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Digital archive of theses discussed at the University of Pisa


Thesis etd-05222023-100841

Thesis type
Tesi di dottorato di ricerca
Thesis title
Design, synthesis, and biological evaluation of allosteric and bitopic ligands for novel strategies targeting G protein-coupled receptors: a special focus on cannabinoid receptors
Academic discipline
Course of study
tutor Prof.ssa Manera, Clementina
correlatore Prof. Laprairie, Robert
  • allosteric ligands
  • bitopic modulation
  • cannabinoid receptors
  • G protein-coupled receptors
Graduation session start date
Release date
G-protein coupled receptors (GPRCs) continue to be the most attractive family of molecular targets for therapeutic drug discovery. Compounds targeting the same site as the endogenous ligand (i.e., orthosteric binding site) have always dominated the GPCR pharmacology landscape, prioritizing the most basic aspects of ligand recognition, which are the agonism and the antagonism. However, there are several obstacles that the orthosteric ligands must overcome, including lower selectivity, limited clinical efficacy, and unfavorable effects on receptor modulation. Over the years, GPCR research has had a sort of renaissance owing new structural insights into ligand recognition which led to the validation of the presence of allosteric binding sites, which are structurally, conformationally, and usually functionally different than the corresponding receptor orthosteric pockets but equally druggable. The allosteric modulators are particularly promising since they lack many of the drawbacks of orthosteric ligands being endowed with greater subtype selectivity, the ability to switch the GPCR response after ligand binding to a specific signaling pathway, fewer on-target side effects, and an overall "safer" pharmacological profile. The repertoire of GPCRs has been furtherly expanded with the advent of a new class of ligands, termed bitopic or dualsteric ligands, which simultaneously target orthosteric and allosteric sites. The idea behind the development of bitopic ligands is to combine the activation properties of orthosteric ligands with the higher selectivity profile of allosteric ligands. The focus of my PhD thesis revolves around these two paradigms in GPCR drug discovery, allosteric and bitopic modulation, which have been applied to cannabinoid receptors (CBR, CB1R and CB2R). These receptors have been long recognized to modulate numerous physiological processes and to be intimately involved in many pathological states. Therefore, this PhD thesis branches into two lines of research:
1. Design, synthesis, and pharmacological evaluation of CBR bitopic ligands. As first goal, my thesis led to the identification of the first hetero-bivalent bitopic CB2R ligand, whose bitopic nature has been demonstrated by extensive biochemical studies. This new compound displayed anti-inflammatory activity in a human microglial cell inflammatory model, antinociceptive activity in vivo in an experimental mouse model of neuropathic pain, and pro-autophagic effects in vitro in an Alzheimer’s Disease model. This discovery has spurred me to design and synthesize a new generation of potential CB2R bitopic ligands, which has culminated in the discovery of the second CB2R hetero-bivalent bitopic ligand beyond providing new insights about the dual orthosteric/allosteric stimulation of CB2Rs. Subsequently, the concept of bitopic modulation has been translated to the other subtype cannabinoid receptor by developing a series of potential bitopic CB1R ligands. Based on binding and functional studies, it has been possible to make assumptions about the bitopic profile of some of them.
2. Design, synthesis, and pharmacological evaluation of CB2R allosteric modulators. A further objective was the rational design and synthesis of a new series of 2-oxo-1,2-dihydropyridine-3-cycloheptanecarboxamide derivatives as analogues of the well-established CB2R PAM EC21a. Competition binding experiments suggested a CB2R PAM behavior for one of them. Collected results will be useful to deepen the knowledge of structural requirements for CB2R allosteric modulation.