Di-aryl guanidinium derivatives: Towards improved α2-Adrenergic affinity and antagonist activity

Compounds with excellent receptor engagement displaying alpha(2)-AR antagonist activity are useful not only for therapeutic purposes (e.g. antidepressants), but also to help in the crystallization of this particular GPCR. Therefore, based on our broad experience in the topic, we have prepared eighteen di-aryl (phenyl and/or pyridin-2-yl) mono- or di-substituted guanidines and 2-aminoimidazolines. The in vitro alpha(2)-AR binding affinity experiments in human brain tissue showed the advantage of a 2-aminoimidazolinium cation, a di-arylmethylene core, a conformationally locked pyridin-2-yl-guanidine and a di-substituted guanidinium to achieve good alpha(2)-AR engagement. After different in vitro [S-35]GTP gamma S binding experiments in human prefrontal cortex tissue, it was possible to identify that compounds 7a, 7b and 7c were alpha(2)-AR partial agonist, whereas 8h was a potent alpha(2)-AR antagonist. Docking and MD studies with a model of alpha(2A)-AR and two crystal structures suggest that antagonism is achieved by compounds carrying a di-substituted guanidine which substituent occupy a pocket adjacent to TM5 without engaging S200(5.42) or S204(5.46), and a mono-substituted cationic group, which favorably interacts with E94(2.65). (c) 2020 Elsevier Masson SAS. All rights reserved.Thanks are given to the School of Chemistry at Trinity College Dublin (M.McM.) and to the Irish Research Council (A.K. -GOIPG/2014/457-and H.B.M. -GOIPG/2017/834-) for postgraduate support. This study was also supported by the Ministerio de Economia y Competitividad of Spain (SAF2013-48586-R) and the Basque Government (IT1211-19). The authors would like to thank the staff members of the Basque Institute of Legal Medicine for their cooperation in the study

Nerve growth factor induces neurite outgrowth of PC12 cells by promoting Gβγ-microtubule interaction

Background: Assembly and disassembly of microtubules (MTs) is critical for neurite outgrowth and differentiation. Evidence suggests that nerve growth factor (NGF) induces neurite outgrowth from PC12 cells by activating the receptor tyrosine kinase, TrkA. G protein-coupled receptors (GPCRs) as well as heterotrimeric G proteins are also involved in regulating neurite outgrowth. However, the possible connection between these pathways and how they might ultimately converge to regulate the assembly and organization of MTs during neurite outgrowth is not well understood. Results: Here, we report that Gβγ, an important component of the GPCR pathway, is critical for NGF-induced neuronal differentiation of PC12 cells. We have found that NGF promoted the interaction of Gβγ with MTs and stimulated MT assembly. While Gβγ-sequestering peptide GRK2i inhibited neurite formation, disrupted MTs, and induced neurite damage, the Gβγ activator mSIRK stimulated neurite outgrowth, which indicates the involvement of Gβγ in this process. Because we have shown earlier that prenylation and subsequent methylation/demethylation of γ subunits are required for the Gβγ-MTs interaction in vitro, small-molecule inhibitors (L-28 and L-23) targeting prenylated methylated protein methyl esterase (PMPMEase) were tested in the current study. We found that these inhibitors disrupted Gβγ and ΜΤ organization and affected cellular morphology and neurite outgrowth. In further support of a role of Gβγ-MT interaction in neuronal differentiation, it was observed that overexpression of Gβγ in PC12 cells induced neurite outgrowth in the absence of added NGF. Moreover, overexpressed Gβγ exhibited a pattern of association with MTs similar to that observed in NGF-differentiated cells. Conclusions: Altogether, our results demonstrate that βγ subunit of heterotrimeric G proteins play a critical role in neurite outgrowth and differentiation by interacting with MTs and modulating MT rearrangement. Electronic supplementary material The online version of this article (doi:10.1186/s12868-014-0132-4) contains supplementary material, which is available to authorized users