From natural products chemistry to HTS by NMR: different approaches in drug discovery.

Abstract

The process of drug discovery, by which new potential medicines are discovered, is a long and intricate path that can be approached with many different strategies. For a long time, drugs have been discovered by studying secondary metabolites produced by plants and other organisms mainly as defense or as a way to interact with competitors and mutualists. During recent years, high-throughput screening (HTS) of large compounds libraries has become very popular since it promised to drastically fasten the process of hit identification though requiring consistent initial investments. However, despite the rise of combinatorial chemistry as an integral part of the lead discovery process, natural products still play a major role as starting material for drug discovery. In fact, a 2012 report found that of the 175 small molecule new chemical entities developed as anticancer agents since 1940s, 48.6% were natural derived or semi synthetic derivatives of natural products. During my PhD, I had the chance to work in different laboratories thus gaining knowledge in different fields ranging from natural products chemistry to HTS by NMR, two approaches that converge in the common purpose of identifying and optimizing pharmacologically active molecules. At the department of Pharmacy, in Naples, under the guidance of Prof. D'Auria and Prof. Zampella, I focused on isolation, characterization and synthetic studies of naturally-occurring compounds working on two different projects: isolation and characterization of secondary metabolites from natural sources; and design, synthesis and pharmacological evaluation of dual agonists of bile acid receptors. The investigation of bioactive natural products from the Indian soft coral Sinularia inelegans led to the isolation of a novel norcembranoid, named 5-epi-norcembrenolide, along with twelve known compounds, which were characterized by means of high resolution mass spectrometry and 1D and 2D NMR experiments. All the isolated compounds, including sinuleptolide and 5-epi-sinuleptolide for which anti-inflammatory and antiviral activities had already been reported, are actually under pharmacological investigation to evaluate their antimicrobial activity and binding to nuclear receptors. As regards dual agonists of bile acid receptors, the project involved a preliminary synthetic study in order to produce the reference compound INT-767 and several analogs that were all synthesized starting from the commercially available chenodeoxycholic and ursodeoxycholic acids. Semi synthetic derivatives were tested in vitro and in vivo to evaluate their activity on the bile acid receptors FXR and GP-BAR1. One of the compounds turned out to be the most potent dual FXR/GP-BAR1 agonist so far reported and docking studies elucidated its binding mode in the ligand binding domains of the two receptors revealing the structural requisites to achieve potent GP-BAR1/FXR dual agonism. This study is relevant for further investigations on the functional mechanism of these two receptors and for the design of novel dual GP-BAR1/FXR agonists, providing new opportunities for the treatment of enterohepatic and metabolic disorders. The second part of my thesis was carried out at Sanford-Burnham Medical Research Institute, in San Diego, California, tutored by prof. Pellecchia and Dr. Barile. I applied a new screening technique known as HTS by NMR - which involves a target-based screening of combinatorial libraries using NMR as detection method - to the study of protein-protein interactions. The research conducted on the BIR3 domain of XIAP, an inhibitor of apoptosis protein, resulted in the identification of an inhibitor with a potency comparable to the one of the clinical candidate GDC-0152 by Genentech (Kd~30 nM), which went through a phase I clinical trial as anticancer agent, but endowed with a more favorable thermodynamic profile and a higher binding specificity. The compound was subjected to a process of medicinal optimization and three prodrugs are currently being tested to evaluate their ability of delivering the active molecule inside the cells. At the same time, I studied the ligand binding domain of EphA3, a tyrosine kinase receptor overexpressed in several types of cancer and involved in stem cells maintenance. The EphA3-LBD has been expressed for the first time in a soluble 13C-Met-labeled form. By using HTS by NMR as screening method we identified first small molecule EphA3 binders endowed with good selectivity between the homolog receptors EphA3 and EphA4, and with dissociation constants against EphA3 in the range of 5-10 µM. These binders will be further optimized in order to afford a useful tool to deepen our knowledge of EphA3 pathological role in cancer development.