Solution structure of a neurotrophic ligand bound to FKBP12 and its effects on protein dynamics

Abstract

The structure of a recently reported neurotrophic ligand, 3-(3-pyridyl)-1-propyl(2S)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidinecarboxylate, in complex with FKBP12 was determined using heteronuclear NMR spectroscopy. The inhibitor exhibits a binding mode analogous to that observed for the macrocycle FK506, used widely as an immunosuppressant, with the prolyl ring replacing the pipecolyl moiety and the amide bond in a trans conformation. However, fewer favourable protein±ligand interactions are detected in the structure of the complex, suggesting weaker binding compared with the immunosuppressant drug. Indeed, a micromolar dissociation constant was estimated from the NMR ligand titration profile, in contrast to the previously published nanomolar inhibition activity. Although the inhibitor possesses a remarkable structural simplicity with respect to FK506, 15 N relaxation studies show that it induces similar effects on the protein dynamics, stabilizing the conformation of solvent-exposed residues which are important for mediating the interaction of immunophilin/ ligand complexes with molecular targets and potentially for the transmission of the neurotrophic action of FKBP12 inhibitors.Keywords: FKBP12; ligand binding; nerve regeneration; NMR structure; protein dynamics.For several years there has been major interest in the development of inhibitors of the FK506 binding protein (FKBP12) because of their potential immunosuppressive effects [1±4]. Recently, FKBP12 was also identified as a target for the promotion of neurite outgrowth [5] and neurotrophic and neuroprotective effects could be associated with the action of different FKBP12 ligands [6,7]. These findings have extended the therapeutic potential of FKBP12-binding drugs to the treatment of nerve injuries and neurodegenerative disorders such as Alzheimer's and Parkinson's diseases.While it is well known that the immunosuppressive effects of FKBP12 are mediated by the interaction of the drug± immunophilin complex with calcineurin, the mechanism of the neuronal activity of FKBP12 has not yet been elucidated. However, it has been shown that the neurotrophic properties of immunophilin ligands are independent of their immunosuppressive action [8] and much effort has been directed toward the design of FKBP12-binding molecules, which are neurotrophic but nonimmunosuppressant, for clinical use [9,10]. One of the most promising compounds was discovered by scientists at Guilford Pharmaceuticals (1, Fig. 1; alternative name: GPI-1046). This ligand was synthesized within a series of small molecules that mimic the FKBP12-binding portion of FK506, a well-known immunosuppressant drug (Fig. 1), but lack the structural requirements for calcineurin inhibition. Among this series, 1 was found to be remarkably potent in inhibiting the peptidyl-prolyl cis±trans isomerase (PPIase) activity of FKBP12 and in promoting nerve regeneration both in vitro and in vivo [11].Given the therapeutic relevance of neurotrophic nonpeptidic molecules, we investigated the interaction of 1 and FKB...

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