Insight into GEBR-32a: Chiral Resolution, Absolute Configuration and Enantiopreference in PDE4D Inhibition.

Valeria Cavalloro,Francesca Vasile,Olga Bruno,Federica Rapetti,Emilio Parisini,Simona Collina,Stefano Donini,Marta S Semrau,Paola Storici,Daniela Rossi,Chiara Brullo,Archimede Torretta,Luca Pignataro,Katia Russo

doi:10.3390/molecules25040935

Abstract

Alzheimer’s disease is the most common type of dementia, affecting millions of people worldwide. One of its main consequences is memory loss, which is related to downstream effectors of cyclic adenosine monophosphate (cAMP). A well-established strategy to avoid cAMP degradation is the inhibition of phosphodiesterase (PDE). In recent years, GEBR-32a has been shown to possess selective inhibitory properties against PDE type 4 family members, resulting in an improvement in spatial memory processes without the typical side effects that are usually correlated with this mechanism of action. In this work, we performed the HPLC chiral resolution and absolute configuration assignment of GEBR-32a. We developed an efficient analytical and semipreparative chromatographic method exploiting an amylose-based stationary phase, we studied the chiroptical properties of both enantiomers and we assigned their absolute configuration by 1H-NMR (nuclear magnetic resonance). Lastly, we measured the IC50 values of both enantiomers against both the PDE4D catalytic domain and the long PDE4D3 isoform. Results strongly support the notion that GEBR-32a inhibits the PDE4D enzyme by interacting with both the catalytic pocket and the regulatory domains.

Highlights

Nowadays, more than 30 million people live with dementia worldwide, and their number is estimated to grow to 65.7 million in 2030 and 115.4 million in 2050 [1]
We have shown previously that the catechol portion of GEBR-32a binds the catalytic domain, while the tail of the molecule extends into the S pocket, where it makes water-mediated contacts with the surrounding residues, and develops toward the entrance of the active site (PDB 6FDC) [10]
Despite the fact that the structure of the PDE4D catalytic domain in complex with GEBR-32a has been determined using the GEBR-32a racemic mixture, the crystallographic analysis provided evidence of a preferential interaction of S-enantiomer with the enzyme (Figure 1B) (PDB 6FDC). This observation suggested that (S)-GEBR-32a may be the eutomer and provided the rational for further careful investigation on the resolved enantiomers. In line with these considerations, we report on the chiral resolution of racemic GEBR-32a, on the absolute configuration (AC) assignment and on the comparison of the inhibitory potency of the two enantiomers against both the PDE4D catalytic domain and the full-length PDE4D3 isoform

Summary

Introduction

More than 30 million people live with dementia worldwide, and their number is estimated to grow to 65.7 million in 2030 and 115.4 million in 2050 [1]. Alzheimer’s disease (AD) represents the most common form of dementia and it can be considered a challenge under both a medical and an economical point of view [3]. Phosphodiesterase (PDE) inhibition has long been considered a promising strategy to avoid the hydrolysis of cAMP and, to treat memory deficits [6]. Inhibitors of the PDE type 4 (PDE4) family have been identified as effective pro-cognitive drugs [7,8], and different drug discovery programs aim at developing an effective PDE4 inhibitor to treat memory disorders. It has been demonstrated that PDE4D is more relevant to cognition enhancement than other PDE4 isoforms [9]. The quest for potent and selective PDE4D inhibitors could open new avenues for memory performance enhancement. By combining X-ray crystallography, molecular dynamic simulations and enzymatic assays, members of our research team have recently provided a structural and functional characterization of several

Methods

Results

Conclusion