3.8 ‘Focal species’ – can this well-known concept in higher-tier risk assessments be an appropriate approach for solitary bees?

Bumble bees and solitary bees have to be considered in addition to honey bees regarding environmental pollinator risk assessments. For solitary bees it is proposed to use Osmia cornuta (LATR., 1805) or O. bicornis (L., 1758) as test organisms. Whereas for higher-tier assessments, semi-field testing of solitary bees has been proved to obtain sound results, experience from current Osmia field studies show that exposure of adults and larvae is not necessarily the case due to the pronounced polylectic feeding behaviour. As an alternative refinement option the ‘focal species’ concept may be used, which is well-known as a kind of first step for higher tier bird and mammal risk assessments. This approach as it applies to solitary bees, as well as its needs, refinement options and limitations is presented.Bumble bees and solitary bees have to be considered in addition to honey bees regarding environmental pollinator risk assessments. For solitary bees it is proposed to use Osmia cornuta (LATR., 1805) or O. bicornis (L., 1758) as test organisms. Whereas for higher-tier assessments, semi-field testing of solitary bees has been proved to obtain sound results, experience from current Osmia field studies show that exposure of adults and larvae is not necessarily the case due to the pronounced polylectic feeding behaviour. As an alternative refinement option the ‘focal species’ concept may be used, which is well-known as a kind of first step for higher tier bird and mammal risk assessments. This approach as it applies to solitary bees, as well as its needs, refinement options and limitations is presented

Biased Signaling of CCL21 and CCL19 Does Not Rely on N-Terminal Differences, but Markedly on the Chemokine Core Domains and Extracellular Loop 2 of CCR7

Chemokine receptors play important roles in the immune system and are linked to several human diseases. Targeting chemokine receptors have so far shown very little success owing to, to some extent, the promiscuity of the immune system and the high degree of biased signaling within it. CCR7 and its two endogenous ligands display biased signaling and here we investigate the differences between the two ligands, CCL21 and CCL19, with respect to their biased activation of CCR7. We use bystander bioluminescence resonance energy transfer (BRET) based signaling assays and Transwell migration assays to determine (A) how swapping of domains between the two ligands affect their signaling patterns and (B) how receptor mutagenesis impacts signaling. Using chimeric ligands we find that the chemokine core domains are central for determining signaling outcome as the lack of β-arrestin-2 recruitment displayed by CCL21 is linked to its core domain and not N-terminus. Through a mutagenesis screen, we identify the extracellular domains of CCR7 to be important for both ligands and show that the two chemokines interact differentially with extracellular loop 2 (ECL-2). By using in silico modeling, we propose a link between ECL-2 interaction and CCR7 signal transduction. Our mutagenesis study also suggests a lysine in the top of TM3, K1303.26, to be important for G protein signaling, but not β-arrestin-2 recruitment. Taken together, the bias in CCR7 between CCL19 and CCL21 relies on the chemokine core domains, where interactions with ECL-2 seem particularly important. Moreover, TM3 selectively regulates G protein signaling as found for other chemokine receptors.publishe

Molecular dynamics-guided discovery of an ago-allosteric modulator for GPR40/FFAR1

The long-chain fatty acid receptor FFAR1/GPR40 binds agonists in both an interhelical site between the extracellular segments of transmembrane helix (TM)-III and TM-IV and a lipid-exposed groove between the intracellular segments of these helices. Molecular dynamics simulations of FFAR1 with agonist removed demonstrated a major rearrangement of the polar and charged anchor point residues for the carboxylic acid moiety of the agonist in the interhelical site, which was associated with closure of a neighboring, solvent-exposed pocket between the extracellular poles of TM-I, TM-II, and TM-VII. A synthetic compound designed to bind in this pocket, and thereby prevent its closure, was identified through structure-based virtual screening and shown to function both as an agonist and as an allosteric modulator of receptor activation. This discovery of an allosteric agonist for a previously unexploited, dynamic pocket in FFAR1 demonstrates both the power of including molecular dynamics in the drug discovery process and that this specific, clinically proven, but difficult, antidiabetes target can be addressed by chemotypes different from existing ligands

Biased signaling and allosteric modulation of G protein-coupled receptor 183 - a 7TM receptor also known as Epstein-Barr virus -induced gene 2

Abstract Background and Purpose The G protein-coupled receptor Epstein Barr virus-induced gene 2 (EBI2, also known as GPR183) is activated by oxysterols and plays a pivotal role for proper B cell migration during immune responses. While the molecular basis of agonist binding has been addressed in several studies, the concept of biased signaling of EBI2 has not been explored. Experimental Approach We investigated the effects of the EBI2 endogenous agonist 7α,25-OHC on G protein-dependent and -independent pathways as well as sodium ion allosterism using site-directed mutagenesis and functional studies. Moreover, we generated a homology model of EBI2 to investigate the structural basis of the allosteric modulation by sodium. Key Results We show that residue N114, located in the middle of TM-III at position III:11/3.35, functions as an efficacy switch. Thus, substituting N114 with an alanine (N114A) completely abolishes Gαi activation by 7α,25-OHC even though the specific binding of the [3H]-7,25-OHC radioligand increases. In contrast, the N114A mutant is still able to recruit β-arrestin and even with enhanced the potency (16-fold) compared to EBI2 wt. Underscoring the key role of N114, we also show that sodium has an negative allosteric effect on oxysterol binding and that this is mediated via N114. This is further supported by molecular modelling of the ion binding site based on a EBI2 homology model. Conclusions and Implications Collectively, our data points to N114 as a key residue for EBI2 signaling controlling the balance between G protein-dependent and -independent pathways and facilitating sodium binding

Molecular dynamics-based identification of binding pathways and two distinct high-affinity sites for succinate in succinate receptor 1/GPR91

SUCNR1 is an auto- and paracrine sensor of the metabolic stress signal succinate. Using unsupervised molecular dynamics (MD) simulations (170.400 ns) and mutagenesis across human, mouse, and rat SUCNR1, we characterize how a five-arginine motif around the extracellular pole of TM-VI determines the initial capture of succinate in the extracellular vestibule (ECV) to either stay or move down to the orthosteric site. Metadynamics demonstrate low-energy succinate binding in both sites, with an energy barrier corresponding to an intermediate stage during which succinate, with an associated water cluster, unlocks the hydrogen-bond-stabilized conformationally constrained extracellular loop (ECL)-2b. Importantly, simultaneous binding of two succinate molecules through either a “sequential” or “bypassing” mode is a frequent endpoint. The mono-carboxylate NF-56-EJ40 antagonist enters SUCNR1 between TM-I and -II and does not unlock ECL-2b. It is proposed that occupancy of both high-affinity sites is required for selective activation of SUCNR1 by high local succinate concentrations.</p