Generation of nanobodies acting as silent and positive allosteric modulators of the α7 nicotinic acetylcholine receptor

Abstract

The α7 nicotinic acetylcholine receptor (nAChR), a potential drug target for treating cognitive disorders, mediates communication between neuronal and non-neuronal cells. Although many competitive antagonists, agonists, and partial-agonists have been found and synthesized, they have not led to effective therapeutic treatments. In this context, small molecules acting as positive allosteric modulators binding outside the orthosteric, acetylcholine, site have attracted considerable interest. Two single-domain antibody fragments, C4 and E3, generated through alpaca immunization with cells expressing the extracellular domain of the human α7-nAChR, are herein described. They bind to the α7-nAChR but not to the other major nAChR subtypes, α4β2 and α3β4. E3 acts as a slowly associating type I positive allosteric modulator, strongly potentiating the acetylcholine-elicited currents, while not significantly altering the desensitization of the receptor. An E3-E3 bivalent construct shows similar potentiating properties but displays very slow dissociation kinetics conferring quasi-irreversible properties. Whereas, C4 does not alter the receptor function, but fully inhibits the E3-evoked potentiation, showing it is a silent allosteric modulator competing with E3 binding. Both nanobodies do not compete with α-bungarotoxin, localizing at an allosteric extracellular binding site away from the orthosteric site. The functional differences of each nanobody, as well as the alteration of functional properties through nanobody modifications indicate the importance of this extracellular site. The nanobodies will be useful for pharmacological and structural investigations; moreover, they, along with the extracellular site, have a direct potential for clinical applications.

An original potentiating mechanism revealed by the cryoEM structures of the human α7 nicotinic receptor in complex with nanobodies

“…Immunolabelling of the extracellular C-terminal Rho1D4 tag show that the MIR and glycosylation mutants are expressed at the cell surface, while mutants of the Nter helix are not. Immunolabelling with E3 and C4, using nanobodies fused to the heavy chain fragment of a human IgG (constructs detailed in 9 , Figure S4), show clear labelling of cells expressing the WT and glycosylation mutant, but no labelling of the MIR mutant. This confirms that the MIR is essential for the nanobody binding, while the glycosylation on Asn23 is not mandatory for E3 high-affinity binding.…”

“…Interestingly, the unsharpened map of the E3/C4- bound structures suggests that glycosylation trees could substantially mask the orthosteric site, thereby plausibly preventing the generation of binders targeting this site and favouring a more apical epitope. Of note, the epitope of C4 and E3 shows weak sequence conservation among the various nAChR subtypes, accounting for their high specificity to the α7-nAChR with no observed binding by immunofluorescence to the α4β2 and α3β4 nAChRs 9 .…”

“…Yields of production were improved around 2-fold in the a7∆ICDcryo construct as compared a7FLcryo. In parallel, we expressed and purified C4 and E3 nanobodies in a monomeric form as described previously 9 (Figure S1 and S3)…”

“…We further investigated the contribution of specific a7 motifs to nanobody binding by mutagenesis of a7FLcryo. On the one hand, we mutated the charged residues of the Nter helix of a7 (R4Q K5Q K9Q triple mutant and E9Q K12Q N13A triple mutant), and on the other hand we replaced the MIR residues to the aligned MIR of the a3-nAChR which does not bind C4 and E3 9 . Finally, we prevented N-linked glycosylation by mutating the serine of the Asn-X-Ser motif (Ser25Ala).…”

“…In a previous study, we immunized alpacas with cells expressing an a7/5HT3 chimera to generate highly potent and a7-specific modulators 9 . Among the resulting camelid antibody fragments called nanobodies, the one called E3 acts as a Type I PAM.…”