher affinity ligands or higher antigen dose promoting T_FH generation (13–15). However, another study examining high and low avidity CD4 T-cell responses during viral infection found significant differences in Th1 but not T_FH generation (16). Sustained TCR–pMHC interactions have also been shown to promote memory T-cell differentiation, which is associated with increased TCR avidity (17, 18). These studies, however, have focused on the development of the Th1 memory compartment, which is phenotypically and functionally distinct from the T_FH memory compartment (19, 20). Thus, although strong TCR signals resulting from high antigen affinity or high antigen dose can clearly affect the extent and quality of T-cell differentiation, whether or not T cells can discriminate these signals, and how this contributes to T-cell differentiation during infection, has not been determined.To address this question, we infected mice with varying concentrations of Listeria expressing either high or low affinity antigens for the TCR. By normalizing the degree of proliferation induced by high and low affinity antigens we were able to discern distinct influences of antigen affinity and antigen dose on Th cell differentiation. We observed a strong positive correlation between antigen affinity and Th1 differentiation that occurs early and is dose independent. Importantly, high antigen dose does not compensate for the low efficiency of Th1 differentiation induced by low affinity antigen. In contrast, early T_FH effector generation was observed after priming with high, intermediate, and low affinity antigen, but was not maintained at later time points under conditions of low antigen dose. In addition, we found that T cells activated by either high or low affinity antigen are equally capable of memory T-cell differentiation. Surprisingly, memory T cells generated by either low antigen affinity or low antigen dose maintained their biased effector lineages following recall activation with high affinity antigen. These data indicate that differential strength of stimulation during primary T-cell activation can imprint unique and long lasting T-cell differentiation programs.     

Warmed,humidified carbon dioxide insufflation versus standard carbon dioxide in laparoscopic cholecystectomy: a double-blinded randomized controlled trial

Background

During laparoscopic cholecystectomy (LCHE), the insufflation with warmed and humidified carbon dioxide (CO₂) may reduce postoperative pain. The aim of the study was to evaluate the positive effects of heated and humidified carbon dioxide gas on patients with regard to postoperative pain after LCHE.

Patients and methods

This is a prospective, randomized, double-blinded, controlled clinical trial. 148 patients (female = 98, male = 50) scheduled for elective LCHE were randomized into two groups: receiving either heated humidified carbon dioxide, or standard gas. Intraoperative core temperature was measured. The perioperative management was identical for both groups. Postoperative pain intensity was assessed using a visual analog pain scale, and the amount of analgesic consumption was recorded. The postoperative pain management was also standardized and equal for both groups.

Results

67 out of 148 received standard gas (group A), and 81 received warmed, humidified gas (group B). The groups were comparable demographically. The amount of analgesic consumption was recorded. Intraoperative core temperature was significant higher in group B than in group A. Pain was significantly less in group B (p = 0.025) 6 h postoperatively. On the first postoperative day, no significant difference in pain between the two groups was detectable (p = 0.437).

Conclusion

The use of warmed and humidified carbon dioxide during LCHE reduces postoperative pain at the day of operation.     

Liquid crystalline networks of polymethacrylates

LC networks on the basis of methacrylate and dimethacrylate components were synthesized via bulk and precipitation copolymerization. Up to an amount of crosslinking component of 1 mol-% a smetic A phase is observed. The networks with 3 and 5 mol-% only show a nematic phase. From X-ray diffraction an orientation of the smectic phase was observed that derives from surface effects or biaxial swelling. It was found that the orientation can be increased by multiple swelling/deswelling cycles.     

Interferon-alpha-induced changes in tryptophan metabolism. relationship to depression and paroxetine treatment.

BACKGROUND: Tryptophan (TRP) degradation into kynurenine (KYN) by the enzyme, indoleamine-2,3-dioxygenase, during immune activation may contribute to development of depressive symptoms during interferon (IFN)-alpha therapy. METHODS: Twenty-six patients with malignant melanoma were randomly assigned in double-blind fashion to receive either placebo or paroxetine, beginning 2 weeks before IFN-alpha treatment and continuing for the first 12 weeks of IFN-alpha therapy. At treatment initiation and at 2, 4, and 12 weeks of IFN-alpha treatment, measurements of TRP, KYN, and neopterin (a marker of immune activation), were obtained, along with structured assessments of depression, anxiety, and neurotoxicity. RESULTS: Regardless of antidepressant treatment status, all patients exhibited significant increases in KYN, neopterin, and the KYN/TRP ratio during IFN-alpha therapy. Among antidepressant-free patients, patients who developed major depression exhibited significantly greater increases in KYN and neopterin concentrations and more prolonged decreases in TRP concentrations than did nondepressed, antidepressant-free patients. Moreover, in antidepressant-free patients, decreases in TRP correlated with depressive, anxious, and cognitive symptoms, but not neurovegetative or somatic symptoms. No correlations were found between clinical and biological variables in antidepressant-treated patients. CONCLUSIONS: The results suggest that reduced TRP availability plays a role in IFN-alpha-induced depressive symptoms, and paroxetine, although not altering the KYN or neopterin response to IFN-alpha, attenuates the behavioral consequences of IFN-alpha-mediated TRP depletion.     

