Implications of dopamine D3 receptor for glaucoma: in-silico and in-vivo studies

Glaucoma is a progressive optic neuropathy and it is considered by the World Health Organization to be the cause of 12% of visual impairment and 2% of blindness. Glaucoma is characterized by alterations of optic disc and visual field. High intraocular pressure (IOP) is the main risk factor of glaucoma. IOP reduction represents the first step in the management of glaucoma which is eventually followed by laser surgery of the trabecular meshwork (TM) and glaucoma-filtering surgery. Currently, there are five main classes of approved ocular hypotensive drugs: beta-blockers, carbonic anhydrase inhibitors, prostaglandin analogs, sympathomimetics and miotics. However, there is still the need to have more potent medications available for this disease. In the panorama of pharmacological targets for regulation of IOP, there are some interesting G protein coupled receptors (GPCRs) such as dopaminergic receptors. The work of the present thesis has been focused on GPCRs and in particular on dopamine D3 receptor as pharmacological target for ocular hypotensive drugs. Cabergoline, bromocriptine, cianergoline and legotrile, classical D2 receptor agonists, have been shown to decrease intraocular pressure. D3 receptor belongs to the D2 class of dopaminergic receptors, along with D2 and D4 receptor. It shares high sequence homology and identity with D2 receptor and several efforts have been carried out in order to design selective ligands for either D3 or D2 subtype. Drug design and discovery, based on structure based approach, need the knowledge of the tertiary structure of the target protein. In 2010 the x-ray structure of human D3 receptor (mutated hD3-lysozime chimera) was solved, then this structure was used to carry out the homology modeling of wild-type (wt) hD3 and hD2L receptors. The homology models of these receptors were not able to discriminate selective ligands by a molecular docking study, thus these structures have been subjected to optimization by means of molecular dynamics in a water-membrane environment. After optimization the structures differentiated in the binding pockets and have been validated, strengthen the validity and reliability of the in silico approach. A similar computational approach was carried out in order to study the structure differences between the D3 receptors and 5HT1A, 5HT2A-C receptors, known to be involved in regulation of intraocular pressure. The role of D3 receptor activation by cabergoline in lowering IOP was confirmed in C57BL/6J wt and D3-/- mice, using a pharmacological approach along with D3 gene deletion. Cabergoline was not effective in ocular hypertensive D3-/- mice, whereas exerted a greater and longer hypotensive effect in ocular hypertensive wt mice, in comparison to normotensive animals. The in silico approach, validated for D3 and D2L receptors, has been used to model and optimize the structures of 5HT1A, 5HT2A-B-C receptors which are other putative ocular targets of cabergoline. In silico results showed that cabergoline binds in a similar way into pockets of D3 and 5HT2A-C and it has higher affinity for D3 receptor in comparison to serotonergic receptors, according to experimental affinity data. Moreover docking revealed that binding of cabergoline into D3 and 5HT1A receptors is associated with a better desolvation energy in comparison to 5HT2A C binding. The structure-based computational approach hereby adopted was able to build, refine, and validate structure models of homologous dopaminergic and serotonergic receptors that may be of interest for structure-based drug discovery of ligands, with dopaminergic selectivity or with multi-pharmacological profile, potentially useful to treat optic neuropathies such as glaucoma. Finally, the present work represents an excellent example of successful integration of two different approaches to biomedical research, in silico and in vivo, which are not in contrast but complementary.