We sequentially analyzed the serum IgG response against normal mouse brain during experimental autoimmune encephalomyelitis in SJL/J mice injected with CFA,
Bordetella pertussis toxin (BPT) and ...proteolipid protein 139–151 peptide, compared with mice that received CFA and BPT or were uninjected. Dynamic changes were observed from day 0 to day 28 in the 3 groups. Six highly discriminant antigenic bands (
κ
=
0.974) were identified. Three non-myelin proteins were characterized (mitochondrial aconitase hydratase 2, phosphoglycerate mutase 1, brain specific pyruvate deshydrogenase). The IgG response against two of them was less frequent in EAE whereas it was associated with multiple sclerosis in our previous work.
The two main myelin proteolipids, PLP (30 kDa) and DM-20 (25 kDa), differ by an internal deletion in DM-20. The deleted fragment, of 35 amino acids (116-150), corresponds to the major hydrophilic ...domain of PLP. Fluorescence anisotropy experiments using diphenylhexatriene as a fluorescent probe were performed to detect the phase separation induced by these two proteolipids in multilamellar vesicles of binary composition. We found that in vesicles composed of 30% L-alpha-PS and 70% DPPC, the PLP boundary layer contained about 18 motionally restricted phospholipids, almost exclusively L-alpha-PS. On the contrary, the DM-20 boundary layer contained only 14 to 15 phospholipids, with a composition no different from that of the bulk vesicle. In mixtures of DMPG and DPPC, the selectivity of PLP for the acidic phospholipid DMPG was maintained, but was lower than that observed for L-alpha-PS. We assume that this selectivity of PLP stems mainly from electrostatic interactions between the charged residues of the 116-150 fragment, deleted in DM-20, and the acidic phospholipids. These results suggest that fragment 116-150 may play a specific role in the interaction of PLP with the lipid bilayer of the myelin membrane.
Super agonists produce greater functional responses than endogenous agonists in the same assay, and their unique pharmacology is the subject of increasing interest and debate. We propose that ...receptor residence time and the duration of receptor signaling contribute to the pharmacology of super agonism. We have further characterized the novel β2 adrenoceptor agonist C26 (7-(R)-2-((1R,2R)-2-benzyloxycyclopentylamino)-1-hydroxyethyl-4-hydroxybenzothiazolone), which displays higher intrinsic activity than the endogenous ligand adrenaline in cAMP accumulation, β-arrestin-2 recruitment, and receptor internalization assays. C26 recruited β-arrestin-2, and internalized the Green Fluorescent Protein (GFP)-taggedβ2 adrenoceptor at a slow rate, with half-life (t1/2) values of 0.78 ± 0.1 and 0.78 ± 0.04 hours, respectively. This was compared with 0.31 ± 0.04 and 0.34 ± 0.01 hours for adrenaline-mediated β-arrestin-2 recruitment and GFP-β2 internalization, respectively. The slower rate for C26 resulted in levels of β-arrestin-2 recruitment increasing up to 4-hour agonist incubation, at which point the intrinsic activity was determined to be 124.3 ± 0.77% of the adrenaline response. In addition to slow functional kinetics, C26 displayed high affinity with extremely slow receptor dissociation kinetics, giving a receptor residence half-life of 32.7 minutes at 37°C, which represents the slowest dissociation rate we have observed for any β2 adrenoceptor agonist tested to date. In conclusion, we propose that the gradual accumulation of long-lived active receptor complexes contributes to the increased intrinsic activity of C26 over time. This highlights the need to consider the temporal aspects of agonist binding and signaling when characterizing ligands as super agonists.
The synthesis of a series of indacaterol analogues in which each of the three structural regions of indacaterol are modified in a systematic manner is described. Evaluation of the affinity of these ...analogues for the β(2)-adrenoceptor identified the 3,4-dihydroquinolinone and 5-n-butylindanyl analogues to demonstrate the most similar profiles to indacaterol. An α-methyl aminoindane analogue was discovered to be 25-fold more potent than indacaterol, and functional studies revealed an atypical β(2)-adrenoceptor activation profile for this compound consistent with that of a slowly dissociating 'super agonist'.
The synthesis of a series of indacaterol analogues in which each of the three structural regions of indacaterol are modified in a systematic manner is described. Evaluation of the affinity of these ...analogues for the β2-adrenoceptor identified the 3,4-dihydroquinolinone and 5-n-butylindanyl analogues to demonstrate the most similar profiles to indacaterol. An α-methyl aminoindane analogue was discovered to be 25-fold more potent than indacaterol, and functional studies revealed an atypical β2-adrenoceptor activation profile for this compound consistent with that of a slowly dissociating ‘super agonist’.
The synthesis of a series of indacaterol analogues in which each of the three structural regions of indacaterol are modified in a systematic manner is described. Evaluation of the affinity of these ...analogues for the beta2-adrenoceptor identified the 3,4-dihydroquinolinone and 5-n-butylindanyl analogues to demonstrate the most similar profiles to indacaterol. An alpha-methyl aminoindane analogue was discovered to be 25-fold more potent than indacaterol, and functional studies revealed an atypical beta2-adrenoceptor activation profile for this compound consistent with that of a slowly dissociating 'super agonist'.
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