Transneuronal delivery of designer cytokines: perspectives for spinal cord injury

We recently achieved significant functional recovery after a complete spinal cord injury,allowing previously paralyzed mice to walk again.This was accomplished by a single,unilateral application of an adeno-associated virus(AAV) carrying the cDNA for the designer cytokine hyper-interleukin-6 (hIL-6) into the sensorimotor cortex after spinal cord injury.     

The impact of COVID-19 on home,social, and productivity integration of people with chronic traumatic brain injury or stroke living in the community

Compare community integration of people with stroke or traumatic brain injury (TBI) living in the community before and during the coronavirus severe acute respiratory syndrome coronavirus 2 disease (COVID-19) when stratifying by injury: participants with stroke (G1) and with TBI (G2); by functional independence in activities of daily living: independent (G3) and dependent (G4); by age: participants younger than 54 (G5) and older than 54 (G6); and by gender: female (G7) and male (G8) participants.Prospective observational cohort studyIn-person follow-up visits (before COVID-19 outbreak) to a rehabilitation hospital in Spain and on-line during COVID-19.Community dwelling adults (≥18 years) with chronic stroke or TBI.Community integration questionnaire (CIQ) the total-CIQ as well as the subscale domains (ie, home-CIQ, social-CIQ, productivity CIQ) were compared before and during COVID-19 using the Wilcoxon ranked test or paired t test when appropriate reporting Cohen effect sizes (d). The functional independence measure was used to assess functional independence in activities of daily living.Two hundred four participants, 51.4% with stroke and 48.6% with TBI assessed on-line between June 2020 and April 2021 were compared to their own in-person assessments performed before COVID-19.When analyzing total-CIQ, G1 (d = −0.231), G2 (d = −0.240), G3 (d = −0.285), G5 (d = −0.276), G6 (d = −0.199), G7 (d = −0.245), and G8 (d = −0.210) significantly decreased their scores during COVID-19, meanwhile G4 was the only group with no significant differences before and during COVID-19.In productivity-CIQ, G1 (d = −0.197), G4 (d = −0.215), G6 (d = −0.300), and G8 (d = −0.210) significantly increased their scores, meanwhile no significant differences were observed in G2, G3, G5, and G7.In social-CIQ, all groups significantly decreased their scores: G1 (d = −0.348), G2 (d = −0.372), G3 (d = −0.437), G4 (d = −0.253), G5 (d = −0.394), G6 (d = −0.319), G7 (d = −0.355), and G8 (d = −0.365).In home-CIQ only G6 (d = −0.229) significantly decreased, no significant differences were observed in any of the other groups.The largest effect sizes were observed in total-CIQ for G3, in productivity-CIQ for G6, in social-CIQ for G3 and in home-CIQ for G6 (medium effect sizes).Stratifying participants by injury, functionality, age or gender allowed identifying specific CIQ subtotals where remote support may be provided addressing them.     

Cardiac magnetic resonance imaging in dilated cardiomyopathy in adults��towards identification of myocardial inflammation

Objective

To assess active myocardial inflammation by cardiovascular magnetic resonance (CMR) and endomyocardial biopsy (EMB) amongst adult patients with dilated cardiomyopathy (DCM).

Methods

We evaluated 23 adults with chronic DCM, who had successfully undergone both CMR and EMB within 3.5?±?2.6?days. EMB was considered the gold standard. CMR assessment of myocardial inflammation used the following parameters as recommended by the recently published ??Lake Louise Criteria??: global myocardial oedema, global relative enhancement (RE), and late gadolinium enhancement (LGE). According to ??Lake Louise Criteria??, myocardial inflammation was diagnosed if two or more of the three above-mentioned parameters were positive.

Results

Myocardial inflammation was confirmed by immunohistology in 12 patients (52.2%). Sensitivity, specificity, and diagnostic accuracy of CMR to detect immunohistologically confirmed myocardial inflammation were 75.0%, 72.7%, and 73.9%, respectively. Sensitivity, specificity, and diagnostic accuracy of the individual CMR parameters to detect myocardial inflammation were as follows: global myocardial oedema, 91.7%, 81.8%, and 87.0%, respectively; global RE, 58.3%, 63.6%, and 60.9%, respectively; LGE, 58.3%, 45.4%, and 52.2%, respectively.

Conclusion

Global myocardial oedema was identified as a promising CMR parameter for assessment of myocardial inflammation in patients with DCM. In these patients, global myocardial oedema yielded superior diagnostic performance compared to ??Lake Louise Criteria??